Effects Of Bufalin On Human Esophageal Cancer Cells Proliferation And Mechanism Study

【Background】 As a common malignancy of the gastrointestinal tract in the human body,ESCC has been one of the leading causes of cancer deaths globally.In the long run,the incidence and mortality of esophageal squamous cell carcinoma(ESCC)has been high,especially in some Asian countries such as China and Singapore.With a cancer diagnosis technology and the improvement of surgical methods make survival had the very big enhancement,but most of the esophageal malignant tumor patients in the hospital the examination once found in cancer,and patients prognosis is relatively poor,reach expected curative effect,treatment effect is still mainly rely on some surgery scheme for treatment.Therefore,the research on the basic pathogenesis and molecular tumorigenicity of esophageal cancer,and the search for natural drugs with precise efficacy,as far as possible to kill normal cells and with fewer side effects,are still urgent problems to be solved by experts and researchers of various countries.In recent years,the potential of Traditional Chinese medicine in the treatment of tumor has attracted extensive attention,showing certain clinical advantages,mainly in the aspects of effectively improving patients’ postoperative resistance,inhibiting tumor recurrence and reducing metastasis.Traditional Chinese medicine(TCM)has a long history and has been widely used to treat cancer.Buflin,a common Chinese medicine extracted from toad venom,is a natural small molecule.The molecular formula is C24H34O4,and the relative molecular mass is 386.5.Bufalin was found to possess various pharmacological properties including cardiac protection,apoptosis induction,and anti-inflammatory effect,and it also has an obvious killing effect on tumor cells.In recent years,reports on the anti-tumor effect and mechanism of bufalin have been increasing year by year.Many scholars have studied the effect of bufalin on cancer.For instance,bufalin increased the apoptosis of human osteosarcoma U-2 OS cells and increased the protein levels of cytochrome c,AIF,and Endo G in cytoplasm,which suggested that bufalin induced apoptosis of U-2 OS cells via endoplasmic reticulum stress,caspase-,and mitochondria-dependent pathways.It has been reported that bufalin can also promote apoptosis of prostate cancer cells.Other studies have found that bufalin can inhibit tumor cells by affecting signaling molecules in related pathways.These findings provide an idea for the antitumor application of bufalin in clinic.However,the effect of bufalin on the proliferation of esophageal cancer and its molecular mechanism have not been reported.Therefore,this study preliminarily observed the effect of bufalin on the proliferation of esophageal cancer cells through in vivo and in vitro experimental model,and preliminarily explored its underlying molecular mechanism.【Objective】 In the present study,esophageal cancer cells were taken as the research object,and the effect of bufalin on the proliferation,ROS level and mitochondrial function of esophageal cancer cells were studied to preliminarily investigate the mechanism of apoptosis induced by bufalin,so as to provide theoretical basis for further research on the application of bufalin in the treatment of esophageal cancer.【Methods】1.In order to detect the effect of bufalin on the growth of ESCC,the effects of different concentrations of bufalin on the survival of esophageal cancer cells were observed under MTT,LDH and microscope.In order to detect the effect of bufalin on tumor formation,subcutaneous tumor formation experiments were conducted in tumor-bearing mice.2.To investigate the mechanism of bufalin affecting the growth of esophageal cancer cells,DAPI staining,TUNEL staining,annexin-v /PI double staining,western blot and q PCR were used to detect the expression of apoptosis-related proteins.3.In order to detect the effect of bufalin on reactive oxygen species(ROS)generation from mitochondria and the mitochondrial function of esophageal cancer cells,the experimental methods of ROS,Glutathione(GSH)levels,GSSG levels,mitochondrial membrane potential detection and ATP generation detection were used.【Results】1.The results of MTT and LDH experiments showed that bufalin significantly inhibited the activity of kyse-70 cells.The results of clone formation and Ed U showed that bufalin significantly inhibited the proliferation of kyse-70 cells.In vitro tumor formation experiments in nude mice showed that bufalin could inhibit the growth of esophageal cancer.2.DAPI staining results showed that chromatin heterogeneity,concentration,aggregation,fragmentation and other apoptotic forms appeared in the nuclei of kyse-70 cells after treatment with bufalin.TUNEL staining and flow cytometry showed that bufalin promoted the apoptosis of kyse-70 cells.The results of q PCR and Western blot showed that the expression of Bax/bcl-2 was increased,and the phosphorylation level of p65 was significantly reduced after bufalin treatment,while the overall level of p65 was unchanged.The results suggested that bufalin could promote the apoptosis of esophageal cancer cells.3.After the treatment of bufalin,the level of ROS increased,GSH was significantly reduced,the level GSSG increased,the mitochondrial membrane potential of KYSE-70 cells showed a significantly decreasing trend,and the production of ATP was significantly reduced.【Conclusion】1.Bufalin can inhibit the proliferation of esophageal cancer cells KYSE-70;2.Bufalin can promote the apoptosis of esophageal cancer cells;3.Bufalin can promote the accumulation of ROS in esophageal cancer cells KYSE-70 and inhibit mitochondrial function.