Cancer Letters 293 (2010) 1–14

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Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

Mini-review

Molecular and traditional chemotherapy: A united front againstprostate cancer

P. Singh c,1, M. Yam a,b,1, P.J. Russell a,b,d,e, A. Khatri a,b,*

a Oncology Research Centre, Prince of Wales Hospital, Randwick, Sydney, NSW 2031, Australiab Faculty of Medicine, University of New South Wales, Kensington, NSW 2036, Australiac Centre for Medicine and Oral Health, Griffith University – Gold Coast GH1, High Street, Southport, Gold Coast, QLD 4215, Australiad Australian Prostate Cancer Research Centre – Queensland, Princess Alexandra Hospital, Woollangabba, QLD, Australiae Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, QLD 4059, Australia

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 October 2009Received in revised form 23 November 2009Accepted 27 November 2009

Keywords:ChemotherapyDocetaxelMolecular chemotherapyPurine nucleoside phosphorylaseGene directed pro-drug enzyme therapyConditionally replicating adenovirus

0304-3835/$ - see front matter � 2010 Elsevier Ireldoi:10.1016/j.canlet.2009.11.019

* Corresponding author. Address: Oncology ReseaWales Hospital, Randwick, Sydney, NSW 2031, Au93822629.

E-mail address: a.khatri@unsw.edu.au (A. Khatri1 Both authors have contributed equally to this ma

Castrate resistant prostate cancer (CRPC) is essentially incurable. Recently though, chemo-therapy demonstrated a survival benefit (�2 months) in the treatment of CRPC. While thiswas a landmark finding, suboptimal efficacy and systemic toxicities at the therapeuticdoses warranted further development. Smart combination therapies, acting through multi-ple mechanisms to target the heterogeneous cell populations of PC and with potential forreduction in individual dosing, need to be developed. In that, targeted molecular chemo-therapy has generated significant interest with the potential for localized treatment to gen-erate systemic efficacy. This can be further enhanced through the use of oncolyticconditionally replicative adenoviruses (CRAds) to deliver molecular chemotherapy. Theprospects of chemotherapy and molecular-chemotherapy as single and as components ofcombination therapies are discussed.

� 2010 Elsevier Ireland Ltd. All rights reserved.

1. Prostate cancer: the current issues best options to address this are through the development

Despite high curative rates of localized prostate cancer(PC) with standard treatments, such as radical prostatecto-my and external beam radiation, one third of patients devel-op advanced PC [1] often with metastases. Initial androgenablation therapy only provides short-term benefit (mediantreatment–response period of <30 months) with inadver-tent recurrence in a castrate-resistant state (CRPC), whichis essentially incurable [2] (Fig. 1). Recently though, for thefirst time, chemotherapy showed activity against CRPC in anumber of landmark trials; docetaxel treatment enhancedsurvival (by �2 months) in 40% of the patients [3,4]. How-ever, the issues of poor tissue-specificity and toxicity atclinical dosages remain significant hurdles. Currently, the

and Ltd. All rights reserved.

rch Centre, Prince ofstralia. Fax: +61 02

).nuscript.

of PC-targeted biological therapies such as targeted genetherapies (via expression of therapeutic genes in transcrip-tionally and transductionally targeted vectors) and immu-notherapy (monoclonal antibodies, cytokines) [5].

However, given the complexity and the heterogeneityof CRPC (pathogenesis reviewed in [6]), there is no singleall-encompassing therapeutic agent. Thus, a new paradigmwhereby beneficial synergistic interactions of multimodalcombined regimens can improve efficacy, patient manage-ment and quality of life, has emerged. Particularly for PC,combinations involving chemotherapy and molecular che-motherapy systems, e.g. gene-derived enzyme pro-drugtherapy (GDEPT), have shown promise and are discussedin this review.

2. Chemotherapy and prostate cancer

Prior to the 1980s, the use of chemotherapeutic agentsfor PC treatment was limited by the chemo-resistant

Fig. 1. A flow schematic outlining the different treatments currently used at the various PC stages. Constructed using information sourced from [7–9].

2 P. Singh et al. / Cancer Letters 293 (2010) 1–14

nature of CRPC and the toxicities in aged patients. How-ever, new chemotherapeutic agents and novel combina-tion-regimens [10–12] have led to significant improvements.Following the success of a key clinical trial involving ad-vanced stage CRPC patients [13], the combination ofmitoxantrone with prednisone as a palliative standard forclinical care, was approved. Though not conferring any sur-vival advantage, it did establish that chemotherapy couldimprove clinical outcome in advanced stage CRPC. Sincethen, successful reduction of prostate specific antigen(PSA) levels and improved survival benefit establishedthe promise of docetaxel for treatment of CRPC [14].

2.1. Docetaxel as a monotherapy

In an early trial [14] involving 35 CRPC patients givendocetaxel (75 mg/m2 every 3 weeks), 46% displayed a de-cline in PSA greater than 50% with an objective responserate of 28% and median survival of 27 months. Subse-quently, another phase II trial [15] concluded that the low-er dose of docetaxel (36 mg/m2) following dexamethasoneinfusion did not compromise response rates, although tox-icity was decreased. Significantly though, a marginal sur-vival benefit in patients with CRPC was noted in TAX326[3] and SWOG9916 [4] trials, over a mitoxantrone plus ste-roids regimen. Since this was effective with no added tox-icity in patients >70 years [16], docetaxel and prednisone

as a first-line treatment for CRPC patients was approvedin 2004. A number of side effects including neutropenia, fa-tigue, dyspnoea, oncholysis and alopecia were reported,though, not requiring cessation of therapy or resulting indeath [14,17]. Whilst docetaxel-based drug regimens area significant milestone in systemic chemotherapy, themedian survival of these patients still remains at most18 months. Further, with the clinical heterogeneity of PC,therapeutic responses to docetaxel are also highly variable[18]. Thus, a clear need for improvement is recognized.

2.2. Docetaxel in combination with other therapies

Despite the success of docetaxel-therapy, significantroom for extending median survival and reducing side ef-fects exists. Consequently, its use in novel combinationswith other treatment modalities has been investigated.Strategically, agents with different cell-killing mechanismsto docetaxel are selected to maximize the potential forsynergy; an important consideration for PC, where variouscancer-cell subpopulations respond uniquely to differenttherapies. For example, molecular targeted therapeuticagents in combination with docetaxel have shown promis-ing results against different forms of PC; these includeangiogenesis [19,20] and EGFR inhibitors [21], vaccines[22] and anti-sense Bcl-2 oligodeoxynucleotides [23].Greatest clinical benefit, thus far, has been shown in com-

P. Singh et al. / Cancer Letters 293 (2010) 1–14 3

bination with the conventional chemotherapeutics, est-ramustine, prednisone and mitoxantrone [3,4] (TAX 327and SWOG 9916 trials). These were the first to demon-strate survival advantage in CRPC patients and hence, areexamined more closely below.

TAX327 compared docetaxel/prednisone (Table 1shows dosing schedule) with mitoxantrone/prednisone asa treatment for metastatic CRPC in an international, ran-domized phase III trial of 1006 patients [3]. A significant in-crease in median survival and 24% reduction in the hazardratio of death (p = 0.009) was demonstrated in the patientsfrom the 3-weekly docetaxel/prednisone treatment arm(18.9 months) compared with those given mitoxantrone/prednisone treatment (16.5 months). Correspondingly,clinically apparent improvements were also recorded inpatient pain response (35% vs. 22%; p = 0.01) and qualityof life (22% vs. 13%; p = 0.09).

Like 3-weekly docetaxel/prednisone treatment, thecombination of docetaxel with estramustine exhibited asurvival advantage over conventional mitoxantrone/pred-nisone regimen in the phase III SWOG 9916 study [4]. Pa-tients in the docetaxel/estramustine arm demonstrated anextension of median survival of 2 months, with a corre-sponding reduction in mortality risk of 20% (p = 0.01).However, these patients also exhibited higher rates ofnausea and vomiting, cardiovascular, neurologic and meta-bolic adverse events when compared with those from

Table 1Single agent and combination therapies involving docetaxel for the treatment of C

Type Docetaxel dose Other agent dose Stagstatu

Single agent (75 mg/m2q)b

every 3 wkscNAd Phas

II75 mg/m2qevery 3 wks

NA PhasII

36 mg/m2q/wkfor 6 of 8 wks

NA PhasII

36 mg/m2q/wkfor 6 of 8 wks

NA PhasII

35 mg/m2q/wkfor 6 of 8 wks

NA PhasII

Combinationtherapy

70 mg/m2qevery 3 wks

Estramustine 280 mg tdsf days 1–5 PhasII

70 mg/m2qevery 3 wks

Estramustine 280 mg tds days 1–5 + hydrocortisone 40 mg/day

PhasII

70 mg/m2qevery 3 wks

Estramustine 280 mg every 6 h � 5doses and Coumadin 2 mg daily

PhasII

TAX 327g 70 mg/m2qevery 3 wks

Prednisone 10 mg/day PhasIII

30 mg/m2q/wkfor 5 of 6 wks

Prednisone 10 mg/day

SWOGh 60 mg/m2qevery 3 wks

Estramustine 280 mg tds days 1–5 PhasIII

Information partially sourced from [47].a PSA response: decline in prostate specific antigen P 50%.b mg/m2q: milligram per meter square.c wks: weeks.d NA: not available.f tds: three times a day.g ASCO: American Society for Clinical Oncology.h SWOG: South West Oncology Group.

mitoxantrone/prednisone arm. Nevertheless, this combi-nation may be important in treatment of CRPC in diabeticpatients [24], who may not benefit from docetaxel/predni-sone treatment, or as a second line therapy in docetaxel-resistant CRPC patients [25]. However, due to their similarmechanisms of action, synergy with a third modality hasalso been explored, with improved cancer cell-killing, orrelief of pain and reduced bone fractures being the conclu-sions of many of these trials. The compounds studied in-clude hydrocortisone [26,27], prednisone [28,29],carboplatin [30,31], calcitriol [32], vinorelbine [33], cele-coxib [34], thalidomide [19], exisulind [35], enoxaparine[36] and zoledronic acid [37].

The success of these trials, has cemented docetaxel-based combination regimens as the standard of care forCRPC treatment. A combination of 3-weekly docetaxel plusprednisone has since been instigated as the standard treat-ment for CRPC patients [38].

Optimized drug scheduling to reduce the toxicity asso-ciated with higher doses of docetaxel has been the focus ofrecent preclinical research; for example an improvedunderstanding of the anti-angiogenic and cytotoxic prop-erties of metronomic docetaxel treatment [39] and itscombinations with 2-methoxyestradiol [40] and dexa-methasone [41] could aid these efforts tremendously.Further, treatments with greater tumour-specificity, thatspare host tissue, are needed to maximize the therapeutic

RPC patients.

e/s

Patients PSAresponse(%)a

Overallresponserate (%)

Mediansurvival(months)

Reference/year

e 35 46 24 27 1999 [14]

e 21 38 29 67% at15 months

1999 [42]

e 60 41 33 9.4 2001 [15]

e 25 46 40 9.7 2001 [43]

e 30 48 28 20 2003 [44]

e – 68 55 77% at 1 year 2000 [45]

e 47 68 50 20 2001 [26]

e 42 45 20 13.5 2002 [46]

e 1006 45 12 18.9 2004 [3]

48 8 17.3

e 770 50 17 18 2004 [4]

4 P. Singh et al. / Cancer Letters 293 (2010) 1–14

response. In this context, targeted gene therapy-based ap-proaches (alone and in combination) could play a signifi-cant role against PC (outlined in Fig. 2) [5]. Thesestrategies rely on the ability of vectors, usually given di-rectly into the primary tumour, to deliver the therapeuticgene. A number of viral and non-viral vectors have beenexplored in gene therapy; the advantages and disadvan-tages of which are outlined in Table 2.

3. Molecular-chemotherapy and prostate cancer

Given the recent success of chemotherapy against CRPC,molecular chemotherapy, engendered by GDEPT based ap-proaches has generated significant interest. Administeredlocally, GDEPT can potentially direct the toxicity of chemo-therapy specifically to the tumour cells, including those indistant locations. Prostate cancer is particularly suitable, asthe prostate is easily accessible for localized treatment andis not-essential for life.

Typically, a GDEPT system consists of two components:(1) a relatively non-toxic pro-drug (clinically relevant),administered systemically, and (2) the gene encoding anenzyme that converts the non-toxic pro-drug to its toxicmetabolites. The unique feature of these systems is the‘‘bystander effect” engendered by in situ spread of cytotox-icity beyond the transduced cell to surrounding and evendistant un-transduced cancer cells. The ‘‘bystander effect”is postulated to involve several mechanisms including dif-fusion of soluble toxic metabolites [48], dissemination of

Fig. 2. A schematic showing several gene therapy approaches explored for treAbbreviations: 1BRCA-1: breast cancer 1, early onset; 2Bcl-2: B-cell lymphoma 2;growth factor receptor 2; 5Bax: Bcl-2 associated X protein; 6HSV–tk/GVC: herpphosphorylase; 8CD/5FC: cytosine deaminase/5-fluorocytosine; 95FU:5-fluorou11NTR: nitroreductase; 12Ad: adenovirus; 13GM-CSF: granulocyte macrophage co16MHC-I: major histocompatibility complex-1; 17CTLA4: cytotoxic T-lymphocyte

apoptotic vesicles [49,50], transduction of endothelial cellsin tumour vessels [51] and stimulation of anti-tumour im-mune responses consisting of cytokines, natural-killer andT cells [52,53]. Thus, in contrast to most gene therapy ap-proaches, GDEPT systems can overcome the limitation oflow cell transduction efficiency and hence, have foundmore widespread applications in PC-targeted gene therapytrials.

For most pro-drug/enzyme combinations, the enzyme isof bacterial or viral origin to minimize the inadvertent hu-man enzyme mediated conversion of the pro-drug inuntargeted areas. This and other criteria defining the selec-tion of enzyme/pro-drug components have been examinedin a number of reviews [54,55].

Since the first successful trial in 1991 [56], severalGDEPT systems have been evaluated against cancers of co-lon, ovary, breast and liver, with varying degrees of suc-cess. By 2007, three of the 109 clinical trials evaluatingGDEPT that had been undertaken were at the phase IIIstage [57]. Unlike for traditional chemotherapy, these trialshave mainly been conducted in PC patients with newlydiagnosed or locally recurrent PC [58] with only a fewexceptions involving late stage PC patients [59]. Thus far,herpes simplex virus–thymidine kinase (HSV–tk) and cyto-sine deaminase (CD)-based GDEPT are the most exploredfor PC treatments and are discussed. The relatively newpurine nucleoside phosphorylase (PNP) GDEPT system pos-sesses a number of advantages that confer a greater futurepotential for its use against PC. Table 3 outlines the fea-

atment of PC and depicts potential combinations with other modalities.3KRAS: Kirsten rat sarcoma viral oncogene; 4Her-2 neu: human epidermales simplex virus-thymidine kinase/ganciclovir; 7PNP: purine nucleoside

racil; 10CDUPRT: cytosine deaminase uracil phosphoribosyl transferase;lony stimulating factor; 14TNF: tumour necrosis factor; 15IFN: interferon;antigen.

Table 2Advantages and disadvantages of different types of vectors used in gene therapy.

Type Advantages Disadvantages

Viral vectorsRetroviruses

(RNA virus)High transfection efficiency; stable integration leading tolong-term gene expression; infect hematopoietic andepithelial cells; absence of immunogenic viral proteins

Very unstable; low titer; integrate into the host cell genome soonly infect dividing cells; risk of malignant transformation inaffected cells due to integration into host genome; relativelysmall amount of genetic information (�9–12 kb)

Adenovirus (DNAvirus)

Well studied; high titer; stable and resistant to physical stress(e.g. freezing); non integrating, transient expression, sideeffects are less severe; infect epithelial cells at high frequency;higher packaging capacity; easy to engineer; infect dividingand non-dividing cells effectively, cellular proliferation notrequired

Immunogenic; potentially hepatotoxic; short term geneexpression as it does not integrate in host genome; virusneutralization with pre-existing antibodies

Adeno-associatedvirus

Stable; integrates into non-dividing cells at low frequency Small capacity for DNA; low titre; requires a helper virus

Herpes virus Infects a wide range of cell types; can achieve high titer; hasrelatively prolonged expression

No integration into genome of infected cells; cytotoxic;immunogenic; difficult to engineer/handle due to complexity;complex packaging system

Reovirus Infection limited to cells with activated ras pathway Not well characterized

Non-viral vectorsDNA cassettes Non-viral; easy to use and develop, can be used for sense and

anti-sense expressionLow efficiency of transfection in vivo; temporary expression,stability

Liposomes Non-viral, easy to develop Low frequency of modification, especially in vivo; cytotoxic tosome cell

Oligonucleotides Non-viral, small in size, potential use in RNA interference Low efficiency of transfection in vivo; temporary expression,stability

P. Singh et al. / Cancer Letters 293 (2010) 1–14 5

tures of the more widely tested non-mammalian enzyme/pro-drug combinations against PC (also reviewed in [60]).

3.1. Herpes simplex virus – thymidine kinase (HSV–tk) GDEPT

HSV–tk enzymatic conversion of the pro-drugs, ganciclo-vir (GCV), acyclovir (ACV) and valcyclovir (VCV), induces celldeath through apoptotic and non-apoptotic mechanisms(reviewed in [75]). Having shown its therapeutic effectsagainst PC in vitro and in vivo [61–64,76,77], the clinical effi-cacy and safety profile of the system has been assessed inseveral phase I and II trials. The toxicity of Adenovirus(Ad)-mediated HSV–tk/GCV was relatively low in patientswith clinically localized PC [64]; no grade 4 toxicities werereported and most other side effects resolved spontaneously.It was also deduced that tumour cell killing was mediated byanti-angiogenic effects, induction of local/systemic immuneresponses and stimulation of apoptosis. Other studies havedemonstrated that low host toxicity is maintained at higherAd-vector doses (up to 2 � 1012 viral particles) [58].

Overall though, clinical therapeutic effects of HSV–tkwere modest [78]. A phase I study in which an Ad-vectorwith osteocalcin promoter-driven HSV–tk was injected di-rectly into PC patient lymph nodes and bone metastases,resulted in limited efficacy due to suboptimal HSV–tkexpression and possibly due to the low tolerable doses ofGCV used (high doses cause bone marrow toxicity) [59].Thus, combination approaches have been explored to com-pensate for these shortfalls. Specifically, additional thera-peutic benefits have been achieved in preclinical andclinical studies when this therapy is combined with castra-tion [79], radiation therapy [80–82], immunotherapy [83–85] and virotherapy [83,86–89].

3.2. Cytosine deaminase (CD)–GDEPT

Similar success has been achieved using E. coli – oryeast-derived CD-based GDEPT, the second most widelyexploited GDEPT system. Here, tumour cell killing is fos-tered by CD-mediated conversion of 5-fluorocytosine (5-FC), into toxic 5-fluorouracil (5-FU), a potent inhibitor ofRNA and DNA synthesis. Both, 5-FC and 5-FU can passivelypenetrate tumour cells, thus propagating the local toxic ef-fects to non-transduced cells. This confers a significantadvantage over the cellular connexins (gap-junction)dependent HSV–tk system. Thus, when CD/5-FC GDEPTwas combined with HSV–tk GDEPT, greater therapeutic re-sponses were demonstrated, with tolerable, non-com-pounded toxicities [90]. In light of this, translation ofother novel combinations of suicide gene therapies to theclinic is a compelling idea. Particularly promising is thecombined use of CD and uracil phosphoribosyl transferase(UPRT), an enzyme which, unlike CD, converts 5-FC to 5-FUdirectly rather than through a series of intermediary stepsincluding the rate limiting production of 5-fluorouridinemonophosphate (5-FUMP) [66,67]. Used in conjunction,these two enzymes sensitize tumour cells to lower dosesof 5-FC. Thus, CDUPRT-GDEPT has been demonstrated tobe more effective than either GDEPT alone against DU145[68] and LNCaP human PC cells [91]. CDUPRT has also dem-onstrated efficacy and synergy with immuno-stimulatoryinterleukin (IL)-12 and IL-18 in RM1 mouse PC cells[66,92]. Enhanced efficacy of combinations involving othermodalities including irradiation have been translated tothe clinic, with success in using CD–GDEPT with irradia-tion, either alone or in combination with HSV–tk GDEPT[70,80].

Table 3GDEPT systems used in prostate cancer gene therapy.

Approach Pro-drug Toxicmetabolite

Type Advantages/disadvantages

HSV–tk1 Ganciclovir Ganciclovirtriphosphate

Antimetabolites � In clinical trials� Associated with significant local and distant

‘bystander effects’� Activated drug is an S-phase specific cytotoxin and

hence, effective on fast dividing cells only� Dependence on cell to cell based gap junctions or

apoptosis vesicles for ‘bystander effects’

[50,51,61–64] Acyclovir Acyclovirtriphosphate

Valacyclovir Valacyclovirtriphosphate

E.coli CDUPRT 2[65,68] 5-Fluorocytosine 5-Fluorouracil Antimetabolite � In clinical trials� Kills dividing and non-dividing cells� Pro-drug activity depends on further metabolism of

enzyme� Development of 5-FU resistance in some cancer cells� Higher toxicity of activated end product (long half life)

HSV–tk and E. coli CDUPRT(double suicide genetherapy)

Ganciclovir Ganciclovirtriphosphate

Antimetabolites � Double suicide gene therapy leads to enhanced cancercell killing without enhancing toxicity to non-targetorgans

[69,70] 5-Fluorocytosine 5-FluorouracilE. coli PNP3(refer to

table IV)6-Methylpurine-2’-deoxyriboside(MeP-dR)

6-Methylpurine(6-MEP)

Antimetabolites � Currently in a phase I clinical trial� Associated with strong ‘bystander effect’ and results

in killing of dividing as well non-dividing cancer cells� 6-MeP exhibits prolonged retention in situ

[60] FludarabinePhosphate

2-Flouroadenine

E. coli Nitroreductase CB1954 (5-aziridin-1-yl)-2,4-dinitrobenzamide

Hydroxylamines Alkylator � Preclinical� Forms DNA cross-links in both cycling and non-cycling

cells� Local and distant ‘bystander effects’� Distant bystander effect may also involve induction of

stress proteins, HSP25 and HSP70[71,72] 2-Nitrobenzyl

carbamatesCytochrome P450

(CYP) [73,74]Cyclophosphamide Phosphoramide

mustardAlkylator � Preclinical

� Moderate local ‘bystander effect’� Cell-killing mechanisms independent of cell-cycle

1 HSV–tk: herpes simplex virus-thymidine kinase.2 CDUPRT: cytosine deaminase uracil phosphoribosyl transferase.3 PNP: purine nucleoside phosphorylase.

6 P. Singh et al. / Cancer Letters 293 (2010) 1–14

3.3. Purine nucleoside phosphorylase (PNP)–GDEPT

In preclinical studies, PNP–GDEPT has exhibited po-tency against a range of cancer types, including PC. Derivedfrom E. coli, this PNP differs from its mammalian counter-part structurally and functionally (reviewed in [93,94]),and uniquely cleaves the pro-drugs, fludarabine-phosphate(Fludara) and 6-methylpurine 2-deoxyriboside (6-MePDR),to toxic metabolites with minimal unwanted toxicityin vivo. Concerns regarding pro-drug conversion by endog-enous PNP, expressed by intestinal bacteria flora, have alsobeen addressed through methods including crystallo-graphic/computer remodeling of the PNP enzyme and con-comitant antibiotic therapy [60]. In comparison to HSV–tkGDEPT, PNP-GDEPT mediates greater cell-killing due to amore extensive ‘‘bystander effect” [95,96].

In-fact, expression of less than 1% expression of PNP ledto near-complete cell killing in a human colon cancer cellline [97], augmented through passive diffusion of toxinsindependent of cell–cell junctions. The cytotoxic purinesproduced after pro-drug conversion disrupt both DNAand RNA synthesis [97], making PNP-GDEPT independentof cell-cycle status. This is particularly relevant to slowgrowing PC where only 2% of cancer cells in a tumour are

dividing [98]. Finally, PNP-GDEPT mediated apoptosis isp53-independent; as demonstrated through its efficacyagainst PC cell lines lacking (PC-3) [99] and containing wildtype p53 (LNCaP) [100]. Given the significance of p53 genemutations in early and late stage PC pathogenesis [101],PNP-GDEPT may be particularly useful in patients whosePC exhibits aberrant p53 expression.

Direct comparisons of PNP with GDEPT other than HSV–tk have not been reported thus far. With respect to PC, itsefficacy has been demonstrated using an ovine atadenovirus(now approved for a phase I trial) in androgen-dependentand CRPC xenografts in nude and immunocompromisedmice (Table 4). Significantly, suppression of PC progressionand improved survival was reported in transgenic adenocar-cinoma of mouse prostate (TRAMP) models, which mimicthe development of human PC [102].

While, its anticipated beneficial effects regardingsafety and efficacy, in the clinical setting, remain unsub-stantiated, in the clinical setting, however, based on thepromising indications from the preclinical data (Table 3)the first two clinical trials evaluating PNP-GDEPT havebeen approved (one against CRPC) and data from thesewill be valuable in determining the potential of this sys-tem [103].

Table 4Studies investigating PNP-GDEPT for the treatment of prostate cancer.

PNP-GDEPT:vector

Pro-drug Title/protocol Outcome/results

Adenovirus[96]

9-(beta-D-2-deoxy-erythropentofuranosyl)6-methylpurine

Relative efficiency of tumour cell killing in vitro bytwo enzyme/pro-drug systems delivered byidentical adenovirus vectors (in vitro)

The PNP-GDEPT was found superior compared toHSV–tk/ganciclovir system in prostate cancer celllines

Adenovirus[104]

6-methylpurinedeoxyriboside (6-MePDR)

In vivo gene therapy of cancer with E. coli purinenucleoside phosphorylase (in vivo)

Improvement in survival of BALB/c nude mice withs.c.a PC-3 tumours when treated with PSAb promoterdriven PNP-GDEPT

Adenovirus[104]

6-MePDR Transcription-targeted gene therapy for androgen-independent prostate cancer (in vivo)

Androgen-independent, prostate-targeting Ad5expressing PNP driven by rat probasin promoterresulted in reduction of PC-3 tumours in nude mice

Adenovirus(ovine)[105]

Fludarabine phosphate Gene therapy for prostate cancer delivered by ovineadenovirus and mediated by purine nucleosidephosphorylase and fludarabine in mouse models(in vivo)

Use of ovine Ad vector containing PNP was effectiveagainst androgen independent, aggressive murineRM1 and human PC3 tumours grown s.c. ororthotically (intraprostatic)

Adenovirus(ovine)[102]

Fludarabine phosphate Gene-directed enzyme/pro-drug therapy forprostate cancer in a mouse model that imitates thedevelopment of human disease (in vivo)

The ovine Ad vector expressing PNP/fludarabineresulted in survival advantage in immune-competentTRAMP mice

Adenovirus[106]

Fludarabine phosphate Purine nucleoside phosphorylase and fludarabinephosphate gene-directed enzyme/pro-drug therapysuppresses primary tumour growth and pseudo-metastases in a mouse model of prostate cancer(in vivo)

Suppression of local prostate cancer growth andreduced lung colony (pseudo-metastases) formationin the RM1 tumour model. Immunostaining showedan increased Thy-1.2(+) cell infiltration into theprostate tumour site; a possible role of immunemediated distant bystander effect

Adenovirus(ovine)[100]

Fludarabine phosphate Preclinical evaluation of a prostate-targeted gene-directed enzyme/pro-drug therapy delivered byovine atadenovirus

Ovine atadenovirus vector (OAdV623) expressingPNP under the control of androgen-independentprostate targeted promoter resulted in tumourgrowth inhibition in LNCaP-LN3 and PC3 lines andtheir xenografts

a s.c.: Subcutaneously.b PSA: Prostate specific antigen.

P. Singh et al. / Cancer Letters 293 (2010) 1–14 7

4. Effective and safe delivery of molecular therapy:development of Conditionally replicative adenoviruses(CRAds)

Despite the in situ amplification, the clinical efficacy ofany GDEPT system is limited due to inadequate and non-specific vector transduction of tumour cells [107,108]. Tothat end, 2nd and 3rd generation adenoviral vectors with

Table 5Different generations of Adenoviral Vectors.

Type of Ad vector Genetic featuresa

First generation [109] E1 and/or E3 regions are deleted

Second generation[110–114]

E1, E2 and E3 and/or E4 regions are deleted

Third generation (highcapacity, gutlessvectors) [115,116]

All essential genes are deleted except ITR and pacsignal sequences

Conditionallyreplicative [109]

Modulated based on cancer cell properties (e.g. pallow cancer-selective replication (type I) or throupromoters linked to viral replication genes (type

a E1–4 regions are expressed mainly prior to DNA replication to produce proteianti-viral immune responses and facilitating viral DNA replication.

improved vector carrying ability and lower immunogenic-ity (see Table 5) have been developed. A recent but signif-icant addition to this repertoire is the oncolytic adenoviralvector, which preferentially replicates in cancer cells to ex-ert their oncolytic activity and thus overcome the limita-tions of inefficient/nonspecific gene delivery.

Although, the first cases of concurrent tumour regres-sion and natural viral infection were recognized in the

Properties

Replication incompetent; capacity for transgene insertionapprox. 8.1 Kb; immunogenic; production in E1complementary cell line e.g. HEK293 cells; risks ofproduction of replication competent viruses duringhomologous recombination with E1 sequences; somereports of replication at very high multiplicity of infectionsReplication incompetent; higher capacity for transgeneinsertion than first generation vectors; improvedtransgene persistence, yet less immunogenic (debatable);lower viral yield; production in E2 or E4 complementarycell lines

kaging Can accommodate up to 37 kb foreign DNA; leastimmunogenic with prolonged transgene expression

53) togh

II)

Replication competent only in cancer cells; geneexpression depends upon promoter activity; selective inexpression

ns responsible for inducing host-cell entry into S-phase, manipulating host

Table 6Some examples of transductional modifications incorporated into adenoviral vectors for gene therapy of prostate cancer.

Method of transductionalmodification

Transductional modification Vector Transgene Status Reference

Bispecific antibodies Conjugate of an anti-adenovirus fiber knob Fab-fragment and aPSMAa monoclonal antibody

Adenovirus eGFPb In vitro [120]

Conjugate of monoclonal anti-adenovirus knob and an anti-FGFc

antibodyAdenovirus LacZd In vitro [121]

Conjugate of monoclonal anti-adenovirus knob and an anti-alpha-323e

Adenovirus LacZ In vitro [121]

Genetic Retargeting Capsids modified to contain Ad serotype 3/5 chimeric knob Adenovirus Luc1f In vitro [122]Incorporation of recombinant RGDg fiber Adenovirus Delta-24h In vitro [123]

a PSMA: prostate specific membrane antigen.b eGFP: enhanced green fluorescent proteins.c FGF: fibroblast growth factor.d LacZ: beta-galactosidase.e anti-alpha-323: antibody against EpCAM (epithelial cell adhesion molecule).f Luc1: Luciferase1.g RGD: Arg-Gly-Asp sequence.h delta-24: CRAd with partial deletion of E1 region.

8 P. Singh et al. / Cancer Letters 293 (2010) 1–14

mid 19th century [117] interest in their use for cancertherapy was reignited in 1990s. Significant progress hasbeen made since then and several viruses are being evalu-ated in the clinic. The specificity and safety of these vectorsis achieved through: (1) capitalising on their biologicalfeatures that restrict replication to cancer cells only e.g.cancer cells that show aberrant expression of p53 will sup-port the replication of ONYX-015 virus [118,119] and/or,(2) through genetic modification to target these transduc-tionally (changed viral tropism) and/or transcriptionally(replication/gene-expression regulated by cancer specificpromoters) to the cancer cells e.g. conditionally replicatingAds (CRAds). (Table 6 outlines the specific transductionalmodifications and Table 7 lists promoters investigated forPC targeting.) The CRAd based approaches have been themost explored for PC in the clinic [109]. The concept is toutilize these either as an alternative or adjunct to GDEPTbased applications.

A phase I trial in twenty patients, of CV706, a CRAd withPSA selectivity reported significant reductions in PSA levels[160] with no significant adverse events. Similar levels ofsafety and efficacy were evident in phase I/II trials of CRAdCG7870 with different elements regulated by prostate-spe-cific, rat probasin and PSA promoters [160]. Promoters de-rived from the prostate specific membrane antigen (PSMA)gene specifically delivered suicide gene therapy to PC le-sions [161]. Unfortunately, these, like most other promot-ers, are active only in androgen-dependent PC. However,there has been progress with the use of osteocalcin pro-moter and the novel chimeric promoter, prostate specificenhancer sequence (PSES). Both of these demonstratedspecificity in androgen dependent and CRPC xenografts[38]. Though, limited spread of the CRAd, under PSES pro-moter control resulted in incomplete tumour eradication,thus, highlighting the need for improvement in these pro-moters/vectors.

4.1. CRAds in combination with other modalities

Use of oncolytic viral vectors may be particularly wellsuited in combination therapies as they are able to killapoptosis-resistant cancer cells [162]. Thus, if armed with

a cytotoxic gene these could further enhance the in situefficacy of treatment through enhanced vector penetrationwithin the tumour mass, which could be of particular sig-nificance against solid tumours such as PC. This factor to-gether with the modest outcomes of the clinical trials,has led to the investigation of CRAds in combination withother therapies. For PC, synergy has been demonstratedin clinical trials with radiation therapy and the CD andHSV–tk GDEPT systems [163,164]. Concurrent use of che-motherapy with oncolytic viruses for the treatment of PChas been the subject of several investigative studies. Onesuch animal study [165] analysed prostate specific CRAd(CV787) in combination with paclitaxel and docetaxeland demonstrated synergistic anti-tumour activity withno increase in toxicity. Indeed, a 1000-fold decrease invirus dose was effective in the combination compared withcontrols. Importantly, animals given the combinationtreatment displayed better health (body weight) comparedwith those given individual treatments. Similarly, tumour-targeted Ad OAS403 combined with doxorubicin in aLNCaP prostate tumour model significantly improved uponresponses recorded for either therapy alone [166].

As mentioned earlier, the concept of CRAds armed withGDEPT has generated great clinical interest with promisingresults from clinical trials evaluating CRAds armed withHSV–tk and CD genes. In patients with intermediate tohigh risk PC (phase I trial), use of CRAds equipped withboth CD and HSV–tk was efficacious and safe [80]. A five-year follow-up of patients given a similar regimen demon-strated an improvement in the PSA-doubling time, whichdelayed the need for salvage androgen suppression – oftenassociated with high morbidity [167]. This trial provides asignificant proof that combination CRAd and molecularchemotherapy has the potential to provide long-termbenefit to patients, with acceptable toxicity. Further tothat, recently, two phase I trials established the feasibilityand safety of a CRAd/radiation therapy combination in PCpatients [163], viral replication was not impaired. Giventhat CRPC patients usually receive radiation and/or chemo-therapy at the time of treatment, such synergistic interac-tions between CRAds and these standard therapies haveadded clinical relevance.

Table 7Promoters used for gene therapy of prostate cancer.

Promoter Vector Transgene Status Reference

Osteocalcin Adenovirus HSV–tk1 phase I/II [59,124]Adenovirus HSV–tk In vitro/in vivo [125,126]Adenovirus E1a (CRAd2) In vitro/in vivo [127]Plasmid (iNOS3) In vitro [128]

PSE-BC Lentivirus eGFP4/Luc5 In vitro/in vivo [129,130]PSMA6 Adenovirus eGFP/CD7 In vitro [131]

Adenovirus eGFP/CDUPRT 8 In vitro/in vivo [91]PSME9 Adenovirus Luc In vitro/in vivo [132]

Adenovirus E1A (CRAd) In vitro/in vivo [133]M6 Adenovirus HSV–tk In vitro/in vivo [134]Rat Probasin, PSA10 Adenovirus eGFP-CAT 11 In vitro [135]ARR(2)PB12 Adenovirus CAT/ Bax13 and Bad14 (pro-apoptotic) In vivo [136]

Lentivirus eGFP/HSV–tk In vitro [137]PSA Lentivirus eGFP/Diptheria Toxin A In vitro/in vivo [138]

Adenovirus HSV–tk In vivo (dogs) [139]Rat Probasin Adenovirus PNP15 In vitro/in vivo [140]Rat Probasin (Pb4) plus

PSME enhancerAdenovirus PNP In vitro/in vivo [100]

PEG-316 Adenovirus eGFP/p53/mda717/ IL-2418 In vitro/in vivo [141]COX-219 Plasmid/ poly-

ethylenimineGFP/Caspase-3 and 9 In vitro [142]

HTERT 20 Adenovirus E1A (CRAd) In vitro [143]Adenovirus E1A (CRAd) In vitro/in vivo [144]Adenovirus E1A (CRAd) and GFP under CMV

promoter in E3 regionIn vitro [145]

Adenovirus GFP/E1A (CRAd) In vitro/in vivo [146]Adenovirus HSV–tk In vivo [147]Retrovirus CXCR421 small hairpin RNA22 In vitro/in vivo [148]Plasmid NAT 23 In vitro [149]

VEGFR224 Adenovirus Caspase 9 In vitro [150]GDEP25 Plasmid In vitro [151]PC-126 Plasmid Luciferase In vitro [152]TARP27 Adenovirus Luciferase In vitro/in vivo [153]Synthetic beta-catenin-

dependent promoterAdenovirus LacZ28 Primary cultures (patient

samples)[154]

PSP9429 Plasmid LacZ In vivo [155]Uroplakin II Adenovirus E1 In vitro (LNCap) [156]Kallikrein 2 Adenovirus eGFP In vitro/in vivo [157]Caveolin-1 Adenovirus HSV–tk In vitro/in vivo [158]Survivin Plasmid GFP In vitro [159]

1 HSV–tk: herpes simplex virus–thymidine kinase.2 CRAd: conditionally replicative adenovirus.3 iNOS: nitric oxide synthase.4 eGFP: enhanced green fluorescent proteins.5 Luc: luciferase.6 PSMA: prostate specific membrane antigen.7 CD: cytosine deaminase.8 CDUPRT: cytosine deaminase uracil phosphoribosyl transferase.9 PSME: PSMA enhancer.

10 PSA: prostate specific antigen.11 CAT: chloramphenicol acetyltransferase.12 ARR(2)PB: androgen response region (2) probasin.13 Bax: Bcl-2 associated X promoter.14 Bad: Bcl-2 associated death promoter.15 PNP: purine nucleoside phosphorylase.16 PEG-3: progression-elevated gene-3.17 mda7: melanoma differentiation associated gene 7.18 IL-24: interleukin-24.19 COX-2: cyclooxygenase-2.20 HTERT: human telomerase reverse transcriptase.21 CXCR4: chemokine receptor 4.22 RNA: ribonucleic acid.23 NAT: nor-adrenaline transporter.24 VEGFR: vascular endothelial growth factor receptor-2.25 GDEP: gene differentially expressed in prostate cancer.26 PC-1: prostate and colon gene-1.27 TARP: T cell receptor gamma-chain alternate reading frame protein.28 LacZ: beta-galactosidase.29 PSP94: prostate secretory protein of 94 amino acids.

P. Singh et al. / Cancer Letters 293 (2010) 1–14 9

10 P. Singh et al. / Cancer Letters 293 (2010) 1–14

5. Conclusion

The non-essential nature and accessibility of the pros-tate for targeted treatment has led to the possibility ofcytoreductive therapies for the treatment of PC. The ensu-ing research has resulted in several clinical trials exploringmolecular chemotherapy (GDEPT) and more recently onco-lytic CRAds with or without GDEPT to treat PC. In that,development of clinically relevant GDEPT systems(through developing new systems, using FDA approvedpro-drugs, improving their design and enzyme activity)and cancer targeted safe vectors (transductional and tran-scriptional targeting) are the continuing focus and willcontribute significantly towards realizing the full potentialof these agents. Further, the success of trials exploringthese in combination or with conventional chemotherapyhave clearly shown that combining these agents may bethe best approach for targeting the heterogeneity of PC,as cancerous cells are targeted through different mecha-nisms, without compounding toxicity. The best feature isthat localized treatment using both agents can potentiallylead to eradication of not only the primary tumour massbut also the metastatic lesions through development ofsystemic anticancer effects. Synergies between conven-tional and new treatments would be a bonus and will facil-itate the translation to clinic, given that conventionaltreatments represent the standard of care for patients.The clinical and preclinical data so far is overall encourag-ing and definitely justifies further development of theseconcepts for PC therapy.

Conflicts of interest

No conflict of interest.

Acknowledgement

Funding from National Health and Medical ResearchCouncil (#510238), Australia.

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