Possible Mechanism Of Microwave Hyperthermia Combined With Gemcitabine Induced Apoptosis And Autophagy In Lung Squamous Cell Carcinoma

Objective:Lung cancer is the main cause of cancer-associated mortality worldwide,and squamous cell lung carcinoma is one of the most common types of non-small-cell lung cancer(NSCLC).Despite treatment with surgical resection combined with chemotherapy and radiotherapy,patients with squamous cell lung carcinoma exhibit an overall 5-year survival of <15%.Many patients with lung squamous cell carcinoma are in the advanced stage of treatment,so it is necessary to actively explore effective methods for the treatment of lung squamous cell carcinoma.Studies have shown that hyperthermia combined with chemotherapy can improve the therapeutic effect of lung squamous cell carcinoma,however,the specific molecular mechanism is not very clear.Our group used the microwave thermotherapy instrument independently developed to investigate the effect of microwave hyperthermia combined with gemcitabine on the proliferation of lung squamous cell carcinoma cell lines in vitro,and further explore its potential molecular mechanism to provide reliable experimental basis microwave sensitization chemotherapy.Methods:(1)The effects of microwave hyperthermia and gemcitabine on the proliferation of lung squamous cell carcinoma were detected by CCK-8 cell activity assay,and the interaction types of microwave hyperthermia combined with gemcitabine in different sequential modes were further analyzed.(2)Cloning formation experiments was to detect the inhibitory effect of microwave hyperthermia combined with gemcitabine on the proliferation of lung squamous cell carcinoma;(2)the effect of microwave thermotherapy combined with gemcitabine on cell cycle was detected by cell cycle flow assay;(3)the effect of microwave thermotherapy combined with gemcitabine on apoptosis was analyzed by apoptotic flow,Caspase-3/ Caspase-8 activity assay and detection of apoptosis-related proteins;(4)Detection by Lyso-Tracker Red staining and autophagy-related proteins(LC3-II/I and P62)to explore the effect of microwave hyperthermia combined with gemcitabine on autophagy;(5)Using DCFH-DA kit to detect ROS production after microwave hyperthermia combined with gemcitabine;(6)Western blot analysis was used to analyzed the expression level of PI3K/AKT/mTOR signaling pathway in heat-treated combined cells;(7)Detection of cell proliferation and induction using autophagy inhibitor(3-MA)and ROS scavenger whether autophagy changes,and the effect on the expression level of the PI3K/AKT/mTOR signaling pathway.Results:Microwave hyperthermia after chemotherapy with gemcitabine can synergistically inhibit the proliferation of human lung squamous carcinoma cells,causing G0/G1 cycle arrest,Caspase-3 dependent apoptosis and autophagy.The combination of heating can induce the production of ROS and down-regulate the PI3K/AKT/mTOR signaling pathway in lung squamous cell carcinoma cells;when ROS scavenger is used,the induced cell proliferation and autophagy are significantly inhibited,and the PI3K/AKT/mTOR signaling pathway Upregulation;when autophagy was inhibited,there was no significant change in cellular ROS production,but the PI3K/AKT/mTOR signaling pathway was up-regulated.The above experimental results indicate that ROS/PI3K/AKT/mTOR is the main pathway for the combined action of heating.Conclusions:Our study demonstrates that microwave hyperthermia after gemcitabine chemotherapy can induce G0/G1 phase arrest in lung squamous carcinoma cells and induce apoptosis and progression of lung squamous cell carcinoma via ROS/PI3K/AKT/mTOR signaling pathway.Phag,therefore,this combination of treatment is of great significance for the treatment of advanced lung squamous cell carcinoma.