The Molecular Mechanism Of Kaempferol On Anti-lung Cancer By Targeting SET7/9 Methyltransferase

ObjectiveLung cancer,a commonmalignant tumor,is one of the important diseases cause serious endangering human health and life.In recent years,although the morbidity of lung cancer has decreased,but the mortality continues to rise and the 5 year survival rate is only about 17%."Early screening,early diagnosis,early detection and prevention" has become an effective measure for prevention and treatment of lung cancer,because most patients have advanced disease at diagnosis.Histone methyltransferases(HMTs),is a regulating the expression of gene and protein,and structure modification through the recruitment of chromatin-remodeling and histone-modifying enzyme,play an important role in the development and progression of lung cancer.Studies have shown that HMTs could promot gene silence or over-expression,its lead to cause cancer germination on account of the microenvironment disorders and imbalance is through catalytic methylation responding in some tumor suppressor and oncogene.So,some propose control methylation is the precondition of controlling cancer.Kaempferol is a kind of flavonols compound,which is rich in a variety of fruits,vegetables and cereals,as well as natural plants.Research shown that kaempferol can promote the regeneration and development of nonnal cells,which protect cells from intrusion,also has the strongly anti-oxidant,anti-inflammatory,antiviral,antibacterial and antitumor and other pharmacological activities.In vitro studies shown that kaempferol can adjustable methylation of suppressor genes(P16 and MGMT)with many tumor cells,including lung cancer cells and others,which restore its activity to prevent further malignancy.Also studies have proved that kaempferol can promote tumor cell apoptosis and necrosis,inhibit proliferation,angiogenesis,inflammation,and invasion and metastasis,through regulation reaction of methylation.However,research of kaempferol is an indispensable part of control cancer in histone methyltransferases attributed to parely reported.Based on the current research,effect and mechanism of kaempferol through inhibit expression of SET7/9 histone methyltransferases was evaluated by using lung cancer cells model and xenograft model on anti-lung cancer in vitro and vivo,respectively.Methods1.Comparative analysis of the SET histone lysine methyltransferases in lung adenocarcinomaSurvival analysis of SET and SET7/9 were performed from TCGA databases with 491 patients of lung adenocarcinoma by using the online database(http://www.oncolnc.org/search_results/).The expression of mRNA and proteins of SET7/9 were comparatively detected using total RNA and proteins from urethane-induced FVB,benzo(a)pyrene-induced A/J mice and nude mice lung tumor and adjacent tissue,as well as human lung adenocarcinoma cells(A549,NCI-H1299,H1975,H1650,H1819)and normal pulmonary bronchial epithelial cell(BEAS-2B)through Real-time qPCR and Western blot assay,respectively.2.Effects of kaempferol on the expression level of SET mRNA and protein family in lung cancer cellsCell viability of A549 and NCI-H1299 cells were evaluated by MTT assay after treated with kaempferol(0-80?M)for 48 h or 5 days,and BEAS-2B cells was evaluated after treated with kaempferol(0-50?M)for 48 h.A549 and NCI-H1299 cells were used as in vitro model,the SET family and SET7/9 gene and total or nuclear protein expression level were detected using Real-time qPCR and Western blot after treated with different concentration of kaempferol(5,10,20 pM)and co-incubation 5-aza(2.5? M)plus TSA(500 nM)(as a positive drug)for 5 days.3.Evaluate of kaempferol on targeting SET7/9SET7/9,whether it is the target of kaempferol was evaluated by using docking(Computer simulation),the Cellular Thermal Shift Assay(CETSA)and co-localization analysis.The affinity(binding energy and Ki value)of kaempferol on SET7/9 were simulated through Docking 4.2 version software.The protein expression of SET7/9 was detected by Western blot analysis with after co-incubation A549 cell lysates plus kaempferol(100 ?M)or DMSO for 30 min at room temperature and heating at different temperatures(44-54?).Then,protein expression was further detected after co-incubation cells lysates and different concentrations of kaempferol(50-800 ?M)at 500C.In vitro,colocalization was analyzed after incubated with A549 cells and kaempferol at different concentrations,and following the combination of primary and secondary antibody.The images were captured by laser confocal microscopy and analyzed.In A549 cells,the protein expression levels of PP2Ac,p-AKT,p-GSK-3? were analyzed by Western blot assay after treated with kaempferol and 5-aza/TSA co-incubation for 5 days.In A549 cells,siRNA and overexpression assay were evaluated by using Lipofectamine TM 2000 transfection reagent.After 12 hours of transfection,the gene and protein expression of SET7/9 downstream(PP2Ac and Cytochrome C)were detected with kaempferol(5,10,20 ?M)treat for 48 h,including PP2Ac and Cytochrome C.In addition,the protein expression levels of PP2Ac and Cytochrome C were detected using overexpression transfection plasmid pEX-4 in BEAS-2B.Following,in A549 and BEAS-2B cells,cell proliferation were investigated byEdU assay after treated different concentrations kaempferol with prior silence or over-expression SET7/9,respectively.4.Evaluate the effect of kaempferol on mitochondrial apoptoticFurthermore,in A549 cells,the combination of kaempferol and mitochondria was investigated by immunofluorescence assay treated with different concentrations of kaempferol(5,10,20 ?M)or 5-aza(2.5 ?M)for 6 h.The changes of mitochondrial membrane potential(MMP)was carried out by rhodamine 123 fluorescence probe substrate,and the proteins expression of Cytochrome C and caspase 9 were delected using Western blot assay.5.Evaluate the anti-tumor effect of kaempferol and verify related mechanism in vivoBalb/c nude mice were injected subcutaneously with A549-luc-C8 fluorescent cell line.After the tumor was grown to 50-1003 mm,the mice were given gavage administration(0.9%saline,K-75 mg/kg and 150 mg/kg)for 30 days.The tumor was removed and the tumor size and indicators were calculated analysis.The gene and protein expression levels of SET7/9,PP2Ac and Cytochrome C in tumor tissue were determined by Real-time qPCR,immunohistochemistry or Western blot.6.Statistical analysisData were expressed as the mean ± standard deviation(SD)of three independent experiments.Statistical significance of the data was conducted by Student' t-test or one-way ANOVA using SPSS 19 software.Statistical significance was determined at p<0.05.Graphs were presented using GraphPad Prism 5.0 software.Results1.The expression of SET gene and protein in lung adenocarcinoma was significantly higher than that in adjacent carcinomaResults of survival analysis shown that prognosis is favourable with low expression of SET and SET7/9 histone methyltransferase in lung adenocarcinoma patients.The gene and protein expression of SET family were significantly increased by comparing the background expression in lung cancer or normal tissues and cells.Furthermore,compared with BEAS-2B cells,the protein expression of SET family in lung cancer cells was significantly increased in A549 cells.2.Kaempferol can significantly inhibited the gene and protein family expression of SET in lung adenocarcinoma cellsAccording to MTT results shown that A549 and H1299 cells were significantly inhibited after treated with low concentration of kaempferol for 5 days.Kaempferol concentrations was determined with low,middle and high-dose group(5,10,20 ?M),and the treatment with 5 days and replace medium once a day.Real-time qPCR and Western blot results showed that kaempferol has a significant inhibitory effect on SET gene and proteins family at 10 or 20 ?M,especially is SET7/9,and the inhibitory efficiency was significantly increased with a dose-dependent manner.3.Kaempferol could selectivity inhibited SET7/9Docking analysis shown that kaempferol have stronger affinity with negative binding energy is-9.55,Ki value is 99.86 nM.CETSA results shown that,compared with DMSO,kaempferol enhanced SET7/9 protein expression with the increase temperature.At 500C,it is proved that kaempferol can well bind with SET7/9,attributed to the gray value was the highest with the increase concentration of kaempferol,following remain unchanged.The similar result was also obtained by laser confocal assay with colocalization.Thus,in summary,it is proved that kaempferol can bind strongly with SET7/9 protein.The results showed that kaempferol significantly up-regulated the proteins expression of PP2Ac,p-AKT and p-GSK-3p by activating the pathway.In addition,in A549 cells,it downstream PP2Ac and Cytochrome C were no significant changes after the SET7/9 silence.Results of EdU shown that the cell proliferation was no obviously attenuated after silence SET7/9 compared with untreated cells.In BEAS-2B cells,the protein expression of PP2Ac and Cytochrome C were significantly down-regulated after with overexpression of SET7/9.Meanwhile,results of EdU shown that the cell proliferation was obviously increased after overexpression SET7/9 compared with untreated cells.Results demonstrated that kaempferol can only against progress of lung cancer by acts on SET7/9.The above two experiments shown that SET7/9 is the only and specific target of kaempferol,which can inhibit the proliferation of lung cancer cells only through SET7/9.4.Kaempferol could induced mitochondrial apoptosisKaempferol could increase mitochondrial membrane potential(MMP)through activat mitochondrial.The protein expression levels of Cytochrome C and caspase 9 were up-regulated 8.25-and 3.35-fold,respectively,promote apoptosis of lung cancer cells.5.Kaempferol inhibition of SET7/9 anti-lung cancer in vivoResults of nude mouse xenograft model experiments shown that kaempferol significantly reduces tumor size.Compared with the model group,kaempferol 75 mg/kg and 150 mg/kg can significantly reduce the volume and weight of tumor.Moreover,compared to model group,the results of immunohistochemistry or Western blot shown that kaempferol significantly decreased by 1.67-fold with protein expression of SET7/9 in tumor tissue and up-regulated by 3.10-fold on protein expression of Cytochrome C.Compared with control group,kaempferol no significantly affect the weight of nude mice in model and drug group.In summary,kaempferol could significant inhibition progress of lung cancer with almost no toxicity in vivo.ConclusionsIn currently study,effects and mechanism were explorated on kaempferol inhibits SET7/9 anti-lung cancer.Results demonstrated that kaempferol selectively targets inhibit SET7/9 methyltransferase and activate by PP2Ac-AKT-GSK-3 ?mitochondria pathway.Then kaempferol could increased mitochondrial membrane potential and promote the release of Cytochrome C from mitochondria,leading to mitochondrial apoptosis.Therefore,this study can provide a theoretical basis that kaempferol become a potential SET7/9 inhibitor to anti-lung cancer.