Reversal Effect and Mechanisms of Recombinant Human Tumor Necrosis Factor-NC Against the Doxorubicin Resistance in Leukemia K562/Doxorubicin Cells

Objective: To explore the reversal effect and mechanisms of recombinant human tumor necrosis factor-NC (rhTNF-NC) against the doxorubicin (Dox) resistance in chronic myelogenous leukemia (CML) K562/Dox cells. Methods: The chemo-sensitivity of tumor cells dealt with different concentrations of rhTNF-NC to Dox was detected by tetrazolium dye assay (MTT). The intra-cellular Dox accumulation represented by fluorescence intensity was determined by flow cytometry (FCM) at the excitation wave length of 488 nm and emission wave length of 550 nm. The expression of multidrug resistance (MDR)-related genes and proteins was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Results: After being exposed to gradually increasing concentrations of Dox for 10 consecutive months, K562/Dox cells were more resistant to Dox (nearly 132 times) than Dox-sensitive K562 cells. The IC50 of Dox for K562 and K562/Dox cells were (0.04 +/- 0.01) and (5.55 +/- 0.08) mu mol/L, respectively. When K562/Dox cells were treated with rhTNF-NC at 500, 2 500 or 5 000U/mL, the IC50 of Dox was decreased to (2.22 +/- 0.34), (1.41 +/- 0.13) and (1.04 +/- 0.09) mu mol/L, respectively. The concentration-response curves were moved upward by the treatment of rhTNF-NC (P< 0.01). FCM analysis displayed that intra-cellular accumulation of Dox was significantly increased when combing Dox with rhTNF-NC. After treatment with rhTNF-NC, the expression of MDR gene (MDR1), MDR-associated protein (MRP), glutathione S transferase pi (GST pi) mRNA, P glycoprotein (P-gp) and protein kinase C alpha (PKC alpha) protein was down-regulated, while topoisomerase II alpha (TopoII alpha) mRNA expression was up-regulated. Conclusion: rhTNF-NC can effectively augment the drug accumulation in tumor cells. This is due to the up-regulation of TopoII alpha and down-regulation of MDR1, MRP and GST pi at mRNA expression as well as reduction of P-gp and PKC alpha expression.