br DHEA and DHT production by PCa
3.3. DHEA and DHT production by PCa cell lines VCaP and LAPC-4 using DHEAS was STS-dependent
VCaP NPS-2143 converted DHEAS to DHEA, whereas, STX64 treatment reduced DHEA production (Fig. 3A). VCaP cells converted both DHEA and DHEAS to produce DHT (Fig. 3B and C, respectively). The amount of DHT produced depended on the concentrations of DHEA or DHEAS. DHT production was low when cells were treated with DHEA or DHEAS at 10 nM, the physiological concentration of circulating DHEA. DHT production was higher when cells were treated with DHEA or DHEAS at 3.5 μM, the physiological concentration of circulating DHEAS. DHEA appeared to be a preferred substrate for DHT production compared to DHEAS since more DHT was produced from DHEA at either treatment concentrations. STX64 inhibited DHT production in the DHEAS treat-ment groups (Fig. 3B&E), and had no eﬀect on DHT production by VCaP cells treated with 3.5 μM DHEA (Fig. 3C). STX64 reduced slightly DHT production by LAPC-4 cells treated with 3.5 μM DHEA (Fig. 3F), but to a lesser extend compared to the reduction of DHT production by LAPC-4 cells treated with 3.5 μM DHEAS. The inhibitory activity of STX64 was specific to DHT production from DHEAS suggesting that STS was critical for DHT production from DHEAS. The ability of LAPC-4 cells to metabolize DHEA, or DHEAS, for DHT production was lower than that of VCaP cells, whereas the sensitivity of LAPC-4 DHT production from DHEAS to the inhibitory eﬀect of STX64, was similar to VCaP cells (Fig. 3D–F).
3.4. Stimulation of AR activity by DHEAS
AR-mediated transcriptional activity in LAPC-4 and VCaP cells was stimulated by incubation with DHEAS (Fig. 4A and B, and STX64 re-versed AR stimulation by DHEAS. T at 1 nM, and DHEAS at 3.5 μM, both activated AR transactivation, with AR activation by the andro-gens/metabolites inhibited by the AR antagonist bicalutamide. Bicalu-tamide alone and STX64 alone did not have eﬀect on AR activity in cells treated in the absence of T or DHEAS (data not shown).
3.5. Stimulation of PCa cell growth by DHEA and DHEAS
\Both DHEA and DHEAS stimulated growth of VCaP cells in the absence of exogenous T in the medium (Fig. 5A). The growth-stimula-tory eﬀect of DHEAS was dose-dependent,” where DHEA has been re-moved. DHEA stimulated growth more eﬀectively than DHEAS when both were used at a 10 nM concentration, whereas, DHEA and DHEAS stimulated growth to a similar degree when both were used at a 3.5 μM concentration. DHEAS-stimulated growth was inhibited by STX64, in-dicating that DHEAS-stimulation of growth required STS conversion of DHEAS to DHEA. Lastly, bicalutamide reversed partially the DHEAS-stimulated growth, indicating that growth stimulation by DHEAS was at least partially AR-dependent (Fig. 5B).
3.6. DHEA sustained growth of VCaP xenograft in castrated mice
The volume of VCaP xenografts growing in castrated nude mice did not increase over the time course of the experiment, while the volume
Fig. 2. DHT production by prostate tissue ex vivo was STS-dependent. Inserted tables showed the exact values of DHT production, in each treatment pre-sented in the respective bar graph at the top in the format of mean and range (lowest value, highest value). DHEAS 3.5 μM and STX64 5 μM were used in the combination treatments.
Fig. 3. DHEA and DHT production by prostate cancer cell lines VCaP (Panels A–C) and LAPC-4 (Panels D–F) using DHEAS was STS-dependent. (A) & (D), DHEA production; (B) & (E), DHT production by cells treated with DHEAS; (C) & (F), DHT production by cells treated with DHEAS. Cells were treated for 3 days. Androgens in the culture medium were normalized against cell numbers, which was indicated by units of OD570 measured using the MTT assay. *p < 0.05.
of VCaP xenografts in mice supplemented with T increased con-tinuously over the time course of the experiment. VCaP xenografts growing in castrated mice supplemented with DHEA maintained a rate of increase similar to in animals supplemented with T (Fig. 6A–C). VCaP xenografts on SCID mice demonstrated the same growth patterns of tumor growth as on nude mice in response to T supplementation, cas-tration without supplementation, and castration in combination with DHEA supplementation, respectively (Fig. 6D–F). Growth rate analysis showed that castration reduced the growth rate of tumors by ∼50% compared to tumor growth on mice treated with T. DHEA increased tumor growth compared to tumor growth on castrated mice, and DHEA supported tumor growth at rates comparable to T (Table 1).
This study reports several unique findings relevant to the ability of adrenal androgens to rescue the loss of testicular androgens due to ADT as measured by production of DHT, maintenance of AR activity and tumor growth. First, STS was expressed both in benign and malignant prostate tissue, in agreement with previous reports of the expression of STS in the prostate (Nakamura et al., 2006; Klein et al., 1988a, 1988b, 1989; Voigt and Bartsch, 1986; Farnsworth, 1973; Cowan et al., 1977). Expression of STS at the mRNA level and protein level were comparable between benign and malignant prostate tissue. It is not clear at the moment whether the expression of STS is diﬀerent in primary PCa and advanced, metastatic PCa. Therefore, further research is needed to