Ethyl Pyruvate(EP) Suppresses The Growth,Invasion And Migration And Induces The Apoptosis Of Non-small Cell Lung Cancer Cells

Background: Lung cancer has been the leading cause of death among tumors in these year and NSCLC accounts for about 80% of all lung cancer.The incidence and mortality of lung cancer have significantly increased worldwide nowadays.The rate of 5-year overall survival of patients treated with traditional chemotherapeutic drugs is only up to 15% due to side effects and drug resistance.Worldwide,1.7 million people die from lung cancer every year.Hence,it is very urgent to find new therapies to prolong the survival of patients suffering lung cancer.Cytokines including inflammatory factors in the microenvironment affect the proliferation and survival of tumor cells.Many inflammatory factors,such as TNF-α,IL-6,IL-8,promote tumor growth through Toll-like receptor(TLR)-mediated signaling pathways which lead to activation of ERK,NFκB,and STAT3.Damage-associated molecular patterns(DAMPs)are released by stressed,injured or dying cells,which initiate noninfectious inflammatory responses.HMGB1(high mobility group protein B1),one of the DAMPs,is released from damaged,inflamed,and tumor cells which in turn promotes tumor cell survival through its receptor RAGE.HMGB1 which organizes the DNA and regulates transcription has many biologic functions inside as well as outside the cell,especially promoting inflammation and tumorigenesis.It is said that HMGB1-RAGE axis can lead to pro-inflammatory gene activation.Because of the enhanced level of HMGB1 in some chronic diseases,this receptor RAGE is thought to have an enhancing effect in most diseases associated with inflammation and even some tumors;NFκB is a heterodimeric protein which is composed by the Rel family.NFκB plays a major role in stress-induced,immune,and inflammatory responses.Nowadays,it has been found that NFκB family members have an important relationship with tumor progression,such as programmed cell death,proliferation control and tumorigenesis;Recently,it is suggested that STAT family proteins play a crucial role,especially STAT3,in carcinogenic inflammatory microenvironment,which is not only at the beginning of malignant transformation but also during cancer progression.Many human malignancies which include lymphomas,leukemias,multiple myeloma and so on,have relationship with STAT3.Thus,it may be a potential approach for treating cancers by inhibiting STAT3 signaling.Ethyl pyruvate(EP),a lipophilic ester,derived from pyruvic acid is a non-toxic food additive.EP has a great many of pharmacological effects,for examples,the alleviation of redox-caused cellular and tissue damage,anti-inflammation,and promoting apoptotic process.EP inhibits the release of HMGB1.As we all know,EP may suppress many tumors in different degrees,yet its effect on lung cancer remains unclear.Here,we studied whether EP has an antitumor effect on lung cancer cells and tried to illustrate the possible mechanism.Objective: 1.To explore the effect of EP on the growth of non-small lung cancer(NSCLC)and the effect of EP on the apoptosis of NSCLC.2.To investigate the effect of EP on the migration and invasion of NSCLC.3.To investigate the mechanism of EP concerning the inhibition of growth,invasion and migration and the induction of apoptosis of NSCLC.Methods: 1.Human non-small lung cancer(NSCLC)cell lines(A549,H520 and PC-9)were treated with different concentrations of EP.MTT assay and Colony formation assay were used to assess the effects of EP on cell growth.2.Human non-small lung cancer(NSCLC)cell lines(A549,H520 and PC-9)were treated with different concentrations of EP.Flow cytometry assays were used to analyze cell apoptosis.3.Human non-small lung cancer(NSCLC)cell lines(A549,H520 and PC-9)were treated with different concentrations of EP.Invasion and migration potential was identified by Transwell assay.4.Human non-small lung cancer(NSCLC)cell lines(A549,H520 and PC-9)were treated with different concentrations of EP.Western blotting assay and PCR were used to explore the expression of PCNA,MMP-9,P53,HMGB1,RAGE.5.Human non-small lung cancer(NSCLC)cell lines(A549,H520 and PC-9)were treated with different concentrations of EP.Western blotting assay was used to test the expression of BCL-2 family protein.6.Human non-small lung cancer(NSCLC)cell lines(A549,H520 and PC-9)were treated with 30mmol/l EP Western blotting assay was used to investigate the expression of STAT-3,NF-ΚB,p-STAT3,p-NFκB(P65)in time-dependent manner.Results: 1.EP could significantly diminish the proliferative activities of A549,H520 and PC-9 cells in a dose and time-dependent manner compared with the control group.The colony formation rate of A549,H520 and PC-9 cells was markedly lower in the EP-treated groups than in the control group.Western Blot and PCR assay showed the decreased amount of PCNA expression in EP treated groups compared with the control group.2.The apoptosis index of A549,H520 and PC-9 cells in the EP-treated groups was markedly higher than in the control group.The protein and RNA level of p53 was increased in the EP-treated groups in comparison with the control group.3.The migration and invasive potential of A549,H520 and PC-9 cells was suppressed in EP treated groups compared with the control group.The protein level of MMP-9 was significantly reduced in the EP-treated groups compared with the control group,and the same results can be found in Real-Time PCR.4.The protein and mRNA expression levels of HMGB1 was significantly reduced in EP treated groups in a dose-dependent manner compared with the control group.The mRNA expression and protein levels of RAGE was decreased in EP treated groups in a dose-dependent manner compared with the control group 5.Western blotting revealed that EP treatment increased the pro-apoptosis protein Bax and decreased anti-apoptosis protein Bcl-2 and Mcl-1.6.We treated A549,H520,PC-9 cell lines with 30mmol/l EP for 2 H,4 H,6 H,8 H to examine the level of NFκB and pNFκB.The protein level of p-NFκB was significantly decreased compared with the control group.We treated A549,H520, PC-9 cell lines with 30 mmol/l EP for 2H,4H,6H,8H to examine the level of STAT3 and p-STAT3.The protein level of p-STAT-3 was significantly decreased compared with the control group.Conclusions: 1.Suppression of NSCLC growth by EP administration,and induction of lung cancer cell apoptosis by EP administration.2.Suppression of lung cancer cell migration and invasion by EP administration.3.Effects of EP administration on expression of HMGBI-RAGE axis and NFκB/STAT3 pathway in lung cancer cell lines.