Effects Of Huaier Aqueous Extract On The Biological Behavior Of Human Lungadenocarcinoma Cells And Underlying Molecular Mechanism

Background: Lung cancer has become one of the most common malignant tumor and the leading cause of cancer-related death.The mortality of lung cancer accounts for the first of all cancer deaths globally.According to the report of the International Agency for Research on Cancer,the number of deaths due to lung cancer has been increased to 1.6 millions worldwide until 2013,which accounts for 19.4% of malignant tumor deaths.Whilst surgical resection before spread can affect a cure the majority of patients with lung cancer present with disease that is beyond remedial surgical intervention.About 80% of lung cancer patients are diagnosed at an advanced stage unfortunately.Despite the chemotherapy,radiotherapy and other medical treatment,the prognosis of patients with advanced lung cancer is still unsatisfactory.Over the past few decades,postoperative adjuvant chemotherapy treatment has been proven to reduce the recurrence rate,but the incidence of other malignancies,such as ovarian cancer,gastrointestinal cancer,and pancreatic cancer,has improved.In recent years,gene targeted therapy has become more and more concerned,however,these treatments can not effectively reduce the suffering of patients and increase the financial burden of patients.Therefore,the research and development of relatively natural and less toxic drugs has become a new topic of cancer treatment.The kind of anti-cancer drugs have fewer side-effects,lower drug resistance and lower cost than other treatments,thus providing a new direction for cancer treatment.Huaier aqueous extract is a type of fungus from China that been approved by the Chinese Food and Drug Administration.The main active ingredient of Huaier aqueous extract is the polysaccharide,which is a binding protein composed of 18 monosaccharides combined with 18 kinds of amino acids.Huaier aqueous extract can induce the differentiation and apoptosis tumor cells,improve the killing ability of immune cell,reverse tumor associated resistance,inhibit the migration and invasion of tumor cells and so on.Huaier aqueous extract can make no damage on the normal cells.A number of studies have found that Huaier aqueous extracthas been used in lung cancer patients and showed unique effect,but the specific mechanism has not been elucidated.In the process of epithelial mesenchymal transition(EMT),the disintegration of the connective complex on the cell surface could cause the reduced contact between the cells and the extracellular matrix.Cells acquire the ability to migrate and penetrate the cell interstitial through the EMT process.EMT which has become one of the key steps of tumor local infiltration is involved in the metastasis of tumor cells.EMT process is regulated by a variety of signaling pathways.The Wnt/β-catenin pathway is a key signaling pathway in the induction of EMT in epithelial tissue.β-catenin,as the core component of Wnt/β-catenin pathway is mainly involved in the tumor invasion and metastasis and the regulation of downstream target genes such as matrix metalloproteinase 9(MMP-9).Blocking or reversing the EMT process plays an important role in the inhibiting of tumor metastasis.Hippo-YAP signaling plays an important role in maintaining cell proliferation/ apoptosis balance,tissue and organ development.The core component is YAP.The abnormality of Hippo-YAP pathway is closely related to the development of various tumors.It seems feasible to directly or indirectly act on Hippo-YAP pathway in the treatment of tumor.Human lung adenocarcinoma cell line A549 is the most widely used human lung adenocarcinoma cell line.It was constructed from human lung cancer tissue culture system since 1972.It was spindle-shaped and had the characteristics and phenotype of alveolar type II epithelial cells under the microscope.The human lung adenocarcinoma cell line M-LAC(Metastastic Lung Adenoarcinoma)with metastastic potienal was successfully established from malignant peritoneal effusion from a patient with adenocarcinoma of lung,100 % transplanting tumors formed in 8 immunodeficient nude mice with 50% lymphatic metastatic rate and 25% hematogenous lung metastatic rate.Compared with A549,M-LAC with metastastic potienal expressed more meshenchymal marker Vimentin,less epithelial marker E-cadherin.The tumor suppressor gene Rb,p53,c-myc and angiogenesis-related factor VEGF-A were higher than those in A549 cells.Therefore,human lung adenocarcinoma cell lines A549 and M-LAC are the ideal cell models for investigation of the development,evolution,diagnosis and treatment of human lung cancer.In this study,in addition to its anti-proliferative and apoptotic effects,the novel possible molecular mechanism that Huaier aqueous extract exerts an anti-invasive effect on A549 and M-LAC cell lines was investigated.Objective:1.To investigate the effect of Huaier aqueous extract on cell proliferation,migration,invasion and apoptosis in human lung adenocarcinoma cell lines A549 and M-LAC.2.To investigate the effect of Huaier aqueous extract on the EMT process in human lung adenocarcinoma cell lines A549 and M-LAC.The target protein includes epithelium transformation markers E-cadherin and interstitial cell markers Vimentin.3.To investigate the effect of Huaier aqueous extract on Wnt signaling pathway in human lung adenocarcinoma cell lines A549 and M-LAC and the target protein is β-catenin.And the expression of matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),vascular endothelial growth factor(VEGFA)and matrix metalloproteinase-1(TIMP-1)were detected in human lung adenocarcinoma cell line A549 and M-LAC to explore the effect of Huaier aqueous extract on the invasion and metastasis potential of human lung adenocarcinoma cells.4.The expression of YAP,P-YAP and the binding factor such as Bax and bcl-2 in Hippo-YAP signaling pathway were detected to investigate the effect of Hippo-YAP signaling pathway on proliferation and apoptosis of human lung adenocarcinoma cell line A549 and M-LAC.Methods:1.Lung cancer cells were treated with different concentrations(0,2,4,6,8,10mg/mL)of Huaier aqueous extract,at the indicated time,cells proliferation was evaluated by CCK-8 colorimetry.And the morphological changes were observed under inverted optical microscope.2.Tumor cell migration was analyzed using the scratching assay: lung cancer cells were incubated without serum for 24 h,then the medium was replaced with DMEM or RPMI 1640 containing different concentrations of Huaier aqueous extract(0,6mg/mL)for an additional 12 h or 24 h.The wound distances were photographed with a microscope.3.Tumor cell invasion was analyzed using a Transwell chamber with an 8μm pore polycarbonate membrane and coated with Matrigel.After lung cancer cells were treated with Huaier aqueous extract(0,6mg/mL)24h,the polycarbonate membranes were analyzed under a light microscope and the migrated cells were counted in five randomly selected fields.4.Lung cancer cells were treated with different concentrations of Huaier aqueous extract(0,2,4,6,8mg/mL)for 48 hours,cell apoptosis was detected by Annexin V-PI staining.5.Morphological detection of apoptotic cells was observed using Hoechst333258 staining after A549 and M-LAC cells were treated with Huaier aqueous extract(0,2,4,6,8,10mg/mL)for 48 h.6.Western blot was used to detect the changes of related pathways in A549 and M-LAC cells treated with Huaier aqueous extract(0,2,4,6,8mg/mL)for 48 h.The detection parameters included epithelial marker E-cadherin,mesenchymal maker Vimentin,key molecule of Wnt signaling β-catenin,matrix metalloproteinase-9(MMP-9),matrix metalloproteinase-2(MMP-2),vascular endothelial growth factor(VEGFA),matrix metalloproteinase inhibitor 1(TIMP-1),yes-associated protein1(YAP1),phosphorylated YAP(P-YAP),B lymphocyte-associated gene(Bcl-2)protein,and Bcl-2 associated X proteiningene(Bax)protein.7.The changes of mRNA in A549 and M-LAC cells treated with Huaier aqueous extract(0,2,4,6,8mg/mL)for 48 h were detected by RT-PCR.The detection parameters included MMP-9,MMP-2,VEGFA,TIMP-1,Bcl-2 and Bax.Results:1.A549 and M-LAC cells were treated with increasing concentrations of Huaier for 48 h.The anti-proliferative effect of Huaier was measured by Cell Counting Kit-8(CCK-8)assay.The inhibition rate at 50%(IC50)of Huaier was 7.01 ±0.501mg/mL and 6.49±0.49 mg/mL respectively in A549 and M-LAC.Viable cell quantitation using the CCK-8 assay revealed decreased cell proliferation after treatment with Huaier aqueous extract at concentrations of 2,4,6,8mg/mL.2.Huaier aqueous extract inhibited human lung cancer cell viability,this growth inhibition phenomenon showed a time-and dose-dependent manner with a morphology change of A549 and M-LAC cells.Compared with the untreated cells,the majority of the Huaier-treated A549 and M-LAC cells became irregular-shaped,spinous and had a vacuolized changed cytoplasm.These morphology changes demonstrated cell damage.3.The results of scratching assay showed that the migration distance of human lung adenocarcinoma cells A549 and M-LAC can be effectively inhibited by Huaier aqueous extract(6mg/mL).In A549 cells,the migration index of Huaier aqueous extract was 23.3%,which was lower than that of the control group(56.1%).In M-LAC cells,the migration index in the test group was 13.3% after treated with Huaier aqueous extract for 12 h,which was not significantly different from 27.6% in the control group.However,after 24 h,the migration index of the test group(26.6%)was significantly different from that of the control group reached 62.9%(t test,P<0.01).4.Huaier aqueous extract(6mg/mL)inhibited the invasion of lung cancer cells.The inhibitory effect of Huaie aqueous extract on M-LAC cells was slightly stronger than that on A549 cells.The results showed that Huaier aqueous extract(6mg/mL)for 48 h can effectively inhibit the number of human lung cancer cell A549 and M-LAC through the membrane,which means that lung cancer cell invasion ability is inhibited.For A549 cells,the number of cells penetrating membranes was 344 ± 7.8 in control group and 115 ± 10.8 in Huaier aqueous extract group(6mg/mL).In M-LAC cells,the number of cells penetrating membranes was 367 ± 13.1 in control group while 54±7.5 in Huaier aqueous extract group(t test,P<0.01).5.Huaier aqueous extract(0,2,4,6,8mg/mL)induced lung cancer cell apoptosis,this pro-apoptotic effect showed a dose-dependent manner(0,2,4,6,8mg/mL,48h).The apoptotic rates of A549 cells were(3.07 ± 0.105)%,(6.167 ± 0.152)%,(9.873 ± 0.307)%,(13.567 ± 0.523)% and(16.643 ± 0.835)%,respectively at different dose(0,2,4,6,8mg/mL,48h).The apoptotic rates of M-LAC cells were(1.835 ± 0.226)%,(4.573 ± 0.345)%,(8.76 ± 0.657)%,(12.511 ± 0.709)% and(15.084 ± 0.543)% respectively.6.With Hoechst333258 staining,changes in cell morphology indicating apoptosis were observed in cells treated with Huaier aqueous extract(0,2,4,6,8mg/mL,48h),also in a dose-dependent manner.Treatment with increasing concentrations of Huaier aqueous extract resulted in the characteristic morphologic indicators of apoptosis,including nuclear condensation and fragmentation.7.Western blot analysis showed that the expression of E-cadherin in A549 and M-LAC cells treated with Huaier aqueous extract(0,2,4,6,8mg/mL,48h)was significantly higher than that in the control group.And Vimentin protein expression levels decreased showing a certain dose-dependent.Changes in the two groups of proteins suggest that reversal of epithelial mesenchymal transition in A549 and M-LAC cells.Huaier aqueous extract may inhibit tumor invasion through inhibiting the EMT process of A549 and M-LAC cells.8.Both of Western blot and RT-PCR results showed that compared with the control group,the expression of VEGFA and MMP-2 in A549 and M-LAC cells treated with Huaier aqueous extract(0,2,4,6,8mg/mL,48h)were significantly higher than those in the control group.The expression of MMP-9 decreased with the increase of the dose of Huaier aqueous extract,and the expression of TIMP-1 was increased in A549 and M-LAC cells,and the relationship between tumor invasion,metastasis and angiogenesis was convinced.The results showed that Huaier aqueous extract could inhibit the invasion and metastasis of A549 and M-LAC cells.9.Western blot analysis showed that the content of β-catenin in A549 and M-LAC cells treated with Huaier aqueous extract(0,2,4,6,8mg/mL,48h)was significantly decreased than that in control group(P<0.05).This result suggests that Wnt signaling pathway is regulated by Huaier aqueous extract.10.Western blot analysis showed that the levels of P-YAP protein in human lung adenocarcinoma A549 and M-LAC cells treated with Huaier aqueous extract(0,2,4,6,8mg/mL,48h)were significantly higher than those in the control group(P<0.05).With the increased dose,the expression of YAP1 is down-regulated.The results suggested that Hippo-YAP pathway is regulated by Huaier aqueous extract in both groups of lung cancer cells.The results of Western blot and RT-PCR showed that the upregulation of bax was promoted by bcl-2,which indicated the apoptosis of both human lung adenocarcinoma A549 and M-LAC cells.This result suggests that Huaier aqueous extract induced apoptosis of A549 and M-LAC cells,and this apoptosis may be related with Hippo-YAP signaling pathway regulation.Conclusion:1.Huaier aqueous extract can greatly influence the biological behavior of A549 and M-LAC cells.It can inhibit proliferation,invasion and migration of lung cancer cells,induce apoptosis in lung cancer cells.The cell line M-LAC,which has a higher metastatic potential,might be more sensitive to the Huaier treatment than A549.2.The results of western blot and RT-PCR showed that Huaier aqueous extract could inhibit the process of EMT,invasion and metastasis of human lung adenocarcinoma cell line A549 and M-LAC.3.Moreover,cell proliferation,apoptosis,cell adhesion and invasion signaling proteins were down-regulated by Huaier treatment.Huaier aqueous extract might induce lung cancer cell growth and cellular invasion inhibition through Wnt/β-catenin and Hippo-YAP signaling pathways,which were found to be related with the proliferation and apoptosis of human lung adenocarcinoma cell line A549 and M-LAC.