Ranibizumab, Conbercept and Aflibercept are the most commonly used angiogenesis inhibitor in treatment of intraocular neovascularization. We used these three anti-VEGF drugs to investigate the effect of autophagy on RF/6A cells, the protein levels of autophagy markers LC3, Beclin-1, Atg7 and p62 were measured by western blotting after 24 h of cell culture. As shown in (Fig. 1), Compared with hypoxia group, the protein level of Beclin-1 and LC3-2/1 in Ranibizumab and Conbercept groups were significantly higher(P < 0.05). While the expression of P62 were decreased (P < 0.05). The autophagic fluxare showed the same results (Fig. 2). In the case of autophagy, the number of yellow punctaes in Ranibizumab and Conbercept groups was more than that in hypoxia group and the red spots are increased when autophagy fused with lysosome. These results indicated that autophagy occurred in Ranibizumab and Conbercept groups. These results suggest that Ranibizumab and Conbercept can triger autophagy of RF/6A cells in hypoxia conditions. However, Aflibercept had a different effect on autophagy which inhibited the expression of Beclin-1 and LC3-2/1.
2. Effects of Conbercept combined with autophagy inhibitior on cell proliferation
In order to further verify the interaction between autophagy and anti-VEGF inhibitors, we chose Conbercept as the representative to do further research. The Cell activity was analyzed by CCK8. As shown in Fig. 3. Compared with control group. Cell proliferation rate was decreased in hypoxia group 85.34% and conbercept group 79.97% (P < 0.05). Autophagy inhibitior 3-MA or CQ can further inhibit cell proliferation (75.41%Vs 73.66) (P < 0.05).
3. Effects of Conbercept combined with autophagy inhibitior on cell apoptosis
The cell apoptosis were performed by flow cytometry and Tunel. The flow cytometry analysis showed that compared with control group, Hypoxia increased the apoptosis of RF/6A cells(2.23 ± 1.25 Vs 3.62 ± 1.31). Compared with hypoxia group, the apoptosis rate of RF / 6A cells in Hypoxia + Conbercept, Hypoxia + Conbercept + 3-MA,
Hypoxia + Conbercept + CQ groups increased in turn. (6.48 ± 1.13 Vs 11.68 ± 2.24 Vs 15.05 ± 2.50) (Fig. 4). The result of TUNEL was showed in Fig. 5. Blue color is the standard nucleus, while red is the nucleus labeled with tunle, which is used to represent apoptotic cells. There results indicated that inhibition of autophagy in RF/6A cells under hypoxic culture could promote apoptosis. The nuclear morphology of the control group was complete, indicating that there was no apoptosis. In hypoxia group, there were red labeled positive nuclei, indicating that apoptosis increased. In the group of Hypoxia + Conbercept, Hypoxia + Conbercept + 3-MA and Hypoxia + Conbercept + CQ, the number of red nuclei increased gradually. The results are consistent with those of flow cytometry.
4. Conbercept combined with autophagy inhibitior on cell migration and tube formation
The effects of RF/6A cells migration and tube formation were detected by scratch wound test and Matrigel assay, respectively. Results indicated that after 36 h of cell incubation, a large number of cells migrated into the bare area of the cell culture plates (Fig. 6). Conbercept significantly inhibited cell migration compared with hypoxic group(633.083 ± 72.52 Vs 546.33 ± 24.61), while autophagy inhibitor group (3-MA or CQ) had more obvious inhibition effect(309.75 ± 86.36 and 263.33 ± 68.67) (P < 0.05). For tube formation, the number of tube formation was decreased significantly in conbercept group (30 ± 2.23) compared to hypoxia group(36 ± 2.2) and even further reduced in autophagy inhibitor group (27 ± 3.42). The length of master segments in hypoxia group was higher than that in control group. (14641.33 ± 1512.3 Vs 9056.67 ± 423.22). Compared with hypoxia, master segments length were decreased in Conbercept (14242.67 ± 942.34) and Conbercept + 3-MA group (12430.33 ± 567.32) (Fig. 7). All these results demonstrated that conbercept combined with autophagy inhibitor (3-MA) could inhibit cell migration and tube formation of RF/6A cells in hypoxia condition more significantly.
5. The effect of concept on the expression of p53 / DRAM
The expression of p53 and DRAM were tested by western blot and qPCR. As shown in Fig. 8,9. The expression of p53 and DRAM were increased in Conbercept and Hypoxia group(P < 0.05). In order to further clarify the mechanism of Conbercept activated autophagy, the effect of Pifithrin-α (PFTα) HBr) on autophagy related proteins was analyzed. The results showed that compare with hypoxia group, the expression of LC-3, LAMP1, Beclin-1 and DRAM were increased in Conbercept group (P < 0.05). However, this trend was attenuated in Pifithrin-α (PFTα) HBr) group (P < 0.05) (Fig. 10). It shows that inhibiting p53 can inhibit the expression of DRAM and autophagy related proteins.