Anti-cancer drug-induced mitochondrial alteration becomes a limitation of metabolic viability-based MTT assay in analyzing growth inhibition

Abstract

Abstract Background High throughput metabolic viability-based colorimetric MTT assay is widely used for cytotoxicity screening of various chemical compounds, anti-neoplastic drugs, and other chemotherapeutic agents. The yellow MTT tetrazolium salt reduces to purple formazan crystals, predominantly by mitochondrial dehydrogenases. The assay assumes all cells have a similar number of mitochondria with equivalent enzymatic activity, resulting in a linear relationship between colorimetric absorbance and cell number. Method Our present study involved the Cisplatin, Etoposide, and Doxorubicin-induced cytotoxicity evaluation using MTT and cell number enumeration in two widely used cancer cell lines, namely human lung epithelial adenocarcinoma cells (A549) and cervix carcinoma (HeLa). Further, Mitochondrial mass was examined to comment on the treatment-induced change in metabolic viability-based MTT assay. Results Drug-induced cell death determined by enumeration of the cell number did not correlate with growth inhibition observed by the MTT assay. Increased protein levels of majorly MTT converting enzyme SDH in both the cell lines following drug treatment were observed. The mitochondrial protein content of the cells was also found to be elevated in response to drug-induced cytotoxic stress. Conclusion In line with our earlier observation about the limitation of MTT assay in estimating radiation-induced cytotoxicity, it was found that certain anti-neoplastic drugs also modulate mitochondrial biogenesis and SDH expression level and enzymatic activity. Therefore, caution should be taken in applying the MTT assay to analyze drug-induced growth inhibition. General significance: Our findings reveal the MTT assay's limitations, which should be considered when determining anti-cancer and chemotherapeutic drugs' pre-clinical cytotoxicity and IC-50.