Inhibitory Effect Of Matrine Combined With Vincristine On The Proliferation Of Eca-109 Cells

Esophageal cancer is a malignant tumor derived from the esophageal epithelium.The incidence and mortality of esophageal cancer have been high in China,which has become a serious threat to the life and health of Chinese residents.The etiology of esophageal cancer is complex,and many factors are involved in the occurrence and development of esophageal cancer.Previous studies on the pathogenesis of esophageal cancer have found that Hippo signaling pathway is one of the important reasons for the formation and development of esophageal cancer.When the upstream protein of the Hippo signaling pathway,Mammalian Ste20-like protein kinase1/2(MST1/2)is activated,the transcriptional activity of Yesassociated protein(YAP),an effector protein of this pathway,is inhibited,resulting in the reduction of cell proliferation and the induction of apoptosis.When it is inactivated,YAP restores its transcriptional activity and promotes the transcription of Connective tissue growth factor(CTGF),which is its downstream connective tissue growth factor,thereby promoting cell proliferation.Under normal conditions,the activation and inactivation of Hippo signaling pathway are in a dynamic balance to regulate the balance of cell proliferation and apoptosis,and promote the normal development of tissues and organs.Abnormal inactivation of Hippo signaling pathway and high expression of YAP can be detected in esophageal cancer.Vincristine(VCR)is a classical chemotherapeutic alkaloid extracted from the Apocynaceae plant Vinca Verna.It not only affects mitosis by inhibiting the polymerization of tubulin,but also directly inhibits the activity of intracellular ribonuclease and causes cell apoptosis,but this inhibitory effect is not specific.The use of large doses of vincristine can cause severe neurotoxicity and other side effects,which leads to the limitation of clinical use of vincristine and can not achieve better therapeutic effect.However,small doses of vincristine combined with other chemotherapy drugs can play a better anticancer effect and reduce the incidence of adverse reactions.Matrine(Ma)is an alkaloid extracted from the rhizome of the legume Matrine,which has anti-inflammatory,anti-viral,anti-fibrosis,antioxidation,anti-tumor and other pharmacological activities.Some studies have found that matrine can inhibit the proliferation and metastasis of tumor cells,block the cell cycle,and induce apoptosis.In addition,matrine,as a chemotherapy adjuvant drug,can reduce the dosage of chemotherapy drugs to a certain extent,reduce the toxic and side effects of chemotherapy,and reduce the incidence of adverse reactions if it is used in combination reasonably.Moreover,matrine has a certain analgesic effect on neuropathic pain induced by vincristine.Therefore,the aim of this study is to investigate the effects of matrine and vincristine on the proliferation of Eca-109 cells and the underlying mechanisms,in order to provide new ideas for the clinical treatment of esophageal cancer.In this study,matrine and vincristine were used alone or in combination to treat Eca-109 cells,and the growth status were observed by inverted microscope.MTT assay was used to detect the effect of matrine and vincristine on the proliferation of Eca-109 cells,and the optimal concentration of the two combined was screened.The subsequent experiments were divided into control group,matrine group,vincristine group,matrine combined with vincristine group.Colony formation assay was used to detect the effect of matrine combined with vincristine on the colony formation ability of Eca-109 cells.The effect of matrine combined with vincristine on the migration ability of Eca-109 cells was detected by wound healing assay.Hoechst33342 assay was used to detect the effect of matrine combined with vincristine on the apoptosis of Eca-109 cells.The effect of matrine combined with vincristine on YAP1 expression in Eca-109 cells was detected by immunocytochemical assay.Western blot was used to detect the effects of matrine combined with vincristine on the expression levels of Hippo signaling pathway-related proteins.The cell growth state was observed by inverted microscope.The results showed that the cells in control group grew densely and in good condition.The cell density and the number of adherent cells decreased slightly after adding matrine or vincristine,while the cell growth was significantly inhibited when adding matrine combined with vincristine.The results of MTT assay showed that matrine and vincristine inhibited the proliferation of Eca-109 cells in a concentration-gradient manner when used alone or in combination,and there was a synergistic effect when matrine(1.0mg/m L)and vincristine(0.2μg/m L)were used together(P <0.01).The subsequent experiments were divided into control group,matrine group(1.0mg/m L),vincristine group(0.2μg/m L),matrine +vincristine group(1.0mg/m L+0.2μg/m L).The results of colony formation assay showed that matrine combined with vincristine significantly reduced the colony formation ability of Eca-109 cells,compared with the control group,matrine and vincristine alone group,the difference was statistically significant(P < 0.01).The results of wound healing assay showed that matrine combined with vincristine significantly inhibited the migration ability of Eca-109 cells,and the difference was statistically significant compared with the control group,matrine and vincristine alone group(P <0.01).The results of Hoechst33342 assay showed that matrine and vincristine could induce apoptosis of Eca-109 cells,but the combination of matrine and vincristine induced apoptosis more significantly.Immunocytochemistry results showed that matrine combined with vincristine could significantly reduce the positive expression of YAP1,compared with the control group,matrine and vincristine alone group,the difference was statistically significant(P < 0.01).The results of Western blot showed that the expression of YAP1 and its downstream CTGF in matrine combined with vincristine group was down-regulated,and the expression of MST1 and p-YAP1 was up-regulated.Compared with the control group,matrine and vincristine alone group,the difference was statistically significant(P < 0.05).The results showed that matrine combined with vincristine inhibits the proliferation of Eca-109 cells,which may be related to the regulation of Hippo signaling pathway.The above experimental results show that:(1)Matrine combined with vincristine can synergistically inhibit the proliferation of Eca-109 cells.(2)The inhibition of matrine combined with vincristine on the proliferation of Eca-109 cells may be related to the regulation of Hippo signaling pathway.