Characterization of Precursor-Dependent Steroidogenesis in Human Prostate Cancer Models.

Castration-resistant prostate tumors acquire the independent capacity to generate androgens by upregulating steroidogenic enzymes or using steroid precursors produced by the adrenal glands for continued growth and sustainability. The formation of steroids was measured by liquid chromatography-mass spectrometry in LNCaP and 22Rv1 prostate cancer cells, and in human prostate tissues, following incubation with steroid precursors (22-OH-cholesterol, pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone). Pregnenolone, progesterone, 17-OH-pregnenolone, and 17-OH-progesterone increased C21 steroid (5-pregnan-3,20-dione, 5-pregnan-3,17-diol-20-one, 5-pregnan-3-ol-20-one) formation in the backdoor pathway, and demonstrated a trend of stimulating dihydroepiandrosterone or its precursors in the backdoor pathway in LNCaP and 22Rv1 cells. The precursors differentially affected steroidogenic enzyme messenger RNA (mRNA) expressions in the cell lines. The steroidogenesis following incubation of human prostate tissue with 17-OH-pregnenolone and progesterone produced trends similar to those observed in cell lines. Interestingly, the formation of C21 steroids from classical pathway was not stimulated but backdoor pathway steroids (e.g., 5-pregnan-3,20-dione, 5-pregnan-3-ol-20-one) were elevated following incubations with prostate tissues. Overall, C21 steroids were predominantly formed in the classical as well as backdoor pathways, and steroid precursors induced a diversion of steroidogenesis to the backdoor pathway in both cell lines and human prostate tissue, and influenced adaptive steroidogenesis to form C21 steroids.

Next-generation steroidogenesis inhibitors, dutasteride and abiraterone, attenuate but still do not eliminate androgen biosynthesis in 22RV1 cells in vitro.

Castration resistant prostate cancer (CRPC) is often lethal and inevitably develops after androgen ablation therapy. However, in the majority of cases it remains androgen dependent. CRPC tumors have the ability to synthesize their own androgens from cholesterol by engaging in de novo steroidogenesis. We investigated the potential of 22RV1 prostate cancer cells to convert the supplemented steroid precursors within this pathway under the effects of current clinical steroidogenesis inhibitors such as abiraterone and dutasteride, either alone or in combination. Under steroid starved conditions, enzymes responsible for de novo steroidogenesis were upregulated. Testosterone and dihydrotestosterone (DHT) were formed by using both dehydroepiandrosterone (DHEA) and progesterone as substrates. Formation of testosterone and DHT was higher following incubation with DHEA compared to progesterone. Progesterone decreased the mRNA expression of enzymes responsible for steroidogenesis. Abiraterone treatment decreased testosterone production but increased several precursor steroids in both classical and backdoor pathways in the presence of progesterone. In contrast, the DHT levels were elevated following treatment with abiraterone when progesterone was absent. Dutasteride decreased the formation of testosterone, DHT and precursor steroids in the backdoor pathway but increased steroid precursors in the classical steroidogenesis pathway. The combination of abiraterone and dutasteride decreased testosterone and DHT in the presence of progesterone but increased DHT in the absence of progesterone. Abiraterone inhibited androgen receptor (AR) activation but not to the same extent as MDV3100. However, abiraterone and dutasteride treatment, either alone or in combination, were more effective in decreasing prostate specific antigen secretion into the media than MDV3100. Thus, while interventions with these drugs alone or in combination fail to completely inhibit steroidogenesis in the 22RV1 cells, the combined inhibition of androgen production and blockade of AR can exceed the effect of MDV3100. Further characterization of bypass mechanisms that may develop as a response to these inhibitors is necessary to achieve optimal suppression of testosterone and DHT synthesis as a part of therapeutic regimens for the treatment of CRPC.

Pomegranate extracts impact the androgen biosynthesis pathways in prostate cancer models in vitro and in vivo.

Castration-resistant prostate cancer (CRPC) remains largely dependent on androgen receptor (AR). Residual tissue androgens are consistently detected within CRPC tumors and play a critical role in facilitating AR-mediated signaling pathways which lead to disease progression. Testosterone and dihydrotestosterone (DHT) are the major androgens detected in tumors. They are produced through three biosynthesis pathways: Δ(4), Δ(5), and backdoor pathways. Both androgens bind to and stimulate AR activation. The current study investigates the effects of pomegranate extracts (POM) and their ability to inhibit androgen biosynthesis using PCa cell lines (22RV1 and LNCaP) in vitro as well as the PTEN knockout mouse model representing prostate cancer. Steroids were extracted using ethyl acetate or solid phase extraction, and then analyzed by UPLC/MS/MS. The results showed that POM (0-12µg/mL) reduced the production of testosterone, DHT, DHEA, androstenedione, androsterone, and pregnenolone in both cell lines. In addition our in vivo data supports this observation with a reduction in serum steroids determined after 20 weeks of POM treatment (0.17 g/L in drinking water). In accordance with these results, Western blotting of cell lysates and tPSA analysis determined that PSA was significantly decreased by the treatment of POM. Interestingly, AKR1C3 and AR levels were shown to be increased in both cell lines, perhaps as a negative feedback effect in response to steroid inhibition. Overall, these results provide mechanistic evidence to support the rationale for recent clinical reports describing efficacy of POM in CRPC patients.

Bioactive compounds from Rhodiola rosea (Crassulaceae).

The methanol extract of the underground part of Rhodiola rosea was found to show inhibitory activity against Staphylococcus aureus. Bioactivity-guided fractionation of a 95% ethanol extract from the stems of R. rosea led to the isolation of five compounds: gossypetin-7-O-L-rhamnopyranoside (1), rhodioflavonoside (2), gallic acid (3), trans-p-hydroxycinnamic acid (4) and p-tyrosol (5). Their structures were elucidated by UV, IR, MS and NMR data, as well as by comparison with those of the literature. Compounds 1 and 2 were evaluated for their antibacterial and antiprostate cancer cell activities. Compounds 1 and 2 exhibited activity against Staphylococcus aureus with minimum inhibitory concentrations of 50 microg/mL and 100 microg/mL, respectively. Cytotoxicity studies of 1 and 2 also displayed activity against the prostate cancer cell line with IC(50) values of 50 microg/mL and 80 microg/mL, respectively.