The Research Of Anti-Proliferation Of Bufalin On Human Esophageal Cancer Cell Line TE13

Esophageal cancer is one of the sixth most common malignant diseasesworldwide. The remarkable geographical distribution is the strikingcharacteristic for esophageal cancer. The incidence and mortality ofesophageal cancer of chain is the highest. Although the Treating techniques ofesophageal cancer improve constantly, still can not stop the increase inincidence of esophageal cancer year by year. Therefore, further study of theeffect of clinical anticancer drug on esophageal cancer and the activation ofrelative signal transduction pathway, is still the goal in the treatment ofesophageal cancer.The c-Jun N-terminal kinase(JNK) is a member of the mitogen activatedprotein kinases superfamily，is also called stress activated protein kinase. In avariety of cellular stress，for instance ionizing radiation, osmotic pressure, heatshock, oxidative damage and so on, the JNK is phosphorylated and activated,when it can transmit signals to the nucleus, and play biological effect, forexample causing cell proliferation, differentiation, apoptosis and so on.Existing study found that the inhibition of JNK can inhibit malignantproliferation of Tumor cell and induce apoptosis.Bufalin is effective anti-cancer substance, extracted from dermal andpostauricular gland of Bufo gargarizans Cantor and Bufo melanostictusSchneider of Bufonidae animals. Existing study have demonstrated thatbufalin can inhibit the growth of In vitro leukemia cell, hepatoma carcinomacell, gastric cancer celland Pancreatic cancer cell. But its antiproliferativemechanism is not yet clear. There is little the study about effect of bufalin onesophageal cancer.We used bufalin in esophageal cancer TE13cells, and detected itsantiproliferative effect and the expression of JNK Protein and the level of activation, to investigate the influence of bufalin on the esophageal cancerTE13cells and its possible molecular mechanism, and then provide the theorybasis for the clinical chemotherapy.Objective: To observe the inhibition of proliferation of bufalin on humanesophageal cancer TE13cells, to investigate the effect of the Bufalin on JNKand P-JNK protein expression of human esophageal cancer cell line TE13, toclarify the mechanism of inhibition proliferation of bufalin on humanesophageal cancer TE13cells, and to offer reasonable experiment evidence forthe mechanism of development of esophageal cancer and novel target in theclinic therapy.Methods: Human Esophageal Cancer Cell Line TE13were treated withdifferent concentrations of bufalin (0、10、25、50、100nmol/l). Cellconcentration change was evaluated by Cell Count method. The inhibition ofproliferation of bufalin on TE13cells were evaluated by MTT, and then drawthe curve chart of proliferation inhibition. The expression of JNK and P-JNKat protein level were measured by Western blotting. The location andexpression of JNK and P-JNK protein in the TE13cells were detected byImmunocytochemistry.Results:1. Cell Count method showed that the growth of human esophagealcancer cell line TE13was inhibited significantly by Bufalin. With the increaseof Bufalin concentration, the cell concentration ((4.35±0.10)×10~5、(4.08±0.06)×10~5、(3.7±0.09)×10~5、(2.7±0.07)×10~5、(2.35±0.09)×10~5cells/ml)decrease gradually, and the difference have statistical signification (P<0.01).Bufalin can inhibit the proliferation of human esophageal cancer TE13cells indoes dependent manner.2. MTT method showed that Bufalin inhibited the growth of esophagealcancer cell line TE13significantly. Different concentrations of bufalin treatedTE13cells for24h、48h、72h. With the increase of Bufalin concentration, theabsorption value (24h:1.004±0.025、0.869±0.012、0.735±0.013、0.562±0.025、0.462±0.021；48h：1.004±0.014、0.760±0.020、0.645±0.010、0.475±0.021、 0.356±0.027；72h：1.001±0.011、0.668±0.024、0.538±0.021、0.413±0.032、0.237±0.037) decrease gradually at the same time, and the discrepancy wassignificant at statistics(P<0.01). With the increase of time, the absorption valuedecrease gradually at the same concentration, and the discrepancy wassignificant at statistics(P<0.01).So the effect of inhibition proliferation ofBufalin on TE13cells enhanced gradually with the increase of Bufalinconcentration and action time in a time-and doses-dependent manner.3. Western bloting result show that the expression of JNK protein (0.786±0.005；0.776±0.005；0.779±0.016；0.792±0.015；0.769±0.014) had nochange obviously, and the difference have no statistical signification(P>0.05)；but the expression of P-JNK protein (0.740±0.007；0.687±0.006；0.662±0.005；0.587±0.007;0.394±0.012) decreased significantly, and thedifference have statistical signification (P<0.01). With the increase ofconcentration, the expression of P-JNK protein decrease gradually in a dosedependent manner.4. The Immunocytochemistry staining result: The JNK protein located atcytoplasm; The P-JNK protein located at cytoplasm and (or) nucleus. Thestandard of the positive cells was that brown-yellow particles or lumpaccumulate in the cytoplasm and (or) nucleus. The percentage of JNK-positivecells (84.44%；85.95%；83.85%；84.16%；84.03%) had no change obviously,and the difference have no statistical signification (P>0.05). With the increaseof concentration, the percentage of P-JNK-positive cells (85.30%；80.52%；72.85%；62.16%；40.03%) decrease gradually in a dose dependent manner.The difference have statistical signification (P<0.01).Conclusion:1. Bufalin may inhibit the proliferation of Esophageal Cancer Cell LineTE13, showing time-and doses-dependent relationship.2. It may be one of mechanism of the inhibition of proliferation of bufalinon human esophageal cancer TE13cells, that the activation of JNK wasinhibited.