| Primary bronchopulmonary carcinoma,which is short for lung cancer,is one of the world’s highest morbidity and mortality of malignant tumor at present. As a common pathological type of lung cancer, the incidence of lung adenocarcinoma is higer than squamous cell carcinomain in many countries and regions in the world recent years,it has become the most common pathological type of lung cancer. Cisplatin is the foundation of non-small cell lung cancer chemotherapy scheme,and its combinations with other chemotherapy drugs such as paclitaxel,gemcitabine are the first-line chemotherapy regimens for advanced non-small cell lung cancer,while chemotherapy resistance is the main cause for treatment failure and cancer recurrence. Hypoxia is a key feature of solid tumors, and involved in chemo-resistance. High mobility group box-1(HMGB1) is a highly conserved nuclear protein and also functions as an extracellular signaling molecule in multiple human diseases. The expression levels of HMGB1 were significantly higher than that of the corresponding normal tissues in a variety of tumor tissues, current study shows that HMGB1 might be related to chemotherapy resistance. Object:This study aim at detecting the level of expression of autophagy and HMGB1 in human lung adenocarcinoma A549 cells under hypoxia,and then exploring their relationship with drug resistance. Method:Human lung adenocarcinoma A549 cells were cultured for 24 hours in CoCl2(0, 200μM). First, the expression of HIF-1 was detected by immunocytochemistry to verify the success of hypoxia model. Then by inverted phase microscope was used to observe the morphology of A549 cells, the growth and proliferation of A549 cells were detected by MTT assay, effects of CCK-8 method for the detection of cisplatin on A549 cell activity, ELISA detection of HMGB1 protein activity, further through Western blot detection of autophagy gene Beclin 1 and LC3-II protein expression. Results:1) The positive A549 positive HIF-1 cells and the expression of HIF-1 protein in the nucleus of hypoxia group were higher than that in the normal oxygen group, which proved that the CoCl2 chemical hypoxia model was successfully established.2) The morphological features of cells growth were observed under inverted microscope,both of the A549 cells in normoxia groups and CoCl2 groups pasted the wall well,while the A549 cells in CoCl2 groups were sparse,and visible multiple pseudopods could been seen.3) A549 cell growth curve showed a S type in normoxia groups,while in CoCl2 groups, the cell growth curve of A549 were lower and cells’ proliferation was slower.4) After treated with same concentration of cisplatin(20μM), the results showed that compared with normoxia groups,the A549 cell viabilities in CoCl2 groups were higher.5) ELISA method detected the HMGB1 protein activity in supernatant of A549 cells, the results showed that compared with normoxia groups,the HMGB1 protein activity in CoCl2 groups were higher..6) The expression of autophagy gene Beclin-1 and LC3-II protein was detected by Western-blot, the results showed that the Beclin-1 and LC3-II protein in CoCl2 groups were higher when compared with normoxia groups.7) Added HMGB1 recombinant protein or specific neutralizing antibodies in CoCl2 groups, treated the cells with same concentration of cisplatin(20μM),the cell viability increased with the increasion of HMGB1 activity and decreased with the decreasion of HMGB1 activity.8) Western-blot detected the expression of autophagy gene Beclin-1 and LC3-II protein, the results showed that Beclin-1 and LC3-II protein increased with the increasion of HMGB1 activity and decreased with the decreasion of HMGB1 activity. Conclusion:Under hypoxia conditions,the levels of HMGB1 expression was inceased,and compared with control group,the chemosensitivity to cisplatin was decreased. Inhibited the expression of HMGB1 could significantly increase the chemosensitivity to cisplatin,and the mechanism may be related to the down-regulation of autophagy. |
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