Effect And Mechanism Of Panaxadiol Saponins On Proliferation And Migration Of Laryngeal Carcinoma Cells

Background: Laryngeal cancer is a common malignant tumor of the head and neck,which not only causes death of patients,but also cancer and treatment methods affect the physiological activities such as swallowing pronunciation of patients.Therefore,seeking more optimal treatment is a hot research topic of laryngeal cancer.In the early stage of the treatment of laryngeal cancer,surgical treatment and radiation therapy are often used,and late treatment is mainly based on radiotherapy and chemotherapy.Hormones are often used in chemotherapy to reduce the side effects of chemotherapy drugs(such as allergies,nausea and vomiting,etc.),but long-term use of hormones can cause side effects such as adrenal hyperfunction,induced or aggravated infection,and central obesity.PDS is a glycol-soluble diol group extract with low toxicity and easy to pass the blood-brain barrier.It is a site drug.Previous studies of PDS have found that PDS has a dexamethasone-like effect on GR binding,and dose-dependently increases GR-mediated luciferase transcription.It also has anti-inflammatory,anti-shock,and protects heart and lung function.Preclinical and clinical studies have shown that ginsenoside has cancer prevention activity against various tumors such as gastric cancer,breast cancer,liver cancer,ovarian cancer,colon cancer,melanoma and leukemia,so whether PDS can fight cancer in laryngeal cancer treatment Role,is its mechanism related to activation of the GR pathway? Therefore,it is necessary to study its anticancer activity in laryngeal cancer.Objective: In vitro study on the effects of ginsengdiol saponin PDS on proliferation,apoptosis,cell cycle and migration of human laryngeal carcinoma Hep2 cells were observed,and the molecular mechanism of its action was explored.Experimental data and research basis for ginsenoside inhibition of tumor growth were provided.Methods and Results:1.The growth of laryngeal carcinoma Hep2 cells was inhibited and the apoptosis increased after 48 hours of PDS treatment.The results of MTT assay showed that the growth of Hep2 cells was significantly inhibited from 0.075 mg/ml after 48 hours of PDS treatment,but there was no cytotoxic effect on myocardial cell lines.Cell growth count and plate colony formation experiments showed that PDS could significantly inhibit cells.Proliferation ability.Flow cytometry showed that G2/M phase arrest occurred in Hep2 cells 48 h after PDS treatment.Western Blot results showed that PDS may affect cell cycle progression by up-regulating the related genes P21 and P27 and down-regulating the expression of Cyclin B1 and CDK1.FITC-Annexin V/PI flow cytometry and Hoechst staining showed that compared with the control group,the apoptosis of Hep2 cells increased after 48 hours of DEX and PDS treatment.The Western Blot test showed that compared with the control group,DEX and The expression of anti-apoptotic protein Bcl-2 was down-regulated after PDS treatment(p<0.05),and the expression levels of pro-apoptotic proteins Bax and Cleaved-caspase3 were increased(p<0.05),further demonstrating that PDS promoted Hep2 through the above mechanism.Apoptosis.The addition of the caspase inhibitor ZVAD to Hep2 cells half an hour before PDS treatment showed that ZVAD attenuated PDS-induced Hep2 cell death but did not completely rescue cell death,indicating that PDS can pass caspase The apoptotic pathway induces cell death,and there are other pathways involved in inducing cell death.2.PDS inhibits NF-?B signaling pathway activity in laryngeal carcinoma Hep2 cells NF?B signaling pathway is the main pathway of tumor cell proliferation.Western Blot results showed that the expression of phosphorylated I?B?(p-I?B?)in Hep2 cells was significantly decreased after 48 hours of PDS and DEX(p<0.05).The cytoplasm and nuclear protein of Hep2 cells were extracted 48 hours after PDS and DEX treatment,and the expression levels of p65 and p50 proteins of NF-?B heterodimer were detected.The results showed that compared with the control group,the expression of p65 and p50 protein in cytoplasm was significantly increased (p<0.05),while the expression in nucleus was significantly decreased(p<0.05),indicating that NF-?B nuclear reduction.Finally,the transcriptional activity of NF-?B was further verified by the dual luciferase reporter gene method.The results showed that the transcriptional activity was significantly decreased after the cells were added to PDS.These results indicate that PDS inhibits p50 and p65 protein entry into the nucleus,and the activation of NF-?B signaling pathway is inhibited,thereby inhibiting the proliferation activity of Hep2 cells.3.PDS inhibits Epithelial-Mesenchymal Transition(EMT)in laryngeal carcinoma Hep2 cells Cell scratches and Transwell results showed that PDS could significantly inhibit the migration ability of cells.Western Blot results showed that PDS could down-regulate the protein expression of MMP2 and MMP9 related to metastasis ability(p<0.05).Morphological analysis of HE staining showed that the surface area and circumference of tumor cells increased significantly after PDS treatment for 48 h(p<0.05),and there was a tendency of epithelial cell differentiation.q PCR was used to detect the m RNA level of EMT-related genes.The results showed that the expression of E-cadherin was up-regulated in the epithelial differentiation(p<0.05),and the mesenchymal marker N-cadherin.The expression of vimentin was down-regulated(p<0.05),and the expression of EMT transcription factor zinc finger protein(Snail)was down-regulated(p<0.05).Western Blot results further confirmed that PDS treatment can affect the expression of EMT-related factors and epithelial and mesenchymal markers at the protein level,and the trend is consistent with the m RNA results,and also with the morphological changes.The above results indicate that PDS inhibits EMT of Hep2 cells.4.PDS inhibits the canonical Wnt/?-catenin pathways The expression of ?-catenin in Hep2 treated with PDS was examined by Western blot and q PCR at the protein and gene levels,respectively.The results showed that PDS could decrease the expression of ?-catenin(p<0.05).These results provide the latest evidence that PDS inhibits Wnt/?-catenin signaling in Hep2 cells,which may be the main molecular mechanism by which PDS inhibits EMT in Hep2 cells.5.PDS enhances chemosensitivity of Hep2 cells to cisplatin MTT results showed that 0.15 mg/ml PDS combined with 2?g/ml cisplatin significantly increased the inhibitory effect of cisplatin on Hep2 cell proliferation compared with control group,PDS group and cisplatin group(p<0.05).Annexin V-FITC flow cytometry showed that compared with the control group,PDS group and cisplatin group,PDS combined with cisplatin group could significantly increase the apoptosis-inducing effect of cisplatin on Hep2 cells(p<0.05).Western Blot showed that compared with the control group,PDS group and cisplatin group,PDS combined with cisplatin group can significantly reduce the expression of Bcl-2 protein in cells;it can significantly enhance the protein expression level of Bax and Cleaved-caspase3(p<0.05).The above results indicate that PDS can increase the anti-proliferative and pro-apoptotic effects of cisplatin on laryngeal carcinoma Hep2 cells,and it is an effective adjuvant drug for chemotherapy.Conclusion: 1.PDS can inhibit the proliferation of laryngeal carcinoma Hep2 cells,cause cell cycle arrest,and induce apoptosis,which may be related to the inhibition of NF-?B signaling pathway.2.PDS can inhibit the migration of laryngeal carcinoma Hep2 cells and inhibit the epithelial-mesenchymal transition of laryngeal carcinoma cells,which may be related to the inhibition of Wnt/?-catenin signaling.3.PDS combined with cisplatin can significantly inhibit the proliferation of laryngeal carcinoma Hep2 cells and promote cell apoptosis.