Treatment Effects of Vorinostat and Letrozole Combination on Breast Cancer Cell Survival and Peripheral Blood Mononuclear Cell Differentiation
Authors: Natarajan Umamaheswari , Samuel Shila , Vijiyaraghavan R , Rathinavelu Appu 
Published: November 2023

HDAC inhibitors are new class of drugs that show promising impact on the cancer growth and metastatic process. Letrozole is one of the aromatase inhibitors that could also elicit strong anticancer effects on breast cancers through direct as well as indirect mechanisms. Our aim was to understand the intracellular mechanism whereby Vorinostat, in monotherapy and in combination with Letrozole, can induce cell cycle arrest and apoptosis in breast cancer cells. In addition, we also attempted to determine how Vorinostat and Letrazole can inhibit Peripheral Blood Mononuclear Cell (PBMC) differentiation into osteoclasts and cause programmed cell death. We performed assays to measure the anti‐proliferative, pro‐apoptotic effects of monotherapy (Vorinostat) and combination (Vorinostat + Letrozole) treatments in cancer cells (MCF‐7) as well as in PBMC, that was isolated from healthy individuals and undergoing osteoclast differentiation in a specialized medium that contained RANKL and M‐CSF (Macrophage – colony stimulating factor). Immediately after treatments, determination of cytotoxicity on MCF‐7 was performed using MTT assay and the pro‐apoptotic effects were assessed by immunoblotting and RT‐PCR analyses. Our results confirmed that both Vorinostat and Letrozole were able to induce cell death in MCF‐7 cells in a dose and time dependent manner. The IC50 value for Vorinostat were around 1.5 μM, and 1.2 mM respectively for MCF‐7 and PBMC. In our experiments, Letrozole showed much stronger effects and the IC50 values were 4 nM and 2.5 nM respectively for the above listed cells. Induction of apoptosis in these cells were evidenced by the elevation of p21, Bax, Caspase 3 and down regulation of Bcl2. The induction of apoptosis was further confirmed by DNA fragmentation in our in vitro experimental systems. Furthermore, our experiments with PBMC using both mono therapies and the combination treatments revealed that, they were able to block the osteoclast differentiation at reduced doses when combined together for treatments. It has been reported that the elevated levels of acetylated histones H3 and H4 and consequent intracellular signals are responsible for the cell cycle arrest and pro‐apoptotic events that are seen when HDAC inhibitors are used. Similar mechanisms may be responsible for the intracellular effects observed in our experimental systems. Thus, the results obtained from our in vitro experiments suggest that combination of Vorinostat and Letrozole is very dynamic and effective, not only in blocking the proliferation of MCF‐7 cells but also in inhibiting the differentiation of PBMC into osteoclasts and reducing their survival capacity. This suggests that, during clinical use, the above indicated combination might provide dual benefits, by blocking cancer cell proliferation as well as reduction of breast cancer associated osteoporosis caused by osteoclasts. In essence, the above discussed treatment outcomes may be achieved by minimizing the differentiation and formation of osteoclasts from PBMC in cancer patients, that appears to be further supported by the induction of programmed cell death.