Triterpenoids from the tirucallic and boswellic acid families induce G0/G1 checkpoint arrest and apoptosis in prostate cancer cells

1920

The gum resins from various Boswellia species contain a complex mixture of mono- and triterpenoids that possess biological activities, including antitumor properties. We have previously shown that systemic application of acetyl-11-keto-β-boswellic acid (AKβBA) inhibited tumor growth and triggered apoptosis in pre-established PC-3 xenografts in nude mice via inhibition of IκB kinase α. Here, we have analyzed the molecular mechanisms of the cytotoxic effects of 3-keto-tirucallic acid (KTA) and AKβBA in human prostate cancer cell lines in vitro. The triterpenoids were isolated from the gum resin of Boswellia Carterii, and purified by reversed phase HPLC to chemical homogeneity; their structures were confirmed by mass spectroscopy. AKβBA and KTA (1-100 μM) exerted concentration-dependent anti-proliferative and cytotoxic effects on the androgen-dependent LNCaP prostate cancer cell line as well as on the androgen-independent, chemoresistant PC-3 prostate cancer cell line as measured either by the XTT assay or by lactate dehydrogenase release. A rapid induction of apoptosis was confirmed by analysis of phosphatidyl serine expression on the cell surface and formation of DNA-laddering. Flow cytometric cell cycle analysis of LNCaP and PC-3 cells treated with KTA and AKβBA (10 μM) for 24 and 48 h demonstrated accumulation of the cells in the G0/G1 phase, whereas the amount of cells in the S-phase was drastically reduced. Accordingly, the cycle-dependent phosphorylation of the retinoblastoma protein was rapidly reduced. Cell division is under strict control of cyclin-dependent kinases (CDKs) and their heterodimeric cyclin partners. Western blot analysis of prostate cancer cells treated with the triterpenoids revealed downregulation of cyclin D1, a crucial cell cycle regulator, but not of cyclin E or Cdk4. Downregulation of cyclin D1 occurred at the transcriptional level. The cyclin D1 promoter is regulated by NF-κB and TCF transcriptional regulators. Accordingly, an early downregulation of the nuclear accumulation of β-catenin and p65 was observed in triterpenoid-treated prostate cancer cells. Overexpression of the NF-κB inhibitor IκBα-SR, inhibited the constitutively active NF-κB signaling in PC-3 cells and reduced the cell proliferation, but did not trigger apoptosis. These results indicate that a sole inhibition of the NF-κB activity is not sufficient to trigger apoptosis in prostate cancer cells. Thus, triterpenoids inducing a G0/G1 checkpoint arrest might provide a novel approach for the treatment of chemoresistant human prostate cancer. This work was supported by the Deutsche Krebshilfe