Establishment Of An Oxaliplatin-induced Human Gastric Cancer Drug Fast Cell Line And The Study Of Its MDR Mechanism

Objective: The death rate of gastric cancer is the highest among all the malignant tumors in our country. Chem the most active adjunctive therapy after operation, play an important role in preventing and curing both palindromia and metastasis. Even though Chem is constant consummated , but the curative effect of Chem to parts of stomach cancer patients is not ideal, and the multidrug resistance(MDR)is the key.MDR have two phaenotypes: one is that the drug fast generate in the first time using chemotherapeutics , which is called primary drug resistance. Another is that the drug fast generate during the stage of Chem, which is called secondary drug resistance. Gastric cancer has the characteristic both primary drug resistance and secondary drug resistance. Some study has confirmed that P-gp is the main reson lead to MDR of gastric cancer. Both Livin and Survivin belong to IAP family and play an important role in apoptosis and MDR of turmor cell. Livin is a new nember of IAP family. Oxaliplatin is an anticancer drug of the third generation and is regarded as the main medicine to treat stomach cancer of progression. Even though different kinds of drug fast cell line of stomach cancer have been established, but oxaliplatin-induced gastric cancer drug fast cell 1ine and the expression of Livin and Survivin in both parent cell 1ine and drug fast cell line of gastric cancer have not been reported.To explore the mechanism of MDR to gastric cancer,we designed this study to establish a oxaliplatin-induced human gastric cancer drug fast cell line, study its biological characteristics, the relationship and mechanism of p-gp, livin and survivin lead to MDR.Methods: High invasion metaptosis cell line OCUM- 2MD3 was raised in DMEM culture fluid under the saturated humidity with 5% CO2 and 37℃, digest by 0.25% parenzyme to passage; OCUM-2MD3/L-OHP cell line was established gradually by large dose of oxaliplatin and intermittent administration;the changes of cell morphology character were determined by using inverted phase contrast microscope and electron microscopy; draw cell growth curve by cytometry, calculate doubing time according to formula: Td(h)=T×[log2/(logN-logN0)]; drug sensitivity determined by MTT assay; The expressions of P-gp,Survivin and Livin were detected by FCM.All statistical analyses were carried out with SPSS 12.0, p<0.05 was considered significant.Results: OCUM-2MD3/L-OHP cell line was developed after 6 months.1 The cytobiology characteristic of drug fast cell line1.1 Characteristic of morphologyUnder the inverted phase contrast microscope, both OCUM-2MD3 and OCUM-2MD3/L-OHP cell lines have no difference as far as cells'morphous concerned. Under the TEM we can see the parent cell has plentiful microvilli on the cell surface; its cell organs have normal morphous; its cell nucleus is big,the morphous is anomalism,with plentiful euchromatin and less heterochromatin in it, the nucleole is big and manifest. Compared with the parent cell some drug fast cells appear typical morphology change of apoptosis.1.2 Population doubling timeThe generation rate of CUM-2MD3/L-OHP cell line step down and population doubling time prolonged for 3 hours(P<0.05).1.3 The disposition of cell cycle and apoptosisCompared with OCUM-2MD3 cell line, the OCUM-2MD3 /L-OHP cell line's cell number of G0/G1-phase and G2/M-phase increased while that of S-phase decreased cell apoptosis rate increased(P<0.05). When dealed by L-OHP for 24 hours, the drug fast cell line's cell number of S-phase increased, while the parent cell line's cell number of G2/M-phase increased, and the cell apoptosis rate of drug fast cell line is less than that of the parent cell line(P<0.05).1.4 The sensitivity and RI of OCUM-2MD3/L-OHP cell line The higher concentration of L-OHP was the stronger inhibition function to OCUM-2MD3 and OCUM-2MD3/ L-OHP cell line we can saw, and the inhibition ratio of the same concentration to drug fast cell line was less than that of the parent cell line(P<0.05). The drug fast cell line show stable resistance to oxaliplatin, the resistance index was 4.3. 1.5 The multidrug resistance and cross-resistance of drug fast cell lineThe inhibition ratio of L-OHP, CDDP, CBDCA, 5-Fu, ADM, MMC, GEM,VCR,IH and PTX to OCUM-2MD3/L-OHP cell line was lower than OCUM-2MD3 cell line(P<0.05). The parent cell line is resistant to MMC, VCR and IH, while the drug fast cell line exhibite cross-resistance to CBDCA, 5-Fu, MMC, GEM,VCR and IH beside its resistance to L-OHP,and its sensitivity to CDDP,ADM and PTX decreased.2 The expression of P-gp, Livin and Survivin in drug fast cell line2.1 The expression of P-gp in drug fast cell line The expression of P-gp in OCUM-2MD3/L-OHP cell line was higher than that in the OCUM-2MD3 cell line(P<0.05), when dealed by L-OHP for 24 hours, the expression capacity of P-gp was decreased in both OCUM-2MD3/L-OHP and OCUM-2MD3 cell line, and the P-gp expression capacity of drug fast cell line was still higher than that of the parent cell line(P<0.05).2.2 The expression of Livin in drug fast cell line The expression of Livin have no difference in both OCUM-2MD3/L-OHP and OCUM-2MD3 cell line(P>0.05), when dealed by L-OHP for 24 hours, the expression capacity of Livin was decreased in both OCUM-2MD3/L-OHP and OCUM-2MD3 cell line,and the Livin expression capacity of drug fast cell line was higher than that of the parent cell line(P<0.05).2.3 The expression of Survivin in drug fast cell line The expression of Survivin in OCUM-2MD3/L-OHP cell line was lower than that in the OCUM-2MD3 cell line(P<0.05), when dealed by L-OHP for 24 hours, the expression capacity of Livin was decreased and had no difference in both OCUM-2MD3/L-OHP and OCUM-2MD3 cell line(P>0.05).Conclusion: 1 OCUM-2MD3 cell line has the charact- eristic of primary drug-resistance, wich are related to P-gp, Livin and Survivin.2 OCUM-2MD3/L-OHP cell line has the character of secondary multidrug-resistance and show stable low resistance to oxaliplatin, the resistance index was 4.3 and exhibite cross-resistance to many other chemotherapeutic agents (CBDCA, 5-Fu, MMC, GEM, VCR and IH).3 The secondary MDR mechanism of OCUM-2MD3 /L-OHP cell line is related to the cell cycle alterationcell,population doubling time prolonging and P-gp expression up-regulation.4 The mechanism of P-gp, Livin and Survivin lead to MDR is related to their depressant effect to apoptosis. In conclusion our study establish OCUM-2MD3/L-OHP cell line succesfully, and it can be used to study the MDR mechanism of stomach cancer. Detect the expression of P-gp, Livin and Survivin in drug fast cell line and also study its MDR mechanism can help us to prophylaxis the appearance of MDR and blindness in Chem course, thus to direct individualized chemotherapy. But as the mechanism of MDR is complicated, there is still have some deeper study and research we shoud do.