| Multidrug resistance (MDR) describes the cross-resistance of tumor cell lines to several structurally unrelated chemotherapeutic agents after exposure to a single cytotoxic drug. Mechanism of MDR of tumor cells is complicated, and it is related with various factors and processes, and more and more researchers focus on it. This phenomenon is often associated with overexpression of several proteins, such as P-glycoprotein (P-gp), multidrug resistance- associated protein (MRP), lung resistance-related protein (LRP), breast cancer resistance protein (BCRP). Among them Pgp and MRP are the most important proteins that belongs to the ABC superfamily of transporters which acts as a drug efflux pump. All of these proteins pump out various structurally unrelated anticancer agents in an ATP dependent manner and decrease the intracellular concentration of anticancer agents in multidrug-resistant cells, mediated resistance to anticancer agents. Additionally, overexpression of genes such as glutathione-S-transferase-pi (GST-π) and O6-methylguanine DNA methyltransferase (MGMT) may contribute to the development of the drug resistant phenotype of tumor cells to anti-neoplastic drugs. GST-πcatalyzes the interactions between glutathione and alkylating drugs, increases the rate of drug detoxification, and plays a significant role in the resistance of cancer cells to a wide range of endobiotic and xenobiotic electrophilic compounds. MGMT is a rapid and error-free DNA repair enzyme that eliminates the alkylating lesion of O6-methylguanine in DNA, and it can protect tumor cell from injury of cytotoxic drug. The overexpresion of anti-apoptotic protein Bcl-2 can resist to apoptosis induced by drugs, so it maybe related to MDR. In addition, NF-κB is a kind of nuclear transcription protein that exist in all of the cell in body. It took part in the regulation of inflammation, immunity and stress. Recently it was found that it played a important role in cell proliferation and regulation of apoptotic related gene. So, it is also related to MDR.Angiotensin II (Ang II) is a main effector molecule of rennin– angiotensin system (RAS), and participates in the regulation of blood pressure and water hemostasis in the body. As a potent mitogen, it has been proven that Ang II participates in the processes of cell proliferation, inflammation and angiogenesis of tumor. Until now, there is not report about relationships between RAS and MDR, and it still needs to be study whether RAS regulates genes or proteins of multidrug resistance related proteins of tumor.Objective:In this study, human acute promyelocytic leukemia (APL) cell line K562 and doxorubicin (DOX) resistant APL cell RK562 was treated with DOX and cisplatin (DDP), and it was determined that resistance index, the concentration of Ang I and Ang II in culture medium, the expression of multidrug associated proteins, apoptosis associated proteins, and Nuclear factor-Kappa B (NF-κB)in both kind of cell line, and it was discussed that mechanism of MDR.Methods:1. Cell morphology was examined using phase-contrast microscope and the inhibition rate of cell was analyzed by MTT method.2. The concentration of Ang I and Ang II in culture medium was analyzed by radioimmunoassay method.3. The expression of P-gp, MRP1, Bcl-2 and NF-κB(p65) was determined with Western blotting method.4. The expression of MDR1, MRP1, MGMT and GST-πmRNA was determined with RT-PCR method.Results:DOX and DDP can inhibit the proliferation of K562 and RK562 cell. IC50 of DOX on K562 and RK562 is 3.21μg/ml, 91.5μg/ml respectively, the resistant index of RK562 cell to DOX is 28.5. IC50 of DDP on K562 and RK562 is 22.97μg/ml, 38.4μg/ml respectively, the resistant index of RK562 cell to DDP is 1.67.The results of radioimmunoassay showed that the concentration of Ang I and Ang II in culture medium increased in the group treated with or without DOX but not in the group treatment with DDP.The results of RT-PCR demonstrated that the expression MDR1, MRP1 mRNA in RK562 cells was higher than that in K562 cells, and their level did not change after DOX or DDP administration. The expression of MGMT mRNA in RK562 cells increased when compared with that in K562 cells, the expression of in K562 but not in RK562 cells was inhibited with DOX, DDP slightly increased the expression of MGMT mRNA in K562 but not in RK562 cells.The results of Western blotting showed that P-gp and Bcl-2 increased in RK562 cells. P-gp level did not change after DOX and DDP administration. DDP induced the increased expression of MRP1 in RK562 cells. NF-κB p65 increased in RK562 cells after DOX and DDP administration,Discussion:The resistant index of RK562 cell to DOX is 28.5, and the resistant index of RK562 cell to DDP is 1.67. It suggested that RK562 is not only resistant to DOX but also resistant to other antitumor drug. There were difference between DOX and DDP in affecting multidrug associated proteins expression, it suggested that the mechanism of RK562 resistant to tumor is different. Ang II participates in the processes of proliferation of tumor besides participating in the regulation of blood pressure and water hemostasis in the body. The concentration of Ang I and Ang II in culture medium increased in the group treated with or without DOX. The expression of Bcl-2 and p65 increased in RK562 cells after DOX and DDP treatment. Angiotensinogen can convert into AngI by rennin, then convert into AngII by ACE. So we supposed that the increased Ang II in tumor cells or local tumor tissue activated and upregulated NF-κB, and then NF-κB induced the Bcl-2 expression, inhibited apoptosis of tumor cells through Bcl-2 signal pathway. Moreover upregulated NF-κB resulted in the rise of P-gp expression in tumor cells, and Ang II can directly upregulate the MRP1 expression to induce multidrug resistance of tumor cells. The change of MGMT mRNA may be involved in the self protection of tumor cells from injury of cytotixic drug.Conclusion:1. Increased expression of MDR, MRP and MGMT was involved in the multidrug resistance of tumor cells resistant to antitumor drug. The resistant index of RK562 cell to DOX is 28.5, and the resistant index of RK562 cell to DDP is 1.67. It suggested that RK562 is not only resistant to DOX but also resistant to other antitumor drug.2. There were difference between DOX and DDP in affecting MRP and MGMT expression, it suggested that the mechanism of RK562 resistant to tumor is different.3. Bcl-2 antiapoptotic proteins upregulated via NF-κB activation inhibited apoptosis of tumor cells, and may participates in the MDR formation of tumor cells.4. Increased NF-κB initiates and regulates the expression of proteins related with MDR and apoptosis.5. The concentration of AngII in RK562 cell culture medium induced by DOX is increased. It suggested that Ang II may participate in the regulation of proliferation, antiapoptosis and multidrug resistance of tumor cells via NF-κB signal pathway.
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