The unreliability of MTT assay in primary cultured glioblastoma cells : A mechanism study

Abstract

MTT assay is commonly used to assess the cellular cytotoxicity caused by the anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay had non-specific intracellular reduction of tetrazolium to underestimate cellular cytotoxicity. The possibility whether MTT assay lead to incorrect information about alcohol-induced cytotoxicity on immortalized and primary cultured glioblastoma cells is defined in this thesis. MTT assay was applied to assess the ethanol-induced cytotoxicity by applying different concentration of 0.1%, 0.5%, 1%, 3% and 5% on immortalized and primary cultured glioblastoma cells. The cellular cytotoxicity on different doses of ethanol was compared with a cellular counting assay. Lactase dehydreogenase (LDH) assay, dead cell protease activity, reverse transcription polymerase chain reaction (RT-PCR), Western Blot Analysis and Cell Counting Kit-8 (CCK-8) assay (WST-8) is applied to evaluate the ethanol-induced cytotoxicity on immortalized and primary cultured glioblastoma cells. Ethanol-induced cytotoxicity is observed in MTT assay on immortalized and primary cultured glioblastoma cells in dose-dependent manner below a 3% ethanol concentration. However, the degree of cellular cytotoxicity is markedly underestimated or wrongfully increased especially in primary cultured glioblastoma cells at a concentration of 5% ethanol in comparison to the control concentration. However, According to dead cell protease activity and the Trypan blue exclusion assay, cytotoxicity of ethanol is increased dose-dependent manner in both cell types. RT-PCR and Western Blot Analysis also show an increase of pro-apoptotic protein expressions and a decrease of anti-apoptotic protein in ethanol dose-dependent manner. We confirm that ethanol have cytotoxicity effect and induce apoptosis in both immortalized cell line and primary glioblastoma cells. We presented possible mechanism for this unreliability of MTT assay result. High concentration of ethanol exposure induces more severe membrane damage on a relatively large primary cultured glioblastoma cells. Increasing the intracellular NADH concentration and membrane damage induced by ethanol can wrongfully enhance the nonspecific reduction of tetrazolium salt in primary cultured glioblastoma cells through the membrane damage. We suggest that the MTT assay can provide inaccurate information in the assessment of ethanol-induced cytoxicity in primary cultured glioblastoma cells. Thus, we have to carefully interpret the cytotoxicity results of a MTT assay on glioblastoma cells.