Drug Resistance Mechanisms in Cancer Cells

INTRODUCTION

A major obstacle in the successful treatment of cancer is the development of resistance mechanisms to drug treatment. Various cellular changes that have been implicated in the development of drug resistance in cancer cells include: the increased expressions of P-glycoprotein (a multidrug transport protein) and multidrug resistance-associated protein (MRP-1); increased levels of the cellular detoxification protein, glutathione; and changes in the expression of apoptosis associated proteins such as Bcl-2, FasL and p53, which generally results in promoting cell survival [1,2,3,4].

P-glycoprotein expression is the mechanism mostly associated with classical multidrug resistance (MDR), as this protein is responsible for the active transport of various drugs out of the cell, thereby decreasing drug accumulation [1,3]. Not much is known in regards to how MRP-1 confers multidrug resistance, a notable exception is that its expression has been associated with a decrease in drug accumulation similar to P-glycoprotein [1]. Glutathione is an important cellular antioxidant and detoxification protein, and its increased levels have been observed to inactivate various chemotherapeutic drugs [2]. Bcl-2 is a known inhibitor of apoptosis and its cellular overexpression has been observed to increase resistance to drugs whose mechanism of action involve apoptotic initiation [2]. FasL (Fas ligand), upon binding, induces apoptosis in cells expressing Fas receptors. As such, FasL interaction is a characteristic mechanism by which cytotoxic T lymphocytes and particular chemotherapeutic agents induce apoptosis [3]. Decreased expressions of FasL mediated apoptosis have been observed to be associated with cell sublines brought up to confer resistance to these chemotherapeutic agents [3]. P53 also induces apoptosis and mutations and alterations in the gene encoding p53 are the most frequent abnormalities detected in malignancies and has been observed to contribute to chemotherapeutic resistance as well [4].

The aim of this experiment is to experimentally observe drug resistant mechanisms associated with leukaemia T-cells (CEM) and in two drug resistance sublines: CEM cells brought up to confer resistance to vinblastine (CEM/VLB100) and cisplastin (CEM/CP800). Observation of the expression of different apoptotic proteins will be conducted by SDS Polyacrylamide Gel Electrophoresis and Western Blot analysis, while cellular glutathione content will be estimated by enzymatic assay. Additionally, observation of drug accumulation of cells by flow cytometric analysis will be conducted to assist in illustrating any involvement that a particular drug resistance mechanism may have within the drug resistant cell sublines employed.

METHODS AND MATERIALS

The materials and methods employed as per UTS 91345 Biochemistry, Genes and Disease (Spring 2004) Experimental Practical Handout entitled 'Drug Resistance Mechanisms In Cancer Cells,' pages 1 - 7.

RESULTS

Experiment A: Western Blot Analysis of proteins associated with apoptosis

Figure 1 - Western blot detecting the presence of Bcl-2 protein. Protein content of cells from a particular cell line (CEM, CEM/VLB100 and CEM/CP800) was extracted, separated by SDS-PAGE, and Bcl-2 expression detected by incubation of a nitrocellulose blot with monoclonal anti-Bcl-2 antibody. Results are blots conducted in duplicate, separated by molecular weight markers for protein size estimation.

Figure 2 - Western blot detecting the presence of FasL protein. Protein content of cells from a particular cell line (CEM, CEM/VLB100 and CEM/CP800) was extracted, separated by SDS-PAGE, and FasL expression detected by incubation of a nitrocellulose blot with monoclonal anti-FasL antibody. Results are blots conducted in duplicate, separated by molecular weight