Ginsenoside CK Enhances Efficacy Of Pemetrexed In Lung Adenocarcinoma By Inhibiting Glycolysis Via EGFR/AKT/G6PC3 Signaling Axis

Background:Lung cancer remains the leading cause of cancer morbidity and mortality worldwide.Non-small cell lung cancer(NSCLC)is the most common type of lung cancer,accounting for 85%of all cases,of which lung adenocarcinoma(LUAD)dominates NSCLC.Currently,the treatment of NSCLC includes surgical excision,radiotherapy,chemotherapy,targeted therapy,immunotherapy,etc.Despite advances in the treatment of non-small cell lung cancer,the prognosis is still unsatisfactory,and the 5-year survival rate of patients with NSCLC is only maintained between 10%and 20%.Most patients are diagnosed in advanced or metastatic states,and are more susceptible to treatment resistance and poor prognosis.Pemetrexed(PEM)is an antifolate synthetic agent that inhibits tumor growth by disrupting the normal folate-dependent metabolic process in cells and inhibiting cell replication,and can be used in combination with platinum for advanced or metastatic non-small cell first-line therapy.However,drug resistance to PEM and side effects caused by high doses of PEM,including myosuppression and renal toxicity,limit its clinical application and therapeutic efficacy.Therefore,it is essential to maintain the effectiveness of chemotherapy and overcome drug resistance.Due to the large amount of time and money required to develop new drugs,reusing existing non-antitumor drugs and drug combinations for potential anti-tumor effects has become a focus of attention.Cancer cells undergo metabolic reprogramming and switch to a "glycolytic-based"metabolic mode to improve their survival rate and meet their energy and macromolecular requirements.This phenomenon is also known as the "Warburg effect".Increased glycolysis can acidify the intracellular microenvironment,reduce drug absorption and promote chemotherapy/radiotherapy resistance.Key ratelimiting enzymes in tumor cell glycolysis include hexokinase II(HKII),platelet type phosphofructokinase(PFKP),and pyruvate kinase M2(PKM2).These three key enzymes have been shown to contribute to the development of tumors.In liver cancer,the Warburg effect of cancer cells is enhanced by activating the EGFR-PI3K-AKT signaling axis,thus promoting the proliferation,invasion and metastasis of liver cancer cells.In human glioblastoma,inhibition of EGFR expression down-regulates the PI3K/AKT pathway and thus inhibits glycolysis.Several recent studies have shown that targeting enhanced glycolysis in cancer cells is a promising strategy to make them more sensitive to other forms of treatment,such as chemotherapy,radiation,etc.However,how to improve the therapeutic effect of lung adenocarcinoma by targeting glycolysis of cancer cells remains to be further studied..Ginsenoside CK,which is the metabolite of ginsenoside Rbl and Rb2 in intestinal bacteria,is a promising antitumor drug,which can effectively inhibit the proliferation,metastasis and invasion of a variety of cancers in vivo and in vitro,and induce their apoptosis,and has the advantages of high safety and diverse biological functions.CK induced glycolytic dysfunction in hepatoma cells by down-regulating key glycolytic enzymes HKII and PKM2.In addition,CK can enhance the sensitivity of tumor cells to cisplatin.However,the effect of CK on the sensitivity of lung adenocarcinoma cells to PEM is not clear.Therefore,this study explored the combined therapeutic effect of ginsenoside CK in combination with pemetrexed and aimed to elucidate the underlying mechanism using human lung adenocarcinoma cell lines A549 and PC9.Objectives:The combined therapeutic effect of ginsenoside CK and pemetrexed was explored and its potential mechanism was elucidated using lung adenocarcinoma cell lines A549 and PC9 as well as a nude mouse transplanted tumor model.Methods:1.The survival activities of A549 and PC9 cells treated with ginsenosides CK and other ginsenosides and PEM were detected by CCK-8 method.2.Compusyn software was used to calculate the combined drug index of CK and PEM.3.Colony formation experiment was conducted to observe the proliferation inhibition effect of CK/PEM combination on A549 and PC9 cells.4.Annexin V/PI double staining was performed to detect the effect of CK/PEM combination on apoptosis of A549 and PC9 cells.5.The effect of CK/PEM combination on cell cycle arrest of A549 and PC9 was detected by flow cytometry.6.The effects of different drug treatments on ROS levels in A549 and PC9 cells were detected by DCFH-DA staining combined with enzyme labeling instrument and fluorescence microscope.7.The effect of CK/PEM combination on migration and invasion ability of A549 and PC9 cells was detected by wound healing experiment and Transwell experiment.8.The content changes of ATP and pyruvate after treatment with different drugs were detected by enzyme label instrument.9.Western Blot analysis of the changes of related protein levels in A549 and PC9 cells and transplanted tumor tissues of nude mice after combined administration of CK,PEM and CK/PEM.10.To explore the effect of CK/PEM combination on lung adenocarcinoma in vivo by establishing B ALB/c A549 cell nude mouse transplanted tumor model.11.HE staining and immunohistochemical staining were used to detect the effects of combined medication on tumor morphology,increment index and important organs..Results:1.Ginsenoside CK and Pemetrexed(PEM)inhibited tumor cell activity in a concentration-dependent and time-dependent manner.Compared with PEM alone,the activity of A549 and PC-9 cell lines decreased by more than 30%when CK/PEM was used in combination.In the treatment of A549 and PC9 cells with 30μM CK and 2μM PEM,the two drugs showed the best synergistic effect,significantly inhibiting colony formation(P<0.01),indicating that CK/PEM synergistically inhibited cell proliferation.2.In A549 and PC9 cell lines,compared with CK and PEM alone,the percentage of apoptotic cells induced by the combination group was significantly increased(P<0.01),and CK promoted the increase of the level of pro-apoptotic protein(CleavedCaspase3,Bax)and the decrease of anti-apoptotic protein(Bcl2)induced by PEM.Moreover,PEM promoted ROS accumulation in CK-induced A549 and PC9 cells.3.After CK treatment,47%A549 cells and 53%PC9 cells were blocked in G1 phase.After PEM treatment,58%of A549 cells and 54%of PC9 cells were blocked in the S phase.In CK/PEM group,70%of A549 and 67%of PC9 were blocked in G1 phase.4.Migration and invasion ability of A549 and PC-9 cells were significantly reduced after CK/PEM combined treatment.After CK/PEM treatment,the expression levels of epithelial markers E-cadherin increased,while the expression levels of interstitial markers N-cadherin and vimentin decreased.These results indicated that CK/PEM inhibited the invasion and migration of A549 and PC-9 cells mainly by inhibiting EMT.5.Transcriptome sequencing results suggested that the combination of CK and CK/PEM could down-regulate the gene expression of glycolysis pathway.However,the gene expression of glycolysis pathway was not affected by PEM treatment.The results of RT-qPCR analysis of the above genes also confirmed that the combination of CK and CK/PEM could significantly down-regulate the glycolysis pathway genes.6.Compared with normal human lung epithelial cell lines BEAS-2B,the levels of key glycolytic enzymes HKII,PFKP and PKM2 in lung adenocarcinoma cell lines A549 and PC9 were significantly increased.The contents of glycolytic end products ATP and pyruvate in lung adenocarcinoma cells treated by CK and CK/PEM were significantly decreased,while no significant changes were observed in PEM group.The protein levels of key glycolysis enzymes HKII,PFKP and PKM2 were downregulated in CK treated cells,but PEM had no significant effect on them.7.Exogenous supplement of sodium pyruvate can significantly reduce the cell death induced by CK and CK/PEM.Moreover,sodium pyruvate reversed the apoptosis related protein levels induced by CK/PEM.8.The subcutaneous transplantation tumor model of balb/c nude mice was established.Compared with the single administration,the combination of CK/PEM significantly reduced the tumor volume and significantly inhibited the expression of Ki67,without obvious toxic and side effects.The results of Western blotting were consistent with the results of in vitro experiments,CK could induce glycolysis dysfunction in xenograft tumors,and the glycolysis dysfunction was further aggravated in the combined treatment group.Compared with the control group and the control group alone,CK/PEM could significantly promote the apoptosis of transplanted tumors and inhibit the epithelial-mesenchymal transformation(EMT).9.CK inhibits glycolysis of lung adenocarcinoma cells by inhibiting the EGFR/AKT/G6PC3 signaling axis,and the presence of PEM enhances this effect.Conclusions:1.Ginsenoside CK and pemetrexed synergically reduce the vitality of A549 and PC9 cells,inhibit cell proliferation,induce cell cycle arrest,promote cell apoptosis,produce ROS,and inhibit cell invasion and migration.2.Ginsenoside CK enhances the antitumor effect of pemetrexed on lung adenocarcinoma cells by inhibiting glycolysis through EGFR/AKT/G6PC3 signaling axis.3.Ginsenoside CK and pemetrexed synergically inhibit the progression of lung adenocarcinoma in vivo.