Effect And Mechanism Of Cinobufotalin On Esophageal Cancer Cell Apoptosis Through ROS/MAPK And NF-?B Signaling Pathway

[Background]Esophageal cancer is one of the most common types of cancer of the digestive tract,yet current therapeutic treatments are inadequate due to a complex disease pathogenesis.However,drug toxicity and the marked side effects of chemotherapy limit the usage and therapeutic results of the treatments.A variety of active ingredients extracted from Chinese herbal medicine have shown good clinical effects in the treatment of cancer in recent years.Cinobufotalin is the major component of Chan-Su(a traditional Chinese medicine)extracts from the venom of Bufo gargarizan.Its main active is indoline alkaloid,with functions of activating blood circulation to disperse blood stasis,clearing heat,reducing swelling by detoxification and anti-inflammatory.At present,cinobufotalin is widely used in clinic as an adjuvant therapy in treating liver cancer,lung cancer and esophageal cancer.Research shows that cinobufotalin could induce the downregulate the anti-apoptotic proteins and upregulate the pro-apoptotic proteins in MDA-MB-231 cells to promote their apoptosis.The mitochondrial-mediated apoptotic pathway begins with mitochondrial membrane potential loss,cytochrome c release,the executioner caspase-3 cleavage,and eventually resulting in the formation of apoptotic bodies.JNK,p38,ERK and NF-?B are activated in response to several stress signals and are associated with the induction of apoptosis.It is becoming one of the key targets in screening treatment agents against cancer.However,the number of investigations concerning the effect of cinobufotalin on esophageal cancer is limited.[Objective]The purpose of this study is to explore the effect of cinobufotalin on esophageal cancer kyse-70 cells proliferation and apoptosis,and to further expiore its possible molecular mechanism.Our study may provide theoretical basis for the therapeutic possibility of cinobufotalin in esophageal cancer treatment.This study is to explore the effect of cinobufotalin on esophageal cancer.[Methods]1.CCK-8,LDH,cloning experiments and EDU assay was used to determine the effects of cinobufotalin on the growth and viability of esophageal cancer cells.KYSE-70 esophageal cancer cells xenograft-bearing nude mice were used to evaluate the therapeutic efficacy.2.TUNEL,DAPI,flow cytometric,western blot and qPCR assay was used to verify the apoptosis rate,apoptosis morphology and expression of apoptotic proteins.3.ATP,mitochondrial membrane potential,ROS,GSH and GSSG were measured to study the effect of the drug cinobufotalin on mitochondrial function in esophageal cancer cells.4.The mechanism of apoptosis was further analyzed by Western blot analysis.[Results]1.Cinobufotalin inhibited the proliferation of esophageal carcinoma cells in vitro and in vivo.2.The cell apoptosis rate was positively associated with drug concentration.3.Cinobufotalin can significantly inhibit ATP production and mitochondrial membrane potential in the mitochondria of esophageal cancer cells.ROS accumulation was excessive and GSH content decreased at the same time.4.Cinobufotalin induces apoptosis by ROS/MAPK?NF-?B signaling pathway in vitro.[Conclusion]Our results show that cinobufotalin induces apoptosis through ROS/MAPK?NF-?B signaling pathway in vitro,which may provide theoretical basis for the therapeutic possibility of cinobufotalin in esophageal cancer treatment.