NVP-BEZ235, Dual Phosphatidylinositol 3-Kinase/MammalianTarget of Rapamycin Inhibitor, Prominently Enhances

Radiosensitivity of Prostate Cancer Cell Line PC-3

Wenjie Zhu,1,* Weijiang Fu,1,* and Likuan Hu1,2

Abstract

Background: Aberrant activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin(mTOR) pathway may account for development of radioadaptation and is not rare in prostate cancer. NeitherPI3K nor mTOR blockade could completely inhibit the pathway owing to paradoxical feedback, so we anticipatedual PI3K/mTOR blockade by NVP-BEZ235 to radiosensitize prostate cancer cells.Methods: We investigated into the radiosensitizing effect of NVP-BEZ235 on PC-3 cells, which are devoid ofandrogen receptors. Clonogenic survival and MTT assays were performed, and to pursue underlying cellularchanges flow cytometric analysis of cell cycle and apoptosis as well as western blot were carried out.Results: Exposure to NVP-BEZ235 and irradiation caused a greater degree of survival inhibition than ionizingradiation (IR) or BEZ235 alone. Dual PI3K/mTOR blockade along with IR induced a G2/M arrest and enhancedproapoptotic effect. NVP-BEZ235 radiosensitized PC-3 cells through counteracting constitutive as well as IR-triggered activation of Akt/mTOR signaling.Conclusions: Our study demonstrated that the dual PI3K/mTOR inhibitor NVP-BEZ235 prominently improvedthe radiosensitivity of PC-3 cells. It sensitized tumor cells to irradiation via interruption of cell cycle progressionand augmentation of cell apoptosis, which was due to its constraint on constitutive and IR-elicited PI3K/Akt/mTOR signaling activation.

Key words: mTOR, NVP-BEZ235, PI3K, prostate cancer, radiosensitivity

Introduction

Prostate cancer is the most common malignant neoplasm inmale urinary system. Combined management for prostate

cancer has been developed, among which radiotherapy is amost important modality of treatment. Nevertheless, the in-trinsic or acquired radioresistance of prostate cancer has pre-sented itself as the main obstacle to achieve long-term control,despite remarkable advances achieved in radiation facilitiesand technology. That provides a strong rationale for explo-ration into potential radiosensitizing agents of prostate cancer.

Phosphatidylinositol 3-kinase (PI3K)/Akt/mammaliantarget of rapamycin (mTOR) make up a vital pathway thatregulates cell cycle, proliferation, apoptosis, and biosynthesis

process in eukaryocytes.1 Indicated by numerous studies andinvestigations in recent years, the aberrant activation ofPI3K/Akt/mTOR pathway, either inherent or induced,plays an indispensable role in neoplastic origination, me-tastasis, and development of radioadaptation.2–4 Over-activation of the pathway often occurs through mutation ofcoding genes (PIK3CA, AKT1, AKT2 etc.), constitutive acti-vation of up-stream molecules, such as epidermal growthfactor receptor (EGFR) and Ras, as well as the depletion ofphosphatase and tensin homologue deleted in chromosome10 (PTEN), most of which are not rare in prostate cancer.5

The kinase mTOR forms two distinct complexes known asmTORC1 and mTORC2. The former consisting of mTORand raptor (regulatory-associated protein of mTOR) can be

1Department of Radiation Oncology, Cancer Center, Qilu Hospital affiliated to Shandong University, Jinan, China.2College of Medicine, Shandong University, Jinan, China.*These authors contributed equally to this work.

Address correspondence to: Likuan Hu; Department of Radiation Oncology, Cancer Center, Qilu Hospital affiliated to Shandong University;107 Wenhua Xi Road, Jinan 250012, ChinaE-mail: studs@yahoo.cn

CANCER BIOTHERAPY AND RADIOPHARMACEUTICALSVolume 28, Number 9, 2013ª Mary Ann Liebert, Inc.DOI: 10.1089/cbr.2012.1443

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inhibited by rapamycin, while the latter, including mTORand rictor (rapamycin-insensitive companion of mTOR) isinsensitive to rapamycin.5 The inhibition of mTORC1 alone,as in the case of rapamycin, can lead to mTORC2-mediatedfeedback activation of Akt on Ser473.6 The existence of theparadoxical feedback loop may abrogate the efficacy ofmTORC1 inhibitors and thus, rationalize the application ofdual inhibitors.

NVP-BEZ235 is a novel antitumor drug developed byNovartis Pharma, which functions as a dual PI3K/mTORinhibitor.7 Preclinical studies have described its anti-proliferative effect in several cell lines, especially those withPI3K or K-Ras mutation.7,8 Of note, NVP-BEZ235 has beendemonstrated to improve the sensitivity of cancer cells tochemotherapy, indicating its potential role as a sensitizingagent in combined modalities of treatment.9

Since the proapoptotic activity of ionizing radiation (IR)can be compromised by IR-triggered activation of PI3K/Akt/mTOR pathway,3,4,10–13 we are justified to hypothesizethat dual PI3K/mTOR inhibition would sensitize prostatecancer cells to radiation therapy. Here in this study we ex-amined the capacity of NVP-BEZ235 to reverse the radiationresistance of prostate cancer cell line PC-314–17 and exploredpossible molecular mechanism. It was concluded that dualPI3K/mTOR blockade by NVP-BEZ235 could synergize withIR in PC-3 cells. The underlying mechanisms may includecell cycle arrest and enhancement of IR-induced apoptosis.

Materials and Methods

Cell culture and reagents

The human prostate cancer cell line PC-3 was obtainedfrom ATCC. Cells were cultured in RPMI-1640 (Hy Clone;Thermo Scientific) with 10% of fetal bovine serum, and in-cubated at 37�C in 5% CO2/95% air.

Dual PI3K/mTOR inhibitor treatment

NVP-BEZ235 was obtained from Novartis Pharma. Theinhibitor was added to mid-log-phase cell cultures at theindicated concentrations. After treatment, the culture me-dium was replaced with drug-free medium. For the controlgroup, cells were incubated with medium containing di-methyl sulfoxide (DMSO) at a concentration correspondingto the highest dose used in inhibitor-treated cells.

Clonogenic survival assay

The exponential growth cells (500-1000/well) were seededinto six-well plates. 12 hours after plating, NVP-BEZ235 wasadministered at concentrations of 50 nM and 100 nM 1 hourbefore irradiation. The cells were then exposed to IR at doses of2, 4, 6, and 8 Gy at an average dose rate of 200 cGy/min.Twenty hours after irradiation, the medium was replaced bybasal culture medium free of the inhibitor. The cells were in-cubated for 7 to 10 days at 37�C in 5% CO2/95% air to allowthe colony formation. The plates were fixed with pure meth-anol and stained with 1% crystal violet. We scored colonies ofmore than 50 cells and graphed the surviving fraction (SF)versus dose of IR. SF was estimated by the following formula:SF = number of colonies formed/number of cells seeded ·plating efficiency. Plating efficiency is the ratio of colonynumber of the control group to cells seeded. Dose–response

clonogenic survival curves were plotted on a log-linear scale.Three cell concentrations were applied for each dose of IR. D0

and Dq values, reflecting the sensitivity and the shoulder of thesurvival curve, were calculated as described.18

MTT assay

Cells (1 · 104/well) were seeded into 96-well plates and in-cubated for 24 hours to allow adherence. NVP-BEZ235 wasadministered at concentrations of 50, 150 and 250 nM 1 hourbefore irradiation. The plates were then exposed to IR at dosesof 2, 4, 6, 8 and 10 Gy at an average dose rate of 200 cGy/min.20 hours after irradiation, the medium was replaced by basalculture medium free of the inhibitor. Thereafter, 10 lL 5 mg/mL MTT was added into each well and the cells were incu-bated for another 4 hours before termination of experiment.The medium was discarded and 100 lL DMSO was added ineach well to dissolve the formazan produced by viable cells.OD values at 490 nm were measured in a microplate reader.Then the rate of inhibition was calculated using the formula:rate of inhibition = 1 - OD value of the experimental group/OD value of the control group.

Flow cytometric analysis for cell cycle and apoptosis

Tumor cells were incubated overnight to allow adherence.The inhibitor was added 1 hour before exposure to IR at asingle dose of 4 Gy. Twenty hours after irradiation, the me-dium was replaced by basal culture medium free of the in-hibitor. The cells were then cultured for another 4 or 28 hoursbefore harvesting. Cell cycle analysis was carried out ac-cording to a standard procedure in Cell Cycle Flow Cyto-metry Testing Kit (Genmed Scientifics, Inc.). Analysis ofapoptosis was performed according to a standard procedurein Annexin V-FITC/PI Apoptosis Detection Kit (ABGAB).Afterwards the samples were subjected to assessment with aFACSCalibur flow cytometer (Becton Dickinson). The datarepresentative of three independent experiments were ana-lyzed with FlowJo v 7.6.5 software.

Western blot

Western blot was performed as described earlier.19 Theexpression of actin was set as the protein loading control. An-tibodies against phosphorylated-(P)-(Ser473)-Akt, P-(Thr308)-Akt, total Akt (T-Akt), P-mTOR, p21cip1, p27kip1, cyclin A,cyclin B, cyclin E, actin as well as horseradish peroxidase-con-jugated secondary antibodies were from Abcam.

Statistical analyses

All data are presented as the average – SD. Experimentswere conducted in triplicate. Significance of differences wasdetermined using two-tailed unpaired Student’s t test or theone-way ANOVA test with SPSS 13.0 software. A valuep < 0.05 was considered statistically significant.

Results

The dual PI3K/mTOR inhibitor NVP-BEZ235 reversedradioresistance of PC-3 cells and sensitized the tumorcells to the proapoptotic effect of IR

To evaluate the radiosensitizing effect of the dual PI3K/mTOR inhibitor, MTT and clonogenic survival assay were

666 ZHU ET AL.

performed. Compared with the control group that excludedtreatment of NVP-BEZ235, tumor cells exhibited higher rateof inhibition under the same IR dose after dual PI3K/mTORinhibition, which demonstrated the radiosensitizing activityof the inhibitor (Fig. 1A). Notably, with the increase of in-hibitor concentration, a climbing degree of inhibition wasobserved, suggesting that the sensitizing effect was also in-hibitor dose-responsive (Fig. 1B). Besides, combination of IR(4 Gy) and NVP-BEZ235 (50 nM) resulted in a greater degreeof inhibition on cell viability than treatment with IR or NVP-

BEZ235 alone ( p < 0.05), implying a synergistic action be-tween the agent and irradiation (Fig. 1C).

The clonogenic survival assay was carried out to estimatethe survival rate of clonogenic tumor cells after differentmeans of interference. The tumor cells were exposed to in-creasing doses of IR (2–8 Gy) with/without the presence ofthe inhibitor. According to preliminary experiments thatwere intended to determine the optimal incubation time withNVP-BEZ235, continuous treatment with the inhibitor led tofailure of colony formation, while incubation for no more

FIG. 1. Effect of combination of NVP-BEZ235 and IR on cell viability in PC-3 cells. Cells were seeded into 96-well plates andwere exposed to NVP-BEZ35 at indicated concentrations 1 hour before irradiation. Twenty hours after IR the medium wasreplaced by basal culture medium and cell viability was assessed by MTT assay. (A) IR dose-responsive curves with differentNVP-BEZ235 concentrations. (B) Drug concentration-responsive curves with different IR doses. (C) Effects of IR (4 Gy) and/or NVP-BEZ235 (50 nM) on cell viability. Data were representative of six independent experiments and expressed asmean – standard deviation (SD). #p < 0.05 between ‘‘IR + BEZ235’’ and ‘‘control,’’ ‘‘IR’’ as well as ‘‘NVP-BEZ235,’’ respectively.

NVP-BEZ235 RADIOSENSITIZES PROSTATE CANCER CELLS 667

than 21 hours didn’t affect the plating efficiency significantly.Therefore, for subsequent assays we exposed PC-3 cells tothe inhibitor 1 hour before IR and limited the total incubationtime to 21 hours. Pretreatment with NVP-BEZ235 resulted inan obvious reduction in the survival rate of tumor cells afterexposure to different doses of IR. An increase of the radio-sensitizing effect was uncovered accompanying the elevationof inhibitor concentration (Fig. 2A).

To derive a quantified evaluation of the effect, the datawere fitted into single-hit multitarget curves and some rele-vant parameters were calculated. Addition of the dual in-hibitor apparently decreased the mean lethal dose D0 as wellas the quasi-threshold dose Dq, suggesting the radiosensi-tivity of PC-3 cells was improved by the inhibitor. Also notsurprisingly, the elevation of the inhibitor concentrationbrought about a further decline in the radioresistance of thetumor cells (Fig. 2B). All those stated above proved thatNVP-BEZ235 was indeed able to sensitize PC-3 cells to IR,and most probably by augmenting DNA damage caused byIR or interfering with the cell cycle checkpoints.

Dual PI3K/mTOR blockade along with IR altered cellcycle distribution and induced a G2/M arrest via cellcycle modulating proteins

To live out whether the dual PI3K/mTOR inhibitorradiosensitized PC-3 cells via redistribution of cell cycle, weconducted cell cycle assay on tumor cells exposed to IR and/or the inhibitor. Cell cycle was detected at 24 and 48 hoursafter irradiation, respectively to help reveal any time-relatedalteration. NVP-BEZ235 (50 nM) alone resulted in growtharrest in the G1 phase of cell cycle, whereas IR alone led tothe G2/M arrest. Strikingly enough, the combination of IRwith the inhibitor induced a further accumulation of PC-3cells in the G2/M phase, which highlights the potentialmechanism of NVP-BEZ235 as a radiosensitizing agent.Different time course points at assay didn’t exert profoundinfluence on the distribution of cell cycle (Fig. 3).

Further western blot detection helped reveal cellularchanges underlying different actions of IR and BEZ235 (Fig.5). In accordance with previous observations, IR alone

caused a G2/M arrest in PC-3 cells. Meanwhile irradiationpromoted the expression of p21 and p27, both of which arevital cyclin-dependent kinase (CDK) inhibitors, resulting indiminished levels of cyclin E and B (Fig. 5B). Unlike IR, NVP-BEZ235 alone elicited G1/S detention. Increased level of p21as well as p27 was observed to concur with dramatic de-crease of cyclin E and A, all of which participate in G1/Scheckpoint (Fig. 5B). Though exerting opposing effect on cellcycle compared with IR, NVP-BEZ235 unexpectedly primedmore cells to G2/M arrest when combined with IR. Con-sistently changes in p21 and cyclin B were detected (Fig. 5B).

NVP-BEZ235 radiosensitized PC-3 cells throughenhancement of proapoptotic effect of IR

Irradiation causes cyto-lethal effect mostly by DNAdamage and programmed cell death, namely apoptosis. Yeton the other hand, IR-induced activation of the PI3K/Akt/mTOR pathway may finally counteract the proapoptoticoutcome of IR by initiation of antiapoptosis cascade. Hence,we were fully convinced that dual PI3K/mTOR blockadewould cut off this pernicious activation and augment proa-poptotic action of IR. Detection of apoptotic PC-3 cells afterexposure to IR and/or NVP-BEZ235 showed that combinedtreatment led to a significantly higher fraction of apoptosis.Tests were done at 24 and 48 hours after IR, respectively.Quite intriguing results were obtained. The DMSO or NVP-BEZ235 group saw no prominent difference between twotime points of detection, while the proportion of apoptoticcells in IR group lowered at 48 hours after irradiation than 24hours, implying the occurrence of anti-apoptosis events, suchas repair of DNA damage. The combination of IR with theinhibitor led to a relatively constant fraction of apoptosisdespite delay of the inspection point (Fig. 4).

NVP-BEZ235 counteracted constitutive as wellas IR-triggered activation of Akt/mTOR signalingin PC-3 cells

Western blot assay was carried out to throw light uponpotential radiosensitizing mechanism of NVP-BEZ235. As

FIG. 2. Clonogenic survival curves (A) and curve-related parameters (B) of PC3 cells after treatment with NVP-BEZ235 at50 and 100 nM. The clonogenic survival assay was done as described in ‘‘Materials and Methods’’ section.

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anticipated, IR induced further phosphorylation of Akt (onboth S473 and T308) and mTOR, while the total content ofAkt remained stable. When applied alone, NVP-BEZ235suppressed intrinsic dysfunction of Akt/mTOR signalingalthough the effect was not a notable one. Combination ofthe agent with IR created a desirable compromise of IR-induced response, cracking down the activity of the pathwayto the basic level (Fig. 5A).

Discussion

As indicated previously, intrinsic and IR-induced activa-tion of PI3K/Akt/mTOR pathway in prostate cancer cells isconsidered to compromise the cytotoxic effect of irradiationand mostly accounts for the development of radio-resistance.20–22 Rapamycin analogues (rapalogs) have beendiscovered to exhibit anti-proliferative effect in some tumorcell lines,23 but with limited efficacy in tumors that havePI3K/Akt and mTOR activation, for they only inhibitmTORC1 rather than mTORC2, which can phosphorylateAkt at Ser473 and eventually restart the deregulation ofPI3K/Akt pathway.24,25 As a dual PI3K/mTOR inhibitor,

NVP-BEZ235 not only inhibits the activity of PI3K andmTORC1 but also effectively blocks mTORC2.7 Therefore,we have every reason to expect NVP-BEZ235 to abrogate thecell survival response elicited by IR and reverse the radio-resistance of prostate cancer.14–17

Former research work has revealed that prostate cancercan be divided into two categories, namely androgen re-ceptor (AR) positive and AR-negative.26 Development ofabnormal AR activation with the deprivation of androgenaccounts for the failure of castration therapy commonly seenin the clinical setting.27 AR activation finally directs to a se-ries of cellular pathways that reportedly cross-talk with thePI3K/Akt/mTOR pathway.28,29 Here in our study PC-3 cells,which are AR-negative,26 were opted so that influence fromAR pathways could be avoided.

In some preclinical models, NVP-BEZ235 have showedpotent antiproliferative activity in tumors, such as renal cellcarcinoma, glioblastoma, colorectal, and breast cancer.8,30 Ithas been proposed as a promising antitumor agent owing toits properties as orally-available and well-tolerated.7 Never-theless description of NVP-BEZ235 being applied to radio-sensitize tumors has been scarce. Our work has provided

FIG. 3. Evaluation of cell cycle distribution in PC-3 cells. Cells were harvested at 24 hour (A and C) or 48 hours (B and D)after irradiation and cell cycle was detected as described in ‘‘Materials and Methods’’ section.

NVP-BEZ235 RADIOSENSITIZES PROSTATE CANCER CELLS 669

insight into this inhibitor as a radiosensitizing agent inprostate cancer, which possesses poor radiosensitivity14–17

and high occurrence of PI3K/Akt/mTOR pathway deregu-lation. Here in this study we demonstrated that when used atminimal concentration NVP-BEZ235 already displayed re-markable radiosensitizing activity, and with the increase ofinhibitor dose such effect was amplified. When administeredalone at low concentration, the dual inhibitor had little im-pact over the proliferative activity of PC-3 cells, supplyingfurther evidence for its role as a radiosensitizing agent.

Actually we did conduct preliminary experiments to de-termine the best treatment schedule for administration ofNVP-BEZ235 (data not shown). The drug was applied 24hours/12 hours/6 hours/1 hour before, coinstantaneously

with and after IR, yet, the latter two arrangements failed tobring about any positive sensitizing effect, revealing thatcomplete blockade of target kinases requests pre-exposure toNVP-BEZ235 and thus, suppresses constitutive as well as IR-induced PI3K/Akt activation. Among all those schedulesgiving NVP-BEZ235 before IR, 1 hour of pre-exposure fol-lowed by IR demonstrated the most prominent radio-sensitizing effect, while preincubation for 12 and 24 hoursbefore IR only caused a mild to moderate degree of cyto-toxicity, similar to that of IR alone. That timing-dependentsensitizing effect of NVP-BEZ235 provided cues for our de-cision on the treatment schedule.

To confirm the cellular changes concurring with theradiosensitizing action of the inhibitor, we implemented

FIG. 4. Analysis of apoptosis in PC-3 cells by flow cytometry. Cells were harvested at 24 hours (A and C) or 48 hours (B andD) after irradiation and cell apoptosis was assessed as described in ‘‘Materials and Methods’’ section. Data were represen-tative of three independent experiments and expressed as mean – standard deviation (SD). **p < 0.01 between ‘‘DMSO’’ and‘‘IR + BEZ235.’’ DMSO, dimethyl sulfoxide.

670 ZHU ET AL.

concerning experiments on cell cycle analysis and detectionof apoptosis, from which we were compelled to concludethat NVP-BEZ235 abated the radioresistance of PC-3 cells byinducing cell cycle arrest and proapoptotic response. In ac-cordance with previous findings about NVP-BEZ235 andother PI3K inhibitors, NVP-BEZ235 alone aroused the G1phase block. More importantly, exposure of PC-3 cells toclinically relevant dose of IR with NVP-BEZ235 pretreatmentresulted in the detention of cancer cells in G2/M phase. Si-milarly, rapalogs, namely rapamycin analogues, have beenadvanced to produce G2/M phase arrest along with irradi-ation.31 Detection of cell cycle regulating proteins suppliedhints as to the observed changes. What should be noted hereis that the main components involved in G1/S and G2/Mcheckpoints were assessed except p53 since PC-3 cells arep53 null. Interestingly, contrary to IR, NVP-BEZ235 aloneinduced G1/S arrest by modulation of p21, p27, cyclin E, andA. Yet, when followed by irradiation, BEZ235 apparentlyfurther promoted IR-triggered G2/M arrest instead of com-bating it. Why such seemingly contradictory consequencesoccurred still needs more pursuit, but it seems safe to con-clude that p53-independent p21 and p27 activity indeed areinducible in PC-3 cells.

In addition, the influence of the dual inhibitor on cellularapoptosis was investigated. Prostate cancer cells were sub-jected to NVP-BEZ235 and/or IR. We also drew a compari-son between cells collected at different time points ofdetection. Here from our statistics it was clearly displayedthat the combination of the dual inhibitor and IR led togreater degree of cellular apoptosis compared with the sep-arate action of the agent or IR. Particularly, when the sam-ples were examined at 48 rather than 24 hours after IR; thus,sparing the cancer cells adequate time to initiate inner pro-survival response, a paradoxical descent in the percent ofapoptosis was observed. This conferred persuasive evidencefor the occurrence of IR-elicited antiapoptotic events, whichpartly offset the cytotoxic action of IR. On the contrary, suchdiminishment of cellular apoptosis with time delay was notseen in the group enduring combined treatment, and thelevel of apoptosis was slightly elevated instead of beinglowered as time went by. We considered this observation avital one, because it provided hints that NVP-BEZ235 most

probably sensitized PC-3 cells to irradiation by constrainingthe IR-evoked cell survival reaction.

Consensus has been reached that the PI3K/Akt/mTORpathway is implicated in cell proliferation and synthesis,32

and IR-induced activation of the pathway may help explainthe acquired insensitivity of PC-3 cells to irradiation,20 whichwas demonstrated in the present study. We initially assumedcomplete and specific blockade of the PI3K/mTOR pathwayby NVP-BEZ235 would ameliorate tumor sensitivity to IRvia suppressing pernicious prosurvival cell response, and thewestern blot results finally bore out that hypothesis. Giventhat cell cycle and apoptosis related proteins belong to PI3K/Akt/mTOR signaling downstream targets, it may be justifiedto conclude that NVP-BEZ235 actually played its radio-sensitising role by direct inhibition of PI3K/Akt/mTORpathway and subsequent restraint of cell cycle progressionand anti-apoptotic events. Yet, to detail the molecularmechanism involved in this interwoven network, such as thechanges in some apoptosis-concerning markers, more fun-damental experiments need to be carried out in our futureexploration into this subject.

Apart from interference of cell cycle regulation and facil-itating proapoptotic events, other molecular mechanismsunderlying the potent radiosensitizing efficacy of NVP-BEZ235 have been proposed. Specific blockade of PI3K andmTOR activity by NVP-BEZ235 has been challenged by arecent study. Mukherjee et al.33 reported that NVP-BEZ235not only effectively inhibited PI3K/mTOR, but also exertedcross-reactivity toward ATM and DNA-PKcs, which belongto the PI3K-like kinase (PI3KK) family and mainly functionin response to IR-induced DNA double-strand breaks(DSBs). This inevitably led to the attenuation of DSB repair,including nonhomologous end joining (NHEJ) and homolo-gous recombination (HR), mediated by DNA-PKcs andATM, respectively. Moreover, phosphorylation of ATMdownstream targets as well as G2/M arrest were also abatedby NVP-BEZ235. Altogether these effects conferred by NVP-BEZ235 resulted in its dramatic radiosensitizing action onglioblastoma. The current study in PC-3 cell line failed tobear evidence of DSB repair defects after NVP-BEZ235 ad-ministration, but whether or not modulation of multiplePI3KK family members by NVP-BEZ235 is ubiquitous in

FIG. 5. Detection of rele-vant proteins involved incellular responses to IR and/or NVP-BEZ235 treatment bywestern blot analysis. (A)Expression level of proteinsin Akt/mTOR signalingpathway in PC-3 cells ex-posed to IR (4 Gy) and/orNVP-BEZ235 (50 nM). Cellswere treated as described in‘‘Materials and Methods’’section. (B) Effects of IR(4 Gy) and/or NVP-BEZ235(50 nM) on cell cycle modu-lating proteins.

NVP-BEZ235 RADIOSENSITIZES PROSTATE CANCER CELLS 671

various classes of carcinoma warrants further investigationnotwithstanding. Taking into account all that has beendiscussed, it’s reasonable to arrive at the conclusion thatNVP-BEZ235 indeed performs a strikingly potent radio-sensitizing effect by disabling numerous cellular signalingpathways correlated with (1) cell cycle arrest owing toPI3K/Akt and ATM inhibition, (2) cell proliferation andsurvival due to PI3K/Akt/mTOR blockade, (3) DSB repairas a result of ATM/DNA-PKcs and less importantly PI3K/Akt inhibition.

In the current setting, NVP-BEZ235 and several other dualPI3K/mTOR inhibitors are still in phase I/II clinical trials. Inthis regard, our work added to its preclinical data and ex-panded its spectrum of application as not only a novel anti-tumor drug but also a radiosensitizing agent. Its radiosensitizingaction has been determined in several cancer cell lines,34 but ourstudy pioneered the investigation of its sensitizing effect onprostate cancer, which is of clinical significance because prostatecancer is generally acknowledged to be radioresistant.

We arrived at a primary conclusion that NVP-BEZ235indeed radiosensitized prostate cancer cells possibly throughinhibition of cellular prosurvival stress response and en-hancement of apoptosis caused by irradiation. For furtherlook into this subject, subsequent experiments are necessary.Our data were gained from AR-negative PC-3 cell to avoidpotential interaction between AR pathway and the targetpathway. In future, we intend to investigate action of NVP-BEZ235 on AR-positive prostate cancer cell lines, such asLNCaP cell to throw light upon crosstalk between these twopathways. Androgen-independent AR activation eventuallyrenders prostate cancer cells resistant to androgen depriva-tion therapy, and to make things worse prostate cancer is bynature insensitive to irradiation, resulting in a complicatedbut commonplace clinical circumstance. Research work sta-ted above will no doubt present solutions of practical im-portance to this dilemma. Last but not least, animal modelscan be established to evaluate the radiosensitizing effect ofNVP-BEZ235 in vivo, which will produce more direct insightinto its practical efficacy.

In summary, our study demonstrated that the dual PI3K/mTOR inhibitor NVP-BEZ235 prominently improved theradiosensitivity of PC-3 cells in which aberrant activationof PI3K/Akt/mTOR pathway was common. It sensitizedtumor cells via interruption of cell cycle checkpoints andaugmentation of cell apoptosis, which was due to its con-straint on IR-elicited prosurvival cellular response. In thefuture, we intend to obtain insight into potential molecularmechanisms associated with the radiosensitizing effect ofdual PI3K/mTOR blockade, and optimize its clinical appli-cation via in vivo experiments.

Disclosure Statement

NVP-BEZ235 produced by Novartis Pharma is pur-chased from LC labs, which does not compromise the studyscientifically.

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NVP-BEZ235 RADIOSENSITIZES PROSTATE CANCER CELLS 673