Study On The Role Of Hyperbaric Oxygen On Biological Behavior Of Chemical Hypoxia Lung Adenocarcinoma Cell A549 And Its Mechanism

Background Lung cancer is one of the most common malignancies,non-small cell lung cancers（NSCLCs）such as squamous cell carcinoma and adenocarcinoma account for nearly 85%of the total lung cancer cases.With the wider application of anti-angiogenesis,targeted therapy and immunotherapy in lung cancer,significant progress have been made in improving the prognosis of lung cancer.For the advanced lung cancer,however,especially in patients with negative gene mutations,radiotherapy and chemotherapy remain the main treatment options,and the mortality rate is still high.It is necessary to constantly seek more effective and new treatment methods that supplement effective adjuvant treatment on the basis of the original treatmen,to improve the prognosis of lung cancer,as well as the quality of life and overall survival of patients.This has been a hot topic in tumor research in the past few years.Some recent studies have shown that the hypoxic tumor microenvironment is the initiating factor that promotes tumorigenesis,metastasis,and increased risk of malignant transformation,making both chemotherapy and radiotherapy less effective on and even tolerated by the hypoxic cells.By altering the oxygen content in the tumor tissue,the hypoxia status of the tumor can be changed,and the defense function of the tumor tissue adaptation to damages can be removed or reversed,while inducing oxidative stress and causing damages to the tumor tissues.This provides a novel way to deliver treatment of tumors.Hyperbaric oxygen therapy（hyperbaric oxygen therapy,HBOT）is a effective way to improve the oxygenation of tumor cells.HBOT refers to use of 100%oxygen under high pressure intermittently to increase the oxygen content dissolved in plasma and therefore increase O₂ delivery to tissues.The therapy is used to manage various conditions,such as inhaled carbon monoxide poisoning,radiation injury,non-healing wounds,ischemic diseases,venous or arterial ulcers,burns,and pressure sores.Recently,research on HBOT and cancer has been focused on whether enhancing the oxygen content in the tumor tissue promotes cancer progression.Moreover,some study suggest that HBOT increases oxygenation to change the hypoxic state of tumor or inhibit the level and activity of HIF-1α,thus reducing the adaptive response of tumor cells to hypoxia and resistance to drug therapy,improving the sensitivity of tumor cells to radiotherapy and chemotherapy.HBOT may be a strong and effective anti-tumor strategy.In this study,cobalt chloride（CoCl₂）was used to induce lung cancer A549 cells to produce hypoxia.Meanwhile,HBOT was used to detect the biological characteristics of tumor cell growth,metabolism,metastasis,and invasion,as well as the impact of HBOT,in the hypoxic microenvironment,clarify the possibility of hyperbaric oxygen in tumor adjuvant therapy and the molecular mechanism.The results would provide a reliable theoretical basis for whether HBOT could play an anti-tumor role by improving the tumor hypoxic microenvironment and be used as an auxiliary treatment for comprehensive treatment of tumor,and develop a new clinical model for the treatment of cancer patients.Objective To explore the effect of HBOT on proliferation,differentiation,apoptosis,migration and invasion ability of CoCl₂ induced hypoxia lung adenocarcinoma cells,and to study its mechanism.Methods THE MTT and CCK-8 methods were used to detect the effects of CoCl₂on the viability and proliferation of lung adenocarcinoma cells,the expression of HIF-1αwas detected and the suitable CoCl₂ concentration which could effectively induce hypoxia but not produce toxicity to cells was screened,then treatead with hyperbaric oxygen.Actate dehydrogenase activity assay,cell scratch assay,Transwell invasion assay,Hoechst staining were used to detect cell differentiation level,cell migration and invasion ability,cell attachment and separation ability,and apoptosis level,respectively.Western blot was used to detect the changes in the expression of proteins related to migration,invasion and apoptosis,such as MLCK,MMP-9,Bcl-2/Bax,EMT markers E-cad/N-cad,GRP78/CHOP and P53,to explore the molecular mechanism of HBOT.The levels of phosphorylation ERK,JNK and P38 were detected to determine whether HBOT played the same role by affecting the activity of MAPK signaling pathway.Results Compared with the normal control group,100μM and 200μMCoCl₂ could both significantly upregulate the expression of HIF-1αand induce hypoxia,because of the concentration of 100μMCoCl₂ had no obvious inhibition on cell proliferation,cell viability,and had no toxicity to cells,so concentration of 100μMCoCl₂ was selected to induce hypoxia.The LDH activity was significantly increased in CoCl₂ treated cells,indicating that the level of cell differentiation decreased,and the LDH activity in HBO+CoCl₂ combined treatment group was significantly lower than that in the CoCl₂treatment group.CoCl₂ could enhance the migration and invasion abilities of lung cancer A549 cells,increased cell detachment and attachment rate,decreased apoptotic cells.The combination of HBOT inhibits migration,invasion,cell detachment and attachment induced by chemical hypoxia,and increased cells apoptosis.When CoCl₂ was used to induce A549 cells,E-cadherin expression decreased significantly,the level of N-cadherin protein increased,and the E-cad/N-cad ratio dropped significantly,MLCK,MMP-9 expression increased.HBOT could restore the ratio of E-cad/N-cad and the MLCK,MMP-9 levels.CoCl₂-induced hypoxia inhibited the anti-apoptotic proteins Bax,P53 and increased the expressions of Bcl-2,GRP78,and CHOP,resulting in a significantly increased Bcl-2/Bax ratio.HBOT lowered the Bcl-2/Bax ratio and the levels of GRP78 and CHOP proteins while increasing the expression level of p53protein,significantly.Meanwhile,the p ERK and p P38 levels in CoCl₂-induced cells were significantly increased,the pJNK level was significantly reduced,the p ERK,p P38,pJNK levels in CoCl2-induced cells were reversed by HBOT.Conclusions Hypoxia-induced lung cancer A549 cells grow with increased malignant phenotype,the degree of cell differentiation decreased,the ability of migration and invasion increased,the ability of detachment and attachment also enhanced,and the proportion of apoptotic cells decreased,these malignant phenotypes were partially inhibited or completely reversed by HBOT.HBOT maybe regulate the expression of protein related to migration,invasion,adhesion and apoptosis,such as MCLK MMP-9,epithelial-mesenchymal transition protein E-cad/N-cad,Bcl-2/Bax,GRP78,CHOP,p53 by regulating the activity of MAPK signaling pathway.The use of HBOT could inhibit or reverse the increase of malignant phenotype of tumor cells caused by tumor hypoxic microenvironment,and could be used as a reliable adjuvant tumor treatment.