| Background:Esophageal cancer is a common and harmful esophageal epithelial malignancies.Nowadays, Chemotherapy is still one of the main methods for the treatment of malignant tumors. The mortality on chemotherapy resistance is urgent for clinical trails. As the malignant tumors occurs drug resistance for one chemotherapeutic drug, simultaneously, it might be cross-resistance for the others drugs which chemical structure and mechanism of action is different from the aforementioned, which is named the tumor multidrug resistance( MDR). Therefore, to explore MDR reversal agents, increasing the sensitivity with clinical first-line chemotherapy drugs, has a potential significance for the cancer treatment.Although some of the compounds have been found as the MDR reversal agents candidate,most of them are due to the large toxic side effects resulting in clinical application.Cepharanthine( CEP), extraction and separation from Stephania cepharantha Hayata, is a double-benzyl isoquinoline alkaloids monomer compound. And the semi-synthetic derivatives of Cepharanthine is Cepharanthine hydrochloride( CEH), CEH has a variety of biological activity. However, it reverse the multidrug resistance of esophageal squamous cell carcinoma cells and what is the mechanism, has not been reported. In this study, we adopt the methods of molecular biology, through establishing the vitro and vivo model model to research the reverse effect of CEH on the drug resistance in esophageal squamous cell carcinoma cells, and to explore the molecular mechanism of its drug resistance.Method:Human esophageal squamous cell carcinoma cell lines Eca109 and cisplatin( cis-Dichlorodiamine platinum( II), CDDP) induced a stable MDR phenotype cisplatin-resistant cell lines Eca109-CDDP were selected in this study. Using MTT method analysis the direct toxic effect of CEH alone or in combination with CDDP treated human esophageal squamous cell carcinoma cell lines, calculating the combined index( CI) by Compusyn software. Annexin V/PI double staining were used to detect apoptosis effects;Western blot to detect the expression and activation of apoptosis related proteins. Western blot and RT-PCR were exerted to determine the change of P-gp and MDR1 m RNA and proteins expression after CEH alone or in combination with CDDP treatment to carcinoma cell linesfor investigating the reversal effect of CEH on multidrug resistance; using Western blot assay was performed to determine the protein expression and phosphorylation of transcription factor c-Jun. Esophageal carcinoma xenograft model was applied to examine the effects and mechanisms of CEH combined with CDDP for observing the inhibitory effect of CEH on tumor growth in vivo. H&E and immunohistochemistry/immunofluorescence were used to detect the changes in protein expression of c-Jun / phosphorylated c-Jun and P-gp induced by CEH or in combination with CDDP in vivo.Result:1. 1-20.0μM concentration of CEH enhance CDDP induced apoptosis of human esophageal squamous cell carcinoma cell lines, and lead to a decrease of Bcl-2 / Bax ratio and increase of the CDDP-induced PARP cut.2. With increasing concentration of CEH, the inhibitory effect of the expression of P-gp and MDR1 m RNA gradually increased with the manner of concentration dependence. CEH can down-regulated the expressions of MDR1 m RNA and P-glycoprotein in concentration manner, the expression of Bcl-2 were gradually decreased and the expression of c-Jun,p-c-Jun and JNK gradually increases.3. In the concentration of 2. 5-10.0-mg/kg CEH combined with CDDP chemotherapy for Eca109-CDDP tumor growth in tumor-bearing mice have different level of inhibition.Compared with positive drug comparison group, the anti-tumor effect in combination therapy group was increased with a dose dependent manner. The anti-tumor effect was the strongest in 10. 0 mg / kg group.4. CEH was also in concentration-dependent down-regulation of P-gp protein expression and up-regulation expression of c-Jun, p-c-Jun and JNK in vivo.Conclusion:1. In vivo and in vitro, CEH could promote the inhibition of CDDP mediated proliferation of human esophageal squamous cell carcinoma cell lines as well as promote the CDDP induced apoptosis.2. CEH could reversed drug resistance in Eca109-CDDP cell lines. The mechanism might related to decreased Bcl-2 and increased expression of p-c-Jun.CEH might regulate the expression of m RNA MDR1 and P-gp by activation of the c-Jun/JNK signaling pathway.3. CEH possibly through inhibiting the expression of P-gp and then play a role in reversal of drug resistance in vivo of drug resistant solid tumor models.Significance:It demonstrates that CEH could effectively reverse the drug resistance of Eca109-CDDP cells. It offers the evidence for the further research and development of this drug for clinical cancer chemotherapy in the future. |
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