Photodiagnosis and Photodynamic Therapy (2014) 11, 213—226

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The role of anti-apoptotic protein kinase C�in response to hypericin photodynamictherapy in U-87 MG cells

Lenka Dzurováa, Dana Petrovajovaa, Zuzana Nadovaa,Veronika Huntosovaa, Pavol Miskovskya,b,Katarina Stroffekovaa,∗

a Department of Biophysics, University of Pavol Jozef Safarik, Kosice, Slovak Republicb Center of Interdisciplinary Biosciences, UPJS, Kosice, Slovak RepublicAvailable online 28 February 2014

KEYWORDSHypericin;Photodynamictherapy;PKC�;Apoptosis;U87 MG cells

Summary Hypericin photodynamic therapy (HypPDT) has been found to be an efficient inducerof cell death. However, there are indications that HypPDT also activates rescuing pathways. Cellresponses to HypPDT are highly dependent on the Hyp intracellular localization and accumu-lation. We have shown previously that in U87 MG cells Hyp localizes mostly in ER and partiallyin mitochondria, lysosomes and Golgi, and that HypPDT resulted primarily in apoptosis via themitochondrial apoptotic pathway. We have also shown that Hyp co-localizes and interacts withanti-apoptotic PKC� in U87 MG cells. To follow up on our previous work, we investigated howHypPDT influences PKC� in U87 MG cells. Here, we show that majority of PKC� present in U87MG cells is already in a catalytically competent form phosphorylated at Thr638, and it is alikely Bcl2 kinase. The presence of Hyp itself does not affect PKC� distribution. HypPDT acuteeffect caused PKC� activation and translocation along the plasma membrane and partially in

the nuclei. The prolonged effect of HypPDT, 5 and 24 h post PDT, results in PKC� located pre-dominantly in cytosol and nuclei. Moreover, we have shown that phosphorylated catalyticallycompetent PKC� is critical for U87 glioma cell viability in response to HypPDT treatment.© 2014 Elsevier B.V. All rights reserved.

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Introduction

Photodynamic therapy (PDT) is based on the concept thatthe combination of a photosensitizing agent, preferablytaken up and retained by tumor cells, with light leads to

∗ Corresponding author. Tel.: +421 552342243;fax: +421 55622124.

E-mail address: katarina.stroffekova@upjs.sk (K. Stroffekova).

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http://dx.doi.org/10.1016/j.pdpdt.2014.02.0101572-1000/© 2014 Elsevier B.V. All rights reserved.

he formation free radicals (ROS) causing selective damageo the target tissue. ROS can be formed by photosensitiza-ion mechanism in the absence (Type I) or in the presence ofolecular oxygen (Type II) with the additional formation of

ighly reactive singlet oxygen. Type I and Type II reactionsan occur simultaneously, and the ratio between these two

rocesses depends on the type of photosensitizing agent,nd the concentrations of substrate and oxygen [40].

Hypericin (Hyp), a naturally occurring photosensitizer,isplays antiproliferative and cytotoxic effects in many

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umor cell lines including the U87 MG line [24]. Hyp’s pho-osensitizing properties together with its selective uptaken tumor tissues, and its minimal dark cytotoxicity, maket promising for clinical use in PDT [27]. The molecu-ar mechanisms underlying PDT, and specifically Hyp-PDT,re not completely understood, although it is clear thatell photosensitization initiates multiple signaling path-ays that ultimately lead to cell death [1,52]. It has been

hown that the sub-cellular Hyp localization and distribu-ion determine which signaling pathway will lead to celleath [1,9,24,31,56]. Malignant gliomas cells, the most com-on brain tumor, are resistant to classical approaches such

s chemotherapy and radiotherapy, and therefore have aoor prognosis. The molecular mechanisms underlying glialeoplastic transformation and migration into surroundingormal brain tissue have been widely studied, and variousignaling pathways including that of protein kinase C (PKC)ave been found to be altered [35,42]. PKC activity has beeneported to be increased in gliomas and glioma cell liness compared with astrocytes, and PKC inhibitors markedlyeduced glioma cell proliferation [10,11,35]. Moreover, dif-erential expression of specific PKC isoforms has beeneported in gliomas and other malignant cells [35,46].

Protein kinase C (PKC) comprises a large family ofer/threonine (Thr) kinases that are activated by manyxtracellular and intracellular stimuli. They are catego-ized into three subfamilies on the basis of their structurend ability to bind diacylglycerol and calcium ions: classi-al, novel, and atypical PKC [41]. PKC plays a fundamentalignaling role in many physiological processes, includingodulating membrane structure, mediating the immune

esponse, and regulating cell proliferation and differenti-tion via phosphorylation of various transcription factors8,39,41,50]. Generally, inactive PKCs are considered to berimarily cytoplasmic. However, upon activation by differ-nt signals, PKC translocate to the plasma membrane, otherembranous organelles and to the nucleus [26,41,43,46].PKC�, one of the classical PKCs, extracellular signal-

elated kinase (ERK), and the stress kinase (JNK) amongthers, have been identified as Bcl2 kinases that promoteurvival in several cancerous cell lines including U87 MG13,18,46].

Members of the Bcl-2 family of proteins are key regulatorsf apoptosis by acting either as promoters or as suppressorsf the cell death process. Bcl-2 proteins are globular pro-eins containing �-helixes and at least one Bcl-2 homologyBH) domain. Anti-apoptotic Bcl-2 proteins (Bcl2, Bcl-xL, andcl1) contain four BH domains and one transmembrane (TM)omain. Pro-apoptotic Bcl-2 proteins are divided into tworoups. The first consists of the effector proteins Bak andax, which contain four BH and one TM domain. The secondonsists of BH3 only proteins (Bid, Bim, Puma, Noxa, Bad,nd Bik), which possess only one BH and no TM domain.

The primary site of action of the Bcl-2 family of proteinss the outer mitochondrial membrane (OMM), where thenteractions between anti- and pro-apoptotic Bcl-2 proteinsontrol OMM permeabilization. However, there is a grow-ng body of evidence that Bcl-2 proteins are also present in

he endoplasmic reticulum (ER) membrane and can regulatepoptosis through regulating of ER Ca2+ homeostasis [7,33].n normal healthy cells, most of anti-apoptotic Bcl-2 pro-eins are found in the OMM, or in the ER. The multi-domain

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L. Dzurová et al.

ro-apoptotic proteins Bak and Bax reside in the OMM, ERnd cytosol, respectively. Even though Bax resides primarilyn the cytosol, there are detectable amounts of Bax alsoound in the OMM and ER. Upon apoptotic signaling, Baknd Bax undergo conformational changes, and Bax translo-ates from cytosol into the OMM. Activated Bak and Bak thenligomerize in the OMM resulting in a point of no return,ermeabilization of the OMM [7,33].

Anti-apoptotic Bcl2 protects cells from a wide range oftress challenges, and thus high Bcl2 levels can predeter-ine cell survival. However, Bcl2 expression levels alone do

ot always correlate with high survival rates and resistanceo cancer therapy. The phosphorylation status of Bcl2 influ-nces its function. It has been shown that phosphorylationf Bcl2 at serine 70 is required for Bcl2’s full and potentntiapoptotic function [13,22,46,32].

We have demonstrated that Hyp colocalizes with PKC�n live U87 MG cells, suggesting a possibility of directnteraction between them [29,30]. The possibility of directnteraction has been further suggested by Hyp binding assaysnd by molecular modeling [29].

The present study represents a continuation of our pre-ious work, in which we examined the role of the PKC�soform in response to HypPDT and apoptosis of U87 MGells. Our results indicate that phosphorylated catalyticallyompetent PKC� is critical for U87 MG cell viability. Thus,egulation of PKC� activity may be important for increasingancer cells’ sensitivity toward PDT. In particular, dephos-horylation of PKC� and pBcl2 may play a role.

aterials and methods

hemicals

hosphate saline buffer (PBS), Trypsin, 0.05% (1×) with EDTANa and Alexa Fluor® 488 F(ab′)2 fragment of goat anti-abbit IgG (H + L) were purchased from Gibco InvitrogenFrance). Anti-PKC alpha antibody and Goat polyclonal sec-ndary antibody to rabbit IgG — H&L (FITC) were purchasedrom Abcam (United Kingdom). MTT (3-[4,5-dimethylthiazol--yl]-2,5-diphenyltetrazolium bromide), carbonyl cyanide-chlorophenylhydrazone (CCCP), phorbol 12-myristate 13-cetate (PMA), propidium iodide (PI), and Annexin-V-FITCrom Becton, Dickinson (Canada).

ell culture

87 MG human glioma cells (Cell Lines Services, Germany)ere plated and maintained according to propagation pro-

ocols onto 35 mm culture dishes with integral No. 0lass cover slip bottoms (MatTek, USA). The U87 MG cellsere grown in Dulbecco’s modified Eagle medium (D-MEM,ibco-Invitrogen, Life Technologies Ltd.) with high glucose

4500 mg L−1) supplemented with 10% FBS or serum sub-titute 2% Ultroser G (Pall Life Sciences, France), in the

resence of 5% CO2 humidified atmosphere at 37 ◦C. Cellsere incubated in the dark. After reaching 40—50% conflu-nce, cells were used in experiments according protocolselow.

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The role of anti-apoptotic protein kinase C�

Hypericin-photoactivation

The photosensitizer Hyp (Gibco Invitrogen, France) was dis-solved in dimethyl sulphoxide (DMSO) and added to theculture medium (2% Ultroser G) at a final concentration of500 nM Hyp. For all experiments the final content of DMSOwas less than 0.1%. Cells were incubated for 1 h under darkconditions. Cell media were then changed to D-MEM con-taining 10% FBS. In the next step, cells were illuminatedby monochromatic homemade diode illuminator at 590 nmwavelength and light dose of 4 J cm−2. Cellular response wasobserved 0, 5 and 24 h after irradiation and compare withnon-treated cells kept in dark conditions (control).

MTT viability assay

The cells were plated into 96-well tissue culture platesat density of 10 000 cells per well and cultivated toreach an optimal 80% confluency. Cells were treated withHypPDT protocol as described above. After the corre-sponding times post irradiation (5 and 24 h), 10 �l oftetrazolium compound MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was added to the wells,and the cells were incubated for an additional 2 h at 37 ◦C.MTT was reduced by metabolically active cells (correspond-ing to cells with active mitochondria) to insoluble purpleformazan dye crystals. To dissolve formazan crystals, acidicisopropanol was added to the wells. The absorbance ofsamples was measured at 570 nm on spectrophotometer(GloMax®-Multi+ Detection System with Instinct Software,USA).

Detection of mitochondrial membrane potential

The mitochondrial membrane potential ��m was monitoredby selective accumulation of 3,3′-dihexyloxacarbocyanineiodide DiOC6(3) (Gibco Invitrogen, France), a membrane per-meable cationic lipophilic probe. 40 nM DiOC6(3) was addedto cells 10 min before analysis by MACSQuant® Analyzer (Mil-tenyi Biotec, Germany). Green emission of DiOC6(3) wasmeasured in the FITC channel (525/50 nm). Cells treatedwith 10 �M CCCP were used as a positive control for totaldepolarization of the ��m.

Analysis of apoptosis

Cells were treated with 500 nM Hyp for 1 h in the dark condi-tion as described above. In order to analyze different typesof cell death, the cells were trypsinated after a definedregeneration time, washed with phosphate buffered saline(PBS), centrifuged and resuspended with Annexin-bindingbuffer. Cells (100 �l) were incubated with 5 �l Annexin-VFITC for 15 min at room temperature. In the same step, PI

was added to distinguish necrotic cells. After the incuba-tion with the markers, 400 �l of Annexin-binding buffer wasadded. The FACS analysis took place within 1 h of staining byBD FACSCaliburTM flow cytometer (BD Bioscience, USA).

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mmunoblot analysis

hole cell lysate was prepared as follows. Briefly, cells werewelled in ice-cold RIPA buffer (150 mM NaCl; 1% Triton X-00; 0,5% sodium deoxycholate; 0,1% SDS; 50 mM Tris at pH; 2 �g/ml aprotinin, 10 �g/ml leupeptin; 1 mM PMSF) for5 min, aspirated repeatedly through a 25-gauge needle (25trokes). Lysate was stored at −20 ◦C. Lysates (60 �g of pro-ein) were subjected to SDS-PAGE (10%) followed by transfero nitrocellulose membranes (Trans-Blot SD, Semi-Dry Elec-rophoretic Transfer Cell, Bio-Rad, USA). The nitrocelluloseembranes were then stained with the primary antibody.esternBreeze® Chromogenic Western Blot Immunodetec-

ion Kit (Invitrogen, USA) was used for immunodetection.

mmunocytochemistry protocol

ell cultures were plated onto 35 mm culture dishes withntegral No. 0 glass cover slip bottoms (MatTek, USA).he cells were fixed with formaldehyde and permeabil-

zed with Triton X-100 according standard protocol [5].ells were then double stained with an appropriate com-ination of specific primary and secondary antibodies (Ab)onjugated with FITC, Alexa 488 or Alexa 546 (Life Tech-ologies Ltd.). Ab used: Ab against PKC� (Anti-PKC alphantibody (ab4124), Abcam, UK), Ab against active PKC�PKC� [pThr638] Antibody (E195ab) (Novus Europe), Abgainst p-Bcl2 (Phospho-Bcl2 (Ser70) (5H2ab) Rabbit mAb,ell Signaling Technology, USA). Cells were then assessed byuorescent confocal microscope.

itotracker protocol

rior immunocytochemistry U87 MG cells were incubated in% UG media with MitoTracker Orange CMTMRos (Invitrogen),nal concentration 400 nM, for 5 min at 37 ◦C. Cells werehen fixed according the protocol above.

luorescence microscopy

ells were placed in PBS/BSA (0.2%), and assessed with a3× oil objective (NA = 1.46) of LSM 700 confocal microscopeystem (LSM 700, Zeiss Germany). The laser line (488 nm) ofhe solid state laser was used to excite FITC or Alexa 488uorophore. Emissions of Alexa 488 were recorded in single-rack configuration with a band-pass filter of 490—555 nm.he laser line (555 nm) of the solid state laser was used toxcite Alexa 546 or MitotrackerOrange fluorophore. Emis-ions of Alexa 546 or MitotrackerOrange fluorophore wereecorded in single-track configuration with a long-pass fil-er of 560 nm. Fluorescence signals were analyzed by theen 2011 software (Zeiss, Germany). Cells were scanned in-stack mode with 0.75 �m slice width. Cells’ total widthsaried from 7 to 12 �m.

o-localization analysis

o-localization analysis was performed on the obtaineduorescence images using the Zen 2011 image processing

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oftware (Zeiss). Mander’s coefficient (M, Eq. (1)), was usedor co-localization analysis:

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-values can vary within the range from 0 to 1. 100% colo-alization corresponds to M = 1. Ri and Gi are the signalntensities of the pixel number ‘‘i’’ obtained in the red andhe green channel, respectively. Mander’s coefficient doesot depend on the relative strengths of each channel, butt can be affected by the background signal [6]. To circum-ent this limit, MRed and MGreen were calculated by settinghe threshold to the value of the background.

tatistical analysis

xperiments under all conditions were done in at least threendependent repetitions. Mean and standard errors (SEM)ere evaluated in at least three independent experiments.tatistical analysis was carried out by Student’s t-test usingigmaplot (Ver. 12.0). A p < 0.05 was considered significant.

esults

ypPDT triggers apoptosis as a primary type of celleath

ig. 1A shows cell death type analysis by flow cytome-ry measurements (Fig. 1A). U87 MG cells were stained bynnexin-V FITC and PI to distinguish apoptotic and necroticells (see Materials and methods). Annexin-V is a Ca2+-ependent phospholipid-binding protein with a high affinityor phosphatidylserine. At the onset of apoptosis, phos-hatidylserine, which is normally found on the inner leafletf the plasma membrane, becomes translocated to the outerayer of the membrane. The binding of Annexin-V to phos-hatidylserine at the outer cell membrane is one of thearliest stages of apoptosis. As apoptosis progresses, theres further loss of the membrane integrity, and Annexin-V isble to bind to phosphatidylserine at the inner leaflet of thelasma membrane. PI is membrane impermeant and gen-rally excluded from viable cells. It binds to cellular DNAnce the integrity of the plasma membrane has been com-romised. Three major populations of cells were observedn the cytotoxicity assay: Annexin Vnegative/PInegative; Annexinpositive/PInegative and Annexin Vpositive/PIpositive. Cells negativeor both fluorochromes represent live healthy cells. Annexinpos/PIneg cells represent early apoptotic cells, and Annexinpos/PIpos cells represent late apoptotic/necrotic cells. Aimilar method and observations for evaluation of the celleath stage in different cell types including glioma cellsave been published by others [4,44,47].

Control cells and cells in the presence of Hyp with-ut irradiation displayed similar viability of around 90%.mmediately after HypPDT (0 h), we observed an increased

ercentage of cells in apoptosis (5.04% early, Annexinpositive/PInegative + 15.23% late, Annexin Vpos/PIpos) as well as

n necrosis (22.63%, Annexin Vneg/PIpos). 5 and 24 h after Hyp-DT, the majority of affected cells were in apoptosis with

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L. Dzurová et al.

ess than 4% in necrosis. These results indicate that HypPDTesulted mostly in cell apoptosis under our conditions.

Flow cytometry experiments are in good agreement withhe MTT assay results (Fig. 1B). MTT is a sensitive in vitrossay for the measurement of cell proliferation or, whenetabolic events lead to apoptosis or necrosis, a reduction

n cell viability. The MTT assay is based on the cleavage ofhe yellow tetrazolium salt MTT to purple formazan crystaly metabolic active viable cells. Thus, live cells and cells inhe early stage of apoptosis, with functional mitochondria,ill give a signal as viable cells in MTT assay.

Cells under control conditions and in the presence ofyp (no irradiation) have similar viabilities with values ∼95%

Fig. 1B). In cells treated with HypPDT, we observed in MTTssay a significant decrease in viability to 53% (5 h afterDT), and to 49% (24 h after PDT), respectively. This cor-esponds to a percentage of live and early apoptotic cellsnder the same conditions measured by Annexin-V FITC/PIssay (Fig. 1A, lower left and right quadrants). Resultsbtained by flow cytometry and MTT tests are in a goodgreement with the results published by others [28].

ypPDT results in apoptosis via mitochondrialpoptotic pathway

ecause PDT is known to increase ROS production and torigger a mitochondrial apoptotic pathway, we examinedhanges in mitochondrial membrane potential (��m) byow cytometry under all experimental conditions in the live87 MG cells (Fig. 2). Cells were stained with potentiomet-ic fluorescent dye DiOC6(3) at 40 nM final concentration.�m was not affected by the presence of Hyp without irra-iation (Fig. 2A, histogram 1) in comparison with controlells without Hyp (data not shown). Mitochondria in cellsnder control conditions and in the presence of Hyp (norradiation) displayed well developed mitochondria networkhroughout the cells as shown by the mitochondria spe-ific marker MitoTracker Orange CMTMRos (Fig. 2B). HypPDTed to depolarization of mitochondrial membrane potentialFig. 2A, histogram 2), and to mitochondria granulation andetwork dissipation shortly after PDT (Fig. 2B). 5 h afterypPDT, there was no significant change in ��m in com-arison with data shortly after PDT (Fig. 2A, histogram 3s 2). However, in 24 h after PDT, there is further signif-cant decrease in ��m (Fig. 2A, histogram 4) with twoeaks. The right peak in histogram 4 represents populationf cells with slightly depolarized mitochondria as in his-ograms 2 and 3. The left peak in histogram 4 correspondso cell population with dissipated mitochondrial potentialimilar to spectra obtained in the presence of 10 �M car-onyl cyanide 3-chlorophenylhydrazone (CCCP) (Fig. 2A,istogram 5). CCCP is known agent to cause rapid mito-hondrial membrane depolarization and is often used as aositive control for dissipation of ��m [25,36]. The lastbservation suggests that 24 h after PDT, the majority of theitochondria in surviving cells are depolarized, and there-

ore dysfunctional. This finding is in good agreement with

iability assays where we found that 24 h after PDT morehan 90% of the cells were apoptotic (Fig. 1A). Monitoringf mitochondrial membrane potential confirmed that Hyp-DT results in mitochondrial membrane depolarization. This

The role of anti-apoptotic protein kinase C� 217

Fig. 1 Cell cytotoxicity before and after HypPDT. (A) Flow-cytometric dot plot analysis of U87 MG cells stained with propidiumiodide (PI) and Annexin-V-FITC after incubation with Hyp (500 nM). The three major populations of cells were observed in thecytotoxicity assay Annexin Vnegative/PInegative; Annexin Vpositive/PInegative, and Annexin Vpositive/PIpositive. Cells negative for both fluo-rochromes represent live cells, Annexin Vpos—PIneg cells represent early apoptotic cells, and Annexin Vpos—PIpos cells represent latestage apoptotic cells. Panels from left to right, control cells, cells after 1 h incubation with Hyp (no irradiation), and cells irradiated(irradiation dose of 4 J cm−2; � = 590 nm) 1 h after incubation with Hyp and then observed at 0, 5 and 24 h. (B) Cell viability beforeand after HypPDT was measured by the MTT test. The cells were plated into 96-well tissue culture plates at a density of 10 000 cellsper well, and cultivated to reach an optimal population density. Cells were treated with HypPDT protocol as described in Materialsand methods. Results of adherent cell numbers are expressed relative to control (100%) and represent mean values. The groupstreated with HypPDT at 5 and 24 h after irradiation were compared with the control and with group treated with Hyp no irradiation.

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finding strongly suggests that HypPDT triggers U87 MG cellapoptosis via a mitochondrial apoptotic pathway under ourconditions.

Most of PKC� present in U87 glioma cells is in thecatalytically competent phosphorylated form

Because increased activity of PKC� has been reported ingliomas and glioma cell lines in comparison with astrocytes[10,11,35,46], we examined distribution of PKC� in U87 MGcells and tested whether the present PKC� in U87 MG cellsis in a catalytically competent form.

It has been shown that phosphorylation at the turn motif

of mature PKC�, which includes Threonin (Thr) 638, is allthat is needed for catalytic function of the enzyme. Phos-phorylation at this position stabilizes PKC� in a catalyticallycompetent, thermally stable and phosphatase-resistant

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onformation. On the other hand, dephosphorylation at thisosition abolishes PKC activity [41]. This mature catalyti-ally competent but inactive PKC resides in cytoplasm, andpon activation by different signals, PKC� may translocateo the plasma membrane, other membranous organelles andhe nucleus [17,41,50,59,60].

We examined the presence and distribution of PKC� in87 MG cells before and after HypPDT by immunocytochem-

stry combined with confocal microscopy and by Westernlot analysis. Cells were double stained with two antibod-es (Abs) against PKC� (ab4124, E195ab) respectively, whichecognized different epitopes respectively (657-672AA; 620-41AA). First Ab (ab4124, Abcam, UK) recognized last 15 AAn C-terminus of PKC�, and could not distinguish between

he catalytically competent or incompetent forms (greenuorescence). Second, Ab (E195ab, Novus Europe) recog-ized 21 AA surrounding Thr 638 (620-641AA of humanKC�), and specifically recognized PKC� as a catalytically

218 L. Dzurová et al.

Fig. 2 HypPDT affects mitochondria function. (A) Flow-cytometric analysis of the frequency histograms of mitochondrial potential��m. U87 MG cells were stained with 40 nM of DiOC6(3). Mitochondrial potential ��m was measured after incubation in thepresence of Hyp without further irradiation (histogram 1). The effects of HypPDT on ��m were examined in 0, 5 and 24 h post PDT(histogram 2, 3 and 4, respectively). 10 �M CCCP was used as a positive control for dissipation of ��m (histogram 5). (B) MitotrackerOrange®CMTMRos fluorescence images (orange fluorescence) displaying mitochondrial network distribution pattern. In control cells,there was well developed extensive mitochondrial network. Please note that Hyp without irradiation did not affect the distributionp et oft

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attern. However, immediately after HypPDT, there was an onso 10 �m.

ompetent form phosphorylated at amino acid residue Thr38 (PKC�(pThr638); red fluorescence). In the control cellsnd in the cells treated with Hyp without irradiation, theajority of PKC� was distributed evenly in the cytoplasm

Fig. 3). This distribution is in good agreement with the pre-iously published results by our group [29] as well as bythers [35]. Immediately after HypPDT, we observed translo-ation of PKC� toward plasma membrane (Fig. 3) similar toMA-treated cells [20,35,58], suggesting that HypPDT acuteffect is similar to PMA activation of PKC�.

As confocal images show (Fig. 3), the majority of PKC�resent in U87 MG cells is already in a catalytically com-etent phosphorylated form. This is in agreement withublished works regarding increased activity of PKC� inliomas and glioma cell lines [35,46].

The finding was confirmed by colocalization analysis

sing Mander’s coefficient (Materials and methods, Eq. (1);able 1), and by Western blot analysis (Fig. 4), where weound catalytically competent PKC�(pThr638) in cell lysatesnder all experimental conditions. The amount of protein

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Table 1 Co-localization analysis of endogenous PKC� with catalEq. (1), Materials and methods) is indicative of co-localization (min100% colocalization).

Mean (near plasmamembrane at 1 �m depth)

Colocalization of PKC˛ with catalytically competent PKC˛ (MandeControl 0.6531 ± 0.0123

Hyp no Irr 0.6560 ± 0.0176

HypPDT 0 h after Irr 0.7013 ± 0.0152**

HypPDT 5 h after Irr 0.6900 ± 0.0164

HypPDT 24 h after Irr 0.7042 ± 0.0219**

** p < 0.05, compared with control.

mitochondrial network granulation. The scale bar corresponds

etected by either Ab against PKC� in Western blot analysisas similar in corresponding cell lysates (Fig. 4), confirming

he assumption that most of PKC� is in a catalytically com-etent phosphorylated form.

Immediately after HypPDT, there was a significant redis-ribution of PKC�. Majority of PKC� was translocated fromytoplasm to the plasma membrane, and a small portionnto nucleus (Fig. 3). The translocation of PKC� towardlasma membrane is similar to PMA-treated cells [20,35,58].lease note, that Ab (ab4124, Abcam, UK), which couldot distinguish between catalytically competent or incom-etent forms, was not able to pick up a PKC� signal inhe cell nucleus (Figs. 3 and 5) in comparison with secondb (E195ab, Novus Europe), which specifically recognized

PKC� catalytically competent form (Fig. 3). The possiblexplanation for this observation may be that ab4124 epi-

ope is part of PKC V5 domain, which serves as a nuclearocalization signal in conventional, novel and atypical PKCs,nd therefore upon nuclear translocation epitope may note accessible for ab4124 [17,41,59,60].

ytically competent PKC�(pThr638). Mander’s coefficient (see. value of 0 means no colocalization, max. value of 1 indicate

Size N Mean (inside cells at 3 �mdepth)

Size N

r’s coefficient)16 0.6338 ± 0.0096 1610 0.6460 ± 0.0154 1024 0.6804 ± 0.0145** 2410 0.6720 ± 0.0104** 1012 0.6733 ± 0.0215 12

The role of anti-apoptotic protein kinase C� 219

Fig. 3 Distribution of PKC� double stained with two antibodies against PKC� (ab4124, E195ab). The figure shows immunofluo-rescence signal of PKC� labeled with Ab (ab4124, Abcam, UK), which could not distinguish between catalytically competent orincompetent form (FITC-PKC˛, green fluorescence), and signal of PKC� labeled with (E195ab, Novus Europe), which specificallyrecognized catalytically competent form of PKC� phosphorylated at amino acid residue Thr 638 (Alexa 546-PKC˛(pThr638); redfluorescence). Please note, that under all conditions, there is a significant overlap of two signals, as indicated by co-localizationplots below the fluorescent images. The scale bar corresponds to 10 �m.

Fig. 4 Western blot analysis of endogenous PKC� distribution pattern in U87 MG cells before and after HypPDT. U87 cells expressingendogenous PKC� under all conditions were harvested. Cell lysates were prepared according the protocol (Materials and methods)and subjected to SDS-PAGE and Western blot analysis. The membranes were probed with either Ab (ab4124, Abcam, UK), which couldnot distinguish between catalytically competent or incompetent form (PKC˛), or with Ab (E195ab, Novus Europe), which specificallyrecognized the catalytically competent form of PKC� phosphorylated at Thr638 (PKC˛(pThr638)). The results are representative offour similar experiments.

220 L. Dzurová et al.

Fig. 5 Distribution of PKC� and pBcl2 in U87 MG cells before and after HypPDT. The figure shows confocal images of U87 MGcells double stained with primary Ab against PKC� (ab4124, Abcam, UK, green fluorescence signal) and Ab against pBcl2S70 (redfl S70 cc to 10

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uorescence signal). Under all conditions, the majority of pBcl2orresponding co-localization plots. The scale bar corresponds

5 and 24 h after HypPDT, PKC� distribution changedgain. 5 h post PDT, PKC� was still localized along the plasmaembrane and some in cytoplasm, but there was a signifi-

ant increase of catalytically competent PKC� in the nucleiFig. 3). Translocation of PKC� into nuclei upon activationy different signals has been reported [17,26,48,59]. 24 host PDT, majority of PKC� detected by either Ab (ab4124r E195ab) was localized in cytoplasm (Figs. 3 and 5) similaro distribution of dephosphorylated PKC� [20]. This suggestshat the prolonged effect of PDT may result in dephospho-ylation and consequently in inactivation of PKC�.

cl2 present in U87 glioma cells is phosphorylatedt Ser70 and co-localizes with PKC�

he PKC� and extracellular signal-related kinase (ERK) have

een identified by Western blot and flow cytometry analysiss Bcl2 kinases that promote survival in several cancerousell lines including U87 MG. In addition, it has also beenhown that phosphorylation of Bcl2 at serine 70 is required

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o-localized with PKC� as indicated by overlay images and their �m.

or Bcl2’s full and potent antiapoptotic function in cancerells [12,32,46]. To investigate this further in U87 MG cells,e simultaneously examined distribution and co-localizationf Bcl2 phosphorylated at Ser70 (pBcl2S70) and PKC� in U87G cells before and after HypPDT by immunocytochemistry.ig. 5 shows confocal images of U87 MG cells double stainedith primary Ab against PKC� (ab4124, green fluorescence

ignal) and Ab against pBcl2S70 (red fluorescence signal).nder all conditions, the majority of pBcl2S70 co-localizedith PKC�, suggesting that PKC� is likely one of the Bcl2inases in U87 MG cells. Under control condition and in cellshere Hyp without irradiation was present, distribution ofBcl2S70 and PKC� was primarily in the cytoplasm (Fig. 5).he presence of Hyp without irradiation did not changehe distribution pattern. However we observed increasedo-localization of pBcl2S70 and PKC� in comparison withontrol cells (Table 2), suggesting that Bcl2 phosphorylation

y PKC� may have increased due to Hyp presence.

Upon HypPDT stimulus, there was a significant redistri-ution of pBcl2S70 and PKC� toward a plasma membranend into discrete foci (presumably mitochondria). 5 and 24 h

The role of anti-apoptotic protein kinase C� 221

Table 2 Co-localization analysis of endogenous PKC� with pBcl2. Mander’s coefficient (see Eq. (1), Materials and methods) isindicative of co-localization (min. value of 0 means no colocalization, max. value of 1 indicate 100% colocalization).

Mean (near plasmamembrane at 1 �m depth)

Size N Mean (inside cells at 3 �mdepth)

Size N

Colocalization of PKC˛ with pBcl-2 (Mander’s coefficient)Control 0.7056 ± 0.0109 9 0.7089 ± 0.0228 9Hyp no Irr 0.7761 ± 0.0134** 18 0.7528 ± 0.0166 18HypPDT 0 h after Irr 0.7943 ± 0.0275** 14 0.7971 ± 0.0157** 14HypPDT 5 h after Irr 0.8370 ± 0.0101**,# 10 0.8000 ± 0.0137** 10HypPDT 24 h after Irr 0.8288 ± 0.0129**,# 8 0.8025 ± 0.0141** 8

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** p < 0.05, compared with control.# p < 0.05 compared with Hyp no irradiation.

after HypPDT, in the cells that did not display morphologicalsigns of apoptosis, there was a further significant increasein co-localization (p < 0.05; Table 2) between pBcl2S70 andPKC� in the vicinity of plasma membrane and in the discretefoci within the cells, suggesting increased activity of PKC�and phosphorylation of Bcl2 to promote cell survival (Fig. 5).

Phosphorylated catalytically competent PKC� iscritical for cell survival in U87 MG glioma cells

Because of the observed activity of PKC� and localizationof pBcl2S70 in surviving U87 MG cells post PDT, and becauseincreased activity of PKC� and Bcl-2 phosphorylation havebeen reported to be responsible for glioma and leukemiccells resistance to anticancer treatments [32,35,46], wetested whether the dephosphorylation of PKC� in U87 MGcells will result in decreased cell viability after HypPDTtreatment.

All conventional PKC isotypes retain the same three prim-ing phosphorylation sites and these are subject to agonist-dependent dephosphorylation [20,41]. The mechanism ofPKC dephosphorylation requires first a PKC-dependent phos-phorylation step that most likely involves membrane trafficof the PKC itself. The temporal restriction imposed on PKCfunction by its induced dephosphorylation and inactivationis anticipated to play a key role in determining cellu-lar responses. There is evidence that PKC� does becomedephosphorylated in an agonist-dependent manner on pro-longed stimulation [14,19].

To dephosphorylate PKC�, we used prolonged exposure ofU87 MG cells (2 h) to PKC� agonist phorbol 12-myristate 13-acetate (PMA) at a final concentration 400 nM followed byHyp treatment as described in Materials and methods. Wethen analyzed cells by immunocytochemistry and analyzedcell viability by MTT assay.

Prolonged exposure to 400 nM PMA did not affectmitochondria network either in the absence or presence ofHyp in U87 MG cells (Fig. 6A) in comparison with cells in theabsence of PMA (Fig. 2B). Fig. 6B shows confocal images ofU87 MG cells after prolonged PMA exposure with and withoutHyp stained with primary Ab E195ab against phosphory-

lated PKC�(pThr638) (green fluorescence signal). Prolongedexposure to 400 nM PMA resulted in different distribution ofphosphorylated PKC� (PKC�(pThr638)) in comparison withcells without PMA treatment (Figs. 6B and 3). In the cells

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reated with PMA only (Fig. 6B), PKC�(pThr638) displayedocalization along the plasma membrane, in the nucleusnd only partially in cytoplasm. This distribution is differenthan PKC�(pThr638) distribution in control cells without anyreatment (Figs. 6B, left most panel and 3(red fluorescenceignal)), and it is also different than in the cells treatedith Hyp without irradiation (Figs. 6B and 3). Distributionf PKC�(pThr638) in cells treated with prolonged exposureo PMA is similar to the distribution in the cells 5 h afterypPDT (Fig. 3 (red fluorescence signal)), indicating thatrolonged PMA treatment triggered translocation of PKC�oward the plasma membrane and into the nuclei. The addi-ion of Hyp and 1 h incubation without irradiation to the PMAretreated cells, resulted in decreased plasma membranend increased cytoplasmic localization of PKC�(pThr638),ut there was still significant localization in the nucleus.his indicates, that Hyp and PMA compete for the same sites suggested in our previous work [29], and that Hyp boundKC� without irradiation does not translocate toward theembrane or into nucleus (Figs. 6B and 3). HypPDT in theMA pretreated cells triggered significant PKC�(pThr638)ranslocation toward the plasma membrane and into theuclei (Fig. 6B), similar to the cells treated with HypPDT onlyFig. 6B) with one difference being that in the PMA prolongedxposure treated cells there is more profound nuclear stain-ng due to PMA induced PKC�(pThr638) nuclear localization.luorescence signal at 0 h post HypPDT from the nucleusf the cells treated with HypPDT only was 12 + 3% (n = 22)raction of total fluorescence. Where in the PMA prolongedxposure treated cells, we observe significant (p < 0.00002)ncrease in fraction of total fluorescence from the nucleuso 69 + 3.4% (n = 17). The results obtained from cells treatedith prolonged PMA exposure indicate that Hyp can compete

or PMA binding site, removing PMA and freeing PKC� intoytoplasm.

Fig. 7 shows effects of HypPDT on viability U87 MG cellsn the absence and presence of 400 nM PMA (2 h). The pres-nce of PMA slightly decreased cell viability by 14% (Fig. 7)rom 100% to 86% (p < 0.001) in comparison with control cellsFig. 7) probably due to PKC� dephosphorylation. The viabil-ty of cells treated with Hyp only without irradiation (Fig. 7)as similar to control cells. Addition of Hyp without irradia-

ion to the PMA pretreated cells improved cell survival backo same values as the control cells. This effect could beaused by either competitive binding of Hyp to a PMA bind-ng site on the PKC�, or it could be independent of PMA. It

222 L. Dzurová et al.

Fig. 6 Dephosphorylation of PKC� affects its distribution pattern in U87 MG cells before and after HypPDT. (A) MitotrackerOrange®CMTMRos fluorescence images (orange fluorescence) displaying mitochondrial network distribution pattern. Prolongedexposure to 400 nM PMA, followed by addition of Hyp without irradiation did not affect mitochondrial network. However, imme-diately after HypPDT, there was an onset of mitochondrial network granulation in 400 nM PMA treated cells. (B) Confocal imagesof U87 MG cells treated with prolonged exposure to 400 nM PMA and then stained with primary Ab against catalytically competentPKC�(pThr638) (green fluorescence signal). Prolonged exposure to 400 nM PMA resulted in a different distribution of PKC�(pThr638)than in control cells without any treatment. In 400 nM PMA treated cells, PKC�(pThr638) displayed localization along plasma mem-brane, in the nucleus and only partially in cytoplasm. The addition of Hyp followed by 1 h incubation without irradiation of PMAtreated cells, resulted in decreased plasma membrane and increased cytoplasmic localization of PKC�. However there was stillsignificant localization in the nucleus. HypPDT in the PMA treated cells triggered PKC� translocation toward plasma membrane withmore profound nuclear staining due to PMA induced PKC� nuclear localization. In cells under control conditions and with Hyp noirradiation, PKC� was mostly cytosolic, distributed evenly in the cytoplasm. Immediately after HypPDT, PKC� translocated towardt r intr

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he plasma membrane and a small portion into the nucleus. (Foeader is referred to the web version of the article.)

as been found that up to 3 �M Hyp in the dark has a stimu-atory effect on cell growth by the known stimulation of therowth pathways of p38MAPK and JNK [31].

In cells treated with Hyp only, viability 5 h after HypPDTas significantly lower (49%; p < 0.001) (Fig. 7) in comparison

ith control and cells treated with PMA and Hyp without irra-iation. PMA treated cells 5 h after HypPDT (Fig. 7) displayedurther significant decrease by 14% (35%; p < 0.001) in viabil-ty in comparison with cells treated with Hyp only 5 h after

D

Ai

erpretation of the references to color in this figure legend, the

ypPDT, suggesting that phosphorylation dependent PKC�ctivity may play a role in response to HypPDT in U87 MGells.

iscussion

lthough in general HypPDT has been found to be an efficientnducer of cell death, some in vivo studies indicate that PDT

The role of anti-apoptotic protein kinase C�

120

100

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60

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Con tro l 400 nMPMA

Hyp no Irr

HypPDTIrr5h after

+ PMA

HypPDTIrr5h after

Hyp no Irr+ PMA

Fig. 7 Dephosphorylation of PKC� plays a role in U87 MG cellsviability before and after HypPDT. Cell viability of U87 MG cellstreated with prolonged exposure to 400 nM PMA before and afterHypPDT was measured by the MTT test. The cells were platedinto 96-well tissue culture plates at a density of 10 000 cellsper well, and cultivated to reach an optimal population den-sity. Cells were treated with HypPDT protocol as described inMaterials and methods. Results of adherent cell numbers areexpressed relative to control (100%) and represent mean values.Cell viability of U87 MG cells treated with prolonged exposure to400 nM PMA decreased significantly in comparison with controlcells. Cell viability in the group treated with HypPDT only 5 hafter irradiation significantly decreased when compared withgroup treated with Hyp no irradiation. In the cells treated withprolonged exposure to 400 nM PMA followed by HypPDT at 5 hafter irradiation, cell viability further decreased in comparisonwith the group treated with HypPDT only 5 h after irradiation.

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also activates survival pathways, which ultimately can leadto tumor survival and recurrence [3,16,23,28,45,57]. Cellresponses to HypPDT are highly dependent on the Hyp intra-cellular localization and accumulation [9,16,21,28]. The Hypdisplays a selective affinity to lipid membranes in general[15], and more specifically, it has been found to accumu-late in the endoplasmic reticulum (ER) and Golgi [9,54],lysosomes [49,53] and mitochondria [2]. Thus the initialphotodamage can involve different organelles with the con-sequent activation of specific death pathways, which maybe dependent or independent on caspase signaling for theirexecution.

We have shown previously that under our conditions(1 h incubation with Hyp followed by PDT), Hyp in U87MG cells localizes mostly in ER, mitochondria, lysosomesand Golgi, and that intracellular distribution of biologicallyactive monomeric forms is highly time dependent [21]. Hyp-PDT in U87 MG cells resulted primarily in apoptotic cell deathtype via mitochondrial apoptotic pathway as it was shownhere by flow cytometry (Fig. 1A). This finding is in good

agreement with our previous work as well with results fromother studies with various cancer cells (C6, HeLa, T24, etc.;[9,16,21,28]). Previously we have also shown that Hyp co-localizes and interacts with anti-apoptotic PKC� in U87 MG

rotp

223

ells [29]. However, little is known about effects of HypPDTn PKC� activity. Here we followed up and investigated howypPDT influences PKC� in U87 MG cells.

In the present work, we have shown by immunocytochem-stry and Western blot analysis that the majority of PKC�resent in control U87 MG cells is already in a catalyticallyompetent form phosphorylated at Thr638 (PKC�(pThr638))Figs. 3 and 4). In agreement with the described localizationf a catalytically competent form of PKC� [41,50], we havehown that under control conditions (Hyp absence) and inhe presence of non-irradiated Hyp, catalytically competentKC�(pThr638) was distributed evenly in the cytoplasm, and

small portion was attached to mitochondria (Figs. 3 and 6)n U87 MG cells. Even though Hyp co-localized with PKC� inive U87 MG cells, and based on molecular modeling, it wasuggested that Hyp can bind with similar affinity to the sameinding site as PMA in the C1B domain of PKC� [29], the Hypresence alone does not seem to enable PKC� translocationoward membranes similar to PMA [35,58] (Figs. 3—6). A pos-ible explanation for this discrepancy may lay in the stericalestriction due to larger size of Hyp molecule in comparisonith PMA. The mechanism of interaction between Hyp andKC� is still unclear and requires further investigation.

HypPDT resulted in a significant redistribution of PKC�.mmediately post PDT, most PKC� translocated from cyto-lasm to the plasma membrane, and a small portion intoucleus (Figs. 3 and 6). The translocation of PKC� toward thelasma membrane was similar to observations of PMA stim-lated cells [20,35,58]. This result indicates that HypPDTcute effect, translocation and activation of PKC�, is simi-ar to PMA effects. The prolonged effect of PDT in U87 MGells resulted in a change of PKC� distribution. 5 h post PDTome of PKC� was still localized along plasma membrane,nd some in cytoplasm, but there was a significant increasep < 0.00002) of catalytically competent PKC�(pThr638) inhe nuclei (Figs. 3 and 6).

Translocation of PKC� into nuclei upon activation by dif-erent signals including PMA has been reported [43,58].KC� seems to play role in the control of transcription fac-ors such as nuclear receptors [26,48,50]. In untreated andnstimulated NIH 3T3 fibroblasts, PKC� is primarily local-zed in the cytoplasm, but it is translocated into the nucleusfter stimulation with 160 nM PMA. The exact pathway andechanism of the PKC� translocation into the nucleus is not

nown, however there is growing evidence that V5 domaint the extreme C-terminus may play a role [17,41,48,59,60].4 h post PDT, the distribution of catalytically competentKC�(pThr638) is similar to PKC� distribution in stimulatedIH 3T3 fibroblasts [48] and in the cells treated with PKC�

nhibitor Gö 6976 [37,38]. This distribution may be indica-ive of a possible role of catalytically competent PKC� inuclei in response to the prolonged effect of HypPDT in U87G cells.

PKC�, extracellular signal-related kinase (ERK), andtress kinase (JNK) among others have been identified ascl2 kinases that promote survival in several cancerous cellines including U87 MG [13,18,46]. However, Bcl-2 expres-ion levels alone do not always correlate with high survival

ates and resistance to cancer therapy [32,46]. Thereforether regulatory mechanisms must be involved such as post-ranslational modifications of Bcl-2. One of them is Bcl-2hosphorylation, and it has emerged as an important device

2

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or modulating the anti-apoptotic properties of Bcl-2. How-ver, the functional significance of Bcl-2 phosphorylationemains highly controversial. Some Bcl-2 phosphorylationsave been reported to enhance the cytoprotective effectsf Bcl-2, whereas others rendered the cells more suscep-ible to apoptosis [12,18,46]. Some studies suggested thatunctional consequences of Bcl-2 phosphorylation might bessociated with mitotic arrest rather than with the regu-ation of apoptosis [34,56]. Nevertheless, it was shown foreveral malignant cell lines that phosphorylation of Bcl-2 aterine 70 is required for Bcl2’s full and potent antiapoptoticunction in cancer cells [12,32,46].

To investigate whether Bcl-2 is a likely PKC� substraten U87 MG cells, we used immunocytochemistry to simul-aneously examine the distribution and co-localization ofcl-2 phosphorylated at Ser70 (pBcl2S70) and PKC� in U87G cells before and after HypPDT. Under all conditions, theajority of pBcl2S70 co-localized with PKC�, suggesting thatKC� is likely one of the Bcl2 kinases in U87 MG cells (Fig. 5nd Table 2). A significant redistribution of pBcl2S70 andKC� together toward a plasma membrane and into dis-rete foci (presumably mitochondria) upon HypPDT stimulusuggests increased activity of PKC� and phosphorylation ofcl-2 which promotes cell survival. One of the possibilitiesy which phosphorylation can enhance Bcl2’s antiapoptoticunction is the finding that phosphorylation appears to main-ain a stable association between Bcl-2 and its proapoptoticartner, Bax [12].

Because of the observed activity of PKC� and the local-zation of pBcl2S70 in surviving U87 MG cells post HypPDT,nd because increased activity of PKC� and Bcl-2 phosphor-lation have been reported to be responsible for gliomand leukemic cells resistance to anticancer treatments32,35,46], we investigated whether the dephosphorylationf PKC� in U87 MG cells will decrease cell viability after Hyp-DT treatment. There is evidence that PKC� does becomeephosphorylated in an agonist-dependent manner with pro-onged stimulation [14,19].

To dephosphorylate PKC�, we used prolonged expo-ure of U87 MG cells (2 h) to PKC� agonist PMA (400 nM).rolonged exposure to PMA resulted in a different distri-ution of PKC� (Fig. 6) in comparison with cells withoutMA treatment (Figs. 6 and 3). PKC� displayed localiza-ion along the plasma membrane, in the nucleus and onlyartially in cytoplasm in the U87 MG cells treated withrolonged exposure to PMA (Fig. 6). This distribution is sim-lar to partially dephosphorylated/inhibited PKC� 5 h postypPDT, and also to PMA-induced PKC� dephosphorylation

n transfected COS-7 [20]. PMA-induced dephosphorylationorrelates with a traffic-dependent step. In transfected COS-

cells, GFP-PKC� showed a diffuse cytoplasmic locationith moderate concentration in a perinuclear compartment.ollowing PMA treatment, cells accumulated GFP-PKC� onembranous structures within 10—30 min. Two hours fol-

owing treatment, when the protein had become largelyephosphorylated, the GFP-PKC� was located predomi-antly in perinuclear and nuclear compartments [20].

Distribution of PKC� in U87 MG cells treated with

rolonged exposure to PMA is indicative of PKC� dephos-horylation. The addition of Hyp without irradiation to theMA prolonged exposure treated cells resulted in decreasedlasma membrane and increased cytoplasmic localization

I2At

L. Dzurová et al.

f PKC�. However, there was still significant PKC� sig-al in the nucleus. This suggests that Hyp and PMA mayompete for the same binding site as indicated previously29], and that Hyp bound PKC� without irradiation does notromptly translocate. Cell viability, due to prolonged expo-ure to PMA, slightly decreased by 14% (Fig. 7) in comparisonith control cells or cells treated with Hyp only proba-ly due to PKC� dephosphorylation. HypPDT significantlyecreased (p < 0.001) cell viability 5 h post PDT (Fig. 7) inhe PMA prolonged exposure and Hyp treated cells in com-arison with cells treated with Hyp only. This finding suggestshat dephosphorylation of PKC� increases HypPDT inducedell death in U87 MG cells. The underlying mechanism, byhich dephosphorylation of PKC� renders U87 MG cells more

ensitive to HypPDT induced cell death, needs further inves-igation.

One also needs to keep in mind that there are otherossible explanations for PMA effects other then dephospho-ylation of PKC�. There are other protein families that haveeen found to be responsive to phorbol ester treatments: (1)rotein kinase D (PKD) family; (2) DAG kinases (DGKs); (3)as guanyl nucleotide releasing proteins (RasGRPs); (4) chi-aerins; (5) Munc13 scaffolding proteins; and (6) myotonicystrophy kinase-related Cdc42-binding kinases (MRCKs).he proteins from above mentioned families are central

n controlling various cellular functions such as cell pro-iferation, apoptosis and/or motility [51]. Further it haseen shown that PMA can also result in the rearrangementf actin-based cytoskeleton and membrane skeleton whichhanges a cell morphology and function [55].

onclusions

aken all together, in the present work we have shown that majority of PKC� present in U87 MG cells is already in catalytically competent form phosphorylated at Thr638.urther, we have shown that PKC� is a likely Bcl2 kinase in87 MG cells. The presence of Hyp without irradiation doesot affect PKC� distribution. Upon HypPDT treatment, acuteffect, PKC� is activated and localized along the plasmaembrane and partially in the nuclei. Prolonged effect ofDT, 5 and 24 h post PDT, results in PKC� localized predom-nantly in cytosol, perinuclear and nuclear compartments.oreover, we have shown that phosphorylated catalyticallyompetent PKC� is critical for cell viability in U87 gliomaells in response to HypPDT treatment. Thus regulation ofKC� activity may be important for increasing cancer cells’ensitivity toward anticancer PDT treatments. In particular,ephosphorylation of PKC� and pBcl2 may play a role.

cknowledgments

his work was supported by the (i) Agency of the Ministry ofducation of Slovak Republic for the Structural funds of theuropean Union, Operational program Research and Devel-pment (Contracts: Doctorant, ITMS code: 26110230013)20%), NanoBioSens ITMS code: 26220220107 (40%), SEPO

I ITMS code: 26220120024 (20%) and CEVA ITMS code:6220120040 (20%), (ii) Slovak Research and Developmentgency under the contracts APVV-0242-11 and (iii) Scien-ific Grant Agency of the Ministry of Education of Slovak

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The role of anti-apoptotic protein kinase C�

Republic under the grant VEGA No. 1/1245/12. OperationProgram Research and Development funded by EuropeanRegional Development Fund, by the grant of Marie CurieActions FP7-PEOPLE-2009-RG, EU (PIRG06-GA-2009-256580)and by FP7 EU project CELIM 316310.

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