Ferroptosis induction, androgen biosynthesis disruption and prostate cancer suppression by androgen and vitamin D combination.

Prostate cancer regression by hormonal vitamin D3(calcitriol) is clinically untenable despite its well-documented anticancer effects in experimental models, since supraphysiologic dosing of calcitriol required in clinical settings causes hypercalcemia and other toxicities. We show that subnanomolar/low-nanomolar calcitriol, while non-inhibitory on its own, inhibited CRPC cells upon co-treatment with androgen (5α-DHT) at a physiologic level, evident from G1/S cell cycle arrest, induction of p21/Cip1, blockade of clonal cell growth, retardation of proliferation, migration and invasion of cells in vitro, and xenograft growth suppression in vivo. AKR1D1, an androgen-inactivating 5β-reductase, was upregulated >30-fold upon calcitriol/DHT co-treatment, while each hormone individually did not induce AKR1D1. In contrast, HSD3β1, a key enzyme for androgen biosynthesis, was upregulated >12-fold by calcitriol, which was blocked by DHT co-treatment. Elevated AKR1D1 and reduced HSD3β1 would lower intracellular androgens and contribute to CRPC repression. Autophagy mediated ferroptosis (ferritinophagy) paralleled CRPC growth inhibition by the hormone combination -indicated by LC3B induction, ferritin reduction, and upregulation of mRNAs encoding NCOA4, ALOX-5 and PTGS2 which are core participants in ferroptosis. The combination treatment caused lipid peroxidation, evident from the fluorescence shift of C11-BODIPY(581/591)-labeled cells from red to green, and from elevated malondialdehyde. GPX4 - an antioxidant peroxidase and ferroptosis regulator - was downregulated. Bipolar androgen therapy employing supraphysiologic testosterone is under clinical evaluation for efficacy against treatment-resistant metastatic CRPC, although the high androgen dose raises safety concerns. Vitamin D and androgen administered concurrently at physiologic levels may offer a superior alternative for arresting CRPC.