Molecular Mechanism Of Ginsenoside Rg3 Sensitizes Lung Cancer To Cisplatin

Background and Objectives:Lung cancer,as the highest mortality rate in the world,has attracted much attention in recent years.Cisplatin is a first-line chemotherapeutic agent for lung cancer,but the problem of reduced sensitivity restricts its clinical application.The decrease of sensitivity may be related to the formation of hypoxic microenvironment in the development of solid tumor of lung cancer.Hypoxia is caused by the proliferation of tumor cells faster than angiogenesis or abnormal neovascularization.In solid tumors,hypoxia alters the microenvironment and is associated with proliferation,metastasis,and drug sensitivity.Hypoxia can promote epithelial mesenchymal transition(EMT)and stem cell differentiation of cancer cells,which is beneficial to the survival of cancer cells.EMT is considered to be a driving force for tumor progression and is also considered to be an important feature of advanced cancer.In the EMT process,mesenchymal markers N-cadherin and Vimentin were up-regulated,while transcription factor Snail activated and the expression of epithelial marker E-cadherin was downregulated.With the occurrence of EMT,a group of tumor stem cells with multiple differentiation potential have been produced.These tumor cells further promote the metastasis of tumors.Stemness markers include transcription factors SOX2,NANOG,OCT4 and surface marker CD44.Hypoxia induced these changes in cancer cells mediated by the activation of NF-κB signaling pathway.The hypoxia-induced desensitization of cisplatin is not clearly elucidated,combination therapy is expected to solve this problem.20(R)-Ginsenoside(Rg3),the traditional Chinese medicine,is extracted from ginseng and has antitumor activities.In this study,we assessed whether the decrease in cisplatin sensitivity is related to hypoxia,and whether Rg3 can improve cisplatin sensitivity by blocking hypoxia.Methods:1.Using Co Cl2 to induce hypoxia,western blot was used to detect the expression of HIF-1 alpha protein.2.The anti-proliferation effect of cisplatin under normoxia and hypoxia was compared by CCK-8 and colony formation assay,and the anti-proliferation effect of Rg3 combined with cisplatin under hypoxia was detected.3.The pro-apoptotic effect of cisplatin under normoxia and hypoxia was compared by DAPI staining analysis,AV-PI apoptosis detection and western blot,and the pro-apoptotic effect of Rg3 combined with cisplatin under hypoxia was detected.4.The occurrence of EMT under normoxia and hypoxia was compared by cell morphology observation,EMT related protein detected by western blot and immunofluorescence,and the inhibitory effect of Rg3 combined with cisplatin were detected.5.The occurrence of stemness under normoxia and hypoxia was compared by spheroid formation assay,stemness related protein detected by western blot and immunofluorescence,and the inhibitory effect of Rg3 combined with cisplatin were detected.6.The activation of NF-κB signaling pathway under normoxia and hypoxia was compared by EMSA,NF-κB signaling pathway-related proteins in nuclear and total proteins detected by western blot,and the inhibitory effect of Rg3 combined with cisplatin were detected.Bay11-7082 is a NF-κB pathway inhibitor,the relationship between NF-κB signaling pathway and occurrence of EMT and stemness were observed by EMT and stemness related proteins detected by western blot.7.The effect of Rg3 combined with cisplatin in vivo was detected in nude mice.Western blot and immunohistochemical test were used to detect the expression of EMT,apoptosis and stemness protein in the tumor tissue to verify the results of the experiment in vitro.Results:1.200 μM of Co Cl2 were used to induce hypoxia in human non-small cell lung cancer(NSCLC)cellsWestern blot results indicated that HIF-1α expression increased over 48 h after treatment with 200 μM Co Cl2 in NSCLC cells.2.Hypoxia reduced the anti-proliferation and pro-apoptosis effects of cisplatin in human NSCLC cellsAfter treatment with cisplatin,the hypoxic NSCLC cells’ viability and colony formation were greater than in normoxia,the degree of nuclear chromatin condensation and apoptosis rate was lower than in normoxia,the pro-apoptotic proteins(caspase-3,-8,-9 and Bax)and apoptosis markers(PARP)were all lower than in normoxia,anti-apoptotic proteins(Bcl-2 and survivin)were both increased.3.Hypoxia induced EMT and stemness in human NSCLC cellsHypoxic cells are interstitial changes,the expression of HIF-1α,Snail,N-cadherin and Vimentin were upregulated,while E-cadherin were downregulated.The spheroid formation rate was higher in hypoxia than in normoxia,and the expression of stemness-related molecules SOX2,NANOG,OCT4 and CD44 were upregulated in hypoxia.4.Hypoxia-induced NF-κB signaling pathway mediated EMT and stemnessHypoxia induced binding to NF-κB DNA,which was greater in hypoxia than in normoxia.Hypoxia activated the expression of p-p65 and p65 in nuclear protein extracts and expression of p-p65,p65,p-IKK and IKK in total protein extracts of cells.Inhibition of the NF-κB pathway,EMT-related molecules including Snail,N-cadherin and Vimentin were down-regulated while E-cadherin protein expression was up-regulated,stemness-related molecules SOX2,NANOG,OCT4 and CD44 were also down-regulated.5.Rg3 + cisplatin inhibited the hypoxia-induced NF-κB signaling pathwayRg3 treatment of hypoxic NSCLC cells decreased the expression of p-p65 and p65 in a concentration-and time-dependent manner.NF-κB DNA binding in the Rg3+ cisplatin group was less than that of the two drugs used individually in hypoxic cells.The expression levels of p-p65 and p65 in nuclear protein extracts and the expression levels of p-p65,p65,p-IKK,and IKK in total protein extracts were lowest in the Rg3+ cisplatin treated group in hypoxia.6.Rg3 sensitized hypoxic human NSCLC cells to cisplatin in vitroHypoxic NSCLC cells treated with Rg3 + cisplatin had decreased cell viability and lower colony formation rate,higher nuclear chromatin condensation and an increase in the total apoptosis rate,higher expression of caspase-3,-8,-9,Bax and PARP,lower expression of Bcl-2 and survivin.7.Rg3 + cisplatin inhibited hypoxia-induced EMT and stemness in NSCLC cellsThe interstitial changes in lung cancer cells in hypoxia were obviously reversed in Rg3 + cisplatin treatment,lower expression of Snail,N-cadherin,Vimentin and higher expression of E-cadherin,lower spheroid formation rate and lower expression of stemness related proteins SOX2,NANOG,OCT4 and CD44.8.Rg3 sensitized human NSCLC cells to cisplatin in a xenograft mouse modelRg3 combined cisplatin significantly reduced the tumor volume and weight.Rg3+ cisplatin decreased the expression of HIF-1α,p-p65,p65,N-cadherin,SOX2,OCT4,Bcl-2 and survivin,and increased the expression of E-cadherin,cleaved-caspase3 and Bax in tumor tissues.Conclusion:1.Hypoxia reduced cisplatin chemosensitivity in lung cancer cells.2.Hypoxia induced EMT and stemness mediated by the NF-κB signaling pathway.3.Rg3 inhibited NF-κB activation in hypoxia in a concentration/time-dependent manner.4.Rg3 + cisplatin inhibited hypoxia-induced NF-κB signaling pathway,which mediated EMT and stemness in vitro and in vivo.5.Rg3 increased cisplatin sensitivity in vitro and in vivo.