Attenuation of Apoptosis by Chromogranin A-Induced Akt and Survivin ...

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Endocrinology 148(9):4489 – 4499 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1748

Attenuation of Apoptosis by Chromogranin A-Induced Akt and Survivin Pathways in Prostate Cancer Cells Junyang Gong,* JuneGoo Lee,* Horiguchi Akio, Peter N. Schlegel, and Ruoqian Shen Department of Urology, Weill Medical College, Cornell University, New York, New York 10021 Multiple studies indicate that neuroendocrine (NE) differentiation in prostate cancer (PC) contributes to androgen-independent progression. Levels of chromogranin A (CgA), which is produced by NE cells, are increased in advanced PC. However, the mechanism by which high levels of circulating CgA contribute to PC progression is unknown. To examine the effects of CgA on PC cells, we first performed proliferation assays in the presence of recombinant CgA (rCgA) in LNCaP and C4 –2 PC cells, and found that rCgA increased cell proliferation in a dose and time-dependent manner. NE differentiated PC cells, also overexpress the antiapoptosis protein survivin. Therefore, we examined survivin expression in the presence of CgA in PC cells. Western blot analysis showed that survivin was significantly increased by rCgA and inhibited by an anti-CgA antibody in both LNCaP and C4 –2 cells. Survivin

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ROSTATE CANCER (PC) is currently the second-leading cause of cancer-related death in men in the United States (1). Understanding the molecular mechanism underlying the development of PC progression is critical for developing novel therapies. Recently, neuroendocrine (NE) differentiation has been suggested as one mechanism contributing to hormone refractory PC progression. NE cells have been found in most prostatic adenocarcinomas. Although several lines of evidence suggested that NE cells may induce proliferation of adjacent cells by paracrine stimulation, the role of NE cells in the progression to androgenindependent growth is still unclear (2–5). Elevated circulating level of chromogranin A (CgA) has been detected after androgen deprivation therapy. However, the single expression of CgA is not the only requisite for the determination of NE-differentiated cells because there exists a regulated secretory pathway in NE cells, suggesting that CgA may, together with other neuropeptides, contribute to PC progression in a hormone starvation condition (6 –12). Although several studies focus on expression of CgA in clinical samples of PC, little data are published on function and biological effect of CgA in PC. First Published Online June 21, 2007 * J.G. and J.L. contributed equally to the work. Abbreviations: AA, Amino acid; CgA, chromogranin A; CS, charcoalstripped serum; FBS, fetal bovine serum; GFX, GF109203X; LNCaP; LY, LY294002; MEK, MAPK kinase; MTS, 3-(4,5-dimethyltiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; NE, neuroendocrine; NT, no treatment; PC, prostate cancer; PKC, protein kinase C; rCgA, recombinant chromogranin A; siRNA, small interfering RNA; TBS-T, Tris-buffered saline Tween; Wort, Wortmannin. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

expression is believed to be regulated by PI3K/Akt pathway. We next assessed the phosphorylation status of Akt and found that Akt phosphorylation was increased by treatment with rCgA. To determine whether Akt phosphorylation is necessary for rCgA-induced survivin expression, we examined the effects of Akt, MAPK kinase, and protein kinase C inhibitors on CgA-induced survivin expression, and found that survivin expression was reduced in the presence of Akt inhibitors, but not MAPK kinase or protein kinase C inhibitors. Furthermore, in the presence of an Akt inhibitor or small interfering RNA of survivin, CgA-enhanced proliferation of C4 –2 and LNCaP cells significantly decreased. Together, our results demonstrate that CgA can increase PC cell survival through Aktmediated survivin up-regulation. (Endocrinology 148: 4489 – 4499, 2007)

CgA is the prototype of granins and belongs to the chromogranin-secretogranin family, a unique group of acidic, soluble secretory proteins. It was discovered in the 1960s, as the major secreted protein of the adrenal medulla, and a major constituent of chromaffin and splenic nerve secretory granules. CgA, consisting of 439 amino acids, is a preprotein as precursors for biologically active peptides, including pancreastatin, vasostatin I and II, and catestatin. Although the sequence of human CgA is known, the function of which is not fully elucidated due to difficulty in purifying these proteins (13–15). Several other putative functions have been ascribed to the chromogranins, such as controlling the intracellular Ca2⫹ concentration, and some yet-undefined function in the nucleus (14). CgA is also reported as an “on/off” switch that is sufficient to drive dense-core secretory granule biogenesis and hormone sequestration in endocrine cells. Down-regulation of CgA expression in the NE cell line PC-12 by antisense RNAs led to profound loss of dense-core secretory granules, impairment of regulated secretion of a transfected prohormone, and reduction of secretory granule proteins (16). CgA stimulates production of nitric oxide and TNF-␣ by microbial action (13–15). It has also been reported that CgA is expressed in PC-3, Du-145, and LNCaP cells, but not in fibroblasts, bladder, or renal cancer cells. Inhibition of PC cell growth and increased apoptosis were reported in PC-3, LNCaP, and Du145 cells in the presence of an antibody to CgA, suggesting that inhibition of CgA may somehow induce apoptosis (17). Recent studies also suggest that NE cells expressing CgA also overexpress the antiapoptosis protein survivin (18), implying that a link may exist between CgA and survivin expressions. Survivin has been cloned and characterized as an important member of the inhibitor of apoptosis family (19). Survivin is

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not normally expressed in the secretory epithelial cells of the prostate but is strongly expressed in PC cells (20). IGF receptor-1 signaling mediates resistance to antiandrogen therapy in PC in part via survivin up-regulation (21). It has been demonstrated that conventional antisense or small interfering RNA (siRNA) of survivin facilitated cancer cell apoptosis and sensitized cells to anticancer agents (22). In the present study, we investigated the regulatory role of the neuropeptide CgA on the downstream effecter-apoptosis pathway in PC cells. We report that CgA induces Akt phosphorylation leading to up-regulation of survivin expression, and attenuation of PC cell apoptosis. Materials and Methods Reagents and antibodies The recombinant CgA (rCgA) was purchased from Spring Bioscience (Fremont, CA). The antibodies used for Western blot analysis include: anti-phospho-Akt, anti-total Akt from Cell Signaling (Beverly, MA); anti-bcl-2 and anti-CgA from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-survivin from Novus (Littleton, CO); and anti-␤-actin from Chemicon Int. (Temecula, CA). Akt inhibitor Wortmannin (Wort), Akt/PI-3K inhibitor, LY294002 (LY), the MAPK [or Erk kinase (MAPK kinase) (MEK)] inhibitor PD 98059, and protein kinase C (PKC) inhibitor GF109203X (GFX) were purchased from Calbiochem Inc. (La Jolla, CA).

Cell culture LNCaP and C4 –2 cells (a kind gift from Dr. W. D. Heston, Cleveland Clinic, Cleveland, OH) were maintained in RPMI 1640 supplemented with 2 mm glutamine, 1% nonessential amino acids, 100 U/ml streptomycin and penicillin, and 10% fetal bovine serum (FBS). Medium was replaced with MEM containing 5% charcoal-stripped serum (CS) 24 h before various treatments. Afterwards, cells were washed with PBS three times and harvested. Cell pellets were frozen at ⫺80 C for future use.

MTS cell proliferation assay The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay was performed with the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit (Promega Corp., Madison, WI) according to the manufacturer’s instructions. Briefly, LNCaP or C4 –2 cells were plated onto 96-well plates with a density of 5 ⫻ 103 per well. The following day, medium was replaced with MEM containing 5% CS. After 24 h, cells were treated with various reagents for a certain time as indicated. The medium was then removed, and culture medium containing MTS and phenazine methosulfate solution were added. Sixty minutes later, the absorbance at 490 nm was measured with a Quant ELISA reader (BIO-TEK Instruments, Inc., Madison, WI). The data were expressed as mean (n ⫽ 4) ⫾ sem. Every MTS assay experiment was repeated at least twice with similar results. The statistical analysis was performed by a one-way ANOVA, followed by Newman-Keuls post hoc analysis.

Cell viability determined by cell counting LNCaP cells were plated into a 96-well plate with 10,000 cells per well with MEM containing 5% CS. On the following day, cells were treated with various concentrations of rCgA. After incubation for an experimental designed period, medium was removed, and 0.25% TrypsinEDTA (Chemicon International) (40 ␮l) was added. After incubation for 2 min at 37 C, trypsinized cells were mixed with the same volume of 0.4% Trypan blue solution (Sigma-Aldrich, St. Louis, MO) and pipetted several times to make a unified suspension. The cell suspension was then transferred onto a hematocytometer counting chamber (Hausser Scientific, Horsham, PA) and counted under a microscope at ⫻100 magnification following the manufacturer’s suggestion. Based on the principal that live cells can exclude dye actively, live cells (clear) are discriminated from dead cells (blue). Briefly, we counted live cells in the four squares of two chambers, and calculated cells per milliliter (23). Relative cell

Gong et al. • CgA, Akt Signaling, Survivin Expression, and PC

viability was calculated by taking control [no treatment (NT)] as 100. The data were expressed as mean ⫾ sem. The cell counting assay experiment was repeated at least three times. The statistical analysis was performed by a one-way ANOVA, followed by Newman-Keuls post hoc analysis.

Apoptosis assay Apoptosis assay was performed using the DeadEnd Colorimetric TUNEL System (Promega Corp.) to detect early apoptotic cells. Briefly, cells were evenly distributed in Lab-Tek chamber slides (Nalgene Nunc Int., Naperville, IL) and treated with various reagents for a designed period as indicated. Cells were washed twice with cold PBS and fixed in 4% paraformaldehyde for 25 min, and then incubated with Triton X-100 (0.2% in PBS) for 5 min at room temperature. Slides were then labeled with the TUNEL reaction mixture according to the instructions of the manufacturer (Promega Corp.). Color development was performed with diaminobenzidine. In each staining procedure, two negative controls (omitting either the terminal deoxynucleotidyl transferase enzyme or biotinylated substrate) were used. Positive controls were the treatment with DNase-I, which caused DNA fragmentation and positive staining. Positively stained cells were enumerated using a light microscope at ⫻100 – 400 magnification. Each data represented the average cell number in six independent microscopic fields of a single experiment. All assays were performed at least three times. The statistical analysis was performed by a one-way ANOVA, followed by Newman-Keuls post hoc analysis.

Western blot analysis Western blot analysis was performed as described previously (24). Cells were lysed in radioimmunoprecipitation buffer [10 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1.2% aprotinin, 5 ␮m leupeptin, 4 ␮m antipain, 1 mm phenylmethylsulfonyl fluoride, and 0.1 mm Na3VO4]. Twenty to 50 ␮g of proteins for each sample were suspended in sample buffer, resolved by SDS-PAGE, and transferred onto nitrocellulose. The membrane was blocked with 25 ml 5% nonfat milk dissolved in Tris-buffered saline Tween (TBS-T) for 1 h, followed by incubation in primary antibody (diluted with TBS-T at a certain concentration as recommended by the manufacturer) for 1 h at room temperature or overnight at 4 C. It was washed with TBS-T three times. The membrane was then incubated in TBS-T containing secondary antibody with a certain dilution for 1 h at room temperature. After washing three times with TBS-T, the membrane was detected with enhanced chemiluminescence and exposed to x-ray film. All Western blot analyses were performed at least twice with three independent preparations of cell lysates with similar results. Results are representative of these independent experiments. Films were scanned using an HP scanner (Hewlett-Packard Co., Palo Alto, CA) and subjected to densitometric analysis using ImageJ (National Institutes of Health, Bethesda, MD).

Real-time PCR Total RNAs from cell samples were extracted using the QIAGEN RNeasy Mini kit (Valencia, CA), followed by DNase-I treatment to prevent amplification of pseudogenes. One microgram of RNA was reverse transcribed into cDNA using random primer and 125 U MultiScribe-reverse transcriptase (TaqMan Reverse Transcription Reagents, Roche, Branchburg, NJ). For real-time PCR, a 5⬘ primer (5⬘-GGCCCAGTGTTTCTTCTGCTT-3⬘) and a 3⬘ primer (5⬘-GCAACCGGACGAATGCTTT-3⬘) for survivin, and a 5⬘ primer (5⬘-TTGCCGACAGGATGCAGAA-3⬘), a 3⬘ primer (5⬘-GCCGATCCACACGGAGTACT-3⬘) for ␤-actin, were prepared from IDT Inc. (Coralville, IA), using the primer designing program Primer Sequence version 1.0 (Applied Biosystems, Foster City, CA), based on the cDNA sequences of human survivin and ␤-actin genes. To avoid amplification of genomic DNA, the forward and reverse primers were designed to represent different exons. Quantitative real-time PCRs were performed with the ABI 7000 system and using SYBR Green PCR Master Mix Kit (Applied Biosystems, Warrington, UK). Thermocycling was performed in a final volume of 20 ␮l, containing a 2 ␮l cDNA sample, 3.5 mm MgCl2, 0.1 ␮m primers, and 10 ␮l SYBR green I Master mix. After a 10-min initial denaturation at 95 C, the 50 cycles run consists of a 15-sec denaturation step at 95 C, and

Gong et al. • CgA, Akt Signaling, Survivin Expression, and PC

an annealing and extension step at 60 C for 1 min. The mean of three repeated PCR values was used in the statistical analysis. Results were normalized to ␤-actin by dividing the individual RT-PCR values by a mean of three repeated ␤-actin test values of the representative samples to reduce variability between RNA amounts introduced into the RTPCRs. To distinguish the specific PCR products from nonspecific products and primer dimers, a dissociation curve analysis was performed. In addition, samples were analyzed by agarose gel electrophoresis to verify that the amplified products possessed proper sizes. Experiments were performed at least three times. Data are presented as mean percent of control ⫾ sem. Statistical analysis was performed by a one-way ANOVA, followed by Newman-Keuls post hoc analysis.

Transfection and application of siRNAs For the siRNA transfection, PC cells were plated on six-well plates in RPMI 1640 containing 5% CS. After 24 h, cells were transfected with 10 ␮l 0.02 mm survivin siRNA (sc-29499; Santa Cruz Biotechnology, Inc.) or siStable Non-Targeting siRNA (Dharmacon, Lafayette, CO) (negative control) using 10 ␮l lipofectamine 2000 per well according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA). Each transfection experiment was performed in triplicate on at least three separate occasions. Results represent an average of three independent experiments, and data are presented as mean ⫾ sem. Statistical analysis of cell proliferation was performed by a one-way ANOVA, followed by Newman-Keuls post hoc analysis.

Results The effect of CgA on PC cell growth

Although several lines of evidence indicate that CgA levels increase during the process of androgen withdrawal in PC (6 –13), the physiological significance of the elevated level of CgA in PC is unclear. Yu et al. (17) reported that apoptotic activity in PC-3 cells treated with a CgA antibody for 24 h was 3.2-fold higher than that in control cells. Therefore, we examined the effect of CgA on proliferation in LNCaP and C4 –2 (25–27) cells. Kadmon et al. (28) measured CgA in the serum of patients with PC and reported that the normal plasma CgA level was in a range of 16 – 86 ng/ml. In patients with stage D2 PC, the plasma CgA level was 142 ⫾ 156 ng/ml. Jongsma et al. (29) measured plasma CgA level in the PC-310 human PC model during castration. The plasma CgA level increased to 3960 ng/ml (47 d after castration). The concentrations of rCgA (1–2 ␮g/ml) we selected are within the range in this mouse model and human samples. BSA (1 ␮g/ml) was used for treatment of the negative control because it is supposed to not induce cell proliferation (30 –32). Cell viability assays revealed that increased concentrations of a rCgA representing a C-terminal portion (from 260 – 439 amino acids, AAs) of the full-length protein resulted in increased cell viabilities in LNCaP cells: a 110% increase 48 h after treatment of 1 ␮g/ml rCgA, and 48% and 62% with 1 and 2 ␮g/ml rCgA for 24 h (P ⬍ 0.01), respectively (Fig. 1, A and B). In addition, we also measured the cell proliferation in C4 –2 cells in the presence of rCgA (Fig. 1, C and D). Increased cell proliferation of 29% and 42% at 1.0 and 2.0 ␮g/ml, respectively, compared with that of the negative control (BSA) C4 –2 cells (P ⬍ 0.01) was observed (Fig. 1C). The time course of rCgA (1.0 ␮g/ml) treatment demonstrated a 30% increase of cell proliferation 48 h after addition of rCgA compared with that BSA (1 ␮g/ml) treatment (Fig. 1D). Together, the results indicate that rCgA can promote proliferation of both LNCaP and C4 –2 cells.

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Expression of Survivin is augmented in the presence of CgA

To elucidate the mechanism underlying CgA-augmented PC cell proliferation, we examined the expression of genes associated with apoptosis pathway. We next examined whether the rCgA stimulates expression of survivin, a member of inhibitors of apoptosis, in LNCaP cells. Western blot analysis revealed that the expression of survivin was induced 12 h after the addition of the rCgA (Fig. 2A). The induction of survivin is dose dependent with the minimal concentration necessary for survivin induction at 1 ␮g/ml (Fig. 2B). Real-time PCR using primers specific to survivin showed that survivin mRNA levels were induced in a time- and dose-dependent manner similar to that of protein levels (Fig. 2, C and D). The levels of survivin mRNA reached 2.7-fold at 6 h after the addition of the rCgA, which corresponded to the levels of survivin protein of approximately 3.0-fold at 12 h after the addition of rCgA, suggesting that the induction of survivin by rCgA is transcriptional. We next tested the effects of an antibody against the rCgA on CgA-mediated survivin expression. Western blot analysis showed that treatment with CgA antibody and rCgA in both LNCaP and C4 –2 cells reduced survivin protein levels gradually at 6 and 12 h (Fig. 3A). However, the levels of survivin expression were recovered 36 h after treatment of CgA antibody in LNCaP cells (Fig. 3B). At first we thought it was probably due to the degradation of the antibody after a longer incubation. To clarify whether the recovered levels of survivin resulted from antibody degradation, we conducted the experiments using medium containing fresh CgA antibodies to treat the cells every 24 h. The similar results were observed after 48-h treatment of a CgA antibody (Fig. 3C), suggesting that the recovered levels of survivin after long-term incubation did not occur from degradation of CgA antibodies (see Discussion). In addition, we also demonstrated that down-regulation of survivin by a CgA antibody was blocked by combined incubation with rCgA. These data demonstrate that CgA up-regulates mRNA and protein levels of survivin, and that elimination of endogenous CgA by a CgA antibody reduces survivin expression in PC cells. Akt is phosphorylated in the presence of CgA

It has been reported that several pro-survival factors upregulate survivin expression via the PI3K/Akt signaling pathway in lung cancer cells (22), and we have also reported previously that the neuropeptides bombesin and ET-1 induce phosphorylation of Akt in PC cells (28). These findings led to investigating if CgA induces the Akt signaling cascade. To examine whether CgA also activates Akt signaling, we performed Western blot analysis to determine the phosphorylation status of Akt in the presence of rCgA. As shown in Fig. 4A, treatment of LNCaP cells with rCgA resulted in a marked increase in Akt phosphorylation. This effect is dose dependent with 1 ␮g/ml rCgA inducing the highest level of Akt phosphorylation compared with control (Fig. 4B, lanes 1 and 4). These data show that Akt phosphorylation is induced in the presence of CgA in PC cells.

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Gong et al. • CgA, Akt Signaling, Survivin Expression, and PC

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FIG. 1. rCgA enhanced proliferation of PC cells. A, Dose response of LNCaP cells in the presence of rCgA. Cell viabilities of LNCaP cells cultured in MEM containing 5% CS were measured when treated with BSA (1 ␮g/ml) (NT) or various concentrations of rCgA (0.1–2 ␮g/ml) by cell counting 24 h after treatment. Medium containing 10% FBS is used as the positive control (FBS). B, Time course of rCgA at a concentration of 1.0 ␮g/ml. LNCaP cells were treated with 1 ␮g/ml BSA (NT) or 1 ␮g/ml rCgA for 4, 12, 24, 48, and 72 h. After treatments, cells were harvested and subjected to cell counting (see Materials and Methods). C and D, Dose response and time course of cell proliferation of C4 –2 cells by MTS assays. Cell proliferation of C4 –2 cells treated with rCgA at various dose 24 h after treatment or various time points with treatment of 1 ␮g/ml rCgA (as described in A and B) were measured by MTS assays. Y-axis shows absorbance at wavelength 490 nm. Relative cell proliferation was calculated by taking control (NT) as 100 (A and B). **, P ⬍ 0.01 represents statistical significance of each group treated with various concentrations of CgA compared with NT (BSA) (A and C) or of each group treated with CgA to the corresponding group treated with BSA (NT) (B and D). All experiments were repeated at least twice with similar results. All data represent the average of one experiment and are presented as mean ⫾ SEM.

CgA-induced survivin expression is mediated by Akt signaling

To determine whether Akt phosphorylation was necessary for CgA-mediated survivin expression, we next assessed the effect of the Akt inhibitor Wort on CgA-induced survivin expression. As shown in Fig. 5A, LNCaP cells cultured in Wort (100 nm) containing media for 20 min resulted in decreased CgA-induced Akt phosphorylation (Fig. 5A, lane 3). In contrast, the inhibition of other kinases such as MEK and PKC, using PD (PD98059) (MEK) or GFX (PKC), did not alter CgA-induced Akt phosphorylation (Fig. 5A, lanes 4 and 5). Correspondingly, CgA-induced expressions of survivin

were reduced in the presence of Wort (Fig. 5A, lane 3), but not PD or GFX (Fig. 5A, lanes 4 and 5). No differences in the expression of bcl-2 were detected (Fig. 5A, panel bcl-2). To confirm that Akt signaling is responsible for CgA-mediated induction of survivin, we tested another Akt/PI-3K inhibitor, LY. Like Wort, LY also eliminated CgA-induced activation of Akt (Fig. 5, B and C). LY suppressed the expression of survivin, but not bcl-2, in LNCaP cells as well (Fig. 5, D and E). Similarly, the decreased CgA-induced Akt phosphorylation and survivin expression were also observed in C4 –2 cells in the presence of Wort or LY, but not GFX (Fig. 5, F and G). Together, these data suggest that CgA-induced survivin ex-

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FIG. 2. Up-regulation of survivin on mRNA and protein levels in the presence of rCgA. A and B, Western blot analysis of survivin induction in the presence of rCgA. A, Time course of survivin induction. LNCaP cells were treated with 1.0 ␮g/ml rCgA at various times (1, 6, 12, 24, and 36 h). Blots were probed with survivin and actin antibodies. B, Dose response of survivin induction. LNCaP cells were treated for 12 h at various concentrations of rCgA (0, 0.1, 0.5, 1.0, and 2.0 ␮g/ml). Blots were probed with survivin and actin antibodies. C and D, Real-time PCR of survivin. mRNAs prepared from LNCaP cells with the same treatments as described in A and B. C, Time course of survivin mRNA induction. D, Dose response of survivin mRNA induction. The treatments of 10% FBS in A, B, and D were used as a positive control (FBS). Relative cell viability was calculated by taking control (0 h) as 100. All experiments were repeated at least twice with similar results. All data represent the average of one experiment and are presented as mean ⫾ SEM. **, P ⬍ 0.01 represents statistical significance compared with the value of 0 h (C) or 0 ␮g/ml rCgA (D).

pression is dependent on Akt activation, but not MEK or PKC signaling. The expression of survivin, but not bcl-2, is induced by CgA in PC cells. The proliferation-promoting effect of CgA is dependent on Akt-mediated expression of survivin in PC cells

We next investigated whether CgA-mediated Akt activation and induction of survivin expression were protective against apoptosis in PC cells. As shown in Fig. 6A, MTS assays revealed that treatment of the rCgA resulted in a significant increase in cell viability compared with that of the negative control (BSA, 1 ␮g/ml) in LNCaP cells (Fig. 6A, lanes NT and CgA). Pretreatment with Wort (100 nm) inhibited the effect of the rCgA, whereas pretreatment with PD (100 ␮m) or GFX (100 nm) had little effect (Fig. 6A, lanes Wort ⫹ CgA, PD ⫹ CgA, and GFX ⫹ CgA). Treatment with these inhibitors alone had no effect on apoptosis (Fig. 6A, lanes Wort, PD, and GFX). Next we assessed the effect of CgA on the paclitaxel-induced apoptosis. TUNEL assays revealed that treatment with 100 nm paclitaxel resulted in a 4.5-fold (P ⬍ 0.01) increase in TUNEL-stained cells compared with untreated control cells in LNCaP cells (Fig. 6B, lanes Pac and NT). Preincubation with rCgA attenuated paclitaxelinduced apoptosis in LNCaP cells (Fig. 6B, lanes Pac and Pac ⫹

CgA; P ⬍ 0.01). Pretreatment with Wort significantly inhibited the survival effects of rCgA (compared with Pac ⫹ CgA; P ⬍ 0.01), whereas pretreatments with PD or GFX did not (Fig. 6B, lanes Pac ⫹ CgA ⫹ Wort, Pac ⫹ CgA ⫹ PD, and Pac ⫹ CgA ⫹ GFX). To confirm whether the cell-survival effects of CgA are mediated by survivin, we assessed the effects of CgA-specific antibody and siRNA against survivin in LNCaP and C4 –2 cells. As shown in Fig. 7, A and B, the anti-CgA antibody blocked the cell-survival effect by rCgA in LNCaP (Fig. 7A) and C4 –2 (Fig. 7B) cells, as previously reported (17). Transfection of survivin siRNA suppressed the expression of survivin in LNCaP (Fig. 7C) and C4 –2 cells (Fig. 7D), and abrogated the cell-survival effect of rCgA in LNCaP and C4 –2 cells (Fig. 7, E and F). Together, these data suggest that CgA attenuates PC cell apoptosis predominantly through Akt phosphorylation, which can modulate survivin up-regulation. Discussion

Previously, we have studied the involvement of the neuropeptides, bombesin, and ET-1 in PC progression, and shown that these neuropeptides induced focal adhesion kinase phosphorylation, ligand-independent phosphorylation of the IGF-I receptor and Akt, cSrc kinase activation, and

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FIG. 3. Expression of survivin was blocked by anti-CgA antibody. A, Western blot analysis showing that reduced levels of survivin in the presence of an anti-CgA antibody (1 ␮g/ml) in PC cells. LNCaP cells cultured in medium containing 5% CS were treated with an anti-CgA antibody for 0, 6, and 12 h. After washed by PBS, cell lysates were prepared and resolved on SDS-PAGE. Blots were probed with antisurvivin and anti-actin antibodies. B, Extended incubation of LNCaP cells with an anti-CgA antibody. Cell lysates of LNCaP treated with an anti-CgA antibody (1 ␮g/ml) for 0, 6, 12, 24, 36, and 48 h were subjected to Western blot analysis with anti-survivin and anti-actin antibodies. C, Extended incubation of LNCaP cells with an anti-CgA antibody with or without CgA. Cell lysates of LNCaP treated with mouse IgG (NT), an anti-CgA antibody (1 ␮g/ml) with (CgA Ab) or without CgA (CgA Ab ⫹ CgA) for 0, 24, and 48 h were subjected to Western blot analysis with anti-survivin and anti-actin antibodies. Experiments were performed at least twice with three independent preparations of cell lysates with similar results. Results are representative of these independent experiments.

rapid degradation of PKC␦ in PC cells (24, 33, 34). We also investigated the effect of these neuropeptides on androgen receptor-mediated transcription (35). Here, we report our study on another important neuropeptide, CgA. Although it has long been proposed that CgA is an excellent marker in detecting NE differentiation in PC, the link between CgA and the cellular apoptotic machinery had not been elucidated. Our present study aimed to elucidate the functional significance of the high level of CgA detected in PC. To examine the function of CgA in PC cells at castrate level of androgen, we performed experiments in LNCaP (an androgen-sensitive cell line) or C4 –2 (an androgen-independent derivative of LNCaP) cells cultured in CS. We demonstrate that CgA possesses direct biological functions in stimulation of specific survival signals that counteract apoptosis in PC cells. This is

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FIG. 4. CgA activates Akt signaling in LNCaP cells. A, Time course of Akt phosphorylation in the presence of rCgA. LNCaP cells treated with rCgA (1.0 ␮g/ml) at 0, 10 min, 60 min, 12 h, and 24 h were lysed and subjected to Western blot analysis. Blots were probed with antiphospho-Akt (p-Akt) and anti-Akt (Akt) antibodies. B, Dose response of Akt phosphorylation in the presence of CgA. LNCaP cells were treated with various concentrations of rCgA (0, 0.1, 0.5, 1, and 2 ␮g/ml). Western blots were probed with anti-phospho-Akt and antiAkt antibodies. The treatment of 10% FBS in B was used as positive control (FBS). Experiments were performed for at least twice with three independent preparations of cell lysates with similar results. Results are representative of these independent experiments.

the first demonstration that a piece of CgA may possess a direct effect on PC cell proliferation, suggesting that a link might exist among NE differentiation, signaling transduction, and PC cell proliferation, which is probably one of the mechanisms underlying androgen-independent PC progression. These results also show that resistance to apoptosis by CgA can be abolished by an Akt inhibitor, which targets survivin a critical inhibitor of apoptosis in PC cells, suggesting CgA may be a potential target in the therapy of androgenindependent PC. Yu et al. (17) studied the effect of CgA using an antibody against a rCgA, covering a region from 210 AA to C terminal (Dako, Inc., Carpinteria, CA). Based on their studies, we assessed the effect of an anti-CgA antibody against a rCgA covering a similar region, 260- to 439-AA of CgA (Santa Cruz Biotechnology, Inc.), and revealed the growth inhibition of PC cells by antibody against this portion of CgA (data not shown), suggesting that the 260- to 439-AA region may consist of effective components to stimulate PC cell proliferation. To test this hypothesis, we performed cell proliferation assays and verified that the rCgA (260 – 439 AA) did attenuate the apoptosis of PC cells. The 260- to 439-AA fragment of CgA, with a molecular mass of around 18 kDa, might be a naturally processed product. Jongsma et al. (29) studied different profiles of NE cell differentiation in a PC-310 human PC model during long-term androgen deprivation and pointed out that clear processing of CgA was evidenced by smaller fragments at 49, 30, and 18 kDa, suggesting that CgA was subjected to specific processing under a castrate condition. However, the identity of these degraded fragments remained to be characterized. A piece of C-terminal half of

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+ 1 ug/ml CgA GFX - Wort LY GFX 0.74 11.12 1.42 2.01 16.41 1.12 2.75 0.90 1.06 2.85 0.99 0.94 0.92 1.04 0.96

* Akt was used as an internal control ** actin was used as an internal control

FIG. 5. Up-regulation of survivin by CgA was mediated by Akt/PI3 K signaling. A, LNCaP cells were treated without or with rCgA (1.0 ␮g/ml) alone, or together with various kinase inhibitors: Akt inhibitor Wort (100 ␮M), MAPK inhibitor PD 98059 (PD) (100 nM), and PKC inhibitor GFX (4 ␮M). Western blot analysis was performed, and blots were probed with antibodies against phospho-Akt (p-Akt), Akt, survivin, bcl-2, and actin. B and C, Time-dependent suppression (0, 10, and 60 min) of CgA-induced phosphorylation by Akt/PI-3K inhibitors Wort and LY (20 ␮M) in LNCaP cells. D, Suppression of Akt phosphorylation down-regulates expression of survivin, but not bcl-2. LNCaP cells were pretreated without or with Akt/PI-3K inhibitors Wort and LY and PKC inhibitor GFX, then treated with CgA for 24 h. Western blots were probed by antibodies of p-Akt, Akt, survivin, bcl-2, and actin. E, The densitometric analysis (ImageJ software) for each protein expressed as factor of control after normalizing the corresponding Akt (*) or actin (**). F, C4 –2 cells were treated without or with rCgA (1.0 ␮g/ml) alone, or together with various kinase inhibitors or various kinase inhibitors alone: Akt inhibitor Wort (100 ␮M), Akt/PI-3K inhibitor LY (20 ␮M), and PKC inhibitor GFX (100 ␮M). Western blot analysis was performed, and blots were probed with antibodies against phospho-Akt (p-Akt), Akt, survivin, bcl-2, and actin. G, The densitometric analysis (ImageJ software) for each protein expressed as factor of control after normalizing the corresponding Akt (*) or actin (**). Experiments were performed for at least twice with three independent preparations of cell lysates with similar results. Results are representative of these independent experiments.

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A LNCaP

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B LNCaP

FIG. 6. Enhanced viability of PC cells by CgA was mediated by PI3K/Akt signaling. A, MTS assays of LNCaP cells in the presence of inhibitors of kinase inhibitors. LNCaP cells cultured in MEM (phenol red free) containing 5% CS were treated with BSA (NT), rCgA alone (CgA), or rCgA plus various kinase inhibitors: Wort ⫹ CgA, PD ⫹ CgA, and GFX ⫹ CgA, or various kinase inhibitors alone. Twenty-four hours later, cells were subjected to MTS analysis. FBS is the positive control showing the effect of the high level of androgen (10% FBS) on PC cell proliferation. The experiment was repeated at least three times with similar results. Relative cell viability was calculated by taking control (NT) as 100. Data represent the average of one experiment and are presented as mean ⫾ SEM. **, P ⬍ 0.01 represents statistical significance compared with NT. B, LNCaP cells cultured in MEM (phenol red free) containing 5% CS were treated with rCgA (1 ␮g/ml) alone (CgA), or together with various protein kinase inhibitors or various kinase inhibitors alone as indicated for 24 h. Cells were then treated with 100 nM paclitaxel (Pac) to induce cell apoptosis or treated with vehicle (NT). Twenty-four hours later, apoptotic cells were measured by the TUNEL staining method. Positively stained cells were numerated and calculated as the percentage of total cell number. The experiment was repeated at least three times with similar results. Data represent the average of one experiment and are presented as mean ⫾ SEM. **, P ⬍ 0.01 represents statistical significance compared with NT. P ⬍ 0.01 (a) represents statistical significance compared with paclitaxel alone (Pac). P ⬍ 0.01 (b) represents statistical significance compared with CgA and paclitaxel (Pac ⫹ CgA).

CgA had been purified from the urine of patients with carcinoid syndrome, the polyclonal antibody generated from which could not recognize the epitopes located on the Nterminal half of CgA (36). The 260- to 439-AA region of the CgA molecule covers sequences of several identified CgA fragments, including WE-14 (324 –337 AA), catestatin (344 – 358 AA), and parastatin (357– 428 AA) (37–39). Few studies examined the function of these CgA fragments, and no clear data exist in PC cells yet (40). To clarify the role of CgA on PC cells, identification of the active components in this 260to 439-AA region and examination of their effects in PC cells are warranted. Our data showed that expression of survivin, but not bcl-2, was induced by CgA in PC cells. The presence of survivin was reported in PC cells, such as PC3 and LNCaP cells (41). The increased survivin expression has been detected in treatments of cytokines, pro-survival factors, anticancer agents, or UV irradiation, suggesting that expression of survivin can be regulated by various reagents (42– 44). Although the precise mechanisms of survivin inhibition of apoptosis have not been conclusively elucidated, recent data suggest a preferential involvement in the upstream initiation of the intrinsic (i.e. mitochondrial) apoptotic pathway. Clear evidence for a role of survivin in counteracting the extrinsic, death receptormediated apoptotic response comes from the fact that survivin effectively blocks TNF-related apoptosis-inducing ligand-induced apoptosis in renal carcinoma cells and suppresses TNF-␣-induced apoptosis in endothelial cells

(45), suggesting a potential interference of survivin in the extrinsic apoptotic cascade (46). Accumulating data point to the participation of survivin in the blocking of apoptosis through its inhibitory effects on caspase-3, caspase-7, and caspase-9. The dominant-negative (T34A) survivin or survivin antisense oligonucleotides induce caspase-dependent apoptosis in glioblastoma multiform (47). Our results demonstrated that CgA antibody could suppress induction of survivin by rCgA. Nevertheless, longterm incubation of CgA antibody partially resulted in recovered levels of survivin. At first we thought it might be due to degradation of CgA antibody. Therefore, we repeated the experiments by replacing medium containing fresh CgA antibody every 24 h, whereas the similar results that after long-term incubation of CgA antibody the levels of survivin were recovered, were observed. Given that CgA antibody can increase apoptotic activity in LNCaP cells in long-term culture (19), the recovered level of survivin implies that other unknown mechanisms, aside from the regulation of survivin expression, might mediate CgA’s action as well. Our previous studies that neuropeptides could affect mRNA splicing and expression of splicing factors (48) suggested that CgA might regulate the apoptosis pathway via mRNA splicing of proapoptosis genes, such as bcl-x. Our data showed that an Akt inhibitor, but not MEK and PKC inhibitors, suppresses CgA-augmented survivin expression, suggesting that Akt function is involved in survivin regulation, which is consistent with previous investigations

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FIG. 7. CgA antibody and survivin-specific siRNA blocked rCgA augmented cell proliferation. A, Relative cell viabilities of LNCaP. LNCaP cells were incubated in 1 ␮g/ml CgA or BSA (NT) treated with anti-CgA antibody (⫹Ab) or normal mouse IgG. Twenty-four hours later, cell viability was measured by cell counting. Relative cell viability was calculated by taking control (NT) as 100. B, Cell proliferation of C4 –2. C4 –2 cells were incubated with the same treatments as described in A. Twenty-four hours later, cell proliferation was measured by MTS assays. Experiments were performed at least twice with similar results. Data represent one experiment and are presented as mean ⫾ SEM. **, P ⬍ 0.01 represents statistical significance compared with the corresponding NT. P ⬍ 0.01 (a) represents statistical significance compared with the corresponding IgG (A and B). C and D, Western blotting showing survivin expression in LNCaP (C) and C4 –2 (D) cells transfected with survivin siRNA and control siRNA. LNCaP or C4 –2 cells were transfected with survivin siRNA (surv siRNA) or nontargeting siRNA (sc siRNA). Forty-eight hours later, cells were treated with rCgA or BSA (NT). Cell lysates were subjected to Western blot analysis and probed with survivin or actin antibodies. Experiments were performed at least twice with three independent preparations of cell lysates with similar results. Results are representative of these independent experiments. E, Relative cell viabilities of LNCaP cells were measured by cell counting with the same treatment as described in C. Relative cell viability was calculated by taking control (NT) as 100. F, Relative cell proliferation of C4 –2 cells was measured by MTS assays with the same treatment as described in D. Vehicles (NT) are treatments with 1 ␮g/ml BSA. Experiments were performed at least twice with similar results. Data represent one experiment and are presented as mean ⫾ SEM. **, P ⬍ 0.01 represents statistical significance compared with the corresponding NT. P ⬍ 0.01 (a) represents statistical significance compared with the corresponding sc RNA (E and F).

(49). Akt is a key mediator of PI3K signaling located at an intersection of multiple pathways implicated in cell proliferation, survival, transcription, and metabolic processes (49). It was first identified as a positive regulator of survivin expression in endothelial cells (50 –52), and subsequent studies pinpointed Akt/survivin signaling as an antiapoptotic pathway in lung cancer, myeloma, leukemia, and PC (40, 50). Kim et al. (45) reported that inhibitor of PI3K/Akt signaling down-regulates survivin expression and enhances TNF-related apoptosis-inducing ligand-mediated apoptosis in neuroblastoma. PI-3K/Akt has also regulated angiotensin IIinduced inhibition of apoptosis in microvascular endothelial

cells through survivin/caspase-3 pathway (53). However, how PC cells regulate the apoptosis pathway via the Akt/ survivin pathway was not fully elucidated. Constitutive PI3K/Akt signaling is maintained by autocrine growth factor loops and promotes anchorage-independent cell growth (54). Our previous studies demonstrated that neuropeptides ET-1 and bombesin stimulate ligand-independent activation of the IGF-I receptor, and this activation is required for neuropeptide-induced Akt phosphorylation (24). Sato et al. (55) reported that in KU19 –20 cells, cell proliferation and survivin expression were significantly induced by IGF-I. Hopfner et al. (56) demonstrated that a novel IGF-I receptor

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tyrosine kinase inhibitor NVP-AEW541 treatment suppressed the expression of the antiapoptotic proteins bcl-2 and survivin, suggesting that CgA might regulate Akt signaling via IGF pathway. Our results of real-time PCR and Western blot analysis suggested that survivin induction by rCgA is transcriptional. It is consistent with recently published data showing that the survivin promoter is cancer specific, and the expression of survivin in cancer is largely controlled at the transcriptional level (56). Current evidence suggests a global deregulation of survivin gene expression in transformed cells that may involve activation of signal transducers and activators of transcription 3 (46). Peng et al. (57) also reported that growth factor signaling activated the PI-3K/AKT pathway, subsequently increasing the level of HIF-1␣ under normoxic conditions. HIF-1␣ then activated survivin gene transcription through direct binding to the survivin promoter. However, the mechanisms by which Akt activates these transcription factors signal transducers and activators of transcription 3 or HIF1␣ remain elusive (58 – 60). In summary, our findings suggest that CgA engagement in PC cells through Akt pathway is associated with upregulation of survivin expression and protection from apoptosis, whereas interference with Akt function using inhibitors reversed the cytoprotective effect of CgA. These findings possessed clinical significance because they implied that CgA might trigger pro-survival events in PC cells.

Gong et al. • CgA, Akt Signaling, Survivin Expression, and PC

9. 10.

11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Acknowledgments We thank Drs. David M. Nanus, Oscar Goodman, Rong Zheng, and Elizabeth Lau for helpful discussion.

23. 24.

Received December 27, 2006. Accepted June 8, 2007. Address all correspondence and requests for reprints to: Ruoqian Shen, Weill Medical College of Cornell University, Department of Urology, Room E-300, Box 23, 1300 York Avenue, New York, New York 10021. E-mail: [email protected]. This work was supported by National Institutes of Health Grants RO1 DK060908-02, RO1 CA80240, the Robert H. McCooey Memorial Cancer Research Fund, Ronald and Susan Lynch Professorship in Urologic Oncology (to J.G.), and Brady Urology Foundation of the Department of Urology. Disclosure Statement: The authors have nothing to disclose.

25. 26. 27. 28. 29.

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