| ObjectiveTo explore the role of integrinαvβ6 in proliferation, apoptosis and drug resistance of gastric cancer AGS cells and the mechanism involved.MethodsThe experiment was divided into two parts.There were three experimental groups in the first part. The control group. The 10D5 group:cells were harvested and re-suspended in RPMI1640, then mAb 10D5 against avP6 was added into the culture solution at the final concentration of 0.1 mg/ml and incubated for 30 min on ice for the sufficient combination of mAb and antigen. Next, cells were rinsed with PBS to end the reaction after culturing for 6h. The IgG2a group:negative controls were performed with the identical concentration of mouse IgG2a in the same way as the 10D5 group. Cells viability was measured by MTT assay, cells apoptosis was detected by FCM, and caspase-3 expression was examined by Western blotting.The second part included four groups. The control group. The 10D5 group:afterαvβ6 was blocked by mAb 10D5 as in the first part, cells were treated with 5-fluorouracil at a specific concentration for 24h. The IgG2a group:negative controls were performed with the identical concentration of mouse IgG2a in the same way as the 10D5 group, then cells were treated by 5-fluorouracil for 24h. The PD98059 group:cells were treated with PD98059 at the final concentration of 20μmol/L and 5-fluorouracil for 24h. Cells inhibition was measured by MTT assay, cells apoptosis was detected by FCM, Bcl-2 and caspase-3 expression was examined by Western blotting.Statistical comparisons were made by One-Way ANOVA and LSD test, and P<0.05 was considered statistically significant.ResultsIn the first part,1. The MTT assay showed that after incubation with mAb 10D5, the survival rate of cells decreased to(73.74±1.7)% compared to the control cells(P<0.05), while in the cells treated with IgG2a(99.4±2.9)%, it was similar to the control cells(P>0.05).2. The results of FCM showed that, the apoptotic rate of cells in the control, 10D5, IgG2a group was (1.93±0.09)%,(11.82±0.45)%,(2.06±0.04)%. By comparison, the apoptotic rate in mAb 10D5 treated cells are direrent from those in the control cells (P<0.05), while in the cells treated with IgG2a, it was similar to the control cells(P>0.05).3. Western blotting analysis demonstrated that after the treatment of mAb 10D5, the expression of caspase-3 increased, comparing to those in the control cells(P< 0.05).There was no difference between control cells and IgG2a treated cells(P>0.05).In the second part,1. The MTT assay showed that,5-FU inhibited the growth of AGS cells in dose-dependent manners. Compared with control conditions (no treatment),0.005, 0.05,0.5,5mmol/L 5-FU resulted in growth inhibition of (17.31±0.78)%,(19.55±0.64)%,(47.56±3.23)%,(80.87±3.4)% of cells, respectively.2. The MTT assay showed that after incubation with mAb 10D5, the inhibition rate of AGS cells increased to (84.49±1.6)% compared to the control cells(28.11±2.7)% (P<0.05), while in the cells treated with IgG2a it was(31.44±5.2)%, similar to the control cells (P> 0.05). Meanwhile after incubation with PD98059, the inhibition rate of AGS cells increased to (86.72±5.2)% (P<0.05).3. The results of FCM showed that the percentage of apoptotic cells in control, IgG2a,10D5 and PD98059 treated cells were(6.63±1.4)%, (8.13±1.3)%, (30.55±2.4)%, (35.99±4.0)%, respectively. Compared with the control cells, the apoptotic rate in mAb 10D5 and PD98059 treated cells increased significantly (P<0.05). There was no difference between control cells and IgG2a treated cells (P>0.05).4. Western blot analysis demonstrated that after the treatment of mAb 10D5 and PD98059, the expression of caspase-3 increased, while the expression of Bcl-2 decreased, comparing to those in the control cells, the difference is significant (P<0.05).There was no difference between control cells and IgG2a treated cells (P>0.05).Conclusions1. Afterαvβ6 was blocked by specific mAb 10D5, the gastric cancer AGS cells proliferated slowlier, the apoptotic rate and caspase-3 expression increased, indicating that avP6 plays a role in the proliferation and anti-apoptosis of gastric cancer AGS cells.2. After cells were incubated with mAb 10D5 and then treated with 5-FU, the inhibition ratio, apoptotic rate and caspase-3 expression of AGS cells all increased than the control and the mouse IgG2a group, while Bcl-2 expression decreased, indicating thatαvβ6 can inhibit cells apoptosis induced by 5-fluorouracil and induce resistance of cells to the drug through up-regulating Bcl-2 expression.3. After ERK function was blocked by PD98059, we also found that the inhibition ratio, apoptotic rate and caspase-3 expression of AGS cells increased than the control and the mouse IgG2a group, while Bcl-2 expression decreased, indicating that PD98059 can abrogate the resistance of cells to 5-fluorouracil mediated byαvβ6, therefore directβ6-ERK binding plays an important role in the process.In conclusion, the experiment demonstrated the role of integrinαvβ6 in malignant biological behaviour of gastric cancer AGS cells, which provides a new way for the mechanism research and treatment of gastric cancer. With the development of research, it is believed there will be of vast potential in treatment of gastric cancer focusing onαvβ6 and interrelated moleculars.
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