Systematic Screening of Histone Deacetylase Inhibitors: Enhancing Immunological Function Against Cancer.

Cancer is the result of a compromised immune system lacking the ability to recognize and eliminate transformed cells. These transformed cells induce an upregulation of negative regulatory tumor cells, which suppress T-cell activation and proliferation while inducing tumor tolerance. Immunotherapy is a novel therapeutic modality that strengthens the immune system against cancer cells, which in turn avoid immunological tolerance of cancer cells and the impedance of transformed cells survival. Histone deacetylase inhibitors (HDACinh) are specific drug compounds that are largely used as anti-cancer drugs. HDACinh have demonstrated control of apoptosis, cell survival, along with immune functionalities. HDAC inhibition also modulates the expression of tumor associated antigens (TAA) and immunosuppressive proteins. Our group has reported that the genetic and pharmacological abrogation of HDACs results in both, a decline in proliferation of tumor cells and important changes in immune regulatory pathways. For this reason, our group is currently focusing on the standardization of methodologies to identify changes in immunological markers without affecting other cellular mechanisms involved in cell survival: such as cell viability, necrosis, and apoptosis, which could interfere with the evaluation of the immune function. In vitro cultured murine SM1 cells were subjected to eight concentrations from clinical grade compounds – Tubastatin A, Nexturastat A, MS275 and LBH589 – to be analyzed within HDAC-Glo and ApoTox-Glo assays. The HDAC-Glo assay provided relative HDAC activity data, while the ApoTox-Glo assay specified the trends of Digitonin (viability control), Ionomycin (cytotoxicity control) and Mitomycin (apoptosis control). Upon inspection, the ApoTox-Glo recommended manufacturer controls compared to a pan-HDAC inhibitor LBH589 were deemed unreliable; LBH589 proved to be a universal control for the quantitative multiplexing protocol outlined. These results were then validated by Western blot protein analysis. Traditional approaches involve the time consuming and costly execution of individual assays. However, multiplex assays allow for quantifiable evaluation of anti-tumor HDACinh affects within cellular pathways by minimizing execution time and maximizing comprehensive statistical analysis of signal outputs from a spectrophotometer. Furthermore, an assay in tandem with our systematic high-throughput platform can accurately evaluate therapeutic doses and innovate treatments for melanoma clinical trials, along with other varieties of cancerous cell lines. The established reproducible and reliable protocol developed from this study will be important for identifying the dose range necessary to achieve immunological effects with minimal toxicity, thus improving the quality of life for treated subjects