| Lung cancer is the most malignant tumor that seriously threaten human health.Its morbidity and mortality rank first in the world.Because the early clinical symptoms of lung cancer are not obvious and it is not easy to diagnose in the early stage,lung cancer patients are often in the advanced or metastatic stage when diagnosed,and the 3-year survival rate is less than 20%.At present,surgery combined with radiotherapy and chemotherapy is still the most effective treatment for lung cancer,but its adverse reactions and complications are also obvious.Therefore,understanding the pathogenesis of lung cancer and elucidating the molecular mechanism of lung cancer development are of great significance for finding new treatments and methods.Hydrogen sulfide（H2S）has long been considered as toxic gas or air pollutant.It was not until the discovery of the physiological function of nitric oxide（NO）as a cardiovascular regulator,the physiological role of H2S,as a new type of small gas signal molecule,has gradually attracted people’s attention.Studies have shown that H2S can widely participate in the regulation progress of the cardiovascular system,digestive system,nervous system,and respiratory system.H2S is closely related to the occurrence and development of tumors as well.H2S in the human body mainly takes L-cysteine as the substrate,and is produced under the catalysis of three enzymes:cystathionine-beta-synthase（CBS）,cystathionine-gamma-lyase（CSE）and3-mercaptopyruvate sulfurtransferase（MPST）.The lungs are the human respiratory organs.H2S,as a gas molecule,enters the body through the nasal cavity from the lungs.So the relationship between H2S and the lungs is the closest.Lung adenocarcinoma is the main component of lung cancer,and the mechanism of H2S in lung adenocarcinoma remains unclear.In this article,we mainly focused on the in-depth research and exploration of the biological function and molecular mechanism of H2S in lung adenocarcinoma.We first examined the expression of endogenous hydrogen sulfide synthase in lung adenocarcinoma tissues and adjacent tissues.Quantitative Real-time PCR（qRT-PCR）and western blot experiments showed that the expression levels of CBS,CSE and MPST in 30 pairs of lung adenocarcinoma tissues were significantly higher than those in the adjacent tissues.Among them,the expression levels of CBS and CSE were significantly related to the tumor size,clinical stage and lymph node metastasis of lung adenocarcinoma.While the expression levels of MPST are mainly related to the tumor size.Similar to the lung adenocarcinoma tissues,the hydrogen sulfide synthase expression and H2S content in lung adenocarcinoma A549,95D and NCI-H1395 cells was significantly higher than that in normal lung epithelial BEAS-2B cells.A549 cell line had the most abundant H2S production,therefore it was chosen for the subsequent experiments.Secondly,sodium sulfide（NaHS）was used as the H2S donor to detect the effect of NaHS on A549 cells.Low concentration of exogenous NaHS（0-50μM）could promote the proliferation of A549 cells,while high concentration of NaHS（2000μM）could promote the apoptosis of A549 cells.Selecting 50μM NaHS which is similar to the content of H2S in the body as the experimental concentration to stimulate A549cells for 24 hours.NaHS could significantly promote the proliferation,migration and invasion of A549 cells,and promote the process of tumor epithelial-mesenchymal transition（EMT）as well.Chemical inhibitors and small interfering RNA（siRNA）silencing methods were used to inhibit CBS and CSE,which mainly produce H2S in lung adenocarcinoma,respectively.The results showed that chemical inhibitors aminooxyacetic acid（AOAA）and propargylglycine（PAG）significantly inhibited cell proliferation,arrested the cell cycle in the G2/M phase,and promoted apoptosis as well.Meanwhile,cell scratch healing rate,transwell invasion cell number and EMT progress were inhibited by AOAA and PAG.These results were further confirmed by siRNA experiments.In addition,as an inorganic electron donor,H2S could promote the energy metabolism process of A549 cells,increased mitochondrial membrane potential,and accelerated ATP production and glycolysis.AOAA and PAG inhibited the activity of CBS and CSE enzymes,which reduced the mitochondrial membrane potential,inhibited the generation of ATP,and the process of glucose decomposition and transformation in A549 cells.Thirdly,western blot experiments showed that H2S increased the expression of hypoxia inducible factor-1α（HIF-1α）in the hypoxia microenvironment of lung tumor.Immunofluorescence experiments showed that H2S increased the accumulation of HIF-1αand promoted the process of HIF-1αentering the nucleus.The effect of H2S on the transcriptional activity of HIF-1αwas detected using a dual luciferase reporter assay.Cobalt chloride（CoCl2）can mimic the chemical hypoxia environment.Results showed that H2S increased the transcriptional activity of HIF-1αunder the condition of CoCl2 treatment.While AOAA and PAG significantly inhibited the transcriptional activity of HIF-1αin A549 cells.Besides,HIF-1αinduced by hypoxia increased the expression of hydrogen sulfide synthase in A549 cells,thereby increasing intracellular H2S content.Both HIF-1αinhibitor 2-MeOE2 and si-HIF-1αsignificantly inhibited the expression of CBS and CSE in A549 cells under hypoxia,thereby inhibited the production of H2S.Therefore,there was a positive feedback regulation between H2S and HIF-1α.Meanwhile,exogenous NaHS further increased the production of endogenous H2S by increasing the expression of endogenous hydrogen sulfide synthase.Finally,the results of in vitro tube formation experiments showed that H2S co-operated with HIF-1αto up-regulate the expression of vascular endothelial growth factor（VEGF）through the PI3K/AKT signaling pathway and promoted angiogenesis in A549 cells.AOAA and PAG significantly reduced the expression of VEGF and inhibited the proliferation,migration and tube formation of human umbilical vein endothelial HUVEC cells.Tumor formation in nude mice also showed that AOAA and PAG inhibited the growth and angiogenesis of transplanted tumors.In conclusion,H2S promoted cell proliferation,migration and EMT process.Meanwhile,H2S increased the transcriptional activity of HIF-1α.Tumor hypoxia environment could promote the production of H2S in A549 cells to form a positive feedback regulation pathway.H2S together with HIF-1α,up-regulated VEGF expression and promoted angiogenesis in lung adenocarcinoma.Overall,our results showed that H2S played a pro-cancer role in lung adenocarcinoma,and inhibition of hydrogen sulfide synthase could effectively inhibit the proliferation of lung adenocarcinoma tumors.This study provided a basis for the study of H2S and new ideas for the prevention,diagnosis and target therapy of lung adenocarcinoma. |
