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Tumor necrosis factor-alpha and expression of the multidrug resistance-associated genes LRP and MRP
U Stein1, W Walther, C M Laurencot
1Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
Background And Purpose:
Cancer cells that express P-glycoprotein, multidrug resistance-associated protein (MRP), or lung resistance protein (LRP) have demonstrated resistance to a wide variety of chemotherapeutic drugs. Recently, we reported that human colon carcinoma cells that express all three proteins exhibit reduced P-glycoprotein gene expression and a loss of multidrug resistance after exposure to tumor necrosis factor-alpha, a hormone-like protein produced by cells of the immune system. In this study, we examined the effects of tumor necrosis factor-alpha on MRP and LRP gene expression in the same colon carcinoma cells.
Methods:
HCT15 and HCT116 colon carcinoma cells were incubated with tumor necrosis factor-alpha at 100 U/mL for 2, 12, 24, 48, or 72 hours; alternatively, cells transfected with an expression vector containing a human tumor necrosis factor-alpha complementary DNA were studied. The effects of tumor necrosis factor-alpha on MRP and LRP messenger RNA expression were evaluated by means of reverse transcription and the polymerase chain reaction; effects on MRP and LRP protein expression were examined by use of specific monoclonal antibodies and flow cytometry. The flow cytometry data were analyzed by use of the two-sided, nonparametric Mann-Whitney rank sum test.
Results:
Treatment with exogenous tumor necrosis factor-alpha reduced the level of LRP messenger RNA in both cell types in an apparently time-dependent fashion; in HCT15 cells, almost no LRP messenger RNA was detected after 48 hours of treatment. In contrast, the level of MRP messenger RNA was increased in HCT116 cells by such treatment, but the level in HCT15 cells was unchanged. Treatment with exogenous tumor necrosis factor-alpha induced changes in LRP and MRP protein expression in the two cell types that paralleled the changes found for messenger RNA. In transfected cells, the endogenous production of tumor necrosis factor-alpha reduced LRP gene expression (both messenger RNA and protein) and increased MRP gene expression (both messenger RNA and protein), regardless of cell type.
Conclusion:
In human colon carcinoma cells, tumor necrosis factor-alpha influences MRP and LRP gene expression in opposite ways. The findings for LRP gene expression parallel our earlier findings for P-glycoprotein expression in these cells.
Implication:
In developing strategies for overcoming multidrug resistance in tumor cells, the possibility that an agent can suppress one or more mechanisms of drug resistance and enhance others should be considered.
Insights
Tumor necrosis factor-alpha differentially affects multidrug resistance genes in colon cancer cells. It reduces lung resistance protein (LRP) expression while increasing multidrug resistance-associated protein (MRP) expression, impacting chemotherapy resistance strategies.
Area of Science:
- Molecular Biology
- Cancer Research
- Immunology
Background:
- Multidrug resistance (MDR) in cancer is mediated by proteins like P-glycoprotein, MRP, and LRP.
- Tumor necrosis factor-alpha (TNF-α) is an immune-derived protein with potential roles in cancer therapy.
- Previous studies showed TNF-α reduced P-glycoprotein expression and MDR in colon carcinoma cells.
Purpose of the Study:
- To investigate the effect of TNF-α on MRP and LRP gene expression in colon carcinoma cells.
- To understand how TNF-α influences key MDR-related proteins in cancer.
Main Methods:
- HCT15 and HCT116 colon carcinoma cells were treated with TNF-α.
- Gene expression (mRNA) was analyzed using RT-PCR.
- Protein expression was assessed via flow cytometry with specific monoclonal antibodies.
- Cells with endogenous TNF-α production (transfected) were also studied.
Main Results:
- Exogenous TNF-α reduced LRP mRNA and protein levels in a time-dependent manner.
- MRP mRNA and protein levels increased in HCT116 cells but remained unchanged in HCT15 cells.
- Endogenous TNF-α production similarly modulated LRP and MRP expression.
Conclusions:
- TNF-α exerts opposing effects on MRP and LRP gene expression in colon carcinoma cells.
- LRP gene expression changes induced by TNF-α are consistent with prior P-glycoprotein findings.
- Therapeutic strategies against MDR should consider agents that modulate multiple resistance mechanisms.