Effect Of Chloride Channel Blockers NPPB In The Cell Injury Of Human Erythroleukemia Cell Line Induced By DDP

Multidrug resistance (MDR) of tumor cell is a major obstacle during the treatment of tumor, especially during the tumor of blood system. Cisplatin (DDP) as one of the most important chemotherapeutics of current clinical using, its inherent or acquired drug resistance reduces its efficacy.Studies have suggested that the ATP-binding cassette transporter superfamily plays an important role in the process of tumor multidrug resistance. Researches have pointed out that the chloride channel as a class of very important anion channel in vivo exerts specific biological functions such as cell volume-regulated functions and endosome - lysosome vesicle acidification and so on. Thereby the chloride channel is involved in the regulation of cell micro-environment. Studies have also indicated that multi-drug resistance protein P-gp may be acted as chloride channels regulator, and it is indicating the relationship between chloride channels and multidrug resistance.K562/DOX cell line is one of the classic multidrug resistance leukemia cell lines which is derived from K562 cells induced by doxorubicin (DOX) long-term. Not only do the K562/DOX cells have a strong resistance to doxorubicin but also have cross-resistance to different clinical commonly used antineoplastic agents which have other chemical structure and mechanism, end the cell line is an important cell line to study the mechanism of multidrug resistance of leukemia and reversal drugs.Objective:Using human erythroleukemia cell line K562 and doxorubicin -resistant human erythroleukemia cell line K562/DOX, we detect resistance index, resistance-associated protein, apoptosis-related protein, chloride channel ClC-3 expression which treated with cisplatin and/or NPPB to explore the related mechanisms between the cisplatin-resistant and the chloride channel. Method:1. Cell culture;2. The viability of cells was measured by MTT assay;3. mRNA levels detectting of MDR1,MRP1,Cyclin D1,ClC-3 by RT-PCR;4. Protein levels detecting of P-gp,Bax,Bcl-2,Cyt-c,caspase-3 by Western blotting;5. Mitochondrial membrane potential and the rate of apoptosis detecting of both K562 cell lines and K562/DOX cell lines by flow cytometry;6. Detecting of endosome - lysosome vesicle acidification by acridine orange.Result and Discussion:1. MTT assay results show cell vitality of K562/DOX treated with cisplatin is increased from the value of the K562 cells suggesting that K562/DOX cell line has cross-resistant to cisplatin.2. MTT assay results show that cisplatin can significantly reduce the vitality of the K562 cells and K562/DOX cells. The vitality of the K562 cells and the K562/DOX cells is significantly increased after treated with NPPB and cisplatin. It is clear that NPPB induces the resistance to cisplatin on K562 cells and K562/DOX cells.3. MTT assay results show that NPPB can not affect the survival rate of K562 cells treated with DOX, VK₃ and H₂O₂. It is indicated that NPPB can affect the cytotoxicity role of some drugs in a certain extent but not widely.4. Western blotting and RT-PCR results show that the expression of multidrug resistance gene MDR1 and its encoded protein, P-gp is decreased when treated with cisplatin only in both cell lines. And the expression is up regulated when treated with cisplatin and NPPB indicating the relationship between P-gp and chloride channel. However there is no difference of the cisplatin resistant role of NPPB between two cell lines indicating that NPPB is not primarily through the regulation of P-gp activity to enhance the cell survival rate.5. Western blotting and RT-PCR results show that using the cisplatin alone increases the ratio of Bax/Bcl-2, the expression of cytochrome C and caspase-3, and decreases cell cycle protein Cyclin D1. Flow cytometry results show that the mitochondrial membrane potential of both cell lines are downregulated and the rate of apoptosis of both cell lines are upregulated. It indicated that DDP mainly through the apoptosis pathway to inhibite both cell lines proliferation.6. Western blotting and RT-PCR results show that using both cisplatin and NPPB decreases the ratio of Bax/Bcl-2, the expression of cytochrome C and caspase-3, and increases cell cycle protein Cyclin D1. Flow cytometry results show that the mitochondrial membrane potential of both cell lines are upregulated and the rate of apoptosis of both cell lines are downregulated. It is indicated that NPPB can protect both cell lines from apoptosis induced by cisplatin.7. The results show that using both cisplatin and NPPB can upregulate the expression of ClC-3 and acidification of endosome - lysosome vesicle. It is indicating the relationship of the ClC-3, endosome - lysosome vesicle acidification and the cisplatin-resistance role of NPPB.Conclusion:1. NPPB can reduce cytotoxicity of cisplatin without effecting cytotoxicity of DOX, VK3 and H2O2. It is indicated that NPPB can affect the cytotoxicity role of some drugs in a certain extent but not widely.2. Cisplatin can induce cell injury of both K562 cell line and RK562 cell line in different extent, and NPPB can reverse the cell injury induced by cisplatin.3. Chloride channel blocker NPPB can upregulate the expression of P-gp indicating the relationship between NPPB and chloride channel. However there is no difference of the cisplatin resistant role of NPPB between two cell lines indicating that NPPB is not primarily through the regulation of P-gp activity to enhance the cell survival rate.4. NPPB can reverse the changes of apoptotic factors, mitochondrial transmembrane potential, expression of ClC-3 and endosome - lysosome vesicle acidification. It is indicated that NPPB can break apoptotic volume decrease and block the cell apoptosis to regulate the cell micro-environment.