Psoralidin, an Herbal Molecule from Psoralea corylifolia, Inhibits Phosphatidylinositol 3-Kinase–Mediated Akt Signaling in Androgen-Independent Prostate Cancer Cells

Wednesday, 18/04/2012  |   Herb or Compound, Prostate Cancer  |  no comments

Kumar R, Srinivasan S, Koduru S, et al. Cancer Prev Res March 2009 2; 234. doi: 10.1158/1940-6207.CAPR-08-0129

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 phosphatidylinositol 3-kinase through lipid phosphatase activity. Recently, we identified a natural compound, psoralidin, which inhibits Akt phosphorylation, and its consequent activation in androgen-independent prostate cancer (AIPC) cells. Furthermore, ectopic expression of Akt renders AIPC cells resistant to 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 phosphatidylinositol 3-kinase activation and transcriptionally represses the activation of nuclear factor-?B 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 that 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 phytochemical-based platform for prevention and treatment strategies for AIPC.

PI3K/Akt signaling is a major component of the cell signaling network, as it is a focal point for a number of prosurvival pathways that modulate 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, an herbal preparation, and have found it to be effective for the treatment of AIPC (30). Psoralidin is one of the active ingredients in the Psoralea corylifolia plant, which is extensively used in traditional medicines against many diseases, including cancer. We found that psoralidin targets PI3K-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 hours), compared with 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 (Ser21/9), 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-3?/? in a dose-dependent manner, thereby revealing that 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 prostate cancer cell lines, suggesting that psoralidin is capable of inhibiting the complete Akt signaling pathway in prostate cancer cells, including its downstream targets. Further, overexpression 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 prostate cancer to apoptosis.

PI3K is one of the key activators of Akt signaling, and accumulating evidence implicates 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 show 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 PI3K 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 the PI3K pathway is currently a major therapeutic target for the treatment of cancer (39, 40); we believe that psoralidin is also one such compound that targets the PI3K/Akt pathway in AIPC cells.

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

In unstimulated conditions, NF-?B is sequestered in the cytoplasm as a heterodimer by the inhibitory protein I?B-? (45). In response to external stimuli, I?B? is rapidly phosphorylated, allowing the active dimers 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 ?B binding sites in the promoter regions of the target genes, thereby inhibiting NF-?B activation in these cell lines (47). Additionally, NF-?B binding as well as promoter activation studies clearly suggested that psoralidin regulates NF-?B at the promoter level.

NF-?B regulates many prosurvival genes such as members of the Bcl-2 and IAP families, which suppress apoptosis (48). Therefore, we speculated that inhibition of NF-?B by psoralidin might result in the down-regulation of antiapoptotic Bcl-2 and Bcl-xL proteins; our results confirmed that there is indeed a complete inhibition of prosurvival 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 to Bax ratio, which could trigger apoptosis through the mitochondrial pathway in these cells. Survivin, also a member of the inhibitor of apoptosis family, is overexpressed 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 12 hours). Survivin has been shown to inhibit apoptosis by binding to active caspase-3 and caspase-7 (50). Thus, we observed that treatment of both the prostate cancer 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 shows that direct modulation of PI3K/Akt/NF-?B signaling activity by psoralidin causes 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 prostate cancer, as a novel chemoprevention strategy, and/or as an effective therapeutic approach. Ongoing studies focus on fully dissecting the mechanism of action of psoralidin in a physiologic setting in the 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.

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