The Unreliability of MTT Assay in the Cytotoxic Test of Primary Cultured Glioblastoma Cells

Abstract

MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.

Novel biomolecule lycopene-reduced graphene oxide-silver nanoparticle enhances apoptotic potential of trichostatin A in human ovarian cancer cells (SKOV3)

“…Abbreviations: con, control; rgO-ag, reduced graphene oxide-silver; Tsa, trichostatin a; lDh, lactate dehydrogenase. 76 GO-AgNPs induced significant leakage of LDH and cytotoxicity than either AgNPs or GO alone in human ovarian cancer cells. 34 Similarly, GO-Ag nanocomposites were more toxic than pristine GO and pristine AgNPs to both tumoral macrophages such as J774 and peritoneal macrophages collected from BALB/c mouse, which significantly induced more ROS production compared to pristine AgNPs.…”

“…LDH activity was determined in the medium by measuring the absorbance at 490 nm using a Multiskan FC multiplate reader. A dead-cell protease activity assay was performed according to the method described by Jo et al 76 CytoTox-Glo cytotoxicity assay (Promega Corp., Fitchburg, WI, USA) was performed to examine dead-cell protease activity by measuring the rGO-Ag-induced cytotoxicity in SKOV3. The cytotoxicity assay was employed to evaluate the cytotoxic effects of rGO-Ag (0.2 µM) or TSA (0.2 µM) or combination of rGO-Ag (0.2 µM) and TSA (0.2 µM).…”

Cell-Type Specific Metabolic Response of Cancer Cells to Curcumin

“…We wanted to explore the effect of curcumin on both isoforms in four different cancer -originating cell lines. Special attention in this current research was dedicated to exploring the potential effect of curcumin and the solvent, absolute ethanol, which was shown as a strong modifier of the results of the MTT assays [29]. Dimethyl sulfoxide (DMSO), a commonly used solvent for curcumin, also was recently shown to be a strong modifier of cellular epigenome and, consequentially, a transcriptome in cardiomyocytes and hepatocytes [30].…”

Andiroba Oil (Carapa guianensis Aublet) Nanoemulsions: Development and Assessment of Cytotoxicity, Genotoxicity, and Hematotoxicity

“…AO and MO were diluted in ethanol, and AN and MON were diluted in water, all in 1%. According to Jo et al [21] ethanol exposures at concentrations lower than 3% do not cause interference in the cell viability in primary glioblastoma cells. In our study ethanol and water were used at 1% as control, which really did not show any evidence of cytotoxicity to NIH/3T3, and showed also similar result with DMEM medium.…”