Synergy between HDAC and PARP Inhibitors on Proliferation of a Human Anaplastic Thyroid...
7
Research Article Synergy between HDAC and PARP Inhibitors on Proliferation of a Human Anaplastic Thyroid Cancer-Derived Cell Line Federica Baldan, 1 Catia Mio, 1 Lorenzo Allegri, 1 Cinzia Puppin, 1 Diego Russo, 2 Sebastiano Filetti, 3 and Giuseppe Damante 1,4 1 Department of Medical and Biological Sciences, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy 2 Department of Health Sciences, University “Magna Graecia” of Catanzaro, 88100 Catanzaro, Italy 3 Department of Internal Medicine and Medical Specialties, University of Roma “La Sapienza”, 00198 Rome, Italy 4 Institute of Medical Genetics, University Hospital “S. Maria della Misericordia”, 33100 Udine, Italy Correspondence should be addressed to Giuseppe Damante; [email protected] Received 3 June 2014; Accepted 9 September 2014 Academic Editor: Ginesa Garc´ ıa-Rost´ an Copyright © 2015 Federica Baldan et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Anaplastic thyroid carcinoma (ATC) is a very aggressive human malignancy, having a marked degree of invasiveness and no features of thyroid differentiation. It is known that either HDAC inhibitors or PARP inhibitors have antiproliferative effects on thyroid cancer cells. erefore, in this study the possible synergy between the two types of compounds has been investigated. e ATC-derived cell line SW1736 has been treated with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the PARP inhibitor PJ34, alone or in combination. In terms of cell viability, the combination index value was always lower than 1 at various tested dosages, indicating, therefore, synergy in a wide range of doses for both compounds. Synergy was also observed in induction of apoptosis. In terms of thyroid-specific gene expression, synergy was observed for TSHR mRNA levels but not for NIS, TTF1, TTF2, and PAX8 mRNA levels. Altogether, these data suggest that the combined use of HDAC and PARP inhibitors may be a useful strategy for treatment of ATC. 1. Introduction yroid cancer is the most common endocrine malignancy, and its incidence has continuously increased in the last three decades all over the world [1]. yroid cancers are typically classified as papillary (PTC), follicular (FTC), medullary (MTC), or anaplastic (ATC) carcinomas. ATC is one of the most aggressive human malignancies. ese tumors have a marked degree of invasiveness and extensive necrosis and there are no features of thyroid differ- entiation [2]. e mechanisms underlying the development of ATCs are incompletely understood. Currently, available therapy for ATCs includes chemotherapy, radiotherapy, and surgery [3]. Nonetheless, patients with ATC still have a median survival of 5 months and less than 20% survive 1 year. Furthermore early tumor dissemination results in 20–50% percent of patients having distant metastases and 90% having adjacent tissue invasion on presentation [2]. HDAC inhibitors (HDACIs) are a group of small mole- cules that promote gene transcription by chromatin remod- eling and have been extensively studied as potential drugs for treating cancer. Luong et al. have established that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), already FDA-approved for the treatment of several neoplastic diseases [4, 5], has antitumor activities against thyroid cancer [6]. Inhibitors of the poly(ADP-ribose) polymerases (PARPs) family are currently being evaluated as potential anticancer drugs. PARPs have a key role in a large number of cell viability processes as DNA repair, genome integrity, regulation of transcription, proliferation, and apoptosis [7]. Different independent studies have demonstrated that the combination of both HDAC inhibitors and PARP inhibitors with other drugs could result in synergistic effects on their antitumor activities if compared to those observed using single agents [8, 9]. Hindawi Publishing Corporation International Journal of Endocrinology Volume 2015, Article ID 978371, 7 pages http://dx.doi.org/10.1155/2015/978371
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Transcript of Synergy between HDAC and PARP Inhibitors on Proliferation of a Human Anaplastic Thyroid...
Research ArticleSynergy between HDAC and PARP Inhibitors on Proliferation ofa Human Anaplastic Thyroid Cancer-Derived Cell Line
Federica Baldan1 Catia Mio1 Lorenzo Allegri1 Cinzia Puppin1 Diego Russo2
Sebastiano Filetti3 and Giuseppe Damante14
1Department of Medical and Biological Sciences University of Udine Piazzale Kolbe 4 33100 Udine Italy2Department of Health Sciences University ldquoMagna Graeciardquo of Catanzaro 88100 Catanzaro Italy3Department of Internal Medicine and Medical Specialties University of Roma ldquoLa Sapienzardquo 00198 Rome Italy4Institute of Medical Genetics University Hospital ldquoS Maria della Misericordiardquo 33100 Udine Italy
Correspondence should be addressed to Giuseppe Damante giuseppedamanteuniudit
Received 3 June 2014 Accepted 9 September 2014
Academic Editor Ginesa Garcıa-Rostan
Copyright copy 2015 Federica Baldan et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Anaplastic thyroid carcinoma (ATC) is a very aggressive humanmalignancy having amarked degree of invasiveness and no featuresof thyroid differentiation It is known that eitherHDAC inhibitors or PARP inhibitors have antiproliferative effects on thyroid cancercells Therefore in this study the possible synergy between the two types of compounds has been investigated The ATC-derivedcell line SW1736 has been treated with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the PARP inhibitor PJ34alone or in combination In terms of cell viability the combination index value was always lower than 1 at various tested dosagesindicating therefore synergy in a wide range of doses for both compounds Synergy was also observed in induction of apoptosisIn terms of thyroid-specific gene expression synergy was observed for TSHRmRNA levels but not for NIS TTF1 TTF2 and PAX8mRNA levels Altogether these data suggest that the combined use of HDAC and PARP inhibitors may be a useful strategy fortreatment of ATC
1 Introduction
Thyroid cancer is the most common endocrine malignancyand its incidence has continuously increased in the last threedecades all over the world [1] Thyroid cancers are typicallyclassified as papillary (PTC) follicular (FTC) medullary(MTC) or anaplastic (ATC) carcinomas
ATC is one of the most aggressive human malignanciesThese tumors have a marked degree of invasiveness andextensive necrosis and there are no features of thyroid differ-entiation [2] The mechanisms underlying the developmentof ATCs are incompletely understood Currently availabletherapy for ATCs includes chemotherapy radiotherapy andsurgery [3] Nonetheless patients with ATC still have amedian survival of 5 months and less than 20 survive 1 yearFurthermore early tumor dissemination results in 20ndash50percent of patients having distant metastases and 90 havingadjacent tissue invasion on presentation [2]
HDAC inhibitors (HDACIs) are a group of small mole-cules that promote gene transcription by chromatin remod-eling and have been extensively studied as potential drugsfor treating cancer Luong et al have established that theHDAC inhibitor suberoylanilide hydroxamic acid (SAHA)already FDA-approved for the treatment of several neoplasticdiseases [4 5] has antitumor activities against thyroid cancer[6]
Inhibitors of the poly(ADP-ribose) polymerases (PARPs)family are currently being evaluated as potential anticancerdrugs PARPs have a key role in a large number of cell viabilityprocesses as DNA repair genome integrity regulation oftranscription proliferation and apoptosis [7]
Different independent studies have demonstrated that thecombination of both HDAC inhibitors and PARP inhibitorswith other drugs could result in synergistic effects on theirantitumor activities if compared to those observed usingsingle agents [8 9]
Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2015 Article ID 978371 7 pageshttpdxdoiorg1011552015978371
2 International Journal of Endocrinology
Current cancer therapy should satisfy requirements fortargeted elimination of cancer cells simultaneously with life-compatible adverse effects [10] One of the main tenetsof cancer therapeutics is that combinations of anticanceragents with different targets or different mechanisms ofaction and varied normal tissue toxicities will produce bettertherapeutic outcomes [11] by decreasing single drugs dosesand minimizing or slowing drug resistance development Inthis study we investigated the possible use of SAHA anHDAC inhibitor and PJ34 a PARP inhibitor in combinationin a cellular model of anaplastic thyroid cancer
2 Material and Methods
21 Cell Line and Treatments SW1736 human cell linederived from anaplastic thyroid cancer was grown inRPMI 1640 medium (EuroClone Milan Italy) supple-mented with 10 fetal bovine serum (Gibco InvitrogenMilan Italy) and 50mgmL gentamicin (Gibco Invitro-gen Milan Italy) in a humidified incubator (5 CO
2in
air at 37∘C) The identity of SW1736 cells was demon-strated by evaluating the following STRs D16S539 THO1vWA D3S1358 D21S11 and D18S51 the obtained genotypewas identical to those reported by the CLS Cell LinesService GmbH (httpwwwcell-lines-servicede) Culturedcells were treated with the following agents either alone orin combination as described in the text SAHA (1ndash4120583M inDMSO) (Cayman Chemical Michigan USA) and PJ34 (5ndash30 120583M in nuclease-free water) (Merck Chemicals Ltd) Theseconcentrations are consistent with those utilized for in vivostudies [12 13] All treatments were done for 72 hours
22 Cell Viability To test cell viability CellTiter-Blue CellViability assay (Promega Milano Italy) was used accordingto the manufacturerrsquos instructions Cells were seeded onto96-well plates in 200120583L medium The next day the growthmedium was replaced with fresh medium containing DMSOas vehicle (untreated cultures) or SAHA and PJ34 alone orin combination For each treatment quadruplicate wells wereused
23 Combination Index (CI Value) Effects of drugs combina-tion used in this study were evaluated using the combinationindex equation based on the multiple drug-effect equation ofChou-Talalay [14 15] In all cases where CI value could bedetermined the following diagnostic rule was applied CI lt1 indicates synergism CI = 1 indicates additive effect andCI gt 1 indicates antagonism The analysis was obtained onCompuSyn software (ComboSyn Inc Paramus USA)
24 Annexin V Staining Cells were treated with appropriatedrugs as described and then they were washed with coldPBS transferred to a polystyrene round-bottomed flow tube(Falcon Becton Dickinson Franklin Lakes NY USA) andresuspended in 195 120583L of 1times binding buffer (BB-10mMHepesNaOH pH 74 140mM NaCl and 25mM CaCl
2)
To the suspension 5 120583L of fluorescein-conjugated AnnexinV (Annexin V-FITC Bender Med Systems Wien Austria)was added and samples were incubated for 10min at room
temperature After washing cells were resuspended in 190120583Lof BB inwhich 10 120583L of propidium iodide stock solution (finalconcentration 1120583gmL) was added Flow cytometry analysiswas done on CyAN Dako Cytomation using the Summitsoftware
25 Quantitative RT-PCR Total RNA from cell line treatedfor 72 h with SAHA 1 120583M and PJ34 15 120583M alone or in com-bination was extracted with RNeasy mini kit according tomanufacturerrsquos instructions (Qiagen Hilden Germany)500 ng of total RNA was reversely transcripted to cDNAusing random exaprimers and MMLV reverse transcriptase(Invitrogen) Real-time PCRs were performed using TaqManUniversal PCR Master Mix (Applied Biosystems) with theABI Prism 7300 Sequence Detection Systems (Applied Biosy-stems Foster City CA USA) The ΔΔCT method by meansof the SDS software (Applied Biosystems) was used to calculate the mRNA levels Oligonucleotide primers were purchased from Life Technologies and were as follows 120573-actin51015840 primer CGAGCGCGGCTACAGCTT 120573-actin probeACCACCACGGCCGAGCGG and 120573-actin 31015840 primer TCC-TTAATGTCACGCACGATTT PAX8 51015840 primer CAACAG-CACCCTGGACGAC PAX8 31015840 primer AGGGTGAGTGAG-GATCTGCC and PAX8 probe CTGACCCCTTCCAAC-ACGCCACTG NIS Hs00166567 m1 TTF1 Hs00968940 m1TTF2 Hs00916085 s1 and TSHR Hs01053846 m1
26 Statistical Analysis Cell viability apoptosis and mRNAlevels were expressed as means plusmn SD and significances wereanalyzed with the t test performed with GraphPAD Softwarefor Science (San Diego CA USA)
3 Results
In a first set of experiments single effects of the HDACinhibitor SAHA and the PARP inhibitor PJ34 on cell viabilityof the human anaplastic thyroid cancer-derived cell lineSW1736 were investigated Cell viability was assessed aftertreatment with different doses of SAHA and PJ34 for 72hours (Figure 1) Both SAHA and PJ34 alone inhibited cellproliferation in a dose-dependent manner however at theutilized doses SAHA seemed to have a greater effect causinga more significant decrease in cell viability compared to cellstreated by PJ34 Thus both compounds alone were able toinhibit proliferation of SW1736 cells We then tested synergyof the two compounds by measuring CI values of differentdrug combinations according to the Chou-Talalay equation[14 15] As indicated in Table 1 all combinations used showeda very high decrease in cell growth compared to untreatedcells (always the CI values were lower than 1) Our resultsindicated that SAHA and PJ34 have a synergic effect indecreasing cell proliferation in a quite high range of utilizeddoses
We focused on the SAHA 1 120583M and PJ34 15 120583M combi-nation which generated the lowest CI value As representedin Figure 2 the treatment with SAHA 1 120583M had only a lighteffect on SW1736 viability while PJ34 15120583Mreduced cell pro-liferation more effectively Using these doses in combination
International Journal of Endocrinology 3
0
20
40
60
80
100
120
SAHA (120583M) PJ34 (120583M)
Cel
l via
bilit
y (a
u)
1 2 3 4 5 10 15 30
lowastlowastlowast
lowast
mdash
Figure 1 Effects of HDAC and PARP inhibitors on SW1736 cellviability Cells were treated for 72 h with SAHA (1120583Mndash4120583M) orPJ34 (5120583Mndash30 120583M) and CellTiter-Blue Cell Viability assay wasperformed as described in Section 2 Bars indicate the percentageof viable cells versus controls (untreated cells) and represent meansplusmn SD of three experiments lowast indicates values significantly differentcompared to control
Table 1 Combination index data for SAHA and PJ34 combination
Dose SAHA(120583M)
Dose PJ34(120583M)
Combination effect( cell viability)lowast CI value
10 50 26 02697910 100 37 06023510 150 12 01387810 300 13 01847320 50 18 03312320 100 17 03341420 150 19 04011120 300 11 02534130 50 13 03650430 100 26 07532030 150 16 04717530 300 14 04541340 50 17 06076340 100 17 06278540 150 10 04031840 300 13 053865lowastMean value of four replicates In each condition standard deviation is lessthan 10
we obtained a strong lowering of cell viability almost 90compared to untreated cells
Subsequently we evaluated apoptosis of SW1736 by mea-surement of Annexin V by fluorescence-activated cell sortingafter treatments with SAHA 1 120583M and PJ34 15 120583M alone orin combination (Figure 3(a)) The percentage of apoptoticcells (AnnexinV-positivePI-negative) was 198with SAHA1 120583M and 199 with PJ34 15 120583Mwhile after the combinationtreatment it reached 816 (Figure 3(b))
We next tested if synergy between the two compoundswas present on expression of several thyroid-specific genesEffects on mRNA levels were evaluated by real-time PCR
0
20
40
60
80
100
120
Cel
l via
bilit
y (a
u)
mdash
lowast
SAHA 1120583M PJ34 15120583M15120583M
SAHA 1120583M+ PJ34
Figure 2 Effect of HDAC and PARP inhibitors combinationon SW1736 cell viability Cells were treated for 72 h with SAHA1 120583M and PJ34 15 120583M alone or in combination CellTiter-BlueCell Viability assay was performed as described in Section 2 Barsindicate the percentage of viable cells versus controls (untreatedcells) and represent means plusmn SD of four experiments lowast indicatesvalues significantly different compared to all other conditions
Among all genes analyzed synergy between the two com-pounds was detectable only for TSHR gene (Figure 4) The72-hour treatment with SAHA 1 120583M induced a marked effecton TSHR mRNA expression while PJ34 15 120583M did not haveany remarkable effect compared to the control cells Howeverby using the two drugs in combination we obtained a strongeffect on TSHR mRNA levels significantly higher than allother conditions with an increment of 36-fold of inductioncompared to the control
4 Discussion
Developing a pharmacological treatment against cancer thecentral issue consists in increasing therapeutic index andat the same time limiting development of resistance Onesolution is to combine multiple drugs that act synergisticallyand in fact a large number of ongoing clinical trials areinvestigating the effects of combined therapy against differ-ent types of cancer [16 17] In developing new strategiesfor treatment of anaplastic thyroid cancer combinations ofHDAC inhibitors and other drugs have been attempted [18ndash24] However neither in preclinical nor in clinical settings ofthyroid cancer treatment the combination between HDACand PARP inhibitors has been investigated It is increasinglyclear that cancer is not only caused by genetic factors but canalso be considered a epigenetics disease [25] and epigeneticenzymes can therefore be considered as novel therapeutictargets Accordingly both HDAC and PARP inhibitors canbe considered as epigenetic drugs Combinations of HDACand PARP inhibitors have been tested in different kindsof neoplastic diseases By using hepatocellular carcinomacell lines a synergistic inhibition of cell growth by SAHAand the PARP inhibitor olaparib has been demonstrated[26] Inhibition of cell proliferation was associated withincrease of apoptosis levels accumulation of DNA damageand modification of the cAMP signaling pathway Combined
4 International Journal of Endocrinology
SAHA + PJ34
104
103
102
101
100
104103102101100
104103102101100 104103102101100
104103102101100
Annexin V Annexin V
Annexin V Annexin V
PI
104
103
102
101
100
PI
104
103
102
101
100
PI
104
103
102
101
100
PI
Q1 Q2
Q3Q4
3764 2763
3424 049
ControlQ1
3315
Q2
4257
Q3198
Q42230
SAHA
Q1
3017
Q2
3159
Q3
199
Q4
3625
PJ34Q1
2571Q2
4146
Q3
816Q4
2467
(a)
0123456789
Apop
tosis
leve
ls (
)
mdash SAHA 1120583M PJ34 15120583M15120583M
SAHA 1120583M+ PJ34
(b)
Figure 3 Combination of SAHA and PJ34 induces apoptosis in SW1736 cell line In (a) Annexin V (FITC) and propidium iodide (PI)staining of anaplastic thyroid cancer cell line after 72 h treatment with SAHA 1120583M PJ34 15 120583Malone or in combination In (b) representationof Annexin V-positivePI-negative cells
International Journal of Endocrinology 5
05
1015202530354045 TSHR
0
02
04
06
08
1
12 TTF1
0
05
1
15
2
25 TTF2
0
05
1
15
2NIS
002040608
1121416
PAX8m
RNA
leve
ls (a
u)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mdash
mRN
A le
vels
(au
)
lowast
lowast
SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583M SAHA+ PJ34 15120583M
1120583Mmdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583MSAHA+ PJ34 15120583M
1120583M
SAHA+ PJ34 15120583M
1120583M
Figure 4 Expression levels of PAX8 NIS TTF1 TTF2 and TSHR genes in SW1736 cell line RNA extraction and real-time PCR are describedin Section 2 For each gene the results were normalized against 120573-actin and expressed in arbitrary unit (2minusΔCt) Each bar represents the meanvalue of three different determinations For each bar standard deviation is not above 10of each valuelowast indicates values significantly differentcompared to all other conditions
effects of SAHA and PJ34 on leukemia cell lines have beeninvestigated [27] Also in that study synergistic effects onproliferation inhibition and apoptosis increase have beenobserved Recently synergy between SAHA and olaparib hasbeen observed even in ovarian cancer cell lines [28] We haverecently obtained similar effects on breast cancer cell lines(unpublished data)
Effects of HDAC and PARP inhibitors alone have beenpreviously investigated by our group We have shown thatHDAC inhibitors affect cell proliferation and expression ofvarious genes in several thyroid cancer cell lines [29 30]Moreover Lavarone et al have recently shown that PJ34inhibits cell growth and increases NIS expression in variousthyroid cancer cell lines [31] In this research we demonstratethat HDAC and PARP inhibitors have a synergistic effect
on proliferation of a human anaplastic thyroid-derived cellline Thus synergy between these two classes of compoundsappears to be a common phenomenon in cancer cell linesof various origins underlying the potential role of thesecombinations as an interesting strategy for cancer therapyOur obtained CI values indicate that synergy between SAHAand PJ34 occurs in a wide range of doses suggesting that thecombined effect could probably be observed also in vivo
In addition to the impact on cell proliferation wehave investigated effects of the SAHA-PJ34 combination onthyroid-specific genes expression Regarding thyroid-specifictranscription factors the SAHA-PJ34 combination induces aTTF1 slight decrease a TTF2 slight increase and no changein PAX8 Such behavior indicates that the control of thesegenes expression occurs through distinct mechanisms This
6 International Journal of Endocrinology
view agrees with previous studies in which control of thyroid-specific transcription factors expression has been investigated[32]
Synergy between SAHA and PJ34 in increasing mRNAlevels of TSHR was observed The TSHR is localized in theplasmamembrane and thus it has been proposed as a targetto direct therapeutic compounds into thyroid cancer cells[33ndash36] Our data would suggest that the combined use ofHDAC and PARP inhibitors may facilitate such approach
Differently from TSHR NIS gene expression is reducedby SAHA alone and in combination with PJ34 Such adifferent effect between these genes expression is not unex-pected TSHR and NIS have a different regulation in termsof gene expression It is well known for example that duringtumorigenesis NIS is one of the earliest downregulated geneswhile TSHR is among the latest ones [37 38]
In conclusion considering our data on cell proliferationand gene expression altogether the combined use of HDACand PARP inhibitors can be a useful strategy for ATCtreatment Preclinical in vivo studies are required to validatesuch a possibility
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was supported by grants to Giuseppe Damantefrom Associazione Italiana per la Ricerca sul Cancro (AIRC)(Project no IG 10296)
References
[1] G Pellegriti F Frasca C Regalbuto S Squatrito and R Vign-eri ldquoWorldwide increasing incidence of thyroid cancer updateon epidemiology and risk factorsrdquo Journal of Cancer Epidemiol-ogy vol 2013 Article ID 965212 10 pages 2013
[2] J P OrsquoNeill and A R Shaha ldquoAnaplastic thyroid cancerrdquo OralOncology vol 49 no 7 pp 702ndash706 2013
[3] S L Kojic S S Strugnell and S M Wiseman ldquoAnaplasticthyroid cancer a comprehensive review of novel therapyrdquoExpert Review of Anticancer Therapy vol 11 no 3 pp 387ndash4022011
[4] P A Marks and W-S Xu ldquoHistone deacetylase inhibitorspotential in cancer therapyrdquo Journal of Cellular Biochemistryvol 107 no 4 pp 600ndash608 2009
[5] D Russo G Damante E Puxeddu C Durante and S FilettildquoEpigenetics of thyroid cancer and novel therapeutic targetsrdquoJournal of Molecular Endocrinology vol 46 no 3 pp R73ndashR812011
[6] Q T Luong JOrsquoKelly GD Braunstein JMHershman andHP Koeffler ldquoAntitumor activity of suberoylanilide hydroxamicacid against thyroid cancer cell lines in vitro and in vivordquo Cli-nical Cancer Research vol 12 no 18 pp 5570ndash5577 2006
[7] A Chen ldquoPARP inhibitors its role in treatment of cancerrdquoChinese Journal of Cancer vol 30 no 7 pp 463ndash471 2011
[8] A J Frew R W Johnstone and J E Bolden ldquoEnhancing theapoptotic and therapeutic effects of HDAC inhibitorsrdquo CancerLetters vol 280 no 2 pp 125ndash133 2009
[9] G Papeo E Casale A Montagnoli and A Cirla ldquoPARPinhibitors in cancer therapy an updaterdquo Expert Opinion onTherapeutic Patents vol 23 no 4 pp 503ndash514 2013
[10] B Brodska A Holoubek P Otevrelova and K KuzelovaldquoCombined treatment with low concentrations of decitabineand saha causes cell death in leukemic cell lines but not innormal peripheral blood lymphocytesrdquo BioMed Research Inter-national vol 2013 Article ID 659254 11 pages 2013
[11] B A Teicher ldquoCombinations of PARP hedgehog and HDACinhibitors with standard drugsrdquo Current Opinion in Pharmacol-ogy vol 10 no 4 pp 397ndash404 2010
[12] M A Smith and P Houghton ldquoA proposal regarding reportingof in vitro testing resultsrdquo Clinical Cancer Research vol 19 no11 pp 2828ndash2833 2013
[13] C Szabo A Biser R Benko E Bottinger and K SusztakldquoPoly(ADP-ribose) polymerase inhibitors ameliorate nephro-pathy of type 2 diabetic Leprdbdb micerdquo Diabetes vol 55 no11 pp 3004ndash3012 2006
[14] T-C Chou and P Talalay ldquoQuantitative analysis of dose-effectrelationships the combined effects of multiple drugs or enzymeinhibitorsrdquo Advances in Enzyme Regulation vol 22 pp 27ndash551984
[15] T C Chou and P Talalay ldquoAnalysis of combined drug effectsa new look at a very old problemrdquo Trends in PharmacologicalSciences vol 4 pp 450ndash454 1983
[16] E Kim M Matsuse V Saenko et al ldquoImatinib enhances doce-taxel-induced apoptosis through inhibition of nuclear factor-120581Bactivation in anaplastic thyroid carcinoma cellsrdquo Thyroid vol22 no 7 pp 717ndash724 2012
[17] J-C Ahn R Biswas and P-S Chung ldquoCombination withgenistein enhances the efficacy of photodynamic therapyagainst human anaplastic thyroid cancer cellsrdquo Lasers in Surgeryand Medicine vol 44 no 10 pp 840ndash849 2012
[18] I Clinckspoor L Verlinden L Overbergh et al ldquo125-Dihy-droxyvitamin D3 and a superagonistic analog in combinationwith paclitaxel or suberoylanilide hydroxamic acid have potentantiproliferative effects on anaplastic thyroid cancerrdquo Journal ofSteroid Biochemistry and Molecular Biology vol 124 no 1-2 pp1ndash9 2011
[19] M G Catalano R Poli M Pugliese N Fortunati and GBoccuzzi ldquoValproic acid enhances tubulin acetylation andapoptotic activity of paclitaxel on anaplastic thyroid cancer celllinesrdquo Endocrine-Related Cancer vol 14 no 3 pp 839ndash8452007
[20] D Russo C Durante S Bulotta et al ldquoTargeting histone deace-tylase in thyroid cancerrdquo Expert Opinion onTherapeutic Targetsvol 17 no 2 pp 179ndash193 2013
[21] P Brest S Lassalle V Hofman et al ldquoMiR-129-5p is requiredfor histone deacetylase inhibitor-induced cell death in thyroidcancer cellsrdquo Endocrine-Related Cancer vol 18 no 6 pp 711ndash719 2011
[22] T Kondo S L Asa and S Ezzat ldquoEpigenetic dysregulationin thyroid neoplasiardquo Endocrinology and Metabolism Clinics ofNorth America vol 37 no 2 pp 389ndash400 2008
[23] C S Mitsiades V Poulaki C McMullan et al ldquoNovel histonedeacetylase inhibitors in thetreatment of thyroid cancerrdquo Clini-cal Cancer Research vol 11 no 10 pp 3958ndash3965 2005
International Journal of Endocrinology 7
[24] M G Catalano N Fortunati M Pugliese et al ldquoValproic acida histone deacetylase inhibitor enhances sensitivity to doxo-rubicin in anaplastic thyroid cancer cellsrdquo Journal of Endocrinol-ogy vol 191 no 2 pp 465ndash472 2006
[25] C A Iacobuzio-Donahue ldquoEpigenetic changes in cancerrdquoAnnual Review of Pathology vol 4 pp 229ndash249 2009
[26] J-X Zhang D-Q Li A R He et al ldquoSynergistic inhibitionof hepatocellular carcinoma growth by cotargeting chromatinmodifying enzymes and poly (ADP-ribose) polymerasesrdquoHep-atology vol 55 no 6 pp 1840ndash1851 2012
[27] E Jasek M Gajda G J Lis M Jasinska and J A LitwinldquoCombinatorial effects of PARP inhibitor PJ34 and histonedeacetylase inhibitor vorinostat on leukemia cell linesrdquo Anti-cancer Research vol 34 no 4 pp 1849ndash1856 2014
[28] P A Konstantinopoulos A J Wilson J Saskowski E Wassand D Khabele ldquoSuberoylanilide hydroxamic acid (SAHA)enhances olaparib activity by targeting homologous recombina-tion DNA repair in ovarian cancerrdquo Gynecologic Oncology vol133 no 3 pp 599ndash606 2014
[29] C Puppin F DAurizio A V DElia et al ldquoEffects of histoneacetylation on NIS promoter and expression of thyroid-specifictranscription factorsrdquo Endocrinology vol 146 no 9 pp 3967ndash3974 2005
[30] C Puppin N Passon J M Hershman et al ldquoCooperativeeffects of SAHA andVPA onNIS gene expression and prolifera-tion of thyroid cancer cellsrdquo Journal ofMolecular Endocrinologyvol 48 no 3 pp 217ndash227 2012
[31] E Lavarone C Puppin N Passon S Filetti D Russo and GDamante ldquoThePARP inhibitor PJ34modifies proliferation NISexpression and epigenetic marks in thyroid cancer cell linesrdquoMolecular and Cellular Endocrinology vol 365 no 1 pp 1ndash102013
[32] G Damante G Tell and R di Lauro ldquoA unique combinationof transcription factors controls differentiation of thyroid cellsrdquoProgress in Nucleic Acid Research andMolecular Biology vol 66pp 307ndash356 2000
[33] J C Morris ldquoStructure and function of the TSH receptor itssuitability as a target for radiotherapyrdquoThyroid vol 7 no 2 pp253ndash258 1997
[34] S El-Kaissi and J R Wall ldquoTargeting the thyrotropin receptorin thyroid diseaserdquo Expert Opinion on Therapeutic Targets vol16 no 7 pp 719ndash727 2012
[35] D Paolino D Cosco M Gaspari et al ldquoTargeting the thy-roid gland with thyroid-stimulating hormone (TSH)-nano-liposomesrdquo Biomaterials vol 35 no 25 pp 7101ndash7109 2014
[36] M DrsquoAgostino M Sponziello C Puppin et al ldquoDifferentexpression of TSH receptor and NIS genes in thyroid cancerrole of epigeneticsrdquo Journal of Molecular Endocrinology vol 52no 2 pp 121ndash131 2013
[37] V Lazar J M Bidart B Caillou et al ldquoExpression of the Na+I- symporter gene in human thyroid tumors a comparisonstudy with other thyroid-specific genesrdquo Journal of ClinicalEndocrinology and Metabolism vol 84 no 9 pp 3228ndash32341999
[38] G Brabant C Maenhaut J Kohrle et al ldquoHuman thyrotropinreceptor gene expression in thyroid tumors and correlationto markers of thyroid differentiation and dedifferentiationrdquoMolecular and Cellular Endocrinology vol 82 no 1 pp R7ndashR121991
2 International Journal of Endocrinology
Current cancer therapy should satisfy requirements fortargeted elimination of cancer cells simultaneously with life-compatible adverse effects [10] One of the main tenetsof cancer therapeutics is that combinations of anticanceragents with different targets or different mechanisms ofaction and varied normal tissue toxicities will produce bettertherapeutic outcomes [11] by decreasing single drugs dosesand minimizing or slowing drug resistance development Inthis study we investigated the possible use of SAHA anHDAC inhibitor and PJ34 a PARP inhibitor in combinationin a cellular model of anaplastic thyroid cancer
2 Material and Methods
21 Cell Line and Treatments SW1736 human cell linederived from anaplastic thyroid cancer was grown inRPMI 1640 medium (EuroClone Milan Italy) supple-mented with 10 fetal bovine serum (Gibco InvitrogenMilan Italy) and 50mgmL gentamicin (Gibco Invitro-gen Milan Italy) in a humidified incubator (5 CO
2in
air at 37∘C) The identity of SW1736 cells was demon-strated by evaluating the following STRs D16S539 THO1vWA D3S1358 D21S11 and D18S51 the obtained genotypewas identical to those reported by the CLS Cell LinesService GmbH (httpwwwcell-lines-servicede) Culturedcells were treated with the following agents either alone orin combination as described in the text SAHA (1ndash4120583M inDMSO) (Cayman Chemical Michigan USA) and PJ34 (5ndash30 120583M in nuclease-free water) (Merck Chemicals Ltd) Theseconcentrations are consistent with those utilized for in vivostudies [12 13] All treatments were done for 72 hours
22 Cell Viability To test cell viability CellTiter-Blue CellViability assay (Promega Milano Italy) was used accordingto the manufacturerrsquos instructions Cells were seeded onto96-well plates in 200120583L medium The next day the growthmedium was replaced with fresh medium containing DMSOas vehicle (untreated cultures) or SAHA and PJ34 alone orin combination For each treatment quadruplicate wells wereused
23 Combination Index (CI Value) Effects of drugs combina-tion used in this study were evaluated using the combinationindex equation based on the multiple drug-effect equation ofChou-Talalay [14 15] In all cases where CI value could bedetermined the following diagnostic rule was applied CI lt1 indicates synergism CI = 1 indicates additive effect andCI gt 1 indicates antagonism The analysis was obtained onCompuSyn software (ComboSyn Inc Paramus USA)
24 Annexin V Staining Cells were treated with appropriatedrugs as described and then they were washed with coldPBS transferred to a polystyrene round-bottomed flow tube(Falcon Becton Dickinson Franklin Lakes NY USA) andresuspended in 195 120583L of 1times binding buffer (BB-10mMHepesNaOH pH 74 140mM NaCl and 25mM CaCl
2)
To the suspension 5 120583L of fluorescein-conjugated AnnexinV (Annexin V-FITC Bender Med Systems Wien Austria)was added and samples were incubated for 10min at room
temperature After washing cells were resuspended in 190120583Lof BB inwhich 10 120583L of propidium iodide stock solution (finalconcentration 1120583gmL) was added Flow cytometry analysiswas done on CyAN Dako Cytomation using the Summitsoftware
25 Quantitative RT-PCR Total RNA from cell line treatedfor 72 h with SAHA 1 120583M and PJ34 15 120583M alone or in com-bination was extracted with RNeasy mini kit according tomanufacturerrsquos instructions (Qiagen Hilden Germany)500 ng of total RNA was reversely transcripted to cDNAusing random exaprimers and MMLV reverse transcriptase(Invitrogen) Real-time PCRs were performed using TaqManUniversal PCR Master Mix (Applied Biosystems) with theABI Prism 7300 Sequence Detection Systems (Applied Biosy-stems Foster City CA USA) The ΔΔCT method by meansof the SDS software (Applied Biosystems) was used to calculate the mRNA levels Oligonucleotide primers were purchased from Life Technologies and were as follows 120573-actin51015840 primer CGAGCGCGGCTACAGCTT 120573-actin probeACCACCACGGCCGAGCGG and 120573-actin 31015840 primer TCC-TTAATGTCACGCACGATTT PAX8 51015840 primer CAACAG-CACCCTGGACGAC PAX8 31015840 primer AGGGTGAGTGAG-GATCTGCC and PAX8 probe CTGACCCCTTCCAAC-ACGCCACTG NIS Hs00166567 m1 TTF1 Hs00968940 m1TTF2 Hs00916085 s1 and TSHR Hs01053846 m1
26 Statistical Analysis Cell viability apoptosis and mRNAlevels were expressed as means plusmn SD and significances wereanalyzed with the t test performed with GraphPAD Softwarefor Science (San Diego CA USA)
3 Results
In a first set of experiments single effects of the HDACinhibitor SAHA and the PARP inhibitor PJ34 on cell viabilityof the human anaplastic thyroid cancer-derived cell lineSW1736 were investigated Cell viability was assessed aftertreatment with different doses of SAHA and PJ34 for 72hours (Figure 1) Both SAHA and PJ34 alone inhibited cellproliferation in a dose-dependent manner however at theutilized doses SAHA seemed to have a greater effect causinga more significant decrease in cell viability compared to cellstreated by PJ34 Thus both compounds alone were able toinhibit proliferation of SW1736 cells We then tested synergyof the two compounds by measuring CI values of differentdrug combinations according to the Chou-Talalay equation[14 15] As indicated in Table 1 all combinations used showeda very high decrease in cell growth compared to untreatedcells (always the CI values were lower than 1) Our resultsindicated that SAHA and PJ34 have a synergic effect indecreasing cell proliferation in a quite high range of utilizeddoses
We focused on the SAHA 1 120583M and PJ34 15 120583M combi-nation which generated the lowest CI value As representedin Figure 2 the treatment with SAHA 1 120583M had only a lighteffect on SW1736 viability while PJ34 15120583Mreduced cell pro-liferation more effectively Using these doses in combination
International Journal of Endocrinology 3
0
20
40
60
80
100
120
SAHA (120583M) PJ34 (120583M)
Cel
l via
bilit
y (a
u)
1 2 3 4 5 10 15 30
lowastlowastlowast
lowast
mdash
Figure 1 Effects of HDAC and PARP inhibitors on SW1736 cellviability Cells were treated for 72 h with SAHA (1120583Mndash4120583M) orPJ34 (5120583Mndash30 120583M) and CellTiter-Blue Cell Viability assay wasperformed as described in Section 2 Bars indicate the percentageof viable cells versus controls (untreated cells) and represent meansplusmn SD of three experiments lowast indicates values significantly differentcompared to control
Table 1 Combination index data for SAHA and PJ34 combination
Dose SAHA(120583M)
Dose PJ34(120583M)
Combination effect( cell viability)lowast CI value
10 50 26 02697910 100 37 06023510 150 12 01387810 300 13 01847320 50 18 03312320 100 17 03341420 150 19 04011120 300 11 02534130 50 13 03650430 100 26 07532030 150 16 04717530 300 14 04541340 50 17 06076340 100 17 06278540 150 10 04031840 300 13 053865lowastMean value of four replicates In each condition standard deviation is lessthan 10
we obtained a strong lowering of cell viability almost 90compared to untreated cells
Subsequently we evaluated apoptosis of SW1736 by mea-surement of Annexin V by fluorescence-activated cell sortingafter treatments with SAHA 1 120583M and PJ34 15 120583M alone orin combination (Figure 3(a)) The percentage of apoptoticcells (AnnexinV-positivePI-negative) was 198with SAHA1 120583M and 199 with PJ34 15 120583Mwhile after the combinationtreatment it reached 816 (Figure 3(b))
We next tested if synergy between the two compoundswas present on expression of several thyroid-specific genesEffects on mRNA levels were evaluated by real-time PCR
0
20
40
60
80
100
120
Cel
l via
bilit
y (a
u)
mdash
lowast
SAHA 1120583M PJ34 15120583M15120583M
SAHA 1120583M+ PJ34
Figure 2 Effect of HDAC and PARP inhibitors combinationon SW1736 cell viability Cells were treated for 72 h with SAHA1 120583M and PJ34 15 120583M alone or in combination CellTiter-BlueCell Viability assay was performed as described in Section 2 Barsindicate the percentage of viable cells versus controls (untreatedcells) and represent means plusmn SD of four experiments lowast indicatesvalues significantly different compared to all other conditions
Among all genes analyzed synergy between the two com-pounds was detectable only for TSHR gene (Figure 4) The72-hour treatment with SAHA 1 120583M induced a marked effecton TSHR mRNA expression while PJ34 15 120583M did not haveany remarkable effect compared to the control cells Howeverby using the two drugs in combination we obtained a strongeffect on TSHR mRNA levels significantly higher than allother conditions with an increment of 36-fold of inductioncompared to the control
4 Discussion
Developing a pharmacological treatment against cancer thecentral issue consists in increasing therapeutic index andat the same time limiting development of resistance Onesolution is to combine multiple drugs that act synergisticallyand in fact a large number of ongoing clinical trials areinvestigating the effects of combined therapy against differ-ent types of cancer [16 17] In developing new strategiesfor treatment of anaplastic thyroid cancer combinations ofHDAC inhibitors and other drugs have been attempted [18ndash24] However neither in preclinical nor in clinical settings ofthyroid cancer treatment the combination between HDACand PARP inhibitors has been investigated It is increasinglyclear that cancer is not only caused by genetic factors but canalso be considered a epigenetics disease [25] and epigeneticenzymes can therefore be considered as novel therapeutictargets Accordingly both HDAC and PARP inhibitors canbe considered as epigenetic drugs Combinations of HDACand PARP inhibitors have been tested in different kindsof neoplastic diseases By using hepatocellular carcinomacell lines a synergistic inhibition of cell growth by SAHAand the PARP inhibitor olaparib has been demonstrated[26] Inhibition of cell proliferation was associated withincrease of apoptosis levels accumulation of DNA damageand modification of the cAMP signaling pathway Combined
4 International Journal of Endocrinology
SAHA + PJ34
104
103
102
101
100
104103102101100
104103102101100 104103102101100
104103102101100
Annexin V Annexin V
Annexin V Annexin V
PI
104
103
102
101
100
PI
104
103
102
101
100
PI
104
103
102
101
100
PI
Q1 Q2
Q3Q4
3764 2763
3424 049
ControlQ1
3315
Q2
4257
Q3198
Q42230
SAHA
Q1
3017
Q2
3159
Q3
199
Q4
3625
PJ34Q1
2571Q2
4146
Q3
816Q4
2467
(a)
0123456789
Apop
tosis
leve
ls (
)
mdash SAHA 1120583M PJ34 15120583M15120583M
SAHA 1120583M+ PJ34
(b)
Figure 3 Combination of SAHA and PJ34 induces apoptosis in SW1736 cell line In (a) Annexin V (FITC) and propidium iodide (PI)staining of anaplastic thyroid cancer cell line after 72 h treatment with SAHA 1120583M PJ34 15 120583Malone or in combination In (b) representationof Annexin V-positivePI-negative cells
International Journal of Endocrinology 5
05
1015202530354045 TSHR
0
02
04
06
08
1
12 TTF1
0
05
1
15
2
25 TTF2
0
05
1
15
2NIS
002040608
1121416
PAX8m
RNA
leve
ls (a
u)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mdash
mRN
A le
vels
(au
)
lowast
lowast
SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583M SAHA+ PJ34 15120583M
1120583Mmdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583MSAHA+ PJ34 15120583M
1120583M
SAHA+ PJ34 15120583M
1120583M
Figure 4 Expression levels of PAX8 NIS TTF1 TTF2 and TSHR genes in SW1736 cell line RNA extraction and real-time PCR are describedin Section 2 For each gene the results were normalized against 120573-actin and expressed in arbitrary unit (2minusΔCt) Each bar represents the meanvalue of three different determinations For each bar standard deviation is not above 10of each valuelowast indicates values significantly differentcompared to all other conditions
effects of SAHA and PJ34 on leukemia cell lines have beeninvestigated [27] Also in that study synergistic effects onproliferation inhibition and apoptosis increase have beenobserved Recently synergy between SAHA and olaparib hasbeen observed even in ovarian cancer cell lines [28] We haverecently obtained similar effects on breast cancer cell lines(unpublished data)
Effects of HDAC and PARP inhibitors alone have beenpreviously investigated by our group We have shown thatHDAC inhibitors affect cell proliferation and expression ofvarious genes in several thyroid cancer cell lines [29 30]Moreover Lavarone et al have recently shown that PJ34inhibits cell growth and increases NIS expression in variousthyroid cancer cell lines [31] In this research we demonstratethat HDAC and PARP inhibitors have a synergistic effect
on proliferation of a human anaplastic thyroid-derived cellline Thus synergy between these two classes of compoundsappears to be a common phenomenon in cancer cell linesof various origins underlying the potential role of thesecombinations as an interesting strategy for cancer therapyOur obtained CI values indicate that synergy between SAHAand PJ34 occurs in a wide range of doses suggesting that thecombined effect could probably be observed also in vivo
In addition to the impact on cell proliferation wehave investigated effects of the SAHA-PJ34 combination onthyroid-specific genes expression Regarding thyroid-specifictranscription factors the SAHA-PJ34 combination induces aTTF1 slight decrease a TTF2 slight increase and no changein PAX8 Such behavior indicates that the control of thesegenes expression occurs through distinct mechanisms This
6 International Journal of Endocrinology
view agrees with previous studies in which control of thyroid-specific transcription factors expression has been investigated[32]
Synergy between SAHA and PJ34 in increasing mRNAlevels of TSHR was observed The TSHR is localized in theplasmamembrane and thus it has been proposed as a targetto direct therapeutic compounds into thyroid cancer cells[33ndash36] Our data would suggest that the combined use ofHDAC and PARP inhibitors may facilitate such approach
Differently from TSHR NIS gene expression is reducedby SAHA alone and in combination with PJ34 Such adifferent effect between these genes expression is not unex-pected TSHR and NIS have a different regulation in termsof gene expression It is well known for example that duringtumorigenesis NIS is one of the earliest downregulated geneswhile TSHR is among the latest ones [37 38]
In conclusion considering our data on cell proliferationand gene expression altogether the combined use of HDACand PARP inhibitors can be a useful strategy for ATCtreatment Preclinical in vivo studies are required to validatesuch a possibility
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was supported by grants to Giuseppe Damantefrom Associazione Italiana per la Ricerca sul Cancro (AIRC)(Project no IG 10296)
References
[1] G Pellegriti F Frasca C Regalbuto S Squatrito and R Vign-eri ldquoWorldwide increasing incidence of thyroid cancer updateon epidemiology and risk factorsrdquo Journal of Cancer Epidemiol-ogy vol 2013 Article ID 965212 10 pages 2013
[2] J P OrsquoNeill and A R Shaha ldquoAnaplastic thyroid cancerrdquo OralOncology vol 49 no 7 pp 702ndash706 2013
[3] S L Kojic S S Strugnell and S M Wiseman ldquoAnaplasticthyroid cancer a comprehensive review of novel therapyrdquoExpert Review of Anticancer Therapy vol 11 no 3 pp 387ndash4022011
[4] P A Marks and W-S Xu ldquoHistone deacetylase inhibitorspotential in cancer therapyrdquo Journal of Cellular Biochemistryvol 107 no 4 pp 600ndash608 2009
[5] D Russo G Damante E Puxeddu C Durante and S FilettildquoEpigenetics of thyroid cancer and novel therapeutic targetsrdquoJournal of Molecular Endocrinology vol 46 no 3 pp R73ndashR812011
[6] Q T Luong JOrsquoKelly GD Braunstein JMHershman andHP Koeffler ldquoAntitumor activity of suberoylanilide hydroxamicacid against thyroid cancer cell lines in vitro and in vivordquo Cli-nical Cancer Research vol 12 no 18 pp 5570ndash5577 2006
[7] A Chen ldquoPARP inhibitors its role in treatment of cancerrdquoChinese Journal of Cancer vol 30 no 7 pp 463ndash471 2011
[8] A J Frew R W Johnstone and J E Bolden ldquoEnhancing theapoptotic and therapeutic effects of HDAC inhibitorsrdquo CancerLetters vol 280 no 2 pp 125ndash133 2009
[9] G Papeo E Casale A Montagnoli and A Cirla ldquoPARPinhibitors in cancer therapy an updaterdquo Expert Opinion onTherapeutic Patents vol 23 no 4 pp 503ndash514 2013
[10] B Brodska A Holoubek P Otevrelova and K KuzelovaldquoCombined treatment with low concentrations of decitabineand saha causes cell death in leukemic cell lines but not innormal peripheral blood lymphocytesrdquo BioMed Research Inter-national vol 2013 Article ID 659254 11 pages 2013
[11] B A Teicher ldquoCombinations of PARP hedgehog and HDACinhibitors with standard drugsrdquo Current Opinion in Pharmacol-ogy vol 10 no 4 pp 397ndash404 2010
[12] M A Smith and P Houghton ldquoA proposal regarding reportingof in vitro testing resultsrdquo Clinical Cancer Research vol 19 no11 pp 2828ndash2833 2013
[13] C Szabo A Biser R Benko E Bottinger and K SusztakldquoPoly(ADP-ribose) polymerase inhibitors ameliorate nephro-pathy of type 2 diabetic Leprdbdb micerdquo Diabetes vol 55 no11 pp 3004ndash3012 2006
[14] T-C Chou and P Talalay ldquoQuantitative analysis of dose-effectrelationships the combined effects of multiple drugs or enzymeinhibitorsrdquo Advances in Enzyme Regulation vol 22 pp 27ndash551984
[15] T C Chou and P Talalay ldquoAnalysis of combined drug effectsa new look at a very old problemrdquo Trends in PharmacologicalSciences vol 4 pp 450ndash454 1983
[16] E Kim M Matsuse V Saenko et al ldquoImatinib enhances doce-taxel-induced apoptosis through inhibition of nuclear factor-120581Bactivation in anaplastic thyroid carcinoma cellsrdquo Thyroid vol22 no 7 pp 717ndash724 2012
[17] J-C Ahn R Biswas and P-S Chung ldquoCombination withgenistein enhances the efficacy of photodynamic therapyagainst human anaplastic thyroid cancer cellsrdquo Lasers in Surgeryand Medicine vol 44 no 10 pp 840ndash849 2012
[18] I Clinckspoor L Verlinden L Overbergh et al ldquo125-Dihy-droxyvitamin D3 and a superagonistic analog in combinationwith paclitaxel or suberoylanilide hydroxamic acid have potentantiproliferative effects on anaplastic thyroid cancerrdquo Journal ofSteroid Biochemistry and Molecular Biology vol 124 no 1-2 pp1ndash9 2011
[19] M G Catalano R Poli M Pugliese N Fortunati and GBoccuzzi ldquoValproic acid enhances tubulin acetylation andapoptotic activity of paclitaxel on anaplastic thyroid cancer celllinesrdquo Endocrine-Related Cancer vol 14 no 3 pp 839ndash8452007
[20] D Russo C Durante S Bulotta et al ldquoTargeting histone deace-tylase in thyroid cancerrdquo Expert Opinion onTherapeutic Targetsvol 17 no 2 pp 179ndash193 2013
[21] P Brest S Lassalle V Hofman et al ldquoMiR-129-5p is requiredfor histone deacetylase inhibitor-induced cell death in thyroidcancer cellsrdquo Endocrine-Related Cancer vol 18 no 6 pp 711ndash719 2011
[22] T Kondo S L Asa and S Ezzat ldquoEpigenetic dysregulationin thyroid neoplasiardquo Endocrinology and Metabolism Clinics ofNorth America vol 37 no 2 pp 389ndash400 2008
[23] C S Mitsiades V Poulaki C McMullan et al ldquoNovel histonedeacetylase inhibitors in thetreatment of thyroid cancerrdquo Clini-cal Cancer Research vol 11 no 10 pp 3958ndash3965 2005
International Journal of Endocrinology 7
[24] M G Catalano N Fortunati M Pugliese et al ldquoValproic acida histone deacetylase inhibitor enhances sensitivity to doxo-rubicin in anaplastic thyroid cancer cellsrdquo Journal of Endocrinol-ogy vol 191 no 2 pp 465ndash472 2006
[25] C A Iacobuzio-Donahue ldquoEpigenetic changes in cancerrdquoAnnual Review of Pathology vol 4 pp 229ndash249 2009
[26] J-X Zhang D-Q Li A R He et al ldquoSynergistic inhibitionof hepatocellular carcinoma growth by cotargeting chromatinmodifying enzymes and poly (ADP-ribose) polymerasesrdquoHep-atology vol 55 no 6 pp 1840ndash1851 2012
[27] E Jasek M Gajda G J Lis M Jasinska and J A LitwinldquoCombinatorial effects of PARP inhibitor PJ34 and histonedeacetylase inhibitor vorinostat on leukemia cell linesrdquo Anti-cancer Research vol 34 no 4 pp 1849ndash1856 2014
[28] P A Konstantinopoulos A J Wilson J Saskowski E Wassand D Khabele ldquoSuberoylanilide hydroxamic acid (SAHA)enhances olaparib activity by targeting homologous recombina-tion DNA repair in ovarian cancerrdquo Gynecologic Oncology vol133 no 3 pp 599ndash606 2014
[29] C Puppin F DAurizio A V DElia et al ldquoEffects of histoneacetylation on NIS promoter and expression of thyroid-specifictranscription factorsrdquo Endocrinology vol 146 no 9 pp 3967ndash3974 2005
[30] C Puppin N Passon J M Hershman et al ldquoCooperativeeffects of SAHA andVPA onNIS gene expression and prolifera-tion of thyroid cancer cellsrdquo Journal ofMolecular Endocrinologyvol 48 no 3 pp 217ndash227 2012
[31] E Lavarone C Puppin N Passon S Filetti D Russo and GDamante ldquoThePARP inhibitor PJ34modifies proliferation NISexpression and epigenetic marks in thyroid cancer cell linesrdquoMolecular and Cellular Endocrinology vol 365 no 1 pp 1ndash102013
[32] G Damante G Tell and R di Lauro ldquoA unique combinationof transcription factors controls differentiation of thyroid cellsrdquoProgress in Nucleic Acid Research andMolecular Biology vol 66pp 307ndash356 2000
[33] J C Morris ldquoStructure and function of the TSH receptor itssuitability as a target for radiotherapyrdquoThyroid vol 7 no 2 pp253ndash258 1997
[34] S El-Kaissi and J R Wall ldquoTargeting the thyrotropin receptorin thyroid diseaserdquo Expert Opinion on Therapeutic Targets vol16 no 7 pp 719ndash727 2012
[35] D Paolino D Cosco M Gaspari et al ldquoTargeting the thy-roid gland with thyroid-stimulating hormone (TSH)-nano-liposomesrdquo Biomaterials vol 35 no 25 pp 7101ndash7109 2014
[36] M DrsquoAgostino M Sponziello C Puppin et al ldquoDifferentexpression of TSH receptor and NIS genes in thyroid cancerrole of epigeneticsrdquo Journal of Molecular Endocrinology vol 52no 2 pp 121ndash131 2013
[37] V Lazar J M Bidart B Caillou et al ldquoExpression of the Na+I- symporter gene in human thyroid tumors a comparisonstudy with other thyroid-specific genesrdquo Journal of ClinicalEndocrinology and Metabolism vol 84 no 9 pp 3228ndash32341999
[38] G Brabant C Maenhaut J Kohrle et al ldquoHuman thyrotropinreceptor gene expression in thyroid tumors and correlationto markers of thyroid differentiation and dedifferentiationrdquoMolecular and Cellular Endocrinology vol 82 no 1 pp R7ndashR121991
International Journal of Endocrinology 3
0
20
40
60
80
100
120
SAHA (120583M) PJ34 (120583M)
Cel
l via
bilit
y (a
u)
1 2 3 4 5 10 15 30
lowastlowastlowast
lowast
mdash
Figure 1 Effects of HDAC and PARP inhibitors on SW1736 cellviability Cells were treated for 72 h with SAHA (1120583Mndash4120583M) orPJ34 (5120583Mndash30 120583M) and CellTiter-Blue Cell Viability assay wasperformed as described in Section 2 Bars indicate the percentageof viable cells versus controls (untreated cells) and represent meansplusmn SD of three experiments lowast indicates values significantly differentcompared to control
Table 1 Combination index data for SAHA and PJ34 combination
Dose SAHA(120583M)
Dose PJ34(120583M)
Combination effect( cell viability)lowast CI value
10 50 26 02697910 100 37 06023510 150 12 01387810 300 13 01847320 50 18 03312320 100 17 03341420 150 19 04011120 300 11 02534130 50 13 03650430 100 26 07532030 150 16 04717530 300 14 04541340 50 17 06076340 100 17 06278540 150 10 04031840 300 13 053865lowastMean value of four replicates In each condition standard deviation is lessthan 10
we obtained a strong lowering of cell viability almost 90compared to untreated cells
Subsequently we evaluated apoptosis of SW1736 by mea-surement of Annexin V by fluorescence-activated cell sortingafter treatments with SAHA 1 120583M and PJ34 15 120583M alone orin combination (Figure 3(a)) The percentage of apoptoticcells (AnnexinV-positivePI-negative) was 198with SAHA1 120583M and 199 with PJ34 15 120583Mwhile after the combinationtreatment it reached 816 (Figure 3(b))
We next tested if synergy between the two compoundswas present on expression of several thyroid-specific genesEffects on mRNA levels were evaluated by real-time PCR
0
20
40
60
80
100
120
Cel
l via
bilit
y (a
u)
mdash
lowast
SAHA 1120583M PJ34 15120583M15120583M
SAHA 1120583M+ PJ34
Figure 2 Effect of HDAC and PARP inhibitors combinationon SW1736 cell viability Cells were treated for 72 h with SAHA1 120583M and PJ34 15 120583M alone or in combination CellTiter-BlueCell Viability assay was performed as described in Section 2 Barsindicate the percentage of viable cells versus controls (untreatedcells) and represent means plusmn SD of four experiments lowast indicatesvalues significantly different compared to all other conditions
Among all genes analyzed synergy between the two com-pounds was detectable only for TSHR gene (Figure 4) The72-hour treatment with SAHA 1 120583M induced a marked effecton TSHR mRNA expression while PJ34 15 120583M did not haveany remarkable effect compared to the control cells Howeverby using the two drugs in combination we obtained a strongeffect on TSHR mRNA levels significantly higher than allother conditions with an increment of 36-fold of inductioncompared to the control
4 Discussion
Developing a pharmacological treatment against cancer thecentral issue consists in increasing therapeutic index andat the same time limiting development of resistance Onesolution is to combine multiple drugs that act synergisticallyand in fact a large number of ongoing clinical trials areinvestigating the effects of combined therapy against differ-ent types of cancer [16 17] In developing new strategiesfor treatment of anaplastic thyroid cancer combinations ofHDAC inhibitors and other drugs have been attempted [18ndash24] However neither in preclinical nor in clinical settings ofthyroid cancer treatment the combination between HDACand PARP inhibitors has been investigated It is increasinglyclear that cancer is not only caused by genetic factors but canalso be considered a epigenetics disease [25] and epigeneticenzymes can therefore be considered as novel therapeutictargets Accordingly both HDAC and PARP inhibitors canbe considered as epigenetic drugs Combinations of HDACand PARP inhibitors have been tested in different kindsof neoplastic diseases By using hepatocellular carcinomacell lines a synergistic inhibition of cell growth by SAHAand the PARP inhibitor olaparib has been demonstrated[26] Inhibition of cell proliferation was associated withincrease of apoptosis levels accumulation of DNA damageand modification of the cAMP signaling pathway Combined
4 International Journal of Endocrinology
SAHA + PJ34
104
103
102
101
100
104103102101100
104103102101100 104103102101100
104103102101100
Annexin V Annexin V
Annexin V Annexin V
PI
104
103
102
101
100
PI
104
103
102
101
100
PI
104
103
102
101
100
PI
Q1 Q2
Q3Q4
3764 2763
3424 049
ControlQ1
3315
Q2
4257
Q3198
Q42230
SAHA
Q1
3017
Q2
3159
Q3
199
Q4
3625
PJ34Q1
2571Q2
4146
Q3
816Q4
2467
(a)
0123456789
Apop
tosis
leve
ls (
)
mdash SAHA 1120583M PJ34 15120583M15120583M
SAHA 1120583M+ PJ34
(b)
Figure 3 Combination of SAHA and PJ34 induces apoptosis in SW1736 cell line In (a) Annexin V (FITC) and propidium iodide (PI)staining of anaplastic thyroid cancer cell line after 72 h treatment with SAHA 1120583M PJ34 15 120583Malone or in combination In (b) representationof Annexin V-positivePI-negative cells
International Journal of Endocrinology 5
05
1015202530354045 TSHR
0
02
04
06
08
1
12 TTF1
0
05
1
15
2
25 TTF2
0
05
1
15
2NIS
002040608
1121416
PAX8m
RNA
leve
ls (a
u)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mdash
mRN
A le
vels
(au
)
lowast
lowast
SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583M SAHA+ PJ34 15120583M
1120583Mmdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583MSAHA+ PJ34 15120583M
1120583M
SAHA+ PJ34 15120583M
1120583M
Figure 4 Expression levels of PAX8 NIS TTF1 TTF2 and TSHR genes in SW1736 cell line RNA extraction and real-time PCR are describedin Section 2 For each gene the results were normalized against 120573-actin and expressed in arbitrary unit (2minusΔCt) Each bar represents the meanvalue of three different determinations For each bar standard deviation is not above 10of each valuelowast indicates values significantly differentcompared to all other conditions
effects of SAHA and PJ34 on leukemia cell lines have beeninvestigated [27] Also in that study synergistic effects onproliferation inhibition and apoptosis increase have beenobserved Recently synergy between SAHA and olaparib hasbeen observed even in ovarian cancer cell lines [28] We haverecently obtained similar effects on breast cancer cell lines(unpublished data)
Effects of HDAC and PARP inhibitors alone have beenpreviously investigated by our group We have shown thatHDAC inhibitors affect cell proliferation and expression ofvarious genes in several thyroid cancer cell lines [29 30]Moreover Lavarone et al have recently shown that PJ34inhibits cell growth and increases NIS expression in variousthyroid cancer cell lines [31] In this research we demonstratethat HDAC and PARP inhibitors have a synergistic effect
on proliferation of a human anaplastic thyroid-derived cellline Thus synergy between these two classes of compoundsappears to be a common phenomenon in cancer cell linesof various origins underlying the potential role of thesecombinations as an interesting strategy for cancer therapyOur obtained CI values indicate that synergy between SAHAand PJ34 occurs in a wide range of doses suggesting that thecombined effect could probably be observed also in vivo
In addition to the impact on cell proliferation wehave investigated effects of the SAHA-PJ34 combination onthyroid-specific genes expression Regarding thyroid-specifictranscription factors the SAHA-PJ34 combination induces aTTF1 slight decrease a TTF2 slight increase and no changein PAX8 Such behavior indicates that the control of thesegenes expression occurs through distinct mechanisms This
6 International Journal of Endocrinology
view agrees with previous studies in which control of thyroid-specific transcription factors expression has been investigated[32]
Synergy between SAHA and PJ34 in increasing mRNAlevels of TSHR was observed The TSHR is localized in theplasmamembrane and thus it has been proposed as a targetto direct therapeutic compounds into thyroid cancer cells[33ndash36] Our data would suggest that the combined use ofHDAC and PARP inhibitors may facilitate such approach
Differently from TSHR NIS gene expression is reducedby SAHA alone and in combination with PJ34 Such adifferent effect between these genes expression is not unex-pected TSHR and NIS have a different regulation in termsof gene expression It is well known for example that duringtumorigenesis NIS is one of the earliest downregulated geneswhile TSHR is among the latest ones [37 38]
In conclusion considering our data on cell proliferationand gene expression altogether the combined use of HDACand PARP inhibitors can be a useful strategy for ATCtreatment Preclinical in vivo studies are required to validatesuch a possibility
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was supported by grants to Giuseppe Damantefrom Associazione Italiana per la Ricerca sul Cancro (AIRC)(Project no IG 10296)
References
[1] G Pellegriti F Frasca C Regalbuto S Squatrito and R Vign-eri ldquoWorldwide increasing incidence of thyroid cancer updateon epidemiology and risk factorsrdquo Journal of Cancer Epidemiol-ogy vol 2013 Article ID 965212 10 pages 2013
[2] J P OrsquoNeill and A R Shaha ldquoAnaplastic thyroid cancerrdquo OralOncology vol 49 no 7 pp 702ndash706 2013
[3] S L Kojic S S Strugnell and S M Wiseman ldquoAnaplasticthyroid cancer a comprehensive review of novel therapyrdquoExpert Review of Anticancer Therapy vol 11 no 3 pp 387ndash4022011
[4] P A Marks and W-S Xu ldquoHistone deacetylase inhibitorspotential in cancer therapyrdquo Journal of Cellular Biochemistryvol 107 no 4 pp 600ndash608 2009
[5] D Russo G Damante E Puxeddu C Durante and S FilettildquoEpigenetics of thyroid cancer and novel therapeutic targetsrdquoJournal of Molecular Endocrinology vol 46 no 3 pp R73ndashR812011
[6] Q T Luong JOrsquoKelly GD Braunstein JMHershman andHP Koeffler ldquoAntitumor activity of suberoylanilide hydroxamicacid against thyroid cancer cell lines in vitro and in vivordquo Cli-nical Cancer Research vol 12 no 18 pp 5570ndash5577 2006
[7] A Chen ldquoPARP inhibitors its role in treatment of cancerrdquoChinese Journal of Cancer vol 30 no 7 pp 463ndash471 2011
[8] A J Frew R W Johnstone and J E Bolden ldquoEnhancing theapoptotic and therapeutic effects of HDAC inhibitorsrdquo CancerLetters vol 280 no 2 pp 125ndash133 2009
[9] G Papeo E Casale A Montagnoli and A Cirla ldquoPARPinhibitors in cancer therapy an updaterdquo Expert Opinion onTherapeutic Patents vol 23 no 4 pp 503ndash514 2013
[10] B Brodska A Holoubek P Otevrelova and K KuzelovaldquoCombined treatment with low concentrations of decitabineand saha causes cell death in leukemic cell lines but not innormal peripheral blood lymphocytesrdquo BioMed Research Inter-national vol 2013 Article ID 659254 11 pages 2013
[11] B A Teicher ldquoCombinations of PARP hedgehog and HDACinhibitors with standard drugsrdquo Current Opinion in Pharmacol-ogy vol 10 no 4 pp 397ndash404 2010
[12] M A Smith and P Houghton ldquoA proposal regarding reportingof in vitro testing resultsrdquo Clinical Cancer Research vol 19 no11 pp 2828ndash2833 2013
[13] C Szabo A Biser R Benko E Bottinger and K SusztakldquoPoly(ADP-ribose) polymerase inhibitors ameliorate nephro-pathy of type 2 diabetic Leprdbdb micerdquo Diabetes vol 55 no11 pp 3004ndash3012 2006
[14] T-C Chou and P Talalay ldquoQuantitative analysis of dose-effectrelationships the combined effects of multiple drugs or enzymeinhibitorsrdquo Advances in Enzyme Regulation vol 22 pp 27ndash551984
[15] T C Chou and P Talalay ldquoAnalysis of combined drug effectsa new look at a very old problemrdquo Trends in PharmacologicalSciences vol 4 pp 450ndash454 1983
[16] E Kim M Matsuse V Saenko et al ldquoImatinib enhances doce-taxel-induced apoptosis through inhibition of nuclear factor-120581Bactivation in anaplastic thyroid carcinoma cellsrdquo Thyroid vol22 no 7 pp 717ndash724 2012
[17] J-C Ahn R Biswas and P-S Chung ldquoCombination withgenistein enhances the efficacy of photodynamic therapyagainst human anaplastic thyroid cancer cellsrdquo Lasers in Surgeryand Medicine vol 44 no 10 pp 840ndash849 2012
[18] I Clinckspoor L Verlinden L Overbergh et al ldquo125-Dihy-droxyvitamin D3 and a superagonistic analog in combinationwith paclitaxel or suberoylanilide hydroxamic acid have potentantiproliferative effects on anaplastic thyroid cancerrdquo Journal ofSteroid Biochemistry and Molecular Biology vol 124 no 1-2 pp1ndash9 2011
[19] M G Catalano R Poli M Pugliese N Fortunati and GBoccuzzi ldquoValproic acid enhances tubulin acetylation andapoptotic activity of paclitaxel on anaplastic thyroid cancer celllinesrdquo Endocrine-Related Cancer vol 14 no 3 pp 839ndash8452007
[20] D Russo C Durante S Bulotta et al ldquoTargeting histone deace-tylase in thyroid cancerrdquo Expert Opinion onTherapeutic Targetsvol 17 no 2 pp 179ndash193 2013
[21] P Brest S Lassalle V Hofman et al ldquoMiR-129-5p is requiredfor histone deacetylase inhibitor-induced cell death in thyroidcancer cellsrdquo Endocrine-Related Cancer vol 18 no 6 pp 711ndash719 2011
[22] T Kondo S L Asa and S Ezzat ldquoEpigenetic dysregulationin thyroid neoplasiardquo Endocrinology and Metabolism Clinics ofNorth America vol 37 no 2 pp 389ndash400 2008
[23] C S Mitsiades V Poulaki C McMullan et al ldquoNovel histonedeacetylase inhibitors in thetreatment of thyroid cancerrdquo Clini-cal Cancer Research vol 11 no 10 pp 3958ndash3965 2005
International Journal of Endocrinology 7
[24] M G Catalano N Fortunati M Pugliese et al ldquoValproic acida histone deacetylase inhibitor enhances sensitivity to doxo-rubicin in anaplastic thyroid cancer cellsrdquo Journal of Endocrinol-ogy vol 191 no 2 pp 465ndash472 2006
[25] C A Iacobuzio-Donahue ldquoEpigenetic changes in cancerrdquoAnnual Review of Pathology vol 4 pp 229ndash249 2009
[26] J-X Zhang D-Q Li A R He et al ldquoSynergistic inhibitionof hepatocellular carcinoma growth by cotargeting chromatinmodifying enzymes and poly (ADP-ribose) polymerasesrdquoHep-atology vol 55 no 6 pp 1840ndash1851 2012
[27] E Jasek M Gajda G J Lis M Jasinska and J A LitwinldquoCombinatorial effects of PARP inhibitor PJ34 and histonedeacetylase inhibitor vorinostat on leukemia cell linesrdquo Anti-cancer Research vol 34 no 4 pp 1849ndash1856 2014
[28] P A Konstantinopoulos A J Wilson J Saskowski E Wassand D Khabele ldquoSuberoylanilide hydroxamic acid (SAHA)enhances olaparib activity by targeting homologous recombina-tion DNA repair in ovarian cancerrdquo Gynecologic Oncology vol133 no 3 pp 599ndash606 2014
[29] C Puppin F DAurizio A V DElia et al ldquoEffects of histoneacetylation on NIS promoter and expression of thyroid-specifictranscription factorsrdquo Endocrinology vol 146 no 9 pp 3967ndash3974 2005
[30] C Puppin N Passon J M Hershman et al ldquoCooperativeeffects of SAHA andVPA onNIS gene expression and prolifera-tion of thyroid cancer cellsrdquo Journal ofMolecular Endocrinologyvol 48 no 3 pp 217ndash227 2012
[31] E Lavarone C Puppin N Passon S Filetti D Russo and GDamante ldquoThePARP inhibitor PJ34modifies proliferation NISexpression and epigenetic marks in thyroid cancer cell linesrdquoMolecular and Cellular Endocrinology vol 365 no 1 pp 1ndash102013
[32] G Damante G Tell and R di Lauro ldquoA unique combinationof transcription factors controls differentiation of thyroid cellsrdquoProgress in Nucleic Acid Research andMolecular Biology vol 66pp 307ndash356 2000
[33] J C Morris ldquoStructure and function of the TSH receptor itssuitability as a target for radiotherapyrdquoThyroid vol 7 no 2 pp253ndash258 1997
[34] S El-Kaissi and J R Wall ldquoTargeting the thyrotropin receptorin thyroid diseaserdquo Expert Opinion on Therapeutic Targets vol16 no 7 pp 719ndash727 2012
[35] D Paolino D Cosco M Gaspari et al ldquoTargeting the thy-roid gland with thyroid-stimulating hormone (TSH)-nano-liposomesrdquo Biomaterials vol 35 no 25 pp 7101ndash7109 2014
[36] M DrsquoAgostino M Sponziello C Puppin et al ldquoDifferentexpression of TSH receptor and NIS genes in thyroid cancerrole of epigeneticsrdquo Journal of Molecular Endocrinology vol 52no 2 pp 121ndash131 2013
[37] V Lazar J M Bidart B Caillou et al ldquoExpression of the Na+I- symporter gene in human thyroid tumors a comparisonstudy with other thyroid-specific genesrdquo Journal of ClinicalEndocrinology and Metabolism vol 84 no 9 pp 3228ndash32341999
[38] G Brabant C Maenhaut J Kohrle et al ldquoHuman thyrotropinreceptor gene expression in thyroid tumors and correlationto markers of thyroid differentiation and dedifferentiationrdquoMolecular and Cellular Endocrinology vol 82 no 1 pp R7ndashR121991
4 International Journal of Endocrinology
SAHA + PJ34
104
103
102
101
100
104103102101100
104103102101100 104103102101100
104103102101100
Annexin V Annexin V
Annexin V Annexin V
PI
104
103
102
101
100
PI
104
103
102
101
100
PI
104
103
102
101
100
PI
Q1 Q2
Q3Q4
3764 2763
3424 049
ControlQ1
3315
Q2
4257
Q3198
Q42230
SAHA
Q1
3017
Q2
3159
Q3
199
Q4
3625
PJ34Q1
2571Q2
4146
Q3
816Q4
2467
(a)
0123456789
Apop
tosis
leve
ls (
)
mdash SAHA 1120583M PJ34 15120583M15120583M
SAHA 1120583M+ PJ34
(b)
Figure 3 Combination of SAHA and PJ34 induces apoptosis in SW1736 cell line In (a) Annexin V (FITC) and propidium iodide (PI)staining of anaplastic thyroid cancer cell line after 72 h treatment with SAHA 1120583M PJ34 15 120583Malone or in combination In (b) representationof Annexin V-positivePI-negative cells
International Journal of Endocrinology 5
05
1015202530354045 TSHR
0
02
04
06
08
1
12 TTF1
0
05
1
15
2
25 TTF2
0
05
1
15
2NIS
002040608
1121416
PAX8m
RNA
leve
ls (a
u)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mdash
mRN
A le
vels
(au
)
lowast
lowast
SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583M SAHA+ PJ34 15120583M
1120583Mmdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583MSAHA+ PJ34 15120583M
1120583M
SAHA+ PJ34 15120583M
1120583M
Figure 4 Expression levels of PAX8 NIS TTF1 TTF2 and TSHR genes in SW1736 cell line RNA extraction and real-time PCR are describedin Section 2 For each gene the results were normalized against 120573-actin and expressed in arbitrary unit (2minusΔCt) Each bar represents the meanvalue of three different determinations For each bar standard deviation is not above 10of each valuelowast indicates values significantly differentcompared to all other conditions
effects of SAHA and PJ34 on leukemia cell lines have beeninvestigated [27] Also in that study synergistic effects onproliferation inhibition and apoptosis increase have beenobserved Recently synergy between SAHA and olaparib hasbeen observed even in ovarian cancer cell lines [28] We haverecently obtained similar effects on breast cancer cell lines(unpublished data)
Effects of HDAC and PARP inhibitors alone have beenpreviously investigated by our group We have shown thatHDAC inhibitors affect cell proliferation and expression ofvarious genes in several thyroid cancer cell lines [29 30]Moreover Lavarone et al have recently shown that PJ34inhibits cell growth and increases NIS expression in variousthyroid cancer cell lines [31] In this research we demonstratethat HDAC and PARP inhibitors have a synergistic effect
on proliferation of a human anaplastic thyroid-derived cellline Thus synergy between these two classes of compoundsappears to be a common phenomenon in cancer cell linesof various origins underlying the potential role of thesecombinations as an interesting strategy for cancer therapyOur obtained CI values indicate that synergy between SAHAand PJ34 occurs in a wide range of doses suggesting that thecombined effect could probably be observed also in vivo
In addition to the impact on cell proliferation wehave investigated effects of the SAHA-PJ34 combination onthyroid-specific genes expression Regarding thyroid-specifictranscription factors the SAHA-PJ34 combination induces aTTF1 slight decrease a TTF2 slight increase and no changein PAX8 Such behavior indicates that the control of thesegenes expression occurs through distinct mechanisms This
6 International Journal of Endocrinology
view agrees with previous studies in which control of thyroid-specific transcription factors expression has been investigated[32]
Synergy between SAHA and PJ34 in increasing mRNAlevels of TSHR was observed The TSHR is localized in theplasmamembrane and thus it has been proposed as a targetto direct therapeutic compounds into thyroid cancer cells[33ndash36] Our data would suggest that the combined use ofHDAC and PARP inhibitors may facilitate such approach
Differently from TSHR NIS gene expression is reducedby SAHA alone and in combination with PJ34 Such adifferent effect between these genes expression is not unex-pected TSHR and NIS have a different regulation in termsof gene expression It is well known for example that duringtumorigenesis NIS is one of the earliest downregulated geneswhile TSHR is among the latest ones [37 38]
In conclusion considering our data on cell proliferationand gene expression altogether the combined use of HDACand PARP inhibitors can be a useful strategy for ATCtreatment Preclinical in vivo studies are required to validatesuch a possibility
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was supported by grants to Giuseppe Damantefrom Associazione Italiana per la Ricerca sul Cancro (AIRC)(Project no IG 10296)
References
[1] G Pellegriti F Frasca C Regalbuto S Squatrito and R Vign-eri ldquoWorldwide increasing incidence of thyroid cancer updateon epidemiology and risk factorsrdquo Journal of Cancer Epidemiol-ogy vol 2013 Article ID 965212 10 pages 2013
[2] J P OrsquoNeill and A R Shaha ldquoAnaplastic thyroid cancerrdquo OralOncology vol 49 no 7 pp 702ndash706 2013
[3] S L Kojic S S Strugnell and S M Wiseman ldquoAnaplasticthyroid cancer a comprehensive review of novel therapyrdquoExpert Review of Anticancer Therapy vol 11 no 3 pp 387ndash4022011
[4] P A Marks and W-S Xu ldquoHistone deacetylase inhibitorspotential in cancer therapyrdquo Journal of Cellular Biochemistryvol 107 no 4 pp 600ndash608 2009
[5] D Russo G Damante E Puxeddu C Durante and S FilettildquoEpigenetics of thyroid cancer and novel therapeutic targetsrdquoJournal of Molecular Endocrinology vol 46 no 3 pp R73ndashR812011
[6] Q T Luong JOrsquoKelly GD Braunstein JMHershman andHP Koeffler ldquoAntitumor activity of suberoylanilide hydroxamicacid against thyroid cancer cell lines in vitro and in vivordquo Cli-nical Cancer Research vol 12 no 18 pp 5570ndash5577 2006
[7] A Chen ldquoPARP inhibitors its role in treatment of cancerrdquoChinese Journal of Cancer vol 30 no 7 pp 463ndash471 2011
[8] A J Frew R W Johnstone and J E Bolden ldquoEnhancing theapoptotic and therapeutic effects of HDAC inhibitorsrdquo CancerLetters vol 280 no 2 pp 125ndash133 2009
[9] G Papeo E Casale A Montagnoli and A Cirla ldquoPARPinhibitors in cancer therapy an updaterdquo Expert Opinion onTherapeutic Patents vol 23 no 4 pp 503ndash514 2013
[10] B Brodska A Holoubek P Otevrelova and K KuzelovaldquoCombined treatment with low concentrations of decitabineand saha causes cell death in leukemic cell lines but not innormal peripheral blood lymphocytesrdquo BioMed Research Inter-national vol 2013 Article ID 659254 11 pages 2013
[11] B A Teicher ldquoCombinations of PARP hedgehog and HDACinhibitors with standard drugsrdquo Current Opinion in Pharmacol-ogy vol 10 no 4 pp 397ndash404 2010
[12] M A Smith and P Houghton ldquoA proposal regarding reportingof in vitro testing resultsrdquo Clinical Cancer Research vol 19 no11 pp 2828ndash2833 2013
[13] C Szabo A Biser R Benko E Bottinger and K SusztakldquoPoly(ADP-ribose) polymerase inhibitors ameliorate nephro-pathy of type 2 diabetic Leprdbdb micerdquo Diabetes vol 55 no11 pp 3004ndash3012 2006
[14] T-C Chou and P Talalay ldquoQuantitative analysis of dose-effectrelationships the combined effects of multiple drugs or enzymeinhibitorsrdquo Advances in Enzyme Regulation vol 22 pp 27ndash551984
[15] T C Chou and P Talalay ldquoAnalysis of combined drug effectsa new look at a very old problemrdquo Trends in PharmacologicalSciences vol 4 pp 450ndash454 1983
[16] E Kim M Matsuse V Saenko et al ldquoImatinib enhances doce-taxel-induced apoptosis through inhibition of nuclear factor-120581Bactivation in anaplastic thyroid carcinoma cellsrdquo Thyroid vol22 no 7 pp 717ndash724 2012
[17] J-C Ahn R Biswas and P-S Chung ldquoCombination withgenistein enhances the efficacy of photodynamic therapyagainst human anaplastic thyroid cancer cellsrdquo Lasers in Surgeryand Medicine vol 44 no 10 pp 840ndash849 2012
[18] I Clinckspoor L Verlinden L Overbergh et al ldquo125-Dihy-droxyvitamin D3 and a superagonistic analog in combinationwith paclitaxel or suberoylanilide hydroxamic acid have potentantiproliferative effects on anaplastic thyroid cancerrdquo Journal ofSteroid Biochemistry and Molecular Biology vol 124 no 1-2 pp1ndash9 2011
[19] M G Catalano R Poli M Pugliese N Fortunati and GBoccuzzi ldquoValproic acid enhances tubulin acetylation andapoptotic activity of paclitaxel on anaplastic thyroid cancer celllinesrdquo Endocrine-Related Cancer vol 14 no 3 pp 839ndash8452007
[20] D Russo C Durante S Bulotta et al ldquoTargeting histone deace-tylase in thyroid cancerrdquo Expert Opinion onTherapeutic Targetsvol 17 no 2 pp 179ndash193 2013
[21] P Brest S Lassalle V Hofman et al ldquoMiR-129-5p is requiredfor histone deacetylase inhibitor-induced cell death in thyroidcancer cellsrdquo Endocrine-Related Cancer vol 18 no 6 pp 711ndash719 2011
[22] T Kondo S L Asa and S Ezzat ldquoEpigenetic dysregulationin thyroid neoplasiardquo Endocrinology and Metabolism Clinics ofNorth America vol 37 no 2 pp 389ndash400 2008
[23] C S Mitsiades V Poulaki C McMullan et al ldquoNovel histonedeacetylase inhibitors in thetreatment of thyroid cancerrdquo Clini-cal Cancer Research vol 11 no 10 pp 3958ndash3965 2005
International Journal of Endocrinology 7
[24] M G Catalano N Fortunati M Pugliese et al ldquoValproic acida histone deacetylase inhibitor enhances sensitivity to doxo-rubicin in anaplastic thyroid cancer cellsrdquo Journal of Endocrinol-ogy vol 191 no 2 pp 465ndash472 2006
[25] C A Iacobuzio-Donahue ldquoEpigenetic changes in cancerrdquoAnnual Review of Pathology vol 4 pp 229ndash249 2009
[26] J-X Zhang D-Q Li A R He et al ldquoSynergistic inhibitionof hepatocellular carcinoma growth by cotargeting chromatinmodifying enzymes and poly (ADP-ribose) polymerasesrdquoHep-atology vol 55 no 6 pp 1840ndash1851 2012
[27] E Jasek M Gajda G J Lis M Jasinska and J A LitwinldquoCombinatorial effects of PARP inhibitor PJ34 and histonedeacetylase inhibitor vorinostat on leukemia cell linesrdquo Anti-cancer Research vol 34 no 4 pp 1849ndash1856 2014
[28] P A Konstantinopoulos A J Wilson J Saskowski E Wassand D Khabele ldquoSuberoylanilide hydroxamic acid (SAHA)enhances olaparib activity by targeting homologous recombina-tion DNA repair in ovarian cancerrdquo Gynecologic Oncology vol133 no 3 pp 599ndash606 2014
[29] C Puppin F DAurizio A V DElia et al ldquoEffects of histoneacetylation on NIS promoter and expression of thyroid-specifictranscription factorsrdquo Endocrinology vol 146 no 9 pp 3967ndash3974 2005
[30] C Puppin N Passon J M Hershman et al ldquoCooperativeeffects of SAHA andVPA onNIS gene expression and prolifera-tion of thyroid cancer cellsrdquo Journal ofMolecular Endocrinologyvol 48 no 3 pp 217ndash227 2012
[31] E Lavarone C Puppin N Passon S Filetti D Russo and GDamante ldquoThePARP inhibitor PJ34modifies proliferation NISexpression and epigenetic marks in thyroid cancer cell linesrdquoMolecular and Cellular Endocrinology vol 365 no 1 pp 1ndash102013
[32] G Damante G Tell and R di Lauro ldquoA unique combinationof transcription factors controls differentiation of thyroid cellsrdquoProgress in Nucleic Acid Research andMolecular Biology vol 66pp 307ndash356 2000
[33] J C Morris ldquoStructure and function of the TSH receptor itssuitability as a target for radiotherapyrdquoThyroid vol 7 no 2 pp253ndash258 1997
[34] S El-Kaissi and J R Wall ldquoTargeting the thyrotropin receptorin thyroid diseaserdquo Expert Opinion on Therapeutic Targets vol16 no 7 pp 719ndash727 2012
[35] D Paolino D Cosco M Gaspari et al ldquoTargeting the thy-roid gland with thyroid-stimulating hormone (TSH)-nano-liposomesrdquo Biomaterials vol 35 no 25 pp 7101ndash7109 2014
[36] M DrsquoAgostino M Sponziello C Puppin et al ldquoDifferentexpression of TSH receptor and NIS genes in thyroid cancerrole of epigeneticsrdquo Journal of Molecular Endocrinology vol 52no 2 pp 121ndash131 2013
[37] V Lazar J M Bidart B Caillou et al ldquoExpression of the Na+I- symporter gene in human thyroid tumors a comparisonstudy with other thyroid-specific genesrdquo Journal of ClinicalEndocrinology and Metabolism vol 84 no 9 pp 3228ndash32341999
[38] G Brabant C Maenhaut J Kohrle et al ldquoHuman thyrotropinreceptor gene expression in thyroid tumors and correlationto markers of thyroid differentiation and dedifferentiationrdquoMolecular and Cellular Endocrinology vol 82 no 1 pp R7ndashR121991
International Journal of Endocrinology 5
05
1015202530354045 TSHR
0
02
04
06
08
1
12 TTF1
0
05
1
15
2
25 TTF2
0
05
1
15
2NIS
002040608
1121416
PAX8m
RNA
leve
ls (a
u)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mRN
A le
vels
(au
)
mdash
mRN
A le
vels
(au
)
lowast
lowast
SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583M SAHA+ PJ34 15120583M
1120583Mmdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15 120583M1120583M
mdash SAHA PJ34 15120583M SAHA+ PJ34 15120583M
1120583M 1120583MSAHA+ PJ34 15120583M
1120583M
SAHA+ PJ34 15120583M
1120583M
Figure 4 Expression levels of PAX8 NIS TTF1 TTF2 and TSHR genes in SW1736 cell line RNA extraction and real-time PCR are describedin Section 2 For each gene the results were normalized against 120573-actin and expressed in arbitrary unit (2minusΔCt) Each bar represents the meanvalue of three different determinations For each bar standard deviation is not above 10of each valuelowast indicates values significantly differentcompared to all other conditions
effects of SAHA and PJ34 on leukemia cell lines have beeninvestigated [27] Also in that study synergistic effects onproliferation inhibition and apoptosis increase have beenobserved Recently synergy between SAHA and olaparib hasbeen observed even in ovarian cancer cell lines [28] We haverecently obtained similar effects on breast cancer cell lines(unpublished data)
Effects of HDAC and PARP inhibitors alone have beenpreviously investigated by our group We have shown thatHDAC inhibitors affect cell proliferation and expression ofvarious genes in several thyroid cancer cell lines [29 30]Moreover Lavarone et al have recently shown that PJ34inhibits cell growth and increases NIS expression in variousthyroid cancer cell lines [31] In this research we demonstratethat HDAC and PARP inhibitors have a synergistic effect
on proliferation of a human anaplastic thyroid-derived cellline Thus synergy between these two classes of compoundsappears to be a common phenomenon in cancer cell linesof various origins underlying the potential role of thesecombinations as an interesting strategy for cancer therapyOur obtained CI values indicate that synergy between SAHAand PJ34 occurs in a wide range of doses suggesting that thecombined effect could probably be observed also in vivo
In addition to the impact on cell proliferation wehave investigated effects of the SAHA-PJ34 combination onthyroid-specific genes expression Regarding thyroid-specifictranscription factors the SAHA-PJ34 combination induces aTTF1 slight decrease a TTF2 slight increase and no changein PAX8 Such behavior indicates that the control of thesegenes expression occurs through distinct mechanisms This
6 International Journal of Endocrinology
view agrees with previous studies in which control of thyroid-specific transcription factors expression has been investigated[32]
Synergy between SAHA and PJ34 in increasing mRNAlevels of TSHR was observed The TSHR is localized in theplasmamembrane and thus it has been proposed as a targetto direct therapeutic compounds into thyroid cancer cells[33ndash36] Our data would suggest that the combined use ofHDAC and PARP inhibitors may facilitate such approach
Differently from TSHR NIS gene expression is reducedby SAHA alone and in combination with PJ34 Such adifferent effect between these genes expression is not unex-pected TSHR and NIS have a different regulation in termsof gene expression It is well known for example that duringtumorigenesis NIS is one of the earliest downregulated geneswhile TSHR is among the latest ones [37 38]
In conclusion considering our data on cell proliferationand gene expression altogether the combined use of HDACand PARP inhibitors can be a useful strategy for ATCtreatment Preclinical in vivo studies are required to validatesuch a possibility
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was supported by grants to Giuseppe Damantefrom Associazione Italiana per la Ricerca sul Cancro (AIRC)(Project no IG 10296)
References
[1] G Pellegriti F Frasca C Regalbuto S Squatrito and R Vign-eri ldquoWorldwide increasing incidence of thyroid cancer updateon epidemiology and risk factorsrdquo Journal of Cancer Epidemiol-ogy vol 2013 Article ID 965212 10 pages 2013
[2] J P OrsquoNeill and A R Shaha ldquoAnaplastic thyroid cancerrdquo OralOncology vol 49 no 7 pp 702ndash706 2013
[3] S L Kojic S S Strugnell and S M Wiseman ldquoAnaplasticthyroid cancer a comprehensive review of novel therapyrdquoExpert Review of Anticancer Therapy vol 11 no 3 pp 387ndash4022011
[4] P A Marks and W-S Xu ldquoHistone deacetylase inhibitorspotential in cancer therapyrdquo Journal of Cellular Biochemistryvol 107 no 4 pp 600ndash608 2009
[5] D Russo G Damante E Puxeddu C Durante and S FilettildquoEpigenetics of thyroid cancer and novel therapeutic targetsrdquoJournal of Molecular Endocrinology vol 46 no 3 pp R73ndashR812011
[6] Q T Luong JOrsquoKelly GD Braunstein JMHershman andHP Koeffler ldquoAntitumor activity of suberoylanilide hydroxamicacid against thyroid cancer cell lines in vitro and in vivordquo Cli-nical Cancer Research vol 12 no 18 pp 5570ndash5577 2006
[7] A Chen ldquoPARP inhibitors its role in treatment of cancerrdquoChinese Journal of Cancer vol 30 no 7 pp 463ndash471 2011
[8] A J Frew R W Johnstone and J E Bolden ldquoEnhancing theapoptotic and therapeutic effects of HDAC inhibitorsrdquo CancerLetters vol 280 no 2 pp 125ndash133 2009
[9] G Papeo E Casale A Montagnoli and A Cirla ldquoPARPinhibitors in cancer therapy an updaterdquo Expert Opinion onTherapeutic Patents vol 23 no 4 pp 503ndash514 2013
[10] B Brodska A Holoubek P Otevrelova and K KuzelovaldquoCombined treatment with low concentrations of decitabineand saha causes cell death in leukemic cell lines but not innormal peripheral blood lymphocytesrdquo BioMed Research Inter-national vol 2013 Article ID 659254 11 pages 2013
[11] B A Teicher ldquoCombinations of PARP hedgehog and HDACinhibitors with standard drugsrdquo Current Opinion in Pharmacol-ogy vol 10 no 4 pp 397ndash404 2010
[12] M A Smith and P Houghton ldquoA proposal regarding reportingof in vitro testing resultsrdquo Clinical Cancer Research vol 19 no11 pp 2828ndash2833 2013
[13] C Szabo A Biser R Benko E Bottinger and K SusztakldquoPoly(ADP-ribose) polymerase inhibitors ameliorate nephro-pathy of type 2 diabetic Leprdbdb micerdquo Diabetes vol 55 no11 pp 3004ndash3012 2006
[14] T-C Chou and P Talalay ldquoQuantitative analysis of dose-effectrelationships the combined effects of multiple drugs or enzymeinhibitorsrdquo Advances in Enzyme Regulation vol 22 pp 27ndash551984
[15] T C Chou and P Talalay ldquoAnalysis of combined drug effectsa new look at a very old problemrdquo Trends in PharmacologicalSciences vol 4 pp 450ndash454 1983
[16] E Kim M Matsuse V Saenko et al ldquoImatinib enhances doce-taxel-induced apoptosis through inhibition of nuclear factor-120581Bactivation in anaplastic thyroid carcinoma cellsrdquo Thyroid vol22 no 7 pp 717ndash724 2012
[17] J-C Ahn R Biswas and P-S Chung ldquoCombination withgenistein enhances the efficacy of photodynamic therapyagainst human anaplastic thyroid cancer cellsrdquo Lasers in Surgeryand Medicine vol 44 no 10 pp 840ndash849 2012
[18] I Clinckspoor L Verlinden L Overbergh et al ldquo125-Dihy-droxyvitamin D3 and a superagonistic analog in combinationwith paclitaxel or suberoylanilide hydroxamic acid have potentantiproliferative effects on anaplastic thyroid cancerrdquo Journal ofSteroid Biochemistry and Molecular Biology vol 124 no 1-2 pp1ndash9 2011
[19] M G Catalano R Poli M Pugliese N Fortunati and GBoccuzzi ldquoValproic acid enhances tubulin acetylation andapoptotic activity of paclitaxel on anaplastic thyroid cancer celllinesrdquo Endocrine-Related Cancer vol 14 no 3 pp 839ndash8452007
[20] D Russo C Durante S Bulotta et al ldquoTargeting histone deace-tylase in thyroid cancerrdquo Expert Opinion onTherapeutic Targetsvol 17 no 2 pp 179ndash193 2013
[21] P Brest S Lassalle V Hofman et al ldquoMiR-129-5p is requiredfor histone deacetylase inhibitor-induced cell death in thyroidcancer cellsrdquo Endocrine-Related Cancer vol 18 no 6 pp 711ndash719 2011
[22] T Kondo S L Asa and S Ezzat ldquoEpigenetic dysregulationin thyroid neoplasiardquo Endocrinology and Metabolism Clinics ofNorth America vol 37 no 2 pp 389ndash400 2008
[23] C S Mitsiades V Poulaki C McMullan et al ldquoNovel histonedeacetylase inhibitors in thetreatment of thyroid cancerrdquo Clini-cal Cancer Research vol 11 no 10 pp 3958ndash3965 2005
International Journal of Endocrinology 7
[24] M G Catalano N Fortunati M Pugliese et al ldquoValproic acida histone deacetylase inhibitor enhances sensitivity to doxo-rubicin in anaplastic thyroid cancer cellsrdquo Journal of Endocrinol-ogy vol 191 no 2 pp 465ndash472 2006
[25] C A Iacobuzio-Donahue ldquoEpigenetic changes in cancerrdquoAnnual Review of Pathology vol 4 pp 229ndash249 2009
[26] J-X Zhang D-Q Li A R He et al ldquoSynergistic inhibitionof hepatocellular carcinoma growth by cotargeting chromatinmodifying enzymes and poly (ADP-ribose) polymerasesrdquoHep-atology vol 55 no 6 pp 1840ndash1851 2012
[27] E Jasek M Gajda G J Lis M Jasinska and J A LitwinldquoCombinatorial effects of PARP inhibitor PJ34 and histonedeacetylase inhibitor vorinostat on leukemia cell linesrdquo Anti-cancer Research vol 34 no 4 pp 1849ndash1856 2014
[28] P A Konstantinopoulos A J Wilson J Saskowski E Wassand D Khabele ldquoSuberoylanilide hydroxamic acid (SAHA)enhances olaparib activity by targeting homologous recombina-tion DNA repair in ovarian cancerrdquo Gynecologic Oncology vol133 no 3 pp 599ndash606 2014
[29] C Puppin F DAurizio A V DElia et al ldquoEffects of histoneacetylation on NIS promoter and expression of thyroid-specifictranscription factorsrdquo Endocrinology vol 146 no 9 pp 3967ndash3974 2005
[30] C Puppin N Passon J M Hershman et al ldquoCooperativeeffects of SAHA andVPA onNIS gene expression and prolifera-tion of thyroid cancer cellsrdquo Journal ofMolecular Endocrinologyvol 48 no 3 pp 217ndash227 2012
[31] E Lavarone C Puppin N Passon S Filetti D Russo and GDamante ldquoThePARP inhibitor PJ34modifies proliferation NISexpression and epigenetic marks in thyroid cancer cell linesrdquoMolecular and Cellular Endocrinology vol 365 no 1 pp 1ndash102013
[32] G Damante G Tell and R di Lauro ldquoA unique combinationof transcription factors controls differentiation of thyroid cellsrdquoProgress in Nucleic Acid Research andMolecular Biology vol 66pp 307ndash356 2000
[33] J C Morris ldquoStructure and function of the TSH receptor itssuitability as a target for radiotherapyrdquoThyroid vol 7 no 2 pp253ndash258 1997
[34] S El-Kaissi and J R Wall ldquoTargeting the thyrotropin receptorin thyroid diseaserdquo Expert Opinion on Therapeutic Targets vol16 no 7 pp 719ndash727 2012
[35] D Paolino D Cosco M Gaspari et al ldquoTargeting the thy-roid gland with thyroid-stimulating hormone (TSH)-nano-liposomesrdquo Biomaterials vol 35 no 25 pp 7101ndash7109 2014
[36] M DrsquoAgostino M Sponziello C Puppin et al ldquoDifferentexpression of TSH receptor and NIS genes in thyroid cancerrole of epigeneticsrdquo Journal of Molecular Endocrinology vol 52no 2 pp 121ndash131 2013
[37] V Lazar J M Bidart B Caillou et al ldquoExpression of the Na+I- symporter gene in human thyroid tumors a comparisonstudy with other thyroid-specific genesrdquo Journal of ClinicalEndocrinology and Metabolism vol 84 no 9 pp 3228ndash32341999
[38] G Brabant C Maenhaut J Kohrle et al ldquoHuman thyrotropinreceptor gene expression in thyroid tumors and correlationto markers of thyroid differentiation and dedifferentiationrdquoMolecular and Cellular Endocrinology vol 82 no 1 pp R7ndashR121991
6 International Journal of Endocrinology
view agrees with previous studies in which control of thyroid-specific transcription factors expression has been investigated[32]
Synergy between SAHA and PJ34 in increasing mRNAlevels of TSHR was observed The TSHR is localized in theplasmamembrane and thus it has been proposed as a targetto direct therapeutic compounds into thyroid cancer cells[33ndash36] Our data would suggest that the combined use ofHDAC and PARP inhibitors may facilitate such approach
Differently from TSHR NIS gene expression is reducedby SAHA alone and in combination with PJ34 Such adifferent effect between these genes expression is not unex-pected TSHR and NIS have a different regulation in termsof gene expression It is well known for example that duringtumorigenesis NIS is one of the earliest downregulated geneswhile TSHR is among the latest ones [37 38]
In conclusion considering our data on cell proliferationand gene expression altogether the combined use of HDACand PARP inhibitors can be a useful strategy for ATCtreatment Preclinical in vivo studies are required to validatesuch a possibility
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This work was supported by grants to Giuseppe Damantefrom Associazione Italiana per la Ricerca sul Cancro (AIRC)(Project no IG 10296)
References
[1] G Pellegriti F Frasca C Regalbuto S Squatrito and R Vign-eri ldquoWorldwide increasing incidence of thyroid cancer updateon epidemiology and risk factorsrdquo Journal of Cancer Epidemiol-ogy vol 2013 Article ID 965212 10 pages 2013
[2] J P OrsquoNeill and A R Shaha ldquoAnaplastic thyroid cancerrdquo OralOncology vol 49 no 7 pp 702ndash706 2013
[3] S L Kojic S S Strugnell and S M Wiseman ldquoAnaplasticthyroid cancer a comprehensive review of novel therapyrdquoExpert Review of Anticancer Therapy vol 11 no 3 pp 387ndash4022011
[4] P A Marks and W-S Xu ldquoHistone deacetylase inhibitorspotential in cancer therapyrdquo Journal of Cellular Biochemistryvol 107 no 4 pp 600ndash608 2009
[5] D Russo G Damante E Puxeddu C Durante and S FilettildquoEpigenetics of thyroid cancer and novel therapeutic targetsrdquoJournal of Molecular Endocrinology vol 46 no 3 pp R73ndashR812011
[6] Q T Luong JOrsquoKelly GD Braunstein JMHershman andHP Koeffler ldquoAntitumor activity of suberoylanilide hydroxamicacid against thyroid cancer cell lines in vitro and in vivordquo Cli-nical Cancer Research vol 12 no 18 pp 5570ndash5577 2006
[7] A Chen ldquoPARP inhibitors its role in treatment of cancerrdquoChinese Journal of Cancer vol 30 no 7 pp 463ndash471 2011
[8] A J Frew R W Johnstone and J E Bolden ldquoEnhancing theapoptotic and therapeutic effects of HDAC inhibitorsrdquo CancerLetters vol 280 no 2 pp 125ndash133 2009
[9] G Papeo E Casale A Montagnoli and A Cirla ldquoPARPinhibitors in cancer therapy an updaterdquo Expert Opinion onTherapeutic Patents vol 23 no 4 pp 503ndash514 2013
[10] B Brodska A Holoubek P Otevrelova and K KuzelovaldquoCombined treatment with low concentrations of decitabineand saha causes cell death in leukemic cell lines but not innormal peripheral blood lymphocytesrdquo BioMed Research Inter-national vol 2013 Article ID 659254 11 pages 2013
[11] B A Teicher ldquoCombinations of PARP hedgehog and HDACinhibitors with standard drugsrdquo Current Opinion in Pharmacol-ogy vol 10 no 4 pp 397ndash404 2010
[12] M A Smith and P Houghton ldquoA proposal regarding reportingof in vitro testing resultsrdquo Clinical Cancer Research vol 19 no11 pp 2828ndash2833 2013
[13] C Szabo A Biser R Benko E Bottinger and K SusztakldquoPoly(ADP-ribose) polymerase inhibitors ameliorate nephro-pathy of type 2 diabetic Leprdbdb micerdquo Diabetes vol 55 no11 pp 3004ndash3012 2006
[14] T-C Chou and P Talalay ldquoQuantitative analysis of dose-effectrelationships the combined effects of multiple drugs or enzymeinhibitorsrdquo Advances in Enzyme Regulation vol 22 pp 27ndash551984
[15] T C Chou and P Talalay ldquoAnalysis of combined drug effectsa new look at a very old problemrdquo Trends in PharmacologicalSciences vol 4 pp 450ndash454 1983
[16] E Kim M Matsuse V Saenko et al ldquoImatinib enhances doce-taxel-induced apoptosis through inhibition of nuclear factor-120581Bactivation in anaplastic thyroid carcinoma cellsrdquo Thyroid vol22 no 7 pp 717ndash724 2012
[17] J-C Ahn R Biswas and P-S Chung ldquoCombination withgenistein enhances the efficacy of photodynamic therapyagainst human anaplastic thyroid cancer cellsrdquo Lasers in Surgeryand Medicine vol 44 no 10 pp 840ndash849 2012
[18] I Clinckspoor L Verlinden L Overbergh et al ldquo125-Dihy-droxyvitamin D3 and a superagonistic analog in combinationwith paclitaxel or suberoylanilide hydroxamic acid have potentantiproliferative effects on anaplastic thyroid cancerrdquo Journal ofSteroid Biochemistry and Molecular Biology vol 124 no 1-2 pp1ndash9 2011
[19] M G Catalano R Poli M Pugliese N Fortunati and GBoccuzzi ldquoValproic acid enhances tubulin acetylation andapoptotic activity of paclitaxel on anaplastic thyroid cancer celllinesrdquo Endocrine-Related Cancer vol 14 no 3 pp 839ndash8452007
[20] D Russo C Durante S Bulotta et al ldquoTargeting histone deace-tylase in thyroid cancerrdquo Expert Opinion onTherapeutic Targetsvol 17 no 2 pp 179ndash193 2013
[21] P Brest S Lassalle V Hofman et al ldquoMiR-129-5p is requiredfor histone deacetylase inhibitor-induced cell death in thyroidcancer cellsrdquo Endocrine-Related Cancer vol 18 no 6 pp 711ndash719 2011
[22] T Kondo S L Asa and S Ezzat ldquoEpigenetic dysregulationin thyroid neoplasiardquo Endocrinology and Metabolism Clinics ofNorth America vol 37 no 2 pp 389ndash400 2008
[23] C S Mitsiades V Poulaki C McMullan et al ldquoNovel histonedeacetylase inhibitors in thetreatment of thyroid cancerrdquo Clini-cal Cancer Research vol 11 no 10 pp 3958ndash3965 2005
International Journal of Endocrinology 7
[24] M G Catalano N Fortunati M Pugliese et al ldquoValproic acida histone deacetylase inhibitor enhances sensitivity to doxo-rubicin in anaplastic thyroid cancer cellsrdquo Journal of Endocrinol-ogy vol 191 no 2 pp 465ndash472 2006
[25] C A Iacobuzio-Donahue ldquoEpigenetic changes in cancerrdquoAnnual Review of Pathology vol 4 pp 229ndash249 2009
[26] J-X Zhang D-Q Li A R He et al ldquoSynergistic inhibitionof hepatocellular carcinoma growth by cotargeting chromatinmodifying enzymes and poly (ADP-ribose) polymerasesrdquoHep-atology vol 55 no 6 pp 1840ndash1851 2012
[27] E Jasek M Gajda G J Lis M Jasinska and J A LitwinldquoCombinatorial effects of PARP inhibitor PJ34 and histonedeacetylase inhibitor vorinostat on leukemia cell linesrdquo Anti-cancer Research vol 34 no 4 pp 1849ndash1856 2014
[28] P A Konstantinopoulos A J Wilson J Saskowski E Wassand D Khabele ldquoSuberoylanilide hydroxamic acid (SAHA)enhances olaparib activity by targeting homologous recombina-tion DNA repair in ovarian cancerrdquo Gynecologic Oncology vol133 no 3 pp 599ndash606 2014
[29] C Puppin F DAurizio A V DElia et al ldquoEffects of histoneacetylation on NIS promoter and expression of thyroid-specifictranscription factorsrdquo Endocrinology vol 146 no 9 pp 3967ndash3974 2005
[30] C Puppin N Passon J M Hershman et al ldquoCooperativeeffects of SAHA andVPA onNIS gene expression and prolifera-tion of thyroid cancer cellsrdquo Journal ofMolecular Endocrinologyvol 48 no 3 pp 217ndash227 2012
[31] E Lavarone C Puppin N Passon S Filetti D Russo and GDamante ldquoThePARP inhibitor PJ34modifies proliferation NISexpression and epigenetic marks in thyroid cancer cell linesrdquoMolecular and Cellular Endocrinology vol 365 no 1 pp 1ndash102013
[32] G Damante G Tell and R di Lauro ldquoA unique combinationof transcription factors controls differentiation of thyroid cellsrdquoProgress in Nucleic Acid Research andMolecular Biology vol 66pp 307ndash356 2000
[33] J C Morris ldquoStructure and function of the TSH receptor itssuitability as a target for radiotherapyrdquoThyroid vol 7 no 2 pp253ndash258 1997
[34] S El-Kaissi and J R Wall ldquoTargeting the thyrotropin receptorin thyroid diseaserdquo Expert Opinion on Therapeutic Targets vol16 no 7 pp 719ndash727 2012
[35] D Paolino D Cosco M Gaspari et al ldquoTargeting the thy-roid gland with thyroid-stimulating hormone (TSH)-nano-liposomesrdquo Biomaterials vol 35 no 25 pp 7101ndash7109 2014
[36] M DrsquoAgostino M Sponziello C Puppin et al ldquoDifferentexpression of TSH receptor and NIS genes in thyroid cancerrole of epigeneticsrdquo Journal of Molecular Endocrinology vol 52no 2 pp 121ndash131 2013
[37] V Lazar J M Bidart B Caillou et al ldquoExpression of the Na+I- symporter gene in human thyroid tumors a comparisonstudy with other thyroid-specific genesrdquo Journal of ClinicalEndocrinology and Metabolism vol 84 no 9 pp 3228ndash32341999
[38] G Brabant C Maenhaut J Kohrle et al ldquoHuman thyrotropinreceptor gene expression in thyroid tumors and correlationto markers of thyroid differentiation and dedifferentiationrdquoMolecular and Cellular Endocrinology vol 82 no 1 pp R7ndashR121991
International Journal of Endocrinology 7
[24] M G Catalano N Fortunati M Pugliese et al ldquoValproic acida histone deacetylase inhibitor enhances sensitivity to doxo-rubicin in anaplastic thyroid cancer cellsrdquo Journal of Endocrinol-ogy vol 191 no 2 pp 465ndash472 2006
[25] C A Iacobuzio-Donahue ldquoEpigenetic changes in cancerrdquoAnnual Review of Pathology vol 4 pp 229ndash249 2009
[26] J-X Zhang D-Q Li A R He et al ldquoSynergistic inhibitionof hepatocellular carcinoma growth by cotargeting chromatinmodifying enzymes and poly (ADP-ribose) polymerasesrdquoHep-atology vol 55 no 6 pp 1840ndash1851 2012
[27] E Jasek M Gajda G J Lis M Jasinska and J A LitwinldquoCombinatorial effects of PARP inhibitor PJ34 and histonedeacetylase inhibitor vorinostat on leukemia cell linesrdquo Anti-cancer Research vol 34 no 4 pp 1849ndash1856 2014
[28] P A Konstantinopoulos A J Wilson J Saskowski E Wassand D Khabele ldquoSuberoylanilide hydroxamic acid (SAHA)enhances olaparib activity by targeting homologous recombina-tion DNA repair in ovarian cancerrdquo Gynecologic Oncology vol133 no 3 pp 599ndash606 2014
[29] C Puppin F DAurizio A V DElia et al ldquoEffects of histoneacetylation on NIS promoter and expression of thyroid-specifictranscription factorsrdquo Endocrinology vol 146 no 9 pp 3967ndash3974 2005
[30] C Puppin N Passon J M Hershman et al ldquoCooperativeeffects of SAHA andVPA onNIS gene expression and prolifera-tion of thyroid cancer cellsrdquo Journal ofMolecular Endocrinologyvol 48 no 3 pp 217ndash227 2012
[31] E Lavarone C Puppin N Passon S Filetti D Russo and GDamante ldquoThePARP inhibitor PJ34modifies proliferation NISexpression and epigenetic marks in thyroid cancer cell linesrdquoMolecular and Cellular Endocrinology vol 365 no 1 pp 1ndash102013
[32] G Damante G Tell and R di Lauro ldquoA unique combinationof transcription factors controls differentiation of thyroid cellsrdquoProgress in Nucleic Acid Research andMolecular Biology vol 66pp 307ndash356 2000
[33] J C Morris ldquoStructure and function of the TSH receptor itssuitability as a target for radiotherapyrdquoThyroid vol 7 no 2 pp253ndash258 1997
[34] S El-Kaissi and J R Wall ldquoTargeting the thyrotropin receptorin thyroid diseaserdquo Expert Opinion on Therapeutic Targets vol16 no 7 pp 719ndash727 2012
[35] D Paolino D Cosco M Gaspari et al ldquoTargeting the thy-roid gland with thyroid-stimulating hormone (TSH)-nano-liposomesrdquo Biomaterials vol 35 no 25 pp 7101ndash7109 2014
[36] M DrsquoAgostino M Sponziello C Puppin et al ldquoDifferentexpression of TSH receptor and NIS genes in thyroid cancerrole of epigeneticsrdquo Journal of Molecular Endocrinology vol 52no 2 pp 121ndash131 2013
[37] V Lazar J M Bidart B Caillou et al ldquoExpression of the Na+I- symporter gene in human thyroid tumors a comparisonstudy with other thyroid-specific genesrdquo Journal of ClinicalEndocrinology and Metabolism vol 84 no 9 pp 3228ndash32341999
[38] G Brabant C Maenhaut J Kohrle et al ldquoHuman thyrotropinreceptor gene expression in thyroid tumors and correlationto markers of thyroid differentiation and dedifferentiationrdquoMolecular and Cellular Endocrinology vol 82 no 1 pp R7ndashR121991
![Synthesis, [18F] radiolabeling, and evaluation of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors for in vivo imaging of PARP-1 using positron emission tomography](https://static.fdokumen.com/doc/165x107/6335c3a302a8c1a4ec01e906/synthesis-18f-radiolabeling-and-evaluation-of-poly-adp-ribose-polymerase-1.jpg)
