Midostaurin inhibits hormone-refractory prostate cancer PC-3 cells by modulating nPKCs and AP-1 transcription factors and their target genes involved in cell cycle

Abstract

BACKGROUND: The development of prostate cancer from a clinically localized, hormone-naive state to a hormone-refractory phenotype involves a complex interplay of protein kinase C (PKC) and activator protein-1 (AP-1). Therefore, the present study aimed to uncover the roles of PKC and AP-1 through midostaurin-mediated regulation—a multi-target protein kinase inhibitor.

METHODS: Androgen Receptor-negative, hormone-refractory prostate cancer cells (PC-3) were used as an in-vitro model system. The effect of midostaurin on cell viability was assessed by an MTT assay. Expression studies on PKC-α, PKC-d, different AP-1 transcription factors, and AP-1 regulating genes were analyzed by semiquantitative RT-PCR, and protein levels of Bcl-2 were evaluated by western blotting.

RESULTS: Midostaurin decreased the viability of hormone-refractory PC-3 cells. Furthermore, midostaurin significantly induced the transcripts of apoptotic-mediated PKC-d, tumor suppressor p53, cell cycle inhibitor p21^cip1/waf1, death receptor TNF-α, pro-apoptotic Bax, and Caspase-8, and eventually inhibited the expression of pro-survival PKC-ε, pro-oncogene c-Jun, c-Fos, Fra-1, positive growth regulator cyclin D1, and anti-apoptotic Bcl-2. In addition, midostaurin also decreased the protein expression of anti-apoptotic Bcl-2.

CONCLUSION: The present study provided evidence that midostaurin suppresses tumor growth and induces apoptosis in hormone-refractory PC-3 cells via modulation of PKC-d and PKC-ε expression, and regulation of PMA-altered c-Jun, c-Fos, and Fra-1 AP-1 transcription factors and their target genes involved in cell cycle regulation (cyclin D1, p53, p21, Bcl-2, and TNF-α). Thus, pharmacological targeting of PKC and AP-1 factors may have therapeutic potential against hormone-refractory prostate cancer.

Keywords

protein kinase C / AP-1 factors / midostaurin / semi-qRT-PCR / western blotting

Introduction

Protein kinase C (PKC) is a familyof serine/threonine kinases involved in the regulation of variouscell functions including proliferation, gene expression, differentiation,cytoskeletal organization, cell migration, and apoptosis (Carter and Kane, 2004). PKCs havebeen linked to carcinogenesis because of PKC activators, such as phorbol12-myristate 13-acetate (PMA), acting as tumor promoters (Blumberget al.,1984). Furthermore,studies have suggested that PKC affects the phenotype of high gradecancers, including skin, colon, ovarian, brain, lung, breast, andprostate cancers, supporting the role of PKC in early carcinogenesisand cancer progression (Teicher 2006). PKCs are classified into conventional (cPKC), novel (nPKC), andatypical PKCs based on their structural and activation characteristics(Schenk and Snaar-Jagalska, 1999). PKCs are capable of promoting opposingresponses, such as survival and growth arrest. This paradigm of functionaldiversity is exemplified by nPKCs, such as PKC-ε that acts asa mitogenic or anti-apoptotic kinase, and PKC-d, whose activation inhibits proliferation or triggersan apoptotic response (Brodie and Blumberg, 2003, Nakagawa et al.,2005). In particular,PKC-ε is implicated in prostate tumor progression and the transitionto androgen-independence (Koren et al., 2004; Aziz et al., 2007). These findingsled to PKC being considered as a potential therapeutic target forhormone-refractory prostate cancer.

The PKC signaling pathway exertsproliferative or anti-proliferative effects through the downstreamtranscription factor, activating protein 1 (AP-1). AP-1 plays a criticalrole in the regulation of prostate cell proliferation, as well ascancer progression (Angel et al., 1987, Ouyang et al., 2008) and is known to determine the fate of cells, life or death, inresponse to variety of extracellular stimuli. AP-1 mediates responsivenessto phorbol ester tumor promoters, and is composed of the cellularhomologs of Jun and Fos oncoproteins, implying its involvement ingrowth control and oncogenesis (Wuet al., 2002). The broad combinatorial possibilitiesprovided by the large number of Jun/Jun or Jun/Fos proteins determinetheir binding specificity and affinity, and consequently, the spectrumof regulated genes (Hess et al., 2004). AP-1 has been implicated as a positive regulator of cell proliferationthrough their ability to mediate expression and function of cell cycleregulators, such as cyclin D1, p53, and p21^cip1/waf1, and can therefore stimulate G1-to-S-phase transition and cell cycleprogression. On the other hand, AP-1 may increase cell survival byregulating genes such as anti-apoptotic Bcl-2 and death receptor TNF-α(Shaulian and Karin, 2001). Despite several studies, the mechanismbehind inhibiting PMA-induced AP-1 factors in hormone-refractory prostatecancer is unclear.

Midostaurin, also known as PKC412,is a protein kinase inhibitor originally developed for use againstPKC, a serine/threonine protein kinase family (Propper et al., 2001). It was latershown that this compound is a multi-target protein kinase inhibitorwith a broad inhibition spectrum, (Fabbro et al., 2000). It has been suggested that midostaurinexhibits its anti-cancer effects through cell cycle arrest and increasedapoptosis (Bahlis et al., 2005, Fischer et al., 2010, Kawai et al., 2015). However, not much is known about the exact mechanisms of midostaurinregulation of nPKC isozymes, expression of AP-1 factors, and AP-1target genes involved in hormone-refractory prostate cancer. Therefore,the present study aimed to demonstrate the anti-proliferative effectof midostaurin on nPKC isozymes, AP-1, and AP-1 target genes usinghormone-refractory prostate cancer cells (PC-3) as a model systemunder in vitro conditions.

Materials and methods

Materials

Human prostate cancer cells (PC-3)were purchased from NCCS (Pune, India). Phorbol 12-myristate 13-acetate(PMA), midostaurin (PKC412), TRIzol, and forward and reverse primersfor PKC-α, PKC-d, differentAP-1 transcription factors, and AP-1 regulating genes (Table 1) werepurchased from Sigma-Aldrich (St Louis, USA). Superscript reversetranscriptase for RT-PCR was purchased from Invitrogen (California,USA). Roswell Park Memorial Institute-1640 (RPMI-1640) medium, fetalbovine serum (FBS), penicillin, streptomycin, glutamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), dimethyl sulfoxide (DMSO), and trypan blue dye and6 and 96-wellplates were purchased from Himedia (Mumbai, India). Primaryantibodies against GAPDH and Bcl-2 were purchased from NeoBiolab (Massachusetts,USA), and goat anti-rabbit HRP-conjugated secondary antibody was purchasedfrom Imgenex India Pvt. Ltd. (Bhubaneswar, India). Taq DNA polymerase(1 U/µL) and Luminata Forte Western HRP substrate were procuredfrom Merck-Millipore (Mumbai, India).

Culturing of PC-3 Cells and treatment

PC-3 cells were grown in 25-cm² culture flasks in RPMI-1640 media supplemented with10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine. Cells were cultured at 37°Cin a humidified atmosphere of 5% CO₂. Flaskscontaining 90%–100% confluent cells were sub-cultured in 96-wellplates (3 × 10³ cells/well) and 6-wellplates (5 × 10⁵ cells/well) for treatmentwith modulators.

MTT assay

PC-3 cells (3 × 10³ cells/well) in 200 µL of medium were seededin 96-well microtiter plates and incubated overnight at 37°C witha supply of 5% CO₂. Cells were treated withmedia containing various concentrations of PMA or midostaurin for48 h, washed with PBS, treated with 20 µL of MTT (5 mg/mL),and then incubated further for 4 h at 37°C in a CO₂ incubator. Live cells take up the yellow MTT compound and mitochondrialenzymes reduce it to insoluble blue formazan products, that are thendissolved in DMSO (100 µL) and visualized. Absorbance was measuredat 540 nm using a multimode plate reader (Massachusetts, USA). Theeffect of PMA and midostaurin on cell viability was calculated andrepresented as the percentage of viable cells compared with control.

RNA isolation and semiquantitative RT-PCR analysis

Overnight cultured PC-3 cells (5× 10⁵ cells/well) in 6-well plateswere treated with or without PMA (10 nM), midostaurin (10 µM),or PMA (10 nM) + midostaurin (10 µM) for 48 h. Total RNA wasisolated from the samples using TRIzol reagent as per manufacturer’sinstructions. Reverse transcription of RNA and PCR analysis was carriedout according to a previously described protocol (Hegde et al., 2016). In brief, totalRNA (2 µg) was reverse transcribed using oligo dT primers andsuperscript reverse transcriptase. The cDNA was subjected to 30 cyclesof PCR using different forward and reverse primer pairs for PKC-d, PKC-ε, several AP-1 factors, andAP-1 regulating genes (Babu et al.,2013) using appropriate annealing temperatures (Table1) in a gradient Eppendorf thermocycler. Amplified PCR products wereanalyzed on 1% agarose gels using 1 × TAE buffer. Relative mRNAlevels were quantified using an image analysis software (ImageJ).Expression of β-actin mRNA was used as a positive control andfor normalization.

Western blot analysis

PC-3 cells (5×10⁵ cells/well) in 6-well plates were treated with orwithout PMA (10nM), or PMA (10nM)+midostaurin (10 μM) for 48 hand were subjected to western blot analysis as described previously(Patil et al., 2016)with minor modifications. Cells were lysed in 0.2 mL cold lysis buffer[50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, and 100 µMPMSF], and protein concentrations of cell lysates were estimated byBradford’s method (Bradford 1976). Equal amounts of protein (40 mg/lane) were electrophoresed on 12% resolving sodium dodecyl sulfate–polyacrylamidegels and transferred onto PVDF membranes. Membranes were blocked with5% fat-free milk (Carnation) for 1 h at room temperature. Blots wereincubated with primary antibody (anti-Bcl-2) in blocking solution(1:500) for 1 h, washed, and then incubated with anti-rabbit HRP-linkedsecondary antibody (1:1000) and further incubated for 1 h at roomtemperature. Proteins were visualized with Luminata Forte WesternHRP substrate (according to manufacturer’s instructions) usinga Syngene gel documentation system (Maryland, USA). GAPDH was usedas an internal control for normalization. Immunoreactive bands werequantified using the image analysis software, ImageJ.

Statistical analysis

All values are expressed as the mean±standarddeviation. Each value represents the mean of at least 3 independentexperiments in each group. MTT assays, images of semiquantitativeRT-PCR gels, and western blotting data were analyzed by one-way ANOVAfollowed by post-hoc Tukey test. Values were considered statisticallysignificant if *p<0.05 and **p<0.005 compared with control, and if#p<0.05 compared with PMA-treatedsamples.

Results

Anti-proliferative effect of midostaurin on hormone-refractoryPC-3 cells

To examine the effect of midostaurinon cell viability, PC-3 cells were treated with or without differentconcentrations of midostaurin (1 nM–10 mM) for 48 h, and then cell viability was determined byMTT assays. PC-3 cells treated with midostaurin at concentrationsof 1 nM and 10 nM had no effect on cell viability, while significantdecreases in cell viability was observed from 50 nM and higher. Amaximum of 40% decrease in cell viability was observed at 10 mM of midostaurin (Fig. 1). Similar resultswere obtained after counting the number of viable cells by the trypanblue exclusion method using a Neubauer counting chamber (data notshown).

Midostaurin induces apoptotic PKC-d and inhibits pro-survival PKC-ε mRNA expression in PC-3 cells

PC-3 cells were treated with PMA(10 nM), midostaurin (10 µM), or PMA (10 nM) in combinationwith midostaurin (10 µM) for 48 h, and the expression levelsof nPKC isozymes (PKC-d and PKC-εmRNA), were analyzed by semiquantitative RT-PCR. Results showed thatPMA exerts almost had no effect on PKC-d expression, but induced PKC-ε mRNA expression by more than38% compared with control (Fig. 2). On the other hand, cells treatedwith midostaurin or midostaurin in combination with PMA showed significantincreases in the expression levels of PKC-d mRNA by 52% and 46%, respectively, compared with control.Furthermore, midostaurin or midostaurin and PMA significantly decreasedmRNA levels of PKC-ε by 30% and 25%, respectively, compared withcontrol, and by 50% and 45%, respectively, compared with PMA-treatedsamples.

Midostaurin inhibits the PMA-induced expression of c-Jun, c-Fos,and Fra-1 mRNA in PC-3 cells

To analyze the effect of midostaurinon the mRNA expression of different AP-1 factors, PC-3 cells weretreated with PMA (10 nM), midostaurin (10 µM), or PMA (10 nM)in combination with midostaurin (10 µM) for 48 h, and the expressionpatterns of AP-1 factors were measured by semi-quantitative RT-PCRto analyze relative mRNA levels. The results showed that cells treatedwith PMA significantly induced mRNA expression of c-Jun by 0.9-fold,c-Fos by more than 2.4-fold, and Fra-1 by 1.8-fold, while the expressionof JunB decreased by 30% compared with the control (Fig. 3). Treatingcells with midostaurin or midostaurin combined with PMA significantlydecreased the PMA-induced expression levels of c-Jun, c-Fos, and Fra-1,implying that c-Jun, c-Fos, Fra-1, and JunB may be involved in thedevelopment of hormone-refractory prostate cancer.

Midostaurin modulates AP-1-regulated cell cycle regulatorsand apoptotic genes

To analyze the effect of midostaurinon mRNA levels of AP-1-regulated cell cycle regulators and apoptoticgenes, PC-3 cells were treated with PMA (10 nM), midostaurin (10 µM),or PMA (10 nM) in combination with midostaurin (10 µM) for 48h. The expression patterns of p53, p21, cyclin D1, TNF˗α, Bax,Bcl-2, and caspase-8 were then measured by semi-qRT-PCR for relativemRNA level analysis. The results showed that cells treated with PMAsignificantly induced the growth regulator cyclin D1 and anti-apoptoticBcl-2 by 30% and 34%, respectively, compared with control, while transcriptlevels of p53, p21, Bax, and caspase-8 were unaffected (Fig. 4). However,midostaurin treatment, either alone or in combination with PMA, resultedin an increase in mRNA levels of tumor suppressors p53 and p21 by1.8- and 1.5-fold, respectively, and induced expression of death receptorTNF-α, pro-apoptotic Bax, and caspase-8 by 2-, 1.1-, and 1.3-foldincreases, respectively. Midostaurin treatment also decreased PMA-inducedexpression of cyclin D1 and Bcl-2 by 60% and 55%, respectively. Therefore,the results confirm the apoptotic effect of midostaurin on hormone-refractoryPC-3 cells.

Midostaurin inhibits PMA-induced expression of AP-1-regulated,anti-apoptotic Bcl-2 protein

The present study also showed thatPMA (10nM) significantly induced the protein expression of anti-apoptoticBcl-2 by 0.8-fold compared with control. On the other hand, treatmentof cells with midostaurin (10μM) or midostaurin combined with PMAsignificantly decreased the expression of anti-apoptotic Bcl-2 by40% and 50%, respectively, compared with control, and by 55% and 65%,respectively, compared with PMA-induced expression (Fig. 5). Theseresults suggest that midostaurin inhibits cell proliferation and promotesapoptosis in hormone-refractory PC-3 cells.

Discussion

In the present study, we revealedthat midostaurin significantly suppresses proliferation of hormone-refractoryPC-3 cells. Hormone-refractory, invasive prostate cancer is an end-stagecancer that accounts for the majority of prostate cancer patient deaths(Edwards and Bartlett, 2005). Knowledge about the molecular mechanismsand regulatory molecules involved in the transition from androgen-dependentto androgen-independent prostate cancer is essential for planningstrategies to prevent and treat prostate cancer. Aberrantly activatedprotein kinase C (PKC) and AP-1 transcription factors are regardedas potential therapeutic targets for hormone-refractory prostate cancer(da Rocha et al., 2002, Ouyang et al., 2008).

Midostaurin, a semi-synthetic alkaloidderived from bacterial staurosporine, is a multi-target protein kinaseinhibitor that inhibits growth or induces apoptosis in several typesof cancer, blocks angiogenesis, and sensitizes cancer cells to ionizingradiation—justifying its use in cancer treatment (El Fitori et al., 2007). Midostaurinhas the advantage of oral administration and a longer half-life becauseof the altered gastrointestinal absorption and plasma protein binding(particularly to AAG) in cancer patients (Propper et al., 2001). Therefore, to understand theanti-proliferative mechanism of midostaurin, human prostate adenocarcinomacells cultured under in vitro conditionswere used as the model system in our study. Midostaurin decreasedthe viability of PC-3 cells as measured by MTT assays, suggestingthat it exhibits cytotoxic effects on androgen-independent prostatecancer cells. Our results are in agreement with an earlier study reportedby Kawai et al.(2015), where midostaurin was found to preferentially suppress proliferationof triple-negative breast cancer cells.

PKC is a family of serine/threoninekinases that are important constituents of signaling pathways thatcontrol mitogenesis, differentiation, survival, adhesion, motility,and apoptosis, among others. At present, the PKC family consists of13 isoforms, and the distribution of different PKC isoforms showsconsiderable tissue- and cell-specificity (Griner and Kazanietz, 2007).PKC isozymes are commonly dysregulated in cancer, and among the differentisoforms, phorbol ester-responsive novel isozymes PKC-d and PKC-ε are key mediators involvedin the regulation of prostate tumorigenesis (Aziz et al., 2007). Our study demonstratedthat PC-3 cells exhibited mere expression of apoptotic-mediated PKC-d and elevated expression of pro-survivalPKC-ε. Previous studies established that PKC-d arrests the G1-to-S phase transition of the cell cycleby controlling the phosphorylation status of retinoblastomas, andis implicated as a negative growth regulator (Xiao et al., 2009). Emerging evidencesuggests that PKC-ε is a transforming oncogene, as PKC-εwas found to contribute to tumorigenesis in prostate cancer throughits stimulatory effects on proliferation, anchorage-independent growth,transition to androgen independence, and invasiveness, as well asits inhibitory effects on cell death (Basu and Sivaprasad, 2007, Meshki et al., 2010). Our resultsshowed that midostaurin significantly induced mRNA expression of theapoptotic-mediated PKC-d isoform,and eventually inhibited expression of the pro-survival PKC-εisoform, confirming the anti-proliferative roles of midostaurin.

PKCs transmit signals to the nucleusvia one or more mitogen-activated protein kinase (MAPK) cascades,which include Raf-1, MEKs, and ERKs (da Rocha et al., 2002). Activated ERKs can activateFos and Jun (AP-1 transcription factors) enabling the expression oftarget genes that encode enzymes required for key metabolic functions,such as cell proliferation and invasion (Seger and Krebs, 1995). However,expression patterns of AP-1 factors during PMA-induced proliferationin hormone-refractory prostate cancer cells and the role of midostaurinin the suppression of AP-1 factors have not been addressed. Our previousstudy reported the expression of the complete set of AP-1 factors(c-Jun, JunB, JunD, c-Fos, Fra-1, Fra-2, and Fos-B) in both LNCaPand PC-3 prostate cancer cells (Kavyaet al., 2017). In the present study, the effect of midostaurinon PMA-induced Jun/Fos mRNA transcripts was demonstrated. Althoughall Jun and Fos transcripts were expressed, only c-Jun, c-Fos, andFra-1 expression was induced by PMA in PC-3 cells. Treatment of PC-3cells with midostaurin significantly inhibited PMA-stimulated c-Jun,c-Fos, and Fra-1 mRNA transcripts. The results suggest that c-Jun,c-Fos, and Fra-1 AP-1 factors are involved in proliferation and thatmidostaurin possesses anti-proliferative activity in hormone-refractoryprostate cancer cells; therefore, further investigation is needed.AP-1 transcription factors regulate downstream genes, such as p53,p21, cyclin D1, and Bcl-2, that are involved in the cell cycle, suggestingimportant regulatory roles of AP-1 in cell proliferation (Shaulianand Karin, 2001). Furthermore, our study showed that midostaurin inducedmRNA expression of tumor suppressor p53 and p21^cip1/waf1, death receptor TNF-α, pro-apoptotic Bax, and caspase-8, andeventually inhibited the expression of growth regulator cyclin D1and anti-apoptotic Bcl-2. Additionally, western blot analysis confirmedthe decreased protein expression of Bcl-2 after midostaurin treatment,indicating the anti-proliferative and anti-cancerous effects of midostaurinon aggressive prostate cancer cells, PC-3.

The present study provided evidencethat midostaurin suppresses tumor growth or induces apoptosis in PC-3cells via modulation of the novel PKC enzymes, PKCε (a pro-survivalkinase) and PKCd (an apoptotickinase), as well as regulation of PMA-altered AP-1 transcription factors(c-Jun, c-Fos, Fra-1, and JunB), that in turn regulate secretion ofAP-1 targeted cell cycle regulators (cyclin D1, p53, p21, anti-apoptoticBcl-2, and death receptor TNF-α). Thus, pharmacological targetingof PKC and AP-1 transcription factors may possess therapeutic potentialfor hormone-refractory prostate cancer. Such approaches may furtherrefine our understanding of the biology and biochemistry of castration-resistantprostate cancer, enabling us to develop new therapeutic opportunitiesagainst this disease.

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