Aconitine Reverse The Drug Resistance Of Human Oral Squamous Cancer Cells Molecular Mechanisms

1 Objective To take the drug resistant human oral squamous epithelium cancer cell(KBv200) as the target cell, to take the aconitine as the experimental drug,to explore the mechanism of drug resistance of aconitine to tumor through observing its cytotoxic effects on cell,detecting the protein expression of P170 of KBv200 by flow cytometry and immunohistochemical methods and gene chip technique. 2 Methods To test the cytotoxic effects of vincristine(VCR) and aconitine to KBv200 and calculate the corresponding IC50 through MTT method; to observe the aconitine concentration at which its inhibition rate to KBv200 is lower than 30%,the cytotoxic effect to KBv200 of aconitine accompanied with VCR,to calculate the sensitization index according to the IC50 of VCR and VCR plus aconitine. After the aconitine with the effect of reversing KBv200 is proved, the Pgp expression on the membrane of KBv200 treated by different doses of aconitine tested by flow cytometer and immunohistochemical methods. The gene expression spectra after treating by aconitine is tested by gene chip technique. 3 Results After the experimental study,the positive results are as follows: ① under the condition of cell culture in vitro,the MTTmethod experiment showed that the IC50 of aconitine at dose of 6.25μg/ml associated with VCR and aconitine at dose of 12.5μg/ml associated with VCR were 0.9185ug/ml,0.2715 ug/ml respectively,the reverse rates were 2.64 and 8.9 respectively. ② the flow cytometer methods experiment showed that the positive cell rate of KBv200 was 43.1%; after adding the aconitine at the dose of 6.25μg/ml and 12.5μg/ml and being cultured after 16h, the positive cell rates were 21.2% and 9.11% respectively. ③ the immunohistochemical study indicated that the brown particles of the positive cells were mainly on the cell membrane and cytolymph. The positive cell rate of the control group was 96%,while the group treated with aconitine at the dose of 6.25μg/ml,12.5μg/ml had the rates of 83% and 30% respectively. ④ the fluorescence signal scan of the gene chip showed that among the 5504 gene clones of the whole chip,there were 208 gene with high expression before treating with the drug,which accounted for 3.8% of the whole clones. There were 5100 gene whose expression without difference after treating with the drug compared with that before using the drug,which accounted for 92.6%. There were 196 gene with high expression after treating with drug,which accounted for 3.6%. after the aggregate analysis,we could see that the effecting target of the aconitine is defined on CDKs and its related genes,c-myc,p53,bcl-2 and related genes,meanwhile,the drug resistance was decreased resulting from the expression of the MDR-1 through effecting the signal transduction system of MAPK. 4 Conclusion The aconitine has the effect of reversing the multi-drug resistance of KBv200 cell. The drug resistance mechanism is related to that the aconitine can decrease the expression of the Pgp protein,accelerate the apoptosis of the tumor cell,effect the signal transduction of MAPK.