Novel two-tiered screening approach identifies synergistic combinations of natural compounds for prostate cancer prevention and treatment.

Prostate cancer (PCa) is the second most common cancer type among American men and it is estimated that in 2023, 34,700 men will die from PCa. Since it can take a considerable amount of time for the disease to progress to clinically evident cancer, there is ample opportunity for effective chemopreventive strategies to be applied for the successful management of PCa progression. In the current study, we have developed a two-tiered metabolomics-based screen to identify synergistic combinations of phytochemicals for PCa chemoprevention. This involves an initial screen for ATP depletion in PCa cells followed by a targeted screen for blocking glutamine uptake in the same cells. One of the phytochemical combinations (enoxolone [ENO] + silibinin [SIL]), identified via this screen, was examined for effects on PCa cell survival, oncogenic signaling and tumor growth in vivo. This combination was found to synergistically reduce cell survival, colony formation and cell cycle progression of PCa cell lines to a greater extent than either agent alone. The combination of ENO and SIL also synergistically reduced tumor growth when administered ad libitum through the diet in a HMVP2 allograft PCa tumor model. Treatment with the combination also significantly reduced STAT3 and mTORC1 signaling pathways in mouse and human PCa cells while significantly reducing levels of critical cell cycle regulatory proteins, contributing to the synergistic inhibition of tumor growth observed. Collectively, the current results demonstrate a novel approach to identifying synergistic combinations of phytochemicals for chemoprevention of PCa and possibly other cancers.

Cysteine depletion sensitizes prostate cancer cells to agents that enhance DNA damage and to immune checkpoint inhibition.

BACKGROUND: Prostate Cancer (PCa) represents one of the most commonly diagnosed neoplasms in men and is associated with significant morbidity and mortality. Therapy resistance and significant side effects of current treatment strategies indicate the need for more effective agents to treat both androgen-dependent and androgen-independent PCa. In earlier studies, we demonstrated that depletion of L-cysteine/cystine with an engineered human enzyme, Cyst(e)inase, increased intracellular ROS levels and inhibited PCa growth in vitro and in vivo. The current study was conducted to further explore the mechanisms and potential combinatorial approaches with Cyst(e)inase for treatment of PCa. METHODS: DNA single strand breaks and clustered oxidative DNA damage were evaluated by alkaline comet assay and pulsed field gel electrophoresis, respectively. Neutral comet assay and immunofluorescence staining was used to measure DNA double strand breaks. Cell survival and reactive oxygen species level were measured by crystal violet assay and DCFDA staining, respectively. Western blot was used to determine protein expression. FACS analyses were preformed for immune cell phenotyping. Allograft and xenograft tumor models were used for assessing effects on tumor growth. RESULTS: PCa cells treated with Cyst(e)inase lead to DNA single and double strand breaks resulted from clustered oxidative DNA damage (SSBs and DSBs). Cyst(e)inase in combination with Auranofin, a thioredoxin reductase inhibitor, further increased intracellular ROS and DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo. A combination of Cyst(e)inase with a PARP inhibitor (Olaparib) also increased DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo without additional ROS induction. Knockdown of BRCA2 in PCa cells increased DSBs and enhanced sensitivity to Cyst(e)inase. Finally, Cyst(e)inase treatment altered tumor immune infiltrates and PD-L1 expression and sensitized PCa cells to anti-PD-L1 treatment. CONCLUSIONS: The current results demonstrate the importance of oxidative DNA damage either alone or in combination for Cyst(e)inase-induced anticancer activity. Furthermore, cysteine/cystine depletion alters the tumor immune landscape favoring enhanced immune checkpoint inhibition targeting PD-L1. Thus, combinatorial approaches with Cyst(e)inase could lead to novel therapeutic strategies for PCa.