THE EFFECTS OF ACIDIC TUMOR MICROENVIRONMENT ON LYMPHOMA CELL RESPONSES TO CHEMOTHERAPEUTICS

Abstract

Acidic tumor microenvironment exists in many types of cancer. Altered glycolytic metabolism of tumor cells and deficient blood supply in tissues are major causes for this phenomenon. Lymphoma cells may have different responses to chemotherapeutics when they are exposed to acidic tumor microenvironment. Bortezomib (BTZ) is a chemotherapeutic drug already approved by FDA for treating multiple myeloma and mantle cell lymphoma, which can inhibit the 26S proteasome and block the protein degradation process. Ramos parental cells (Human Burkitt's lymphoma cell line) and BTZ-resistant Ramos cells (obtained after chronic selection using low concentration of BTZ) were used in the study. Cells were treated from 2h to 24h with RPMI culture media buffered to pH 7.4 and pH 6.4, with or without chemical therapeutics. Cancer therapeutics tested in this study were BTZ, Cyclophosphamide, Doxorubicin, and ABT-737. MTT assay was utilized to detect cell viability under each treatment condition. Flow cytometry was used to examine changes on apoptosis and cell cycle. Western blots were performed to measure the changes of cellular protein levels. The MTT assays showed the acidic microenvironment could decrease cell proliferation rate, and if combined with BTZ treatment it could notably increase the cytotoxic efficacy of BTZ. After chronic selection under low concentration of BTZ, BTZ-resistant Ramos cells were obtained and were able to survive and grow in the presence of BTZ. However, acidic pH with BTZ could sensitize the BTZ-Resistant Ramos cells to BTZ again. Apoptosis assays demonstrated that acidic pH itself did not significantly induce cell apoptosis within 24h. But the combination of acidic pH with BTZ treatment caused much more apoptosis than BTZ alone. Cell cycle analysis showed that acidic pH could block the cell cycle, which led to cell cycle arrest and reduced percentage of cells in the G2/M phase. Western blots illustrated that acidic pH alone could upregulate the level of p53, Phospho-p53 at Ser15, Phospho-CHK1, Phospho-CHK2, pro-apoptotic proteins of the mitochondrial Bcl-2 family, and the pro-apoptotic Caspase family. In addition, if combined with BTZ treatment, acidosis could further increase phosphorylation of p53 at Ser15, expression of the Bcl-2 family proteins, and level of cleaved caspases. In conclusion, the combination of acidosis and BTZ treatment induce higher level of apoptosis and decrease Ramos cell proliferation. Our study demonstrates that arrested cell proliferation and increased apoptosis occur by the inhibition of cell cycle regulators and the activation of p53-mitochondria-caspase apoptosis pathways respectively.