| Background and objectiveNowadays,lung cancer has been the most common malignancy in China,with the highest morbidity and mortality,and new cases still increasing year by year.According to pathological features,it can be divided into small cell lung cancer and non-small cell lung cancer,and the latter in 75-80 percentage.NSCLC can be further divided into Squamous carcinoma,adenocarcinoma,adenosquamous carcinoma and large cell carcinoma,with adenocarcinoma accounts for half of NSCLC.Up to now,operation is still considered as the first choice in the treatment of lung adenocarcinoma in early or middle stage.But based on more and more evidence,it indicates that actually lung cancer is a kind of systemic disease,which calls for multidisciplinary interventions and comprehensive therapy.Thus it is quite important and worthful to deeply investigate the etiopathogenesis of lung adenocarcinoma,find out those key steps and molecules involved in the process,providing new ideas in targeting drugs.It has been verified that the abnormal regulation of epigenetics plays a key role in tumorigenesis and progress,bears close relation to the activation of oncogenes and deficiency of suppressor genes,and among that,histone methylation modification is a main mechanism in chromatin structure alteration and gene transcription regulation.The methylation of histone is a reversible dynamic modification process,coordinated by histone methyltransferases(HMT)and histone demethylase(HDM).As a member of SET domain family,histone methyltransferase G9a can catalyze the monomethylation and dimethylation of histone H3K9,which has been reported over-expression in many types of tumors,such as hepatic carcinoma,colon cancer and prostatic cancer,bearing some relation to tumor malignant progression and poor prognosis.We applied G9a inhibitor BIX-01294 in human lung adenocarcinoma cell lines,then observed cell proliferation,further analyzed the alterations in cell apoptosis,those apoptosis-related proteins and the expression of G9a catalytic products,H3K9meand H3K9me2.Then cells were treated with pan Caspases inhibitor,Z-VAD-FM,we then further explored whether the apoptosis induced by BIX-01294 is caspase pathway-dependent,and investigate the effect and underlying mechanism of BIX-01294 on attenuating cell proliferation in human lung adenocarcinoma.Experiment methodsThe lung adenocarcinoma cell lines A549,H1650 and H1975 were cultured in vitro.MTT assay was used for observing the cell proliferation after treated by BIX-01294 in different concentrations for 24 hours.The proliferation capacity of A549,H1650 and H1975 cells after BIX-01294 treated for 7 days was analyzed by colony forming assay.After the treatment of BIX-01294 for 24 hours,the apoptosis rates of A549,H1975 cells and the expression of H3K9me,H3K9me2,apoptosis-related proteins were analyzed by Annexin V-FITC/PI double staining and Western blot,respectively.Then we pre-treated A549 cells with pan caspases inhibitor Z-VAD-FM for 2 hours,to inhibit the activity of endogenous caspase,to further investigate the role of caspase cascade reaction in the BIX-01294 induced cell apoptosis and the expression of related proteins in A549.ResultThe inhibition by BIX-01294 in cell proliferation of lung adenocarcinoma A549,H1650 and H1975 cells was observed in a dose-dependent manner.The survival rates of cells after treated by BIX-01294 for 24 hours in different concentrations(1,2.5,5 and 10 μM,respectively)were 106.3%±7.2%,81.1%±4.8%,52.9%±5.6%,19.8%±13.6%in A549;91.2%±4.4%5 78.2%±5.1%,41.6%±4.8%,16.8%±3.5%in H1650 and 102.4%±6.3%,73.3%%±4.3%,36.4%±2.8%,12.5%±2.4%in H1975.In colony forming assay,after BIX-01294(10μM)treated for 7 days in A549,there were 42.5±8.7 colony forming units in BIX-01294 group,172.7±23.0 units in control group,the colony forming in A549 was greatly suppressed(p<0.05),and a similar effect was observed in H1975,165.2±42.8 units in control group,27.8±6.5 units in BIX-01294 group(p<0.05).After the treatment of BIX-01294(100μM)for 24 hours，the result of Annexin V-FITC/PI double staining showed A549 cell apoptosis rate increased from 7.2%±3.6%to 47.6%±18.4%(p<0.05),and that in H1975 increased from 8.9%±5.2%to 62.8%±8.2%(p<0.05).After BIX-01294 24 hours treatment,the H3K9me and H3K9me2 expression significantly decreased,mitochondrial apoptosis pathway proteins,Bax,Bak and cleaved caspase9 increased,anti-apoptoasis protein Bcl-2 decreased,and cleaved caspase3,cleaved PARP increased.Furthermore,in the presence of Z-VAD-FMK pretreatment,the A549 cells apoptosis and the expression of apoptosis-related proteins were inhibited significantly.ConclusionThe histone methyltransferase G9a targeted inhibitor BIX-01294 could attenuate cell proliferation in lung adenocarcinoma,it may upregulate Bax and Bak expression,downregulate Bcl-2 expression,activate the mitochondrial pathway,thus initiate caspase cascade reaction to induce cell apoptosis. |
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