CYP2E1 Activity in Cisplatin Resistant Cancer Cells.
(CP) is an anticancer drug that kills tumor cells by interfering with DNA replication.
One of the main obstacles limiting its use is intrinsic or acquired resistance
development. Reactive oxygen species (ROS) have a known role in cancer cell apoptosis
upon CP treatment. An excessive increase in ROS generation leads to other modes of cell
death such as necrosis. Microsomal ... read moreCytochrome P450 2E1(CYP2E1), known for its ROS
generation upon CP treatment, might be a mechanistic link to hepatotoxicity and
nephrotoxicity associated with CP exposure. The increase of metabolically active
microsomal CYP2E1 enzyme could generate more ROS with CP that also might be exploited to
sensitize resistant cancer cells to CP. Therefore, the aim of this study was to evaluate
the role of microsomal CYP2E1 on cancer cells exhibiting resistance to CP. Thus, the
main hypothesis was that microsomal CYP2E1 sensitizes CP-resistant extrahepatic and
hepatic cancer cells by generating ROS upon treatment with CP. Materials and Methods:
Two extrahepatic cancer cell lines, A2780/R and MOR/R, and the hepatic Hep G2 cell line,
which are resistant to CP, were transfected with microsomal CYP2E1 cDNA and treated with
CP. High performance liquid chromatography (HPLC) was used to measure the metabolic
activity of microsomal CYP2E1 using chlorzoxazone (CHZ) hydroxylation as an index
reaction. Fluorescence activated cell sorting (FACS) measured ROS in transfected cells.
The microculture tetrazolium viability assay (MTT assay) was used to evaluate the effect
of CP treatment with different concentrations on transfected cell death. Results: An
increase in CYP2E1 mRNA expression occurred in all transfected cells. However, no CYP2E1
metabolic activity was detected in extrahepatic cells when compared to microsomal
CYP2E1-transfected Hep G2 cells. There were no significant changes in cell viability
among Hep G2 cells transfected with microsomal CYP2E1 when compared to controls. In
conclusion, despite an increase in ROS with exposure to CP in Hep G2 cells transfected
with CYP2E1, the cells tolerated the changes in ROS and did not demonstrate any change
in CP sensitivity. Extrahepatic CP-resistant cancer cells lack some essential cellular
biology to support active microsomal CYP2E1
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Pharmacology & Experimental Therapeutics.
Advisor: David Greenblatt.
Committee: Margery Beinfeld, Martin Beinborn, Jeffrey Blumberg, and Michael Court.
Keywords: Pharmacology, and Chemical engineering.read less