Bufalin Plus Cisplatin Affect Esophageal Cancer Cells ECA109 Under Hypoxic Environment

BackgroundTumor hypoxia is one of the characteristics of the solid tumor,studies reported that tumor hypoxia microenvironment is the important factor of drug resistance.How to reverse the drug resistance of tumor,and further induce tumor cell apoptosis.it is a key link in the process of anti-tumor therapy.Bufalin has anti-tumor effect,it is the major active ingredient component of Chan Su.Bufalin is an traditional Chinese medicine and belongs to cardiac steroids,it has been proofed to the promotion of apoptosis,induced differentiation,inhibition of tumor angiogenesis inhibition,enhancing the efficacy of chemotherapy drugs and so on.Studies on bufalin induced of esophageal cancer apoptosis are rarely done in hypoxic environment.ObjectiveUnder chemical cobalt chloride?CoCl₂?simulated hypoxic environment,we investigated the effect of bufalin on proliferation and apoptosis of esophageal squamous cell carcinoma?ESCC?,we investigated the effect of bufalin combined with DDP on the anti-tumor activities,and explore its underlying molecular mechanism.Methods1.ECA109 cells were divided into four groups: the control group?A?,Bufalin group?B?with 5 concentration group?B-a 10nmol/L?B-b 25nmol/L?B-c 50nmol/L?B-d 100 nmol/L?B-e 200nmol/L?,DDP group?C?with 5 concentration group?C-a 2.5mg/L?C-b 5.0mg/L?C-c 7.5mg/L?C-d 10mg/L?C-e 20mg/L?,Bufalin +DDP group?D?with the concentration of Bufalin group D10nmol/L?combined a group?combined b group?combined c group?combined d group?combined e group?.2.Cell Counting Kit assay?CCK-8?was used to measure cell proliferation inhibition rate in different time segment?24h?48h?,10nmol/L of Bufalin was selected as the followup experimental concentration.3.Flow Cytometry was used to assess apoptosis of each experimental group after 48 h exposure.4.Hoechest 33342 staining method detect cell apoptosis after 48 h.5.The expression of HIF-1??NF-KBp65?Bcl-2 and Bax were detected with Western Blot on protein level after 48 h exposure.Results1.The CCK-8 results showed that Bufalin had a higher rate of proliferation inhibition than that of control group?P<0.05?,with a concentration dependent manner?P<0.05?;in Bufalin group,the inhibitory rate of 48 h exposure was higher than that of 24h?P<0.05?.Inhibitory rates of DDP plus 10 nmol / L bufalin group were higher than that single DDP group,in a time and dose-dependent manner,the difference was statistically significant?P <0.05?.2.Our flow cytometry results showed that the apoptotic rate was?46.72±32?%in the bufalin plus 5.0mg/L DDP group,obviously higher than that than single 5.0mg/L DDP group?32.84±42?%.showing statistical difference?P<0.05?;the apoptotic rate was?61.22±2.12?% in the bufalin plus7.5mg/L DDPgroup,obviously higher than that than single 7.5mg/L DDP group?43.17±4.03?%.showing statistical difference?P<0.05?.3.Hoechest 33342 staining under fluorescence microscope detect apoptotic morphology display: control cells have uniform shape and size,the cells appear concentrated nucleus,apoptotic bodies after 48 h in DDP group.The apoptosis were higher than the control group?P<0.05?,in a dose-dependent manner?P<0.05?,DDP plus 10nmol/L bufalin group were higher than the same concentration DDP group?P<0.05?.4.Western Blot results showed that,Hypoxic environment,compared to group I,the expression of HIF-1??NF-KBp65?Bcl-2 protein in group II?III?IV and V was significantly reduced?P<0.05?,Bax protein in group II?III?IV and V was significantly increased?P<0.05?.ConclusionsHypoxic environment,Bufalin can inhibit the proliferation of esophageal cancer ECA109 cells in a time and dose-dependent manner;Bufalin ECA109 can increase the sensitivity of ECA109 cells to cisplatin chemotherapy;Bufalin enhance the anti-tumor effect of cisplatin on anti-ESCC through its inhibition of the NF-KBp65/ HIF-1? pathway activated and inhibition of the Bcl-2 protein expression.