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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Prev Res (Phila). Author manuscript; available in PMC 2010 March 1.
Published in final edited form as:
PMCID: PMC2652513
NIHMSID: NIHMS65627

This article has been retractedRetraction in: Cancer Prev Res (Phila). 2012 June 2; 4(11): 1945    See also: PMC Retraction Policy

Psoralidin, An Herbal Molecule Inhibits PI3K Mediated Akt Signaling In Androgen Independent Prostate Cancer (AIPC) Cells

Abstract

The protein kinase Akt plays an important role in cell proliferation and survival in many cancers, including prostate cancer. Due to its kinase activity, it serves as a molecular conduit for inhibiting apoptosis and promoting angiogenesis in most cell types. In most of the prostate tumors, Akt signaling is constitutively activated due to the deletion or mutation of the tumor suppressor PTEN, which negatively regulates PI3K through lipid phosphatase activity. Recently, we identified a natural compound, psoralidin, which inhibits Akt phosphorylation and its consequent activation in androgen independent prostate cancer cells (AIPC). Furthermore, ectopic expression of Akt renders AIPC cells resistant chemotherapy; however, psoralidin overcomes Akt-mediated resistance and induces apoptosis in AIPC cells. While dissecting the molecular events, both upstream and downstream of Akt, we found that psoralidin inhibits PI3 kinase activation and transcriptionally represses the activation of NF-κB and its target genes (Bcl-2, Survivin, and Bcl-xL, etc.), which results in the inhibition of cell viability and induction of apoptosis in PC-3 and DU-145 cells. Interestingly, psoralidin selectively targets cancer cells, without causing any toxicity to normal prostate epithelial cells. In vivo xenograft assays substantiate these in vitro findings, and show psoralidin inhibits prostate tumor growth in nude mice. Our findings are of therapeutic significance in the management of prostate cancer patients with advanced or metastatic disease, as they provide new directions for the development of a phyotochemical-based platform for prevention and treatment strategies for AIPC.

Introduction

Prostate cancer (PCa) is the second leading cause of cancer-related death in men. An estimated 186,320 new cases will be diagnosed in 2008 in the United States (1). Androgen ablation therapy (AAT) and bilateral orchidectomy have been the main therapeutic options for locally advanced and/or metastatic AIPC (2). While most patients initially respond to AAT (~30% have AAT refractory disease), a majority of these patients transit from androgen dependent prostate cancer (ADPC) to androgen independent prostate cancer (AIPC), for which there is no curative therapy (3). Most patients with AIPC show resistance to current chemotherapeutics, and even docetaxel-based combination therapy yield only a limited improvement in patient survival (4), suggesting that novel targeted therapies are much needed. Recent evidences suggest that Akt plays an important role in the conversion of ADPC to AIPC.

Accumulating evidence suggests that Akt is the focal point of a number of signaling pathways that regulate cell growth, survival, proliferation, immune activation, apoptosis (5) and tumor progression in many cancer types (6), including AIPC (7, 8). Akt is activated by the lipid kinase, phosphatidylinositol 3-kinase (PI3K) (9), which plays an important role in the pro-survival mechanism in a number of cell types and in a majority of cancers. PI3K is a heterodimeric protein composed of a catalytic subunit (p110α/β/γ/δ) and a regulatory subunit (p85α/β) that is involved in cell growth, transformation, and differentiation. Concurrent phosphatase and tensin homolog (PTEN) inactivation negatively regulates Akt activation, however PTEN is frequently mutated or deleted in many cancers including in PCa (10).

Three isoforms of Akt (Akt-1, -2, and -3) have been identified, and all three share a similar domain structure, which is widely expressed in most organs and tissues. Akt-1 and Akt-3 is also expressed to some extent in PCa cell lines and human PCa samples (11). Evidence suggests that over expression of Akt decreases the cellular levels of p27, thereby causing cell proliferation (12). Conversely, activation Akt increases expression of cyclin D1 which is an established driving force for cell cycle progression, through the phosphorylation and resultant inactivation of GSK-3β (13). Down regulation of constitutively active Akt (by PI3 kinase inhibitors, Wortmannin, or LY 294002) reverses both cell survival and resistance to chemotherapeutic agents. In a transgenic mouse model, a constitutively activated Akt has been shown to promote prostate intraepithelial neoplasia (PIN) (14). Similarly, in vitro studies in PCa suggest that over expression of Akt inhibits the apoptotic response and releases the cells from cell-cycle arrest, ultimately rendering these tumor cells resistant to current treatments (15). Phosphorylation of Akt (ser473) has been shown to correlate with higher Gleason score which is a prognostic marker for poor clinical outcome in PCa patients (16). Many studies indicate that activation of this key survival kinase plays an important role in tumor development by activating NF-κB/Bcl-2 pro-survival signal transduction pathway.

Chemoprevention as well as chemotherapeutic intervention by phytochemicals provides new dimensions for the management of AIPC. Psoralidin is one of the active ingredients (Figure 1) in Psoralea corylifolia, a herbal plant which is extensively used in traditional medicine against gynecological bleeding, vitiligo and psoriasis (17). Moreover, it possesses hepatoprotective properties (18), osteoblastic proliferation stimulating activity (19) and anti-bacterial activity (20). The molecular components of Psoralea corylifolia include coumarins and flavones, such as psoralen, isopsoralen, bavachalcone, etc. (21). Sporadic reports detail the cytotoxic effect of psoralidin on MCF-7 (breast), HT29 (colon) (22) SNU-1 and SNU-16 (stomach carcinoma) cell lines (23), with no significant effects observed in A549 (lung) and HepG2 (liver hepatoma) cell lines (22). This study provides the first evidence of the anticancer effect of psoralidin and enables mechanistic insights into antitumor action of this compound against AIPC cells. Our data suggests that psoralidin exhibits a potent anti-cancer effect on AIPC cells also, in combination psoralidin may enhance the anticancer activity of the current chemotherapeutic agents by inhibiting the PI3 kinase mediated pro-survival signaling and inducing apoptosis in AIPC cells.

Figure 1
Structure of Psoralidin

Materials and methods

Cell lines, plasmid constructs and natural compounds

PC-3, DU-145, LNCaP, 22Rv-1 and PzHPv-7 cells were purchased from American Type Culture Collection (Manassas, VA). The cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% glutamine and antibiotics. The plasmids pCB6+, pcDNA3 flag HA Akt1, and pcDNA KD-Akt were kindly given by Naoya Fujita, Japanese foundation for cancer Research (24). Psoralidin was isolated in Jurgen Rohr’s laboratory (University of Kentucky); high performance liquid chromatography (HPLC), Nuclear magnetic resonance (NMR) and elemental analysis were performed to determine the purity of the compound.

Cell viability and apoptotic assays

Cells were treated with Psoralidin (indicated concentrations) or vehicle (DMSO) for 24h to determine cell viability as described earlier (25). Also, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Annexin V-FITC staining assays were performed following treatment of PC-3, DU-145, 22Rv1, LNCAP, PC-3/Neo and PC-3/Bcl-2 cells with psoralidin as described earlier (25).

Transfection and luciferase assays

For NF-κB promoter assays and ectopic expression of Akt, PCa cells (PC-3 and DU-145 at 50-60% confluency) were transiently transfected using lipofectamine reagent (Life technologies, Carlsbad, CA) with 2.5μg of plasmid DNA. The plasmids included the pcDNA3 flag HA Akt1 vector, contains the same hemagglutinin-tagged myr-Akt or a kinase-inactive mutant of Akt pcDNA (KD-Akt) or pCB6+ (control vector). Each construct was co-transfected with NF-κB-luciferase reporter plasmid (containing two tandem NF-κB responsive sites) in the presence of pRL-TK (renilla plasmid control) to normalize transfection efficiency and promoter activation studies were performed as described earlier (26).

Binding studies for NF-κB activity

PC-3 and DU-145 cells were treated with psoralidin for different time intervals and different doses as indicated and ELISA was performed for the activity of NF-κB using universal EZ-TFA transcription factor assay kit (Millipore, Bedford, MA) as per the manufactures instructions.

IκB-α activation studies

PC-3 and DU-145 cells were treated with psoralidin for indicated time intervals and IκB-α activity was quantified using IκB-α ELISA kit (Calbiochem, Gibbstown, NJ) as per the manufacturer’s instructions. Briefly, standards and samples (cell lysates) were incubated in a streptavidin coated plate for 2 hours, then an anti-IκB-α detector antibody was used to detect IκB-α present in the sample which was then detected using a second antibody conjugated with HRP. Finally a TMB substrate was added to detect the activity in each sample and the absorbance was read at 450nm. The OD was directly proportional to IκB-α activity in the samples.

Western blot analysis

PC-3 and DU-145 cells were treated with psoralidin for varying time intervals or doses, whole cell lysates or nuclear and cytoplasmic extracts were obtained and subjected to Western blot analysis using antibodies Akt, NF-κB p50, p65, IκB-α, pIkB-α, IKKα, IKKβ, pIKKα/β, NIK, Bcl-2, , cyclin-D1, GSK-3 and Bax from Santa Cruz Biotechnology (Santa Cruz, CA), PI3 kinase p85, p110, and pAkt from Cell Signaling technology (Danvers, MA) and Bcl-xL from MBL International (Woburn, MA). β-actin and h (Santa Cruz Biotechnology) were used as the internal loading control.

Immunoprecipitation studies

PC-3 and DU-145 cells were treated with psoralidin or vehicle (DMSO) at different time intervals, cell lysates were immunoprecipitated using PI3 kinase antibody from Upstate USA, Inc. (Chicago, IL) and the immunoprecipitated samples were subjected to Western blot analysis using PI3 kinase p85 and p110 antibodies from Cell Signaling technology (Danvers, MA).

Kinase assays

PI3 kinase assay was performed in psoralidin treated and vehicle treated PC-3 and DU-145 cells. Briefly, cells were treated with psoralidin or vehicle for 24h, cell lysates were subjected to immunoprecipitation and kinsae assay was performed as per the manufacturer’s instruction from Echelon (Salt Lake City, UT) using PI (4, 5) P2 as a substrate and PI (3, 4, 5) P3 as a primary and (K-SEC1) as a secondary detector. The kinase reaction was performed in streptavidin coated detection plate and incubated with TMB solution. The reaction was stopped by adding 1 N H2SO4. Absorbance was read at 450nm on a plate reader.

Similarly, Akt kinase assays were performed using kinase assay kits from Cell Signaling technology (Danvers, MA). As per the manufacturer’s protocol, cell lysates were immunoprecipitated with immobilized Akt. The kinase assay was performed using purified proteins that were incubated with GSK-3 fusion protein in the presence of ATP for 30 minutes at 30°C. The reaction was stopped using 3× SDS sample buffer and the samples were subjected to Western blot analysis using p-GSK-3α/β (ser 21/9) antibody.

Xenograft studies

We tested the effect of psoralidin on tumors derived from PC-3 cells in 5-6 week male nude (nu/nu) mice from Harlan (Indianapolis, IN), in accordance with the University of Kentucky Animal Care and Use Committee guidelines. About 5 × 106 cells were injected subcutaneously and tumors were allowed to grow until they reached a volume of 50 mm3. At this time, the animals were randomized into three groups, with 9 mice in each group, and psoralidin (15 and 25 mg/kg body weight), dissolved in corn oil) or same amount of corn oil was administrated intratumorally five days a week for up to four weeks. Tumor volume was measured daily over the observation period.

Following completion of the study, regressed tumors were obtained and TUNEL assay was performed to determine whether the tumor regression observed in the animals was a result of the induction of apoptosis (25).

Statistical analysis

All experiments were performed three times to ascertain the reproducibility of the results. The Student’s t test was used to calculate statistical significance.

Results

Psoralidin targets Akt signaling in prostate cancer

To investigate whether psoralidin targets Akt signaling in AIPC cells, we treated PC-3 and DU-145 cells with psoralidin in dose and time-dependent manner and the activity of Akt was assessed using phosphorylation site specific antibodies. Psoralidin inhibits the phosphorylation of Akt at ser473 without altering total Akt levels in PC-3 (IC50: 60 μM) and DU-145 (IC50: 45 μM) cells (Figure 2A). We used LY294002 (20 μmol/L) as the positive control for determining the phosphorylation status of Akt at ser473 in PC-3 and DU-145 cells and treatment with LY294002 resulted a decrease in Akt phosphorylation in both the cell lines, that was comparable to psoralidin treatment (Figure 2A). Similarly, dose dependent inhibition of pAkt was seen in both PC-3 and DU-145 cells (Figure 2B). Additionally, cyclin-D1, one of the major downstream targets of Akt was also down regulated in a dose dependent fashion in both the cell lines following treatment with psoralidin (Figure 2B).

Figure 2
Psoralidin inactivates Akt kinase in PC-3 and DU-145 cells

GSK-3 is a direct substrate of Akt kinase, so we assessed the kinase activity of Akt, based on the expression of phospho-GSK-3 (Ser21/9) in AIPC cells treated with psoralidin. As expected, in PC-3 and DU-145 cells the phospho-GSK-3β expression was down regulated and total GSK-3 levels remained constant following treatment with psoralidin (Figure 2C). We subsequently investigated whether over expression of Akt could abrogate the anti-cancer effect of psoralidin on AIPC cells, for which we transiently over expressed myr-Akt, KD-Akt or pCB6+ followed by treatment with psoralidin and treated the cells were subjected to Western blot or apoptotic assays. The results revealed that KD-Akt and pCB6+ transfected AIPC cells were sensitized to psoralidin due to the inhibition of pAkt, however, the myr-Akt over expressed PC-3 cells renders resistance to psoralidin; where a complete inhibition of pAkt and a significant level of apoptosis was seen only at higher concentrations (100 μM) (Figures 2D and 2E). Similar results were observed in DU-145 cells suggesting psoralidin overcomes Akt-mediated resistance in AIPC cells (data not shown).

Effect of psoralidin on PI3K activation in AIPC cells

To investigate the role of PI3K-mediated Akt activation, we treated PC-3 and DU-145 cells with both psoralidin and pharmacological inhibitors. Pretreatment with psoralidin rapidly inhibited PI3 kinase activation in a dose (Figure 3A) and time dependent manner (Figure 3B) in both cell lines; these results are similar to the inhibition effected by the commercially available PI3K inhibitor, wortmannin (data not shown). PC-3 and DU-145 cells were treated with psoralidin and whole cell lysates were immunoprecipitated with PI3 kinase antibody and subjected to Western blot analysis. Psoralidin treatment resulted in a decrease in the expression of PI3K p110 and p85 in time dependent fashion for up to 24 hours (Figures 3C and 3D-upper panel). To confirm PI3 kinase activity levels, we used commercially available PI3 kinase kits which revealed that psoralidin inhibits PI3 kinase activation in a time-dependent (3-24h) fashion (Figures 3C and 3D-lower panel). Statistical analysis of the data in the control and psoralidin-treated population yielded a P value of 0.0001 indicating a significant difference between the groups.

Figure 3
Psoralidin down regulates PI3 kinase activity in PC-3 cells

Psoralidin Inhibits constitutive NF-κB activation in AIPC cells

We subsequently analyzed the downstream effectors of Akt signaling specifically examining the effect of psoralidin on the constitutive activation of NF-κB in PC-3 and DU-145 cells. Our results showed that psoralidin significantly down regulates (P <0.003) the binding activity of NF-κB in both the cell lines in a time and dose dependent manner when compared to the control cells (Figures 4A and 4B). Next, we determined the effect of psoralidin on nuclear translocation of the NF-κB subunits p65 and p50 in both cell lines. Immunoblot analysis with the nuclear and cytosolic extracts from psoralidin-treated cells revealed a downregulation of p65 in both the cytosolic and nuclear fractions of PC-3 and DU-145 cells. Interestingly, a complete downregulation of the p50 expression in the cytosolic fraction in PC-3 and DU-145 cells was observed; however, the p50 levels in the nuclear fraction either upregulated (PC-3) or downregulated (DU-145) as is seen in Figures 4C and 4D. Similar results of NF-κB expression and localization were observed via confocal microscopy (data not shown).

Figure 4
Psoralidin inhibits NF-κB signaling in prosatate cancer cells

To ascertain whether psoralidin regulates NF-κB at the promoter level we transiently transfected PC-3 and DU-145 cells with NF-κB-Luc followed by treatment with psoralidin. Interestingly, NF-κB promoter was down regulated by ~ 92% (P<0.0003) from 3h onwards in both PC-3 (Figure 4E) and DU-145 (data not shown) suggesting that psoralidin regulates NF-κB at the promoter level in both the cell lines. To establish Akt mediated NF-κB regulation, we over expressed myr-Akt, KD-Akt and pCB6+ in PC-3 cells and co-transfected them with NF-κB promoter luc followed by treatment with psoralidin. Over expression of myr-Akt increased the constitutive activation of NF-κB, which in turn renders PC-3 cells resistant to psoralidin treatment by ~68% (P<0.001) (Figure 4F). In contrast, psoralidin completely inhibited NF-κB promoter activation in KD-Akt and pCB6+ transfected PC-3 cells by ~90% (P<0.0001) (Figure 4F). Similar observations were made in DU-145 cells (data not shown) and these results indicate that NF-κB signaling plays an important role in the treatment of AIPC.

Psoralidin-mediated NF-κB inhibition acts via the IκB-α pathway

To clarify whether psoralidin acts by blocking the phosphorylation of IκB-α thereby sequestering the NF-κB dimers in the cytoplasm, we determined the expression profile of key regulatory proteins in the NF-κB signaling pathway. We determined the expression levels of NIK (NF-κB inducing kinase) which phosphorylates IKKα and/or IKKβ, thereby initiating NF-κB activation in cells. Psoralidin caused a time-dependent decrease in NIK expression in both PC-3 and DU-145 cells suggesting that NIK-inducible NF-κB activation is inhibited by psoralidin in both the AIPC cell lines. A time-dependent decrease of IKKα and a constant level of IKKβ expression were seen in PC-3 cells whereas vice versa was observed in DU-145 cells indicating the preferential binding of IKKs to the NF-κB dimers in AIPC cells. Also, phosphorylated IKKα/β (Thr23) was found to be decreasing in a time-dependent manner in PC-3 cells while it maintained its level of expression in DU-145 cells (Figure 5A).

Figure 5
Psoralidin inhibits NF-κB signaling in prosatate cancer cells via IκB pathway

We then examined the total and phosphorylated levels of IκB-α following treatment with psoralidin which revealed a decrease in the levels of phosphorylated IκB-α in both PC-3 and DU-145 cells. In contrast, non-phosphorylated level of IκB-α either remained constant or upregulated in both the cell lines. Collectively these results suggest that psoralidin has an inhibitory effect on the phosphorylation and degradation of IκB-α which is preceded by the inhibition of the IKKs and NIK resulting in the complete inhibition of NF-κB activation in both the AIPC cell lines (Figure 5A). The net inhibitory effects of psoralidin have been found to be a complete inactivation of NF-κB in AIPC cells. Following observation of constant or increased IκB-α levels by Western blotting, we determined IκB-α activity which revealed that in both the AIPC cells, non-phosphorylated IκB-α levels either remained constant or increased following treatment with psoralidin which was in accordance with the Western blot results (Figure 5B). As IκB degradation is responsible for the nuclear translocation of NF-κB, we infected the AIPC cells with Ad5-IκB Super-Repressor. Twenty four hours after infection, AIPC cells were treated with either vehicle control (DMSO) or psoralidin, and the results of Western blotting and DNA binding assays suggests that psoralidin sensitized the AIPC cells infected with Ad5-IκB Super-Repressor more significantly when compared to the controls (Data not shown).

Bcl-2 and inhibitor of apoptosis (IAP) family members, many of which are regulated by the PI3K/Akt/NF-κB pathway are the major regulators of apoptosis in both normal and transformed cells. Therefore, we explored the impact of psoralidin on the Bcl-2 and IAP family of proteins and found that psoralidin completely down regulated pro-survival proteins such as Bcl-2, Bcl-xL and survivin while maintaining constant levels pro-apoptotic protein Bax in both PC-3 and DU-145 cells (Figure 5C).

Over expression of Bcl-2 is reported in a variety of human cancers, including prostate cancer and is associated with tumor aggressiveness. Since psoralidin inhibits Bcl-2 levels in AIPC cells, we over expressed Bcl-2 in PC-3 cells and then explored whether psoralidin effectively suppresses the growth of PC-3/Bcl2 cells. As expected Bcl-2 over expressed PC-3 cells imparts resistance to psoralidin treatment when compared to PC-3/Neo control cells harboring empty vector, however, higher concentration of psoralidin sensitizes PC-3/Bcl-2 over expressed cells by down regulating ectopically over expressed as well as basal levels of Bcl-2 in PC-3 cells (Figure 5D) resulting in the induction of apoptosis (Figure 5E).

Psoralidin inhibits cell viability and induces apoptosis in AIPC cells

To explore the potential anti-tumor properties of psoralidin, androgen-negative (PC-3 and DU-145), androgen-positive (22Rv1 and LNCAP) and normal immortalized prostate epithelial PzHPV-7 cell lines were selected to perform cell viability assays (trypan blue dye exclusion). At 24 hours post-treatment, psoralidin (25-100 μM) significantly inhibited (P<0.001) viability of AIPC and ADPC cells when compared to normal prostate epithelial cells (Figure 6A). These findings suggest that psoralidin may selectively inhibit the survival of PCa cells. To address the question of whether psoralidin induces apoptosis, we performed two different apoptotic assays (Annexin V-FITC and TUNEL) using PC-3, DU-145, 22Rv1, and LNCaP cells which showed that psoralidin induces significant (P<0.0001) apoptosis in both androgen-negative and androgen-positive cell lines (Figure 6B).

Figure 6
Psoralidin inhibits cell viability and induce apoptosis in prostate cancer cells

Effect of psoralidin on xenograft models

To explore the in vivo effects of psoralidin on AIPC, we tested psoralidin on PC-3 xenografts in male nude mice. The animals were observed for 1-2 weeks until the tumors reached a volume of 50 mm3. At this time, a total of 27 mice were randomized into three groups (n=9 per group). Psoralidin (15mg/kg and 25 mg/kg body weight) or vehicle (corn oil) was administered intratumorally, five days a week for four weeks and tumor regression studies were performed over a four week period. The data was analyzed using Longitudinal Analysis and computations were carried out using the SAS program PROC MIXED, which yielded a statistically significant difference in tumor volumes between the control and treatment groups at 2wks, 3wks, and 4wks, with respective P-values of 0.006, 0.0001, and < 0.0001 (Figure 6C). Upon observing the tumor growth inhibition effected by psoralidin we investigated whether the tumor regression by psoralidin was due to induction of apoptosis and not necrosis by using TUNEL staining in the tumor sections and a more significant number of TUNEL positive cells were seen in psoralidin treated tumors compared to corn oil -treated controls (Figure-6 C, see insert). The strong induction of TUNEL-positive cells in psoralidin treated tumors indicates that psoralidin is a potent compound that inhibits the growth of AIPC. We sacrificed three animals for gross morphological changes, such as color or texture of organs (liver, lung, kidney, heart, prostate and bladder), and tissues were weighed and compared with healthy, non-tumor bearing animals; we noted no significant difference between the groups indicating that psoralidin is a non-toxic agent in animal models.

Discussion

PI3K/Akt signaling is a major component of the cell signaling network, as it is a focal point for a number of pro-survival pathways, which modulates numerous transcriptional factors and genes involved in the regulation of cell proliferation, cell survival, angiogenesis and tissue invasion (27). PTEN is a negative regulator of Akt, which often gets mutated or deleted in AIPC resulting in Akt mediated survival signaling which confers chemotherapeutic resistance in AIPC (28). To effectively target PTEN negative prostate tumors, more selective therapeutic approaches are needed, however, the low degree of specificity of compounds that are currently under investigation has been an impediment in realizing this goal (29). In this study, we have identified a chloroform extractable natural compound, psoralidin from Rasagenthi Lehyam, a herbal preparation and has found it to be effective for the treatment of AIPC (30). Psoralidin is one of the active ingredients in Psoralea corylifolia plant which is extensively used in traditional medicines against many diseases including cancer. We found that psoralidin targets PI3 kinase mediated Akt signaling resulting in the inhibition of cell survival and induction of apoptosis in AIPC cells.

Our findings suggest that psoralidin inhibits pAkt (ser473) in PC-3 and DU-145 cells without altering total levels of Akt. Our results also indicate that psoralidin inhibits pAkt after shorter exposure (3 hrs), compared to commercially available Akt inhibitors. The complete down regulation of pAkt by psoralidin suggests that it could be an excellent candidate to inhibit Akt-mediated signaling in prostate cancer. Psoralidin also inhibits Akt kinase activation, and as a result its direct substrate GSK-3 (31). Akt has been shown to phosphorylate GSK-3 (ser 21/9) which via the regulation of genes involved in cell cycle progression and survival plays a role in cell proliferation. Psoralidin inhibits the kinetics of pGSK-3α/β in a dose dependent manner, thereby revealing GSK-3 is directly regulated by Akt in AIPC cells. Further, GSK-3 is involved in the phosphorylation and degradation of the cell cycle regulatory protein, cyclin D1 (32) and our results showed that psoralidin down regulates cyclin D1 in both the PCa cell lines suggesting that psoralidin is capable of inhibiting the complete Akt signaling pathway in prostate cancer cells including its downstream targets. Further, over expression of myr-Akt induces Akt phosphorylation and enhances cell growth in PC-3 and DU-145 (results not shown). Psoralidin however overcomes Akt-mediated resistance and induces apoptosis in Akt-overexpressed AIPC cells. Recently, it was found that phosphorylation and activation of Akt correlates with prostate tumor invasiveness (33), and high Gleason grade prostate cancers (34). As we know that Akt is involved in tumor aggressiveness and metastasis psoralidin-mediated inhibition of Akt signaling might possess a therapeutic potential in sensitizing PCa to apoptosis.

PI3-kinase is the one of the key activator of Akt signaling and accumulating evidence implicate the involvement of the PI3K/Akt signaling as having a critical role in the development of several human malignancies including prostate cancer (35, 36). Our results demonstrate that psoralidin inhibits the constitutive levels of PI3K p110 and p85 in a dose- and time-dependent manner. Our results also reveal that psoralidin blocks PI3 kinase activation in both PC-3 and DU-145 cells. Several studies suggested that inhibition of the PI3K signaling results in the induction of apoptosis in AIPC (37, 38) and PI3K pathway is currently a major therapeutic target for the treatment of cancer (39, 40) and we believe that psoralidin is also one such compound that targets the PI3K/Akt pathway in AIPC cells.

We have previously demonstrated that activation of NF-κB confers resistance to current treatments in AIPC (25), as activated NF-κB promotes tumor growth and curtails induction of apoptosis in AIPC (41, 42). Interestingly, psoralidin-mediated inhibition of NF-κB activation occurred in both PC-3 and DU-145 cells suggesting that psoralidin not only selectively inhibits Akt but it also targets other pro-survival signaling in prostate cancer. Although Akt-driven NF-κB activation is well established in many cancer cell types, EGFR and Her-2 mediated NF-κB activation via Casein Kinase II (CK-2) has also been reported in AIPC (43, 44). Inhibition of NF-κB activation by psoralidin suggests that not only Akt-mediated activation of NF-κB but also EGFR- or Her-2-mediated NF-κB might be suppressed in PCa.

In unstimulated conditions, NF-κB is sequestered in the cytoplasm as a heterodimer by the inhibitory protein IκB-α (45). In response to external stimuli, IκBα is rapidly phosphorylated, allowing the active dimmers to translocate to the nucleus thereby activating the target genes. In our results we observed that psoralidin degrades the p65 in the cytosolic and nuclear fractions of both PC-3 and DU-145 cells. Recently, it was reported that PDLIM2 causes ubiquitination and degradation of p65 in several cell types (46) and hence, it might be possible that psoralidin activates PDLIM2 thereby degrading p65 in AIPC cells. Interestingly, decreased expression of p50 was observed only in cytosol but not in nuclear extract of both the cell lines. This may be due to the fact that p50 is constitutively bound to the κB binding sites in the promoter regions of the target genes thereby inhibiting NF-κB activation in these cell lines (47). Additionally, NF-κB binding as well as promoter activation studies clearly suggested that psoralidin regulates NF-κB at the promoter level.

NF-κB regulates many pro-survival genes such as members of the Bcl-2 and IAPs families which suppress apoptosis (48). Therefore we speculated that inhibition of NF-κB by psoralidin might result in the downregulation of anti-apoptotic Bcl-2 and Bcl-xL proteins; our results confirmed that there is indeed a complete inhibition of pro-survival signaling in both PC-3 and DU-145 cells. Down regulation of endogenous as well as ectopic expression of Bcl-2 protein might lead to an alteration in the Bcl-2: Bax ratio which could trigger apoptosis through the mitochondrial pathway in these cells. Survivin also, a member of the inhibitor of apoptosis family is over expressed in the AIPC (49) and treatment with psoralidin resulted in a decrease in survivin expression over time (undetectable levels in both PC3 and DU-145 cells after 12hours). Survivin has been shown to inhibit apoptosis by binding to active caspase-3 and caspase-7 (50). So, we observed that treatment of both the PCa cell lines with psoralidin resulted in increased expression of active caspase-3 and caspase-7, which resulted in the induction of apoptosis.

In summary, this study demonstrates that direct modulation of PI3K/Akt/NF-κB signaling activity by psoralidin towards apoptosis induction in AIPC cells which could provide the molecular basis for therapeutic targeting of advanced prostate cancer with this compound. Considering the pivotal role of PI3K/Akt signaling in the pathogenesis of human prostate cancer, these findings may have significant clinical relevance; in the context that psoralidin could be developed as an agent for the management of PCa, as novel chemoprevention strategy and/or effective therapeutic approach. Ongoing studies focus on fully dissecting the mechanism of action of psoralidin in physiological setting in AIPC models, and functionally linking the antitumor action of this (relatively safe and well-tolerated) phytochemical with the prevention of prostate cancer during prostate tumor progression to metastatic disease using in vivo model systems. The observations made in our in vivo studies may also enable us to conduct clinical trials with psoralidin to determine its chemotherapeutic and chemopreventive effects in human subjects.

Acknowledgments

This study was supported by an NIH R01 (R01 AT002890) and University of Kentucky Research Foundation grants (awarded to C.D.). We are grateful to Dr. Naoya Fujita, Japanese foundation for Cancer Research, for kindly providing Akt plasmids.

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