Effects of Vorinostat, Letrozole and RG7388 Treatments on Cell Cycle Arrest and Survival of Breast and Prostate Cancer Cells

Abstract

Alterations in gene expressions are often due to epigenetic modifications that can lead to significant influence on cancer development, growth, and progression. In this respect, HDAC inhibitors such as Vorinostat, which can exert epigenetic alterations, are emerging as a new class of drugs with promising impact on the cancer growth and metastatic process. Letrozole is one of the commonly used aromatase inhibitors that can elicit strong anti‐cancer effects on breast cancers through direct as well as indirect mechanisms. On the other hand, RG7388 is a newly developed inhibitor for an oncogene‐derived protein called MDM2, which is in clinical trials for the treatment of various cancers. Our aim for this study was to understand the molecular mechanism whereby Vorinostat can induce cell cycle arrest and trigger necrosis or apoptosis, when used in monotherapy or in combination with Letrozole or RG7388. We performed assays to measure the cell cycle arrest effects of individual drug treatments alongside of combination treatments with Vorinostat + Letrozole, and Vorinostat + RG7388 using MCF‐7 and LNCaP cancer cells. The cytotoxicity, cell cycle arrest and pro‐apoptotic effects of the treatments were assessed by using Trypan Blue dye exclusion method, MTT assay, immunoblotting, RT‐PCR, and qRT‐PCR methods. Our results from MCF‐7 and LNCaP cells confirmed that Vorinostat, Letrozole and RG7388 treatments were able to induce cell death via combination of cell cycle arrest and cytotoxic mechanisms in a dose and time dependent manner. The IC50 value for Vorinostat and RG7388 were determined to be around 7.5 μM and 2 μM respectively. Although, Letrozole showed stronger cytotoxic effects with the IC50 value of 100 nM in MCF‐7 and LNCaP cells, combination of Letrozole with SAHA did not produce synergy or additive effects. On the other hand, when SAHA was used in combination with RG7388, a significant increase in the cytotoxic effect was obtained. Induction of cell cycle arrest in the cancer cells were evidenced by the elevation of p21 protein levels following SAHA and RG7388 treatments. In addition, the SAHA treatment in LNCaP cells showed significant up regulation in the expression of other cell cycle related genes such as, p27, and p53 and down regulation of AURKB, CDC25C, and CDK1. The effects of SAHA monotherapy and combinations were assessed further by using SYTOX and Ac‐DEVD‐amc [Ac (N‐acetyl)‐DEVD‐AMC (7‐amino‐4‐methylcoumarin)] staining method, to determine the extent of necrotic cell death or apoptosis. Our results confirmed that the cell death caused by SAHA treatment was primarily through induction of necrosis. On the other hand, the RG7388 treatment was able to induce apoptosis by elevating Caspase 3 activity. It appears that, during combination treatments with SAHA and RG7388, two parallel pathways are induced simultaneously that could lead to increased cancer cell death. It has been reported in the literature that the elevated levels of acetylated histones (H2 and H3) and consequent activation of intracellular signals are responsible for the cell cycle arrest and necrosis that are typically observed when HDAC inhibitors (HDACi) are used. So far, the results of SAHA and RG7388 combination experiments have confirmed the abilities of these two agents for eliciting additive or synergistic effects in MCF7 and LNCaP experimental models. We are speculating that our findings could lead to the development of newer treatments for breast and prostate cancers using this type of combinations.Support or Funding InformationThis research was supported by the Royal Dames of Cancer Research, Fort Lauderdale, Florida, USAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.