Betulinic acid inhibits constitutive activation of NF-κB and induces apoptosis in human prostate cancer cells

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The transcription factor, Nuclear Factor-kappaB (NF-κB) and its signaling pathways play critical role in cancer development, progression and therapy. Aberrant or constitutive NF-κB activation mediates inhibition of apoptosis by increasing the expression of survival factors leading to chemotherapeutic resistance. Recently, NF-κB has turned out to be an interesting therapeutic target and have become a focal point for intense drug discovery and development efforts. In this study we investigated the role of NF-κB in betulinic acid (BetA)-induced apoptosis in human prostate cancer. BetA is a pentacyclic triterpene found in many plant species, among others in the bark of white birch Betula alba. BetA has been reported to display a wide range of in vitro cytotoxic activities against various tumor cell lines and in vivo anti-tumor activities in human tumor xenografts that may have potential for prevention and therapy of prostate cancer. Treatment of human prostate cancer cells, viz. LNCaP, DU145 and PC-3 at 5-80 μM concentration of BetA for 24 h caused cell growth inhibition and apoptosis in these cell lines. Immunoblot analysis revealed that BetA suppresses NF-κB activation involved accumulation of NF-κB/p65 in cytoplasm and concomitant decrease of NF-κB/p65 in nuclear fractions in dose and time dependent manner in all three cell lines. BetA decreased IKKalpha expression and inhibited the phophorylation of IκBalpha at serine 32/36 and degradation of IκBalpha. Importantly, electrophoretic mobility shift assay demonstrated BetA-mediated inhibition of NF-κB DNA binding activity. Furthermore, BetA-induced apoptosis was detected by an increase in caspase activity in these cells which triggered a series of effects associated with apoptosis involving cleavage of poly(ADP-ribose) polymerase, shift in Bax:Bcl-2 rheostat, upregulation of p21/WAF1 in favor of apoptosis, but with no effect on p53, suggesting activation of apoptotic response, different from that induced by conventional chemotherapeutic agents. Taken together, our results provide the evidence that BetA induces cell growth inhibition and apoptosis through inhibition of constitutive activation of NF-κB indicating its potential for further development in prevention and therapy of prostate cancer.