| BackgroundLung cancer is the leading cause of death in patients with cancer.Non-small cell lung cancer(NSCLC)accounts for over 85%of total lung cancer and is prone to invasion and metastasis.After 5 years of surgery or radiotherapy,about 20%and 34%of patients developed local or distant recurrent metastases.Despite the availability of many current treatments,drug resistance poses a challenge to clinical prognosis.Therefore,the study of the mechanism of invasion,metastasis,and drug resistance in NSCLC is still an urgent problem to be solved.A key metabolic characteristic of cancer cells is the reprogrammed bioenergetic state,where hypoxia triggers increased glycolysis and reduced mitochondrial respiration capacity.The glycolysis rate of tumor cells is 200 times higher than that of normal cells,and the high glycolysis phenotype changes the tumor microenvironment(TME)by activating hypoxia-inducing factor-1α(HIF-1α)signaling pathway to promote tumor invasion,migration,and drug resistance.These results suggest that targeting key metabolic enzymes in aerobic glycolysis could be an effective way to treat cancer.Studies found that the development of drug resistance is associated with the Epithelial-mesenchymal transition(EMT)induced by cancer stem cells.Phosphoglycerate kinase 1(PGK1)is both a phosphorylated protein kinase and a key metabolic enzyme in aerobic glycolysis.PGK1 could catalyze the reversible transfer of the phosphate group from 1,3-diphosphoglycerate(1,3-BPG)to ADP and produces 3-phosphoglyceric acid(3-PG)and ATP.In addition to regulating cellular metabolism,PGK1 is also involved in several biological activities,including angiogenesis,autophagy,and DNA repair.Studies have confirmed that PGK1 is highly expressed in a variety of tumors and is linked to cancer occurrence,resistance to chemotherapy and poor prognosis of patients with cancer.However,whether PGK1 can affect proliferation,migration,and chemotherapy resistance of lung cancer has not been reported.This study aims to explore the effects and regulatory mechanism of PGK1 on lung cancer proliferation,migration,and drug resistance to find potential targets for treating lung cancer and lay the foundation for the development of new drugs.Methods and Results1.PGK1 could regulate the EMT process and metabolic reprogramming to promote the proliferation and migration of NSCLC.1.1 Bioinformatics analysisSamples from TCGA online database were used to find the PGK1 expression in a variety of tumors.The results showed that PGK1 was not only significantly higher in tumor tissues than in normal tissues but also significantly higher in lung cancer than in other tumors.In lung cancer,the high expression of PGK1 was negatively associated with overall survival(OS),post progression survival(PPS),and first progression survival(FP).Next,the expression of PGK1 in different stages of lung cancer was further investigated by TCGA online database,and it was found that the expression of PGK1 gradually increased with the malignant progression of lung cancer.1.2 Construction of the PGKl knockdown modelA panel of NSCLC cells(H157,H460,CALU-1,A549,and H1299)and normal lung epithelial cells(BEAS-2B)were cultured and the different expression of PGK1 was determined by Western blotting.The results showed that the expression of PGK1 was relatively high in A549 cells.According to the endogenous expression of PGK1 in lung cancer cell lines,siPGK1 was transfected into A549 cells with high PGK1 expression.Western blotting was used to detect the transfection efficiency from the protein level.The results showed that the PGK1 expression decreased after siPGK1 transfection,indicating that the PGK1 knockdown model was successfully constructed.1.3 PGK1 could promote the proliferation,migration,and invasion of NSCLC.After siPGK1 was transfected into A549 cells,it was found that PGK1 knockdown could inhibit cell proliferation,migration,and invasion.1.4 PGK1 inhibition could inhibit proliferation,migration,invasion and promote the process of NSCLC apoptosis.To further explore the role of PGK1,the effects of inhibiting PGK1 enzyme activity on the proliferation,migration,invasion,and apoptosis of NSCLC were examined by using the PGK1 inhibitor NG52.First,the survival rate of different cells treated with NG52 for 48h was examined by MTT experiments.It was found that A549/siPGK1 cells with reduced PGK1 expression were less sensitive to NG52 than their parent A549 cells which suggest that NG52 is a specific PGK1 inhibitor.NG52(4,8,16,32,64,and 128μM)was applied on A549 and H460 cells and the survival rate was calculated by MTT assay.NG52 could inhibit the proliferation of different cells and had different IC50 at 24h or 48h,which was mainly related to the different PGK1 expression.NG52(10,20,and 40μM)was shown to inhibit the migration and invasion of A549 and H460 cells for 24h by using Transwell assay.AnnexinV-FITC apoptosis detection kit and flow cytometry were used to detect the effect of NG52 on apoptosis.The effects of NG52(10,20,and 40μM)on the apoptosis-related protein Bax/Bcl-2 were detected by Western blotting assay.We found that NG52 promoted apoptosis of A549 cells but had no significant effect on H460 cells.The above results indicated that the knockdown of PGK1 expression and inhibition of PGK1 enzyme activity could effectively inhibit the proliferation,migration,and invasion of NSCLC,1.5.PGK1 could promote EMT and metabolic reprogramming of NSCLC.After siPGK1 was transfected into A549 cells,Western blotting showed that PGK1 knockdown caused an increased expression of E-Cadherin,and a decreased expression of N-Cadherin,lactate dehydrogenase A(LDHA),and M2 pyruvate kinase(PKM2).Immunofluorescence experiments further confirmed that PGK1 knockdown decreased N-Cadherin expression and increased E-Cadherin expression.Glucose and lactate assay kits were used to demonstrate that PGK1 knockdown inhibited glucose uptake and lactate production under hypoxic and oxygen conditions.1.6 Inhibition of PGK1 could inhibit EMT and metabolic reprogramming.NG52(10,20,and 40μM)was applied on A549 and H460 cells and Western blotting revealed that NG52 was able to downregulate the expression of E-Cadherin,and upregulate the expression of N-Cadherin,LDHA,and PKM2 in both A549 and H460 cells.Immunofluorescence assays showed that NG52 could decrease the fluorescence intensity of N-Cadherin and increase that of E-Cadherin.Next,we treated A549 and H460 cells with NG52 and cultured them in oxygen and hypoxic incubators for 48h,and found that NG52 inhibited glucose uptake and lactate production by using glucose and lactate assay kits.The results showed that PGK1 promoted the proliferation and migration of NSCLC by regulating EMT and metabolic reprogramming.1.7 PGK1 could mediate the PI3K/Akt/mTOR signaling pathway to regulate NSCLC proliferation and migration.Western blotting confirmed that A549 and H460 cells treated with NG52(10,20,and 40μM)could inhibit the protein phosphorylation of PI3K,Akt,and mTOR without affecting the total protein expression.A549 cells were treated with 1,2,and 4μM MK-2206(Akt inhibitor)for 24h and could inhibit the phosphorylation of Akt when the concentration was bigger than 2μM,MK-2206 could inhibit the phosphorylation of Akt in H460 cells when the concentration was 4μ.Therefore,4 μM MK-2206 was used as the subsequent experimental concentration.A549 cells and H460 cells were treated with 20 μM NG52,4 μM MK-2206,and 4μM MK-2206+20μM NG52,and found that MK-2206 partially reversed the migration inhibition produced by NG52 by inhibiting the phosphorylation of Akt.This result indicated that NG52 inhibited cell migration by inhibiting PI3K/Akt/mTOR signaling pathway.1.8 In vivo experimentsA549 cells(4 × 105 cells/mouse)were inoculated subcutaneously into the left axilla of male Balb/c-nu mice(6 weeks of age).Then nude mice were divided into control group,NG52 low-dose group(10mg/kg),NG52 medium-dose group(20mg/kg),and NG52 high-dose group(40mg/kg),with 5 mice in each group.Each nude mouse was gavaged with 0.1mL/10g NG52 or saline once a day for 10 days.The changes in body weight and tumor growth were observed and recorded.Ten days after administration,the nude mice were sacrificed and subjected to tumor dissection and body weight measurements.The results showed that NG52 inhibited lung cancer growth.NG52 had little effect on the body weight of nude mice,indicating that NG52 had a high safety.Next,the isolated nude mouse tumor tissues were selected for HE staining and IHC experiments.It was found that with the increase of NG52 dose,the number of tumors in the mouse lung cancer model significantly decreased,and the tumor activity decreased.The results of IHC experiments showed that E-Cadherin expression in mouse lung cancer model was increased and N-Cadherin expression was decreased with the increase of NG52 dose.This indicated that PGK1 inhibitors could inhibit lung cancer growth in nude mice by inhibiting the EMT process.Glucose uptake and lactic acid production were gradually inhibited with the increase of NG52 dose.The results showed that consistent with the in vitro results,PGK1 could promote NSCLC growth by regulating EMT and metabolic reprogramming.2.The effect of PGK1 on NSCLC resistance2.1 Construction of the PGK1 knockdown model in A549/Cisplatin cellsThe expression of PGK1 in resistant and non-resistant cells was determined by Western blotting assay,and the results found that PGK1 expression in A549/Cisplatin cells was significantly higher than in A549 cells,indicating that PGK1 may mediate the development of drug resistance,A549/Cisplatin cells were transfected using siPGK1,and Western blotting was used to detect the transfection efficiency from the protein level,and the results showed that the PGK1 knockdown model was successfully constructed.2.2 The effect of PGK1 knockdown on cisplatin resistanceAfter siPGK1 was transfected into A549/Cisplatin cells,the results showed that PGK1 knockdown inhibited the proliferation by MTT assay.A549/Cisplatin and A549/Cisplatin/siPGK1 cells were all treated with 12μM cisplatin,which found that A549/Cisplatin/siPGK1 cells were more sensitive to 12μM cisplatin treatment.The cell viability of different cells treated with cisplatin for 48h was determined by MTT assay,calculating IC50 and the results showed that PGK1 knockdown in A549/Cisplatin cells increased cisplatin sensitivity(Cisplatin IC50 from 38.7μM to 10μM).Furthermore,the cisplatin IC50 in A549 cells was 3.57μM,and the relative resistance index of drug-resistant and non-drug-resistant cells was 10.84,indicating cisplatin resistance was in A549/Cisplatin cells.2.3 The effect of PGK1 on cellular transport proteinsAfter siPGK1 was transfected into A549/Cisplatin cells,the results showed that PGK1 knockdown significantly caused a decrease in P-glycoprotein(P-gp)and multidrug resistance-associated protein 1(MRP1),but the effect of breast cancer resistance protein(BCRP)is not obvious,and increased expression of P-gp and MRP 1 in resistant cells compared with non-resistant cells.Rodanmin 123 uptake assay was used to detect the change of fluorescence intensity in A549/Cisplatin cells after transfection with siPGK1 for 48h.The results showed that the fluorescence intensity of A549/Cisplatin was decreased,and P-gp efflux activity increased compared with A549 cells.PGK1 knockdown inhibited P-gp efflux activity in A549/Cisplatin cells.Next,Western blotting was used to detect the effect of inhibiting PGK1 enzyme activity on drug transporters.The results found that NG52 could reduce the expression of P-gp at lower concentrations and had no obvious effect on MRP1 and BCRP.Rhodamine 123 assay showed that the fluorescence intensity of cells was gradually increased,and the efflux activity of P-gp was gradually inhibited with the increased concentration of NG52.2.4 The effect of PGK1 on cellular autophagyWestern blotting assay showed that compared with non-resistant cells,increased LC3-II expression and decreased P62 expression in resistant cells and PGK1 knockdown significantly inhibited the expression of LC3-Ⅱ and increased expression of P62 in A549/Cisplatin cells.Western blotting assay showed that when NG52 was greater than 10μmol/L,LC3-Ⅱ expression in A549/Cisplatin cells could be significantly down-regulated and P62 expression increased.Next,Western blotting assay was performed to detect the effects of 10μM NG52,0.4mM hydrogen peroxide(autophagy inducer)and their combination on autophagy related proteins.The results showed that,compared with the 0.4mM hydrogen peroxide group alone,the combination of NG52 reduced the expressions of LC3-II and P-gp and promote the expression of P62.These results indicated that PGK1 promoted the development of drug resistance by mediating autophagy.2.5 The effect of PGK1 on metabolic reprogrammingA549/Cisplatin/siPGK1 cells were growed in normoxic incubators or hypoxic incubators,respectively.Western blotting assay showed that the PGK1 knockdown significantly inhibited the expressions of LDHA and PKM2 under normoxic conditions.Meanwhile,glucose and lactic acid assay kits were used to detect the effect of down-regulated PGK1 expression on cell metabolism.The metabolic reprogramming process was enhanced in resistant cells compared with non-resistant cells and PGK1 knockdown inhibited glucose uptake and lactic acid production under hypoxia and oxygen conditions.Western blotting confirmed that NG52 reduced the expression of LDHA and PKM2 in drug-resistant cells after being treated with NG52(5,10,and 20μM)for 48h.To determine the effects of NG52 on glucose uptake and lactate production in A549/Cisplatin cells,we used the glucose and lactate assay kit.We found that NG52 inhibited glucose uptake and lactate production under hypoxia and oxygen conditions which are the same as the trend of PGK1 knockdown.The results indicated that PGK1 could promote the development of resistance by regulating metabolic reprogramming.2.6 The effect of PGK1 on EMTWestern blotting assay showed that NG52 significantly inhibited N-Cadherin expression and increased E-Cadherin expression which are same as the trend of PGK1 knockdown.N-Cadherin was up-regulated and E-Cadherin was down-regulated in resistant cells compared with non-resistant cells.The results indicated that PGK1 could promote the development of resistance by regulating the EMT process.Furthermore,the Western blotting assay confirmed that NG52 inhibited the phosphorylation of PI3K,Akt,and mTOR without affecting the total protein expression.2.7.In vivo experimentsA549/Cisplatin cells(8 × 105 cells/mouse)were inoculated subcutaneously into the left axilla of male Balb/c-nu mice(4 weeks of age).Then,nude mice were randomly divided into control group,NG52 low-dose group(5mg/kg),NG52 mediumdose group(10mg/kg)and NG52 high-dose group(20mg/kg),with 5 mice in each group.Each mouse was gavaged with 0.1mL/10g NG52 or normal saline once a day for 15 consecutive times.After 15 administrations,nude mice were sacrificed.The results showed that NG52 effectively inhibited tumor growth in nude mice in a dosedependent manner and had little effect on body weight,indicating that NG52 had a high safety.Next,the isolated nude mouse tumor tissues were selected for HE staining and IHC experiments.It was found that with the increase of NG52 dose,the number of tumors in the mouse lung cancer model decreased significantly.IHC experiments found that increased E-Cadherin and P62 expression,and decreased N-Cadherin and P-gp expression in the lung cancer transplantation model of nude mice with the increase of NG52 dose.The results showed that consistent with the in vitro results,PGK1 promoted lung cancer growth by upregulating P-gp expression,mediating autophagy,regulating EMT,and metabolic reprogramming.Conclusions1.PGK1 could regulate the EMT and metabolic reprogramming via the PI3K/Akt/mTOR signaling pathway to promote NSCLC proliferation and migration.2.PGK1 could upregulate the expression of drug transporter P-gp,mediate autophagy,and regulate EMT and metabolic reprogramming via the PI3K/Akt/mTOR signaling pathway to promote the development of NSCLC drug resistance.Research significanceIn this study,the molecular mechanism and related signaling pathways of PGK1 in promoting proliferation,migration,and invasion were verified from in vitro and in vivo experiments.The results found that PGK1 promoted NSCLC proliferation,migration,and drug resistance by promoting EMT and metabolic reprogramming.These results suggested that PGK1 may play an important role in promoting metabolic reprogramming and EMT,making it an attractive molecular target for the treatment of lung cancer.The potential clinical application of small-molecule inhibitors targeting PGK1 is feasible. |
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