| Objective: This study make the experimental study by selecting human oral squamous cell carcinoma cell line KB/ADM and its parental cell line KB in the previous task group,and studying the reversal effect of nitidine chloride(NC)on KB/ADM cells and the expression of related genes and key proteins in PI3K/Akt/mTOR signaling pathway by NC,and meanwhile,exploring whether NC reversal is related to PI3K/Akt/mTOR signaling pathway.Methods:(1)Culture and identification of multi drug resistant cells of human oral squamous cell carcinoma:(1)Determination of sensitivity of KB/ADM and KB cells to chemotherapeutic drugs through the methods of MTT,preparation of adriamycin(ADM)and etoposide(VP-16)and vincristine(VCR),and each has six concentration group,which were 200,100,50,25,10 seperately,5mg/L as well as the calculation of IC50 and multiple drug resistance cells;(2)The expression of MDR1 gene was detected by Real-time PCR;(3)The expression of P-gp protein was detected by flow cytometry.(2)The study on reversal of multidrug resistance in human oral squamous cell carcinoma by NC:(1)Determination of resistance reversal effect of NC on the KB/ADM through MTT method,the experiment was divided into KB blank control group,KB+VP-16 group,KB/ADM blank control group,KB/ADM+VP-16 group,KB/ADM+VP-16+VRPgroup,KB/ADM+VP-16+NCgroup and drug group,each with 5 different concentrations(200,100,50,25,10 mg/L),and to calculate the cell’s IC50 and multiple drug resistance;(2)In vitro scratch test,whose grouping method is the same as with "(2)(1)",is to calculate cell migration rate;(3)The Transwell invasion experiment,whose grouping method and dosing mode is the same with "(2)(2)",the inverted microscope through the Transwell chamber at the end of the film has been stained cells,were photographed at ramdom and been counted;(4)Flow cytometry was used to detect the apoptosis rate of KB/ADM and KB cells by NC;(5)Detection of expression of Bax,Bcl-2 and Survivin by Real-time PCR.(3)The effect of NC on the expression of PI3K/AKT/mTOR pathway related genes and proteins:(1)Effects of NC on the expression of P53 and PTEN in Real time PCR;(2)The effect of NC on the expression of AKT[Y89],AKT(S473),mTOR,4EBP1[Y329],4EBP1(T37),S6K1[E175] and S6K1(S235 +S236)protein by Western blot.Results:(1)(1)According to the experimental results,the KB/ADM cells’ IC50/mg·L-1 of the three chemotherapeutic drugs on ADM、VP-16、VRP were(158.2±1.21),(73.21±4.83)and(84.48±2.48),the KB cells’ IC50/mg·L-1 of the three chemotherapeutic drugs on ADM、VP-16、VRP were(44.72±3.08)、(38.78±3.56)and(36.53±0.73),the resistance of KB/ADM cells to ADM,VP-16 and VRP were 3.54,1.89 and 2.42 respectively;(2)The relative expression of MDR1 gene in KB/ADM and KB cells was(1.01±0.03)、(0.22±0.01);(3)The relative expression of P-gp protein in KB/ADM and KB cells(95.58±3.03),(0.23±0.05).(2)(1)The results of drug resistance test: the IC50/mg·L-1 of KB/ADM+ VP-16 group,KB/ADM+VP-16+VRP group and KB/ADM+VP-16+NC group were(121.14±8.31)、(34.75±1.36)、(7.37±1.30)respectively,the reversal index KB/ADM+VP-16+VRP group and KB/ADM+VP-16+NC group were 3.49 and 16.44,respectively;(2)Scratch after 48 h,the migration rate of sensitive cell groups were(30.51±0.88)%,(40.89±0.05)% and(23.17± 1.49)%,and there was significant difference between KB group and blank group(P<0.01);drug-resistant cell groups were(29.83±1.78)%、(28.05±1.05)% 、(17.45±1.82)% and(14.12±0.26)%,compared with KB/ADM control group with significant difference(P<0.01);(3)KB 25mg/L VP-16 group(27.70± 0.76)and KB100mg/L VP-16 group(17.50±0.50)respectively compared with KB blank group(85.80±1.68),the difference was statistically significant(P<0.01);KB/ADM25mg/L VP-16 dosing group(59.20±2.60),KB/ADM25 mg/L VP-16+VRP group(47.10±4.24)and KB/ADM25mg/LVP-16+NC group(29.9±3.10)in comparison with KB/ADM blank group(100.00±1.50),the difference was statistically significant(P<0.01);(4)Adding 48 h after treatment by flow cytometry,the apoptosis rates were(0.87±0.41)% 、(7.00±1.40)%、(24.37±4.92)%、(0.33±0.21)%、(7.97±2.90)%、(9.57±4.97)% and(48.37±7.45)%;(5)The relative expression of the bax gene in the resistance group was significantly different(P<0.01),and showed an upward trend,and the expression level of Bax gene was significantly increased in the sensitive cell groups;Under the premise of control group,the expression of Bcl-2 gene was significantly down regulated,but the expression of the sensitive strains was not statistically significant.Compared with KB/ADM group,the expression of Survivin gene in the VRP group and NC group was decreased,and the difference was statistically significant(P<0.01).(3)(1)The relative expression of PTEN gene,the expression level of PTEN in sensitive cell groups were significantly high,and there was no significant difference;in the resistant groups showed an upward trend(P<0.01).The relative expression of P53 there was no statistical significance.In the resistant group were up-regulated(P<0.01,P<0.05,P<0.01);(2)Non phosphorylated Akt,expressed in the sensitive group increased and the difference have no sense of statistics,the resistance groups except the KB/ADM25mg/L VP-16+NC group were increased,the difference is statistically significant(P<0.01),the phosphorylated Akt expressed in drug-resistant group there was no statistically significant difference in turn reduce,sensitive to low dose group had significant difference(P<0.05);mTOR was significantly different in KB/ADM25mg/L VP-16+NC group(P<0.01);non-phosphorylated 4EBP1 was not statistically significant in each group,and the phosphorylated 4EBP1 was significantly different in the VRP and NC groups(P<0.01,P<0.01);non-phosph orylated S6K1 decreases in the expression of the sensitive cell groups(P<0.01,P<0.01),phosphorylation S6K1 in sensitive cell expression in high dose group have statistical significance(P<0.01),The resistance cell group expression decreased in turn(P<0.01,P<0.01,P<0.01).Conclusion:(1)KB/ADM cells were identified as MDR cell lines,which can be used as MDR tumor cell model for experimental study;(2)NC can reverse the effect of KB/ADM on MDR cells,Bax,Bcl-2,Survivin and the apoptosis gene of may be related to the apoptosis of KB/ADM cells induced by NC;(3)the mechanism by which NC reverses KB/ADM MDR may not be mediated by inhibition on PI3K/Akt/mTOR signaling pathway. |
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