Metformin Inhibits IL-6-induced Epithelial-mesenchymal Transition(EMT)and Metastasis In Lung Adenocarcinoma

BackgroundNon-small cell lung carcinoma(NSCLC)is the leading cause of cancer death worldwide with an overall 5-year survival rate of less than 15%.Adenocarcinoma is the most widespread histological subtype of lung cancer,accounting for more than half of all NSCLC.Despite advances in treatments,recurrence and distant metastasis are still the main cause of death in the patients with lung adenocarcinoma.Growing Evidence reveals that epithelial-mesenchymal transition(EMT)plays a pivotal role in tumorigenesis,drug resistance,relapses,as well as in metastasis in various cancers.Numerous literatures have suggested EMT as poor prognosis in the patients with NSCLC.Therefore,elucidating the underlying mechanisms of EMT and identifying the molecule target and efficient drug to block this process is the key to reverse drug resistance and reduce metastasis,thereby improving overall survival of lung adenocarcinoma patients.EMT is a complex process including dissolution of cell-cell junctions and loss of apicobasolateral polarity,resulting in transition of epithelial cells into migratory mesenchymal cells with invasive properties.Migratory mesenchymal cells after transition are endowed with mesenchymal markers such as vimentin and N-cadherin,y-catenin and E-cadherin expression was depressed.Loss of E-cadherin expression is generally accepted as a hallmark of the EMT process.During this process,various transcription factors,such as SNAIL,ZEB1,ZEB2,Twist and so on,are key controller through directly and indirectly repressing E-cadherin.On the other hand,MicroRNAs(miRNAs),such as miR-200 family,are also involved EMT regulation by targeting the key transcription factors for direct repressor,ZEB1 and ZEB2.Indeed,the above mentioned intrinsic regulation,either transcription factors activation or miRNAs expression,they are all controlled by extrinsic signaling,such as the mediators from tumor microenvironment.Transforming growth factor beta(TGF-?),which is implicated in various tumor metastasis,has been proved the main factor involving EMT in the tumor microenvironments.Interleukin-6(IL-6)is another key factor in the tumor microenvironments,which is involved in tumorigenesis and progression.IL-6 activates the IL-6 receptor(IL-6R)to initiate signaling through the Janus kinase(JAK)/signal transducers and activators of transcription(STAT)signaling pathway and NF-kappa B(NF-?B)Elevated levels of IL-6 correlate with poor prognosis for cancer patients,such as breast cancer and lung cancer.Recently,some literatures have reported that IL-6 contributes to tumor metastasis and the process of EMT in breast cancer and ovarian cancer,through JAK/STAT3 signaling pathway.However,the role of IL-6 during EMT process in lung adenocarcinoma remains to be poorly defined.Metformin,an anti-diabetic drug,is associated with a reduced risk of many cancers.Studies have found that type 2 diabetics taking metformin experienced reduced cancer incidence and improved survival.Regarding pharmacological mechanism for its anti-tumor function,previous studies have found that metformin could inhibit the expression of pro-inflammatory mediators which play an important role in the tumor development,such as IL-6 and IL-17,by reducing activation of NF-?B.Moreover,metformin could inhibit cell growth and induce apoptosis in triple-negative breast cancers via blocking STAT3 phosphorylation.Thirdly,recent studies have showed that metformin could regulate breast cancer stem cell EMT processes and significantly down regulate the expression of several EMT markers through decreased expression of key drivers of the EMT machinery including the transcription factors ZEB1,TWIST1 and Slug.Taking the evidence that IL-6 might be involved in EMT process through JAK/STAT3 signaling in lung adenocarcinoma,metaformin could inhibit EMT program and the inhibition of STAT3 phosphorylation by metaformin,we hypothesized that metaformin could inhibit IL-6-induced EMT and metastasis in lung adenocarcinoma.ObjectiveEpithelial-mesenchymal transition(EMT)plays an important role in cancer metastasis.However,the underlying mechanisms of EMT in lung adenocarcinoma and how to inhibit this process remain to be explored.This study investigated the role of IL-6 in lung adenocarcinoma cell EMT and explored the potential effects of metformin on this process.Methods1.Invasion assayFor the matrigel invasion assay,filters were precoated with 30?1 Matrigel for 3h.Cells were starved in serum-free medium overnight,then trypsinized,centrifuged,and resuspended in serum-free RPMI 1640,and then seeded onto the upper wells with 2×104 cells each well supplemented with various concentrations of rhIL-6(50,100 and 500ng/mL),and rhIL-6 was replaced on alternative days.To determine the effect of metformin on cell invasion,various concentrations(1,5 and 10mmol/L)of metformin was added in cell culture with 50ng/ml of rhIL-6.Lower wells of the transwells contained the same medium as the up well with 2%FBS.After 18 h of incubation,the cells on the upper well and the matrigel were swabbed with a q-tip gently,fixed with 4%cold paraformaldehyde,and stained with 0.1%crystal violet.The cells that were attached to the lower surface of the polycarbonate filter were counted under a light microscope.Each experiment was repeated three times.2.Immunofluorescence and ImmunohistochemistryImmunofluorescence analysis was performed on 8-?m-thick frozen sections,or treated cells.Cells were cultured in 6-well chamber-slides,treated with hrIL-6 and with or without metformin for the indicated time,fixed with ice-cold 4%paraformaldehyde for 15 minutes at 37?,and then blocked with normal serum for 20 minutes at room temperature before that was incubatied with the primary antibodies(E-cadherin 1:200,vimentin 1:50)overnight in the dark at 4?.After three washes,slides were then stained with Cy3-labelled secondary antirabbit antibody(1:500)for 60 min at normal room temperature.Stained cells were visualized with an Olympus confocal microscope.The tumor specimens were fixed in 10%formalin,embedded in paraffin,and cut into 6?m-thick sections.For immunohistochemistry,the sections were deparaffinized,rehydrated,incubated with 3%H2O2 for 10 min,and subjected to heat-induced antigen retrieval by boiling for 10 min in 0.01 M sodium citrate.Then the specimens were blocked with normal goat serum for 1 hour and incubated overnight at 4? with anti-E-cadherin(1:400),anti-vimentin(1:100)or anti-phosphospecific Stat3(Tyr705)antibody(1:400).The immune complex was visualized using a kit according to the manufacturer's instructions.3.Enzyme-linked immunosorbent array(ELISA)STAT3-pY705 and IL-6 protein level were measured by Elisa according to the manufacturer's protocol.Absorbance at 450 nm was measured by a microplate reader.Each measurement was performed in triplicate.4.RNA extraction and Reverse transcription(RT)-PCR analysisTotal RNA from the cells and tissues was extracted from the cells with Trizol Reagent following the manufacturer's protocols,and then reverse-transcribed using random hexamers to generate cDNA.The PCR reaction was carried out in 25?L reactions with 10 pmol primers,the expression level of E-cadherin,vimentin,snail and IL-6 was detected with real-time PCR on ABI prism 7500 sequence detection system.The reaction conditions were:95? for 30 seconds,followed by 40 cycles at 95? for 5 seconds,60? for 34 seconds,and 72? for 45 seconds.GAPDH was used as a reference gene.Experiments were performed three times in triplicate.According to the 2(-Delta Delta Ct)method,the relative mRNA levels of E-cadherin,Vimentin,Snail,and IL-6 were calculated.5.Western blot analysisCells were lysed with M-PER Mammalian Protein Extraction Reagent ice-cold lysis buffer containing PMFS and phosphatase inhibitors for 30 min on ice,sonicated for 15 seconds to get complete cell lysis.SDS-PAGE(with the concentration of 8%)was used to separate cellular protein,The separated protein was then transferred to PVDF membranes.After blocking with 5%fat-free milk for 1h,the membranes were incubated with p-Stat3(Tyr705)(1:2,000),total Stat3(1:2000),E-cadherin(1:1,000),vimentin(1:1,000),snail(1:1,000)and ?-actin(1:2,000)antibodies at 4? overnight,washed three times with 0.1%Tween 20-TBS,and incubated in the secondary antibody(1:2000)for 60 min at normal room condition.Bands were visualized via ECL according to the manufacturer's instructions.6.In vivo xenograft experimentsFour-week-old nude female mice were purchased from the Chinese Academy of Medical Sciences and housed and maintained in laminar flow cabinets under specific pathogen-free conditions.These mice were divided into three groups with 6 mice in each group:group HCC827 and group HCC827-pSB388 received pure drinking water;group HCC827-pSB388+Met received drinking water with metformin(250 mg/kg body weight)for 2 days before tumor cells inoculations,and until mice sacrifice.2×106 HCC827(group 1)and HCC827-pSB388(group 2 and group 3)lung cancer cells were injected subcutaneously.Engrafted mice were inspected biweekly for tumor appearance by visual observation and palpation.Tumor volume(mm3)was calculated as length × width2/2.Mouse care and use was performed in accordance with local ethical guidelines.Results1.rhIL-6 induces A549 and HCC827 EMT and enhances tumor cell invasion in vitro.A549 and HCC827 cells have the typical features of epithelial:cobblestone shape and cluster formation.After one-week stimulation with rhIL-6 at the concentration of 50ng/ml,A549 and HCC827 cells were changed into scattered and fibroblast-like shapes,which are characteristic of mesenchymal-like morphology.These changed morphology suggested EMT of A549 and HCC827 under rhIL-6 stimulation.We subsequently determined the effect of IL-6 on cell invasion ability.The results showed that rhIL-6 treatment could significantly enhance invasive capacity in a dose-dependent manner both for A549 and HCC827 cell lines.More importantly,western blotting,quantitative PCR and immunofluorescence showed that 50ng/mL of rhIL-6 treatment both on A549 and HCC827 reduced the expression of epithelial cell marker(E-cadherin),and increased the expression of mesenchymal cell markers(vimentin and snail).2.A positive relationship between IL-6 production and vimentin expression is observed in lung adenocarcinoma tissues.The expression of IL-6,E-cadherin and vimentin mRNA expression in 18 cases of lung adenocarcinoma tissues and 6 cases of counterpart paracancerous tissues were detected with quantitative PCR,we found that IL-6 mRNA expression in paracancerous tissues is much lower than in lung adenocarcinoma tissue.There is a positive correlation between IL-6 and vimentin expression,and there is a negative correlation between IL-6 and E-cadherin expression.Immunofluorescence assay results showed weak vimentin expression but strong E-cadherin expression in low IL-6-expressing cancer tissues,whereas strong vimentin expression but weak E-cadherin expression in high IL-6-expressing cancer tissues.3.IL-6 activates STAT3 phosphorylation in vitro and a positive correlation between IL-6 production and p-STAT3 expression is observed in lung adenocarcinoma tissues.We found that rhIL-6 at the concentration of 50ng/mL could really activates STAT3 and induces STAT3 tyrosine phosphorylation at site of Tyr 705 in A549 and HCC827 cells.At the 30 minutes time point,p-STAT3 reached to the highest level and then declined to the basal line at 120 minutes.Also,we detected p-STAT3 expression in HCC827-pSB388 cells which stably express hIL-6.HCC827-pSB388 cells stably kept at higher p-STAT3 level,but not in HCC827 and HCC827-pLVT7 cells.These results suggested the possible role of p-STAT3 in IL-6-induced EMT.Importantly,the IL-6 production level is positively associated with the p-STAT3 level in cancer tissues when the values for each individual patient were plotted.Moreover,we found significantly higher p-STAT3 expression in high IL-6-producing tumor tissues than that in low IL-6-producing tissues.4.Metformin reverses IL-6-induced EMT in lung adenocarcinoma cells.Our study revealed that metformin could significantly reduced cell invasive capacity induced by rhIL-6 at the concentration of 5mmol/mL(p<0.05)or 10mmol/mL(p<0.005)both in A549 and HCC827 cell lines.The effect of metformin(10mmol/mL)on invasion capacity was even similar to that with a selective STAT3 inhibitor cucurbitacin Q(Cuc)at the concentration of 2?mol/L.Additionally,the results with quantitative PCR,western blotting and immunofluorescence showed that metformin could significantly repress vimentin and snail expression,and recover E-cadherin expression,which were changed by rhIL-6 treatment.5.Metformin suppresses IL-6-enhanced tumor growth and metastasis in nude mice.First,we established stable IL-6-expressing HCC827 cells(HCC827-pSB388)with lentivirus infection.We then detected the E-cadherin and vimentin protein expression of HCC827-pLVT7 and HCC827-pSB388 with western blotting,and the cell invasive capacity was also examined.The results showed that over-expressing IL-6 in HCC827-pSB388 cells could depress the E-cadherin expression and elevate vimentin expression,and enhanced cell invasive capacity in vitro.These results are similar to the findings in rhIL-6 stimulation experiments.With xenograft experiments,we found that hIL-6 transfection significantly promoted the growth of HCC827 tumors,However,this enhanced tumor growth by IL-6 transfection was partially reversed by metformin treatment(250 mg/kg body weight),we found the metastatic node in the lung and liver under microscope both in metformin-treated and PBS-treated HCC827-pSB388-bearing mice,but much fewer in the former than the latter(p<0.05).Consequently,we examined the EMT markers expression in HCC827,HCC827-pSB388 and metformin-treated HCC827-pSB388 tumor tissues with immunohistochemistry.We found the reduced E-cadherin expression but enhanced vimentin expression in HCC827-pSB388 tumors compared with HCC827 tumors,Interestingly,metformin could partially reverse EMT caused by IL-6 over expression.6.Metformin reverses lung adenocarcinoma EMT through inhibition of IL-6-induced STAT3 tyrosine phosphorylation.We determined the effect of metformin on IL-6-induced STAT3 tyrosine phosphorylation.The results showed that metformin could significantly reduce p-STAT3 expression induced by IL-6 in A549,HCC827 and HCC827-pSB388 lung adenocarcinoma cells in a dose-dependent manner.Moreover,the p-STAT3 expression in HCC827-pSB388,HCC827-pSB388+Met and HCC827-pSB388+Cuc cells were detected with immunofluorescence,results showed that the p-STAT3 expression was inhibited by both metformin and Cue.Immunohistochemistry showed us that more p-STAT3 expression was observed in HCC827-pSB388 cells grafted tumors than in HCC827 cells grafted tumors.However,the expression of p-STAT3 was significantly reduced in HCC827-pSB388 cells grafted tumors when treated with metformin.Conclusions1.IL-6 can induce lung adenocarcinoma cell A549 and HCC827 cell lines EMT and enhance tumor cells invasion.2.IL-6 induces lung adenocarcinoma EMT and metastasis by the mechanism of promoting STAT3 phosphorylation.3.Metformin can inhibit IL-6-induced EMT process by blocking STAT-3 phosphorylation.4.There is a potential clinical use for Metformin in inhibiting lung adenocarcinoma growth and metastasis.