Evaluation of Ferroptocide (FTC) As a Potential Immunogenic Cell Death Inducing Agent in Prostate Cancer Cells

Prostate cancer (PCa) is one of the most common types of cancer in men, and its incidence continues to rise in many countries. PCa is the sixth most common cancer in the world. It is the third most common cancer in men and the second most leading cause of death in American males. The American cancer society's estimated 191,930 new cases and 33,330 death from prostate cancer. The existing treatments for PCa include surgery, radiation therapy, chemotherapy, and hormonal therapy. However, these treatments have limited efficacy and patients often experience side effects. Although these treatment options show effects in the beginning stages subsequently, the majority of the patients develop resistance. Therefore, effective treatments for PCa are urgently required. A novel synthetic small molecule compound ferroptocide (FTC), a derivative of diterpene pleuromutilin is one such potential therapeutic agent against PCa. It displays potent tumor-suppressive effects in a variety of cancer models. Immunogenic Cell Death (ICD) pathways result in a regulated activation of tumor immunity that could potentially eliminate PCa. Therefore, agents that promote ICD is highly desirable. We hypothesize that ferroptocide (FTC) would be an effective anticancer agent against prostate cancer cells. In this study, we have evaluated the ICD inducing potential of FTC using human-derived LNCaP and mouse-derived TRAMP-C1 PCa cellular models. Our results showed that FTC potently inhibits the cell viability of LNCaP and TRAMP-C1 cells. Interestingly, FTC elevated the levels of ROS by utilizing DCFH-2DA probe and analyzed by confocal and flow cytometry, intracellular ROS time point study by flow cytometry depicted that FTC robustly induces ROS within 1hr. Mitochondrial superoxide was generated using MitoSox probe suggesting FTC induces oxidative stress and leading to G2/M cell cycle arrest and activation of apoptosis in LNCaP cells. Notably, FTC treatment promoted the secretion of Adenosine Triphosphate (ATP), in both LNCaP and TRAMP-C1 cells in a dose-dependent manner. Furthermore, Western blot analysis of LNCaP cells treated with FTC showed increased expression of ICD markers such as HMGB1 and HSP70. Confocal immunofluorescence analysis confirmed the higher expression of these ICD markers in both the cellular models. Hence, this is the first study to demonstrate that FTC targeting mitochondria induce mitophagy as well as its ability to induce ICD in prostate cancer cells. Our data suggest that FTC is a promising anticancer agent with the potential to activate ICD to effectively combat PCa.