PD10: Prostate Cancer: Advanced (including Drug Therapy) I
PD10-01: Preclinical efficacy & mechanism of action of combination of nitric oxide donor and growth hormone-releasing hormone antagonists for castration-resistant prostate cancer
Introduction: Castration resistant prostate cancer (CRPC) is unresponsive to androgen therapy and thus, other therapeutic agents and molecular events are being explored. We have shown in our studies that increased NO levels affect receptors for GnRH, LH and T through the hypothalamic-pituitary-gonadal axis, while simultaneously increasing overall oxidative/nitrosative stress. In the same context, several studies have used inhibition of GHRH-R (receptor) as an approach to target PCa. Therefore, considering that both increased NO levels and GnRH antagonists could be targeting the PCa via the same mechanistic channel, in the present study, we evaluated the impact of increased NO levels with or without GnRH antagonists on CRPC tumor progression to establish their preclinical efficacy for treatment of androgen independent prostate cancer.
Methods: Proliferation assay was performed using 22RV1 cells in the presence of GHRHR antagonist (MIA-602) at 1uM concentration, in the absence or presence of increasing concentration of GSNO (from 25, 50 or 100uM respectively). Expression of down terminal markers of CRPC (PSA, AR TMRPSS2, AR, ARV7 and pERK respectively) was checked at RNA and protein levels after treating 22RV1 cells with MIA-602 at 1uM concentration, in the absence or presence of increasing concentration of GSNO (from 10, 25, 50 or 100uM respectively). For in-vivo experiments, castrated SCID mice were grafted with 22RV1 cells to generate CRPC murine models. Once the tumors became palpable, the animals were divided into three groups. Group 1 received vehicle control, group 2 received GSNO treatment at the dosage of 10mg/kg/day intraperitonially (IP), Group 3 received GSNO (10mg/kg/day IP) and MIA-602 (5ug/kg/day IP) combination treatment. Treatment was continued for 30 days. Weight of the animals and tumor volume were measured 3 times per week during the 30 days treatment period. Post treatment, tumors and blood were extracted from the mice. Proteins were harvested from the tumors to study expression of markers of cancer progression. Animal experiments were carried out in compliance with the Institutional Animal Care and Use Committee of University of Miami. Molecular analyses were performed using standard procedures. GraphPad Prism (GraphPad Software) was used for statistical analysis. All data were presented as the means ± SEM. The statistical significance between two groups was estimated by unpaired two-tailed t test.
Results: MTT assay showed that MIA-602 compounds were able to significantly reduce the cell proliferation of 22RV1 cells (p<0.05). GSNO did not interfere with ability of MIA compounds towards reducing cell proliferation. Additionally, the increasing concentration of NO levels potentiated the effects of MIA-602 by decreasing PSA, AR and TMRPSS2 at RNA levels and AR, ARV7 and pERK at protein levels respectively. In-vivo results showed that both GSNO and the combinatorial treatment of GSNO and MIA-602 showed a significant decrease (p<0.05) in tumor volume and weight compared to the control. Proteins from tumors showed a reduction in androgen receptor (AR) and AR-V7 compared to individual treatments of control and GSNO respectively.
Conclusions: This study shows that the combination of GSNO and MIA-602 work to decrease CRPC tumor burden and cellular proliferation. GSNO is known to act on the tumor microenvironment so when it is paired with a GHRHR antagonist the results show significant reduction in CRPC tumor burden. Further studies with active NO donors and GHRHR antagonists are in process to establish the preclinical efficacy and mechanism of action of the combination against CRPC. Source of
