Bevacizumab Induces Lung Cancer A549 Cells Apoptosis Through The Mechanism Of Endoplasmic Reticulum Stress

Objective:To observe the effect of bevacizumab on lung cancer A549 cells and explore its mechanism.Methods: 1. Bevacizumab interfere A549 cells. A549 cells are cultivated by PRMI-1640 which containing 10% of FBS and are put in the incubator whose temperature is 37°C and the concentration of CO2 is 5%. Bevacizumab is attenuated by sterile water for injection. The cells are interfered respectively for 12-hr, 48-hr and 72-hr with Bevacizumab of 0 μM, 1 μM, 5 μM and 25 μM.2. CCK-8 assay detects cell proliferation. We inoculate A549 cells in a plate with 96 holes. Then we respectively use bevacizumab in different concentrations such as 0 μM, 1 μM, 5 μM, 25 μM to process the cells for corresponding 12-hr, 24-hr, 48-hr, 72-hr. Next, we add CCK-8 solution(10 μL) to each hole. Finally the absorption value at 450 nm is measured by a plate reader after the plate being put in an incubator for another 4 hours. We repeat the procedure for three times and then calculate the average value.3. Flow cytometry technology detects cells apoptosis. A549 cells are inoculated in a 60-mm-plate overnight. We respectively use bevacizumabin different concentrations(0 μM, 1 μM, 5 μM, 25 μM) to process the cells for 24-hr, and then use PBS buffer solution to wash them for 3 times as well as using tryptan-EDTA(0.25%) to digest. After being centrifuged, the cells will suspend in PBS(0.5 ml). And the cells are not only dyed by Annexin V and Propidium Iodide(PI) but also processed by RNA enzyme and Triton X-100(0.1%) at indoor temperature for 30 minutes. In the end, flow cytometric analysis can be carried out by using fluorescence activated cell sorter.4. Real-time quantitative PCR detects RNA expression. The extraction of total m RNA were from cells treated by different concentration(0 μM, 1 μM, 5 μM, 25 μM) of bevacizumab with using the Ambion RNA-queous kit. The high capacity c DNA archive kit used to get the first chain c DNA of m RNA. The m RNA level of CHOP, caspase-4, IRE1 and XBP-1 were determined through specific gene kit and real-time polymerase chain reaction(Real-time PCR) detection system.5. Western blotting technology detects protein expression. The expression of protein(CHOP, caspase-4) are detected by Western blotting. Briefly, after cytolysis, we collect the supernate and then extract the plasmosin or nucleoprotein in an already-known-way. At the voltage of 120 V, hydrochloric acid polyacrylamide gel can make protein transfer to the blotting membrane, on which we use specific polyclonal antibody to probe, of PVDF. We can conduct electrophoretic analysis of equal quantity from protein lysate with β-actin being the internal reference.Results: 1. Bevacizumab treat A549 cells for 12 hours, showing the inhibition of cell proliferation mild, but after 24 hours showing significant induction of cell apoptosis in a dose dependent manner.2. We adopt flow cytometry to assess the influence on A549 cells apoptosis exerted by bevacizumab. The results show that bevacizumab processed for 24-hr can obviously induce cells apoptosis which further indicates that bevacizumab participates the signal channel.3. We further study the possibility that Bevacizumab induces A549 cell apoptosis through ERS. In the unfolded protein response(UPR) caused by ERS, we choose inositol requiring enzyme 1(IRE-1) and X-box binding protein 1(XBP1) as our test index while in the signal channel of endoplasmic reticulum stress-induced apoptosis(ERSIA), we select C/EBP-homologous protein(CHOP)and caspase-4 as our test index. We can come to the conclusion that, in terms of the RNA level, expression of XBP-1 has increased obviously in each group(1 μM, 5 μM, 25 μM)(P<0.01), the figures were 2.196±0.101, 4.605±0.413, 2.532±0.185; the expression of CHOP(25 Μm) and caspase-4(5 μM) have increased slightly(P<0.05), the figures were 2.191±0.112, 1.588±0.167(Figure 3A). In terms of the protein level, the expression of CHOP has increased obviously in each group(1 μM, 5 μM, 25 μM) when compared with the control group(0 μM)(P<0.05), the figures were 0.67±0.08, 1.52±0.24, 1.32±0.31. As for caspase-4(5 μM, 25 Μm), the expression have increased slightly when compared with the controlgroup(0 μM)(P<0.05), the figures were 0.65±0.15, 0.63±0.23. Our data shows that bevacizumab activates cascade reaction of the endoplasmic reticulum stress induced apoptosis.Conclusion: 1. Bevacizumab is showing the inhibition of lung cancer A549 cells proliferation in vitro. 2. Bevacizumab can obviously induce lung cancer A549 cells apoptosis in vitro. 3. When bevacizumab inducing apoptosis, the signal channel of endoplasmic reticulum stress is activating. 4. Bevacizumab can induce A549 cell apoptosis through the mechanism of endoplasmic reticulum stress.