| Esophageal cancer has become the sixth leading cause of death and the eighth most frequently diagnosed cancer worldwide. Esophageal squamous cell carcinoma is predominant in most parts of the world, especially in high risk regions such as China where it accounts for over90%of the total cases of esophageal cancer. Despite significant advances in the diagnosis, staging and treatment of esophageal squamous cell carcinoma in recent decades, few significant improvements in overall survival have been achieved:the5-year overall survival rate remains below14%. The traditional chemotherapy drugs have many side effects, such as liver and kidney damage, and drug resistance is very common, which make traditional chemotherapy drug effect is not ideal for the treatment of esophageal cancer. Therefore, to find chemotherapy drugs with strong specificity, good curative effect and less side effects is urgent in the clinical treatment of esophageal cancer.Isodon xerophilus (C. Y. Wu et H. W. Li) H. Hara is a special Labiatae plants of Isodon genus in Yunnan province of China which branches and leaves can be used as pharmaceutical raw materials to produce natural ent-kaurane diterpenoids. In this study, ten known natural ent-kaurane diterpenoids were isolated from Isodon species, among which sxpl-3(xerophilusin B), showed antitumor activity in human ESCC cell lines.To study the effect and mechanism of the growth inhibition of human esophageal squamous cell carcinoma by sxp1-3, we used a series of methods both in vitro and in vivo. After being treated by sxpl-3in different density (0μM,0.1μM,0.5μM,1μM,2μM,5μM and10μM) during different time (0h,6h,12h,24h,48h and72h), the rates of the growth inhibition of human esophageal squamous cell carcinoma cell line KYSE-150and KYSE-450were detected by Cell Counting Kit-8(CCK-8), and the characteristic morphological features of apoptosis were examined under an inverted light microscope; Using double-dye assay of Annexin V and propidium iodide staining, the rate of the apoptotic cells and the phrase distribution of cell cycle were detected by flow cytometry; The expression changes of proteins of apoptosis related signal pathway were detected though Western blot analysis; Using normal cell lines, tumor-bearing nude mice, the antitumor activity and safety were observed.Compound sxpl-3was shown conspicuously cytotoxicity against both KYSE-150and KYSE-450cell lines. The inhibition ratio reached99.1%and95.3%with sxp1-3in a density of5μM after72hours for KYSE-150and KYSE-450cells, respectively. Flow cytometric analysis demonstrated that sxpl-3induced cells apoptotic events in a dose-dependent manner (0-5μM). Cell cycle analysis showed that sxp1-3induced cell cycle arrest at the G2/M phase. Western blot analysis of apoptosis-related proteins revealed that sxpl-3induced cell apoptosis through mitochondrial cytochrome c-dependent activation of the caspase-9and caspase-3cascade pathway (intrinsic pathway). An in vivo experiment using tumor-bearing nude mice showed that treatment with sxpl-3at7.5mg/kg per day decreased the KYSE-150and KYSE-450cells tumor mass by62%and74%, respectively. Pathological and blood biochemical test showed that sxpl-3have no hepatotoxicity and renal toxicity on nude mice.In conclusion, sxpl-3could induce apoptosis on KYSE-150and KYSE-450cell lines by the mechanism of mitochondrial apoptotic pathway probably, and showed conspicuously antitumor activity with low toxicity. Therefore, sxpl-3has potential as novel therapeutic agent for the treatment of ESCC. |
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