title: 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.
authors.affiliation: Department of Neurosurgery, Seoul National University College of Medicine, Seoul 03082, Korea. ; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03082, Korea. ; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 03082, Korea.
authors.forename: Hwa Yeon
authors.initials: HY
authors.lastname: Jo
authors.affiliation: Department of Neurosurgery, Seoul National University College of Medicine, Seoul 03082, Korea. ; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03082, Korea. ; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 03082, Korea.
authors.forename: Yona
authors.initials: Y
authors.lastname: Kim
authors.affiliation: Department of Neurosurgery, Seoul National University College of Medicine, Seoul 03082, Korea. ; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03082, Korea. ; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 03082, Korea.
authors.forename: Hyung Woo
authors.initials: HW
authors.lastname: Park
authors.affiliation: Department of Neurosurgery, Seoul National University College of Medicine, Seoul 03082, Korea. ; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03082, Korea. ; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 03082, Korea.
authors.forename: Hyo Eun
authors.initials: HE
authors.lastname: Moon
authors.affiliation: Department of Neurosurgery, Seoul National University College of Medicine, Seoul 03082, Korea. ; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03082, Korea. ; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 03082, Korea.
authors.forename: Seongtae
authors.initials: S
authors.lastname: Bae
authors.affiliation: Department of Neurosurgery, Seoul National University College of Medicine, Seoul 03082, Korea. ; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03082, Korea. ; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 03082, Korea.
authors.forename: JinWook
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Neurosurgery, Seoul National University College of Medicine, Seoul 03082, Korea. ; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03082, Korea. ; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 03082, Korea.
authors.forename: Dong Gyu
authors.initials: DG
authors.lastname: Kim
authors.affiliation: Department of Neurosurgery, Seoul National University College of Medicine, Seoul 03082, Korea. ; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03082, Korea. ; Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 03082, Korea.
authors.forename: Sun Ha
authors.initials: SH
authors.lastname: Paek
keywords: Tetrazolium salts; ethanol; non-specific intracellular reduction; primary cell