Reversal of multidrug resistance by transduction of cytokine genes into human colon carcinoma cells

Abstract

Background: Multidrug resistance can be a major obstacle to successful cancer chemotherapy and is often associated with increased expression of the mdr1 (also known as P-glycoprotein) gene. Some of the proteins produced by the body's immune system, i.e., cytokines such as tumor necrosis factor-α (TNF) and interleukin 2 (IL-2), have been shown to modulate multidrug resistance. However, cytokines administered by the conventional intravenous method can cause severe side effects. Transduction of cytokine genes into tumor cells constitutes an alternative approach for production and release of the cytokine proteins in the local tumor microenvironment, which may reduce problems of toxicity associated with systemic administration. Purpose: In this study, we investigated the therapeutic potential of a combination of gene therapy and chemotherapy on the basis of cytokine-mediated modulation of multidrug resistance in human colon carcinoma cells. Methods: Human colon carcinoma cell lines HCT15 and HCT116 were transduced with TNF or IL-2 carrying murine leukemia virus (MLV)-based retroviral vectors. Tumor cell clones were analyzed for cytokine expression by reverse transcriptase- polymerase chain reaction (RT-PCR) and by cytokine-specific enzyme-linked immunosorbent assays (TNF-ELISA or IL-2-ELISA). Expression of mdrl messenger RNA (mRNA) was investigated using RT-PCR, and P-glycoprotein (Pgp) expression was determined by immunoflow cytometry with the monoclonal antibodies MRKI6 and C219. The function of Pgp was analyzed by measuring accumulation of the fluorescent drug doxorubicin by flow cytometry. The XTT-(i.e., [2,3-bis(2- methoxy-4-nitro-5-sulfophenyl)]-5-[(phenylamino)-carbonyl-2H-tetrazolium hydroxide]-colorimetric cytotoxicity assay was used to determine chemosensitivity of cytokine gene-transfected tumor cells to doxorubicin and vincristine. Statistical significance was determined by the nonparametric Mann-Whitney rank sum test for the flow cytometry experiments (Pgp detection as well as drug uptake assays) and the parametric Student's t test for the chemosensitivity assay (XTT cytotoxicity assay). All P values reported were derived from two-sided statistical tests. Results: Transduction and expression of human TNF and IL-2 in HCTI5 and HCT116 human colon carcinoma cell lines were found to reverse multidrug resistance. Both TNF and IL-2 secretion reduced mdrl expression on the mRNA and Pgp levels (P