Effect Of Polyene Phosphatidylcholine On The Sensitivity Of Oxaliplatin In Human Gastric Cancer Cell

BackgroudChina is high incidence area of gastric cancer.gastric cancer is endangering the lives of the people.Chemotherapy plays an important role in the comprehensive treatment of gastric cancer,but the efficiency of chemotherapy is one of the important factors affecting the prognosis of patients with gastric cancer.Oxaliplatin(L-OHP)is a third-generation derivative of platinum-based anticancer drugs and has no obvious cross-resistance to cisplatin.It is widely used in the treatment of cisplatin-resistant tumors.The complexes act on DNA to form interchain and interchain crosslinks,thereby inhibiting DNA synthesis and replication of tumor cells.With the use of oxaliplatin in clinical work,it is found that the induced liver toxicity by oxaliplatin is also more common.As the mechanism of liver injury induced by oxaliplatin is not yet clear,routine use of liver protection drugs or the use of what kind of liver drug in the clinical work for the oxaliplatin-based chemotherapy is inconclusive.Polyene phosphatidylcholine(PPC)is one of the main active components of essential phospholipids.It has high bioavailability and high affinity for cell and subcellular membrane.It can combine the whole molecule with cell membrane and organelle membrane,increase membrane Integrity,mobility and stability,and reduce oxygen stress,the inflammatory response and lipid peroxidation,inhibit apoptosis and other effects.For liver tissue,PPC can liver alleviate fibrosis by increasing hepatic collagen degradation,preventing mitochondrial abnormalities.At present,PPC is a kind of hepatoprotective drug commonly used in clinic.It is also a commonly used drug for treating chemotherapy-induced liver injury during chemotherapy.However,the effect of polyene phosphatidylcholine on the anti-tumor effect of oxaliplatin is unknown.At present,oxaliplatin-containing chemotherapy has been widely used in the treatment of gastric cancer.Although the development of various anti-cancer treatment has made great progress,the resistance to oxaliplatin has become increasingly evident,regardless of primary drug resistance or secondary resistance.The mechanism of oxaliplatin resistance has become the key to the treatment of gastric cancer,it involves the microenvironment of tumor growth,mitochondria,cell membrane and gene,protein and signal transduction and many other factors..At present,the most popular mechanism of drug resistance is about the ABC superfamily proteins and tumor stem cells related to drug resistance.For the above problem,this subject is to be carried out in two parts,first of all,to explore the effect of polyene phosphatidylcholine on the efficacy of oxaliplatin chemotherapy.The results suggest that low concentrations of polyenephosphatidylcholine can be synergistic with oxaliplatin Anti-tumor effect.In the discovery of polyene phosphatidylcholine and oxaliplatin can produce synergistic anti-tumor efficacy,in the second part of the study,we induced gastric cancer cell line of oxaliplatin-resistant gastric cancer.And the expression of ABC superfarmily protein and tumor stem cell protein were detected.The results showed that Nanog and ABCF2 were highly expressed.After being treated with polyene phosphatidylcholine,the oxaliplatin sensitivity index increased obviously,and the expression of Nanog and ABCF2 in drug-resistant cells decreased.Suggesting that polyene phosphatidylcholine can reverse the resistance of gastric cancer drug-resistant cells against oxaliplatin.Method:1.Cell proliferation assay The cell proliferation index was determined by MTT assay:SGC-7901 were seeded in 96-well plates(200 ?l/well containing 6,000 cells/well).After 24 h,the medium was replaced with complete culture medium supplemented with different concentrations of L-OHP and/or PPC.Following treatment for 24,48 or 72 h,Drug combination group,Cells were cultured in complete medium supplemented with 3.5 ?g/ml L-OHP combined with various concentrations of PPC(0-64 ?mol/l)for 48 h..(Group ?,?,?a,?b,?c,?d,?e and ?f),respectively.After 48 hours of incubation in the incubator,the cells were incubated with 20?l MTT in each well.2.Drug combination analysis.The combination index(CI)was calculated based on the Chou-Talalay equation(12,18).CI values were calculated using the formula:CI =(D)1/(Dx)1+(D)2/(Dx)2 for mutually exclusive drugs,where D refers to the drug dose.In the denominator of the equation,(Dx)1 represents the D1 'alone'that inhibits a system by certain percentage and(Dx)2 represents the D2 'alone' that inhibits a system by certain percentage.In the numerator of the equation,(D)1+(D)2 'in combination' also inhibit a system by certain percentage.The.CI values were calculated according to the different percentages of inhibition,from 0.05 to 0.95(which represents 5-95%cell death).Briefly,CI<0.85,0.85<CI<1.15 and CI>1.15 indicated a synergistic,additive and antagonistic effect,respectively.3.Cell cycle analysis.The cell cycle was analyzed by flow cytometry.Cells were cultured in complete medium supplemented with 3.5 ?g/ml L-OHP combined with various concentrations of PPC(0-64 ?mol/1)for 48 h.4.Cell apoptosis assay.Cell apoptosis was evaluated by flow cytometry.SGC-7901 cells were treated with 3.5 ?g/ml L-OHP combined with various concentrations of PPC(0-64 ?mol/l)for 48 h.Cells were digested by 2.5 g/1 trypsin,washed with 0.01 mol/1 PBS twice,fixed with cold 95%alcohol at 4? for 30 min,stained with PI and annexin V-fluorescein isothiocyanate.5.Cell cycle-related protein detectionWestern blotting was used to detect the expression of cyclin D1 and Cyclin E in the combined group6.Detection of apoptosis-related proteinsThe expressions of Cytochrome c,Bax,Bak,Bcl-2,cleaved caspase-3 and caspase-9 were detected by Western blotting in the combined group7.Reactive oxygen species(ROS)detectionThe activity of SOD,GSH-Px and the content of malondialdehyde(MDA)were measured to evaluate the intracellular oxidative and antioxidant status of the combined group.ROS system in the joint function of the difference between groups.8.Establishment of SGC-7901/L-OHP cell line and its proliferative activitySGC-7901 gastric cancer cells were treated with oxaliplatin in a step-by-step cell culture medium,and the SGC-7901/L-OHP cells were induced and screened in vitro The inhibitory rate of oxaliplatin was determined by MTT assay.The results showed that the established cell lines had good drug resistance and stability9.The expression of ABC family protein and tumor stem proteinThe expression of ABCB1,ABCC1C,ABCF2 and Sox2 and Nanog were detected by Western blotting.in resistant cell lines10O.Effect of polyene phosphatidylcholine on oxaliplatin-sensitive index of drug-resistant cellsThe IC50 of SGC-7901/L-OHP to L-OHP was measured after 48 hours of treatment with 0.25umol/L,0.5umol/L,1umol/L and 2umol/L polyene phosphatidylcholine.Then calculate The drug reversal index.11 The proportion SP cellsSGC-7901 and SGC-7901/L-OHP cells were incubated with Hoechst 33342 for 90 min,and the percentage of SP cells was calculated by flow cytometry12.Polyene phosphatidylcholine on the expression of ABCF2 protein and Nanog Western blotting was used to detect ABCF2 protein and Nanog expression in drug-resistant cell lines.Results:Oxaliplatin inhibited the growth of tumor cells,and the inhibitory intensity was related to drug concentration and time(F=194.193,P=0.0027;F=12.428,P=0.01).The proliferation activity of SGC-790 cells was different in different concentrations of polyenephosphatidylcholine(P<0.01).Low concentration of polyphosphatidylcholine could promote the proliferation of gastric cancer cells(F = 373.769,P<0.01)Inhibitory effect of phosphatidylcholine on proliferation of gastric cancer cells.The cell viabilities of ?.?,?a-f groups were 100±0.18%,67.8±4.0%,38.9± 4.7%,34.9±4.7%,39.3±5.5%,38.6±4.60%,37.7±2.6%,36.1 ± 5.1%respectively,after being treated with different concentrations of polyene phosphatidylcholine and oxaliplatin..The drug combination index in the combination group was 3.1,2.89,1.7,1.41,0.94 and 0.94.The percentages of G0/G1 phase cells in ?,?,?a-f groups were 45.5±1.68%,68.1±1.02%,86.5±1.21%,87.2±1.25%,87.8±1.07%,86,1±1.60%.87.9±The percentage of apoptosis was 2.75±1.23%,9.5 ± 1.45%,23.2±0.97%,27.5±1.66%,22.2 ±1.39%,23.4 ± 1.23%,24.1±1.31%.21.2±0.31%1.13%.There was no significant difference between the different concentrations of polyene phosphatidylcholine(P=0.46;P=0.75).SOD activity were 40.8±1.81,23.5± 1.68,22.5 ± 1.21,21.8 ± 1.76,22.7± 1.70,20.7 ± 1.35 and 19.2±1.76,21.6±1.59 U/mg prot,respectively,in ?,,?,?a-f,group;the activities of glutathione peroxidase(GSH-Px)were11.9± 1.57,10.9 ±1.10,11.2±1.85,10.4±1.97,11.1±1.60,11.9±0.97,10.4±1.34 25.3 ± 1.15,Mg prot U/L;MDA level were 4.1±0.13,8.3±0.36,8.2±0.76,8.6±0.59,7.9±1.03,8.1±0.65,8.0±0.77,7.6±0.89nmol/mg prot.PPC greatly promoted cell apoptosis induced by L-OHP,and remarkably promoted G0/G1 phase arrest induced by L-OHP.PPC and L-OHP could upregulate the expressions of cytochrome c and activate the following downstream caspases,downregulate Cyclin D1 and E expressions.But all the promotive effects were not PPC dose-related(P=0.37).Induced gastric cancer drug resistant cell SGC-7901/L-OHP,so that it can be 10?g/ml of oxaliplatin in the stable survival,Western blot analysis showed:that ABCC1 gene was highly expressed in SGC-7901/L-OHP cells and SGC-7901/L-OHP cells,while ABCB1 and ABCC1 were down-regulated in SGC-7901/L-OHP cells?Stem cell marker Sox2 were not expressedinSGC-7901/L-OHP cells and SGC-7901 cells,Nanog protein was in the overexpressed in SGC-7901/L-OHP cells.The percentage of SPC in SGC-7901 and SGC-7901/L-OHP cells was 0.12±0.035%and 85.7± 9.66%,respectively.The difference between SGC-7901 and SGC-7901/L-OHP was statistically significant(P<0.01)The sensitivity of SGC-7901/L-OHP cells to oxaliplatin increased with the combination of 0.25umol/L,0.5umol/L,1umol/L and 2umol/L polyene phosphatidylcholine.The drug reversal index was:4.24,6.09,3.80,2.15.Meanwhile,the expression of Nanog and ABCF2 protein in SGC-7901/L-OHP cells were significantly decreased.(P=0.023).Conclusion:1.Oxaliplatin can inhibit the proliferation of SGC-7901 cells in a dose-and time-dependent manner.2.The proliferation activity of SGC-790 cells in different concentrations of different concentrations of phosphatidylcholine showed a double-sided effect on the proliferation of SGC-790 cells.Low-dose polyphosphatidylcholine promoted tumor cell proliferation,low-dose polyphosphatidylcholine inhibition Tumor cell proliferation.3.The drug combination formula showed that low-dose polyphosphatidylcholine(2-16?mol/L)and oxaliplatin showed a synergistic effect on the proliferation of gastric cancer.High-dose polyene phosphatidylcholine(32-64 ?mol/L)and oxaliplatin showed a simple additive effect of anti-gastric cancer proliferation.4.SOD and GSH-Px activity and MDA level in combination group and single drug group had no difference,There was no significant correlation between anti-tumor effect and reactive oxygen species(ROS).5.Oxaliplatin plays an important role in antiproliferation by arresting cell cycle arrest and inducing cell apoptosis.6.PPC stimulated the growth of SGC-7901 cells and greatly promoted their apoptosis induced by L-OHP,which was supported by the upregulation of cytochrome c and the downstream activation of caspases 3 and 9.and the downregulate the expression of cyclins D1 and E.7.SGC-7901/L-OHP cells expressed higher levels of SP than SGC-7901 cells,and high expression of Nanog and ABCF2 protein8.Polyenephosphatidylcholine can restore the sensitivity of SGC-7901/L-OHP cells to oxaliplatin,in which the drug resistance index of 0.5umol/l polyene phosphatidylcholine is the strongest9.polyene phosphatidylcholine can reverse the resistance of SGC-7901/L-OHP cells to oxaliplatin by Which may be related to the down-regulation of Nanog and ABCF2 protein expression in drug-resistant cells.