Abstract B75: Herbal extract amalgam Zyflamend suppresses prostate cancer cell growth and survival through inducing androgen receptor degradation and inhibiting AR signaling

Background: Prostate cancer is a public health problem due to its high incidence and mortality rates. The intervention of androgen receptor (AR) signaling is one of the methods for prostate cancer chemoprevention [eg. prostate cancer prevention trail (PCPT)] and therapy (androgen deprivation therapy). It is now being increasingly recognized that natural herbal and phytochemical agents can be crucial to decease the morbidity and mortality of prostate cancer for both chemoprevention and therapy. Zyflamend is an amalgam comprised of ten different herbal extracts (rosemary, turmeric, ginger, holy basil, green tea, hu zhang, Chinese goldthread, barberry, oregano, and Scutellaria baicalensis) and preliminary data showed that it reduced serum PSA after 18‐month treatment in patients who has a prior biopsy showing HGPIN. We hypothesize that Zyflamend can induce anti‐cancer effects through affecting AR signaling and androgen antagonist Casodox can sensitize cells to Zyflamend. Methods: We performed MTT, colony formation and soft agar assays to test the anti‐cancer effects of Zyflamend. The alterations in cell cycle were analyzed by flow cytometry and further confirmed by Western blotting. AR and its downstream targets were analyzed at mRNA and protein level. Half‐life of AR protein was tested by cycloheximide assay. The nuclear localization and activation of AR were detected by immunofluorescence and luciferase assays. Finally, the combination of androgen antagonist Casodex with Zyflamend was tested in LNCaP cells. Results: Zyflamend showed cytotoxicity on AR expressing human prostate cancer cells (LNCaP, VCaP and 22Rv1) and mouse prostate cancer cells (CASP1.1 and CASP2.1) in a dose and time dependent manner. Long term exposure to Zyflamend also reduced colony formation capacities of prostate cancer cells in both anchorage dependent and independent assays. Flow cytometry assay revealed that Zyflamend induced G1 phase arrest and apoptosis, manifested by the induction of p21waf1 and p27kip1 protein levels and cleavage of PARP and caspase‐3. Of note, Zyflamend reduced AR expression in human and mouse prostate cancer cells, regardless of their androgen responsiveness, indicating that it is not restricted to specific cell line. We found that Zyflamend can reduce AR expression level at mRNA level by semi‐quantitative RT‐PCR and at protein stability level by cycloheximide assay. Zyflamend reduced AR protein level, induced by DHT treatment and inhibited DHT‐induced cell proliferation. Consistently, AR downstream targets genes (PSA and NKX3.1) were reduced and luciferase assay revealed that PSA and probasin promoter activities were reduced by Zyflamend in dose dependent manner. Interestingly, co‐treatment with 25 uM casodex sensitized LNCaP cells to the cytotoxicity of Zyflamend. Conclusions: These data indicate that Zyflamend suppressed cell growth through AR signaling, and suggested that the co‐treatment of androgen antagonist casodex with Zyflamend may be a promising approach for chemoprevention and therapy. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B75.