摘要
我国食管癌发病率居世界首位,并且是全球食管癌死亡率最高的国家；其中约95%为鳞癌,与欧美国家70%左右为腺癌相比具有明显差异。我国食管鳞癌(Esophageal Squamous Cell Carcinoma, ESCC)在城市全部恶性肿瘤中发病率占第4位,死亡率占第5位；在农村中其发病率和死亡率均居第4位,是危害我国人民健康的最主要恶性肿瘤之一。在过去的几十年里,对食管鳞癌的预防、诊断以及治疗进行了大量研究,但食管鳞癌的总体生存率仍然没有明显改善,总体5年生存率仍然不到14%,而现有的传统化疗药物均存在着不同程度的毒副作用例如肝脏与肾脏损伤等,并且由于肿瘤细胞对化疗药物耐药性的产生,从而使得传统化疗药物治疗食管癌的效果并不理想。因此,寻找特异性强、疗效显著且副作用较小的治疗药物是提高食管鳞癌治疗效果的有效手段。
旱生香茶菜[Isodon xerophilus(C. Y. Wu et H. W. Li)H. Hara]是中国云南省特有的香茶菜属物种,为唇形科(Labiatae)香茶菜属植物。其枝叶部位可作为药用原材料,在对该植物有效抗肿瘤化合物成分的研究中发现,其有效成分中主要含有对映-贝壳杉烷型二萜类(ent-kaurane diterpenoid)化合物。
在本研究中,我们从旱生香茶菜中提取出10个已知对映-贝壳杉烷二萜类化合物(sxpl-1-10)并在食管鳞癌细胞中进行活性筛选,发现化合物sxpl-3即旱地香茶菜素B(xerophilusins B)能够有效抑制食管鳞癌细胞生长,因此我们进一步用不同浓度梯度的sxpl-3分别作用人食管鳞癌KYSE-150与KYSE-450细胞,应用CCK-8(Cell Counting Kit-8)法检测不同时间点的细胞生长抑制率；倒置显微镜观察细胞形态和结构变化；利用Annexin V/碘化丙啶(PI)双染流式细胞术检测细胞凋亡率和细胞周期；Western blot法检测加药前后凋亡相关信号通路蛋白表达变化；利用正常细胞系NIH-3T3, HEK-293以及裸鼠皮下移植瘤模型,检验sxpl-3体内抑瘤活性及其安全性。
实验结果发现,sxpl-3对KYSE-150与KYSE-450细胞的生长有显著的抑制作用,5μM的sxp1-3作用72h的细胞生长抑制率分别为99.1%与95.3%；2μM与5μM的sxpl-3作用24h后,显微镜下观察到细胞形态结构发生改变；流式细胞仪检测sxp1-3诱导的凋亡具有时间与剂量依赖性,并且细胞周期图像显示明显的G2/M期阻滞及凋亡峰；同时Western blot显示细胞色素c释放,caspase-9、caspase-3酶原蛋白的激活,凋亡过程中伴有Bcl-2表达降低和Bax上调；体内实验结果显示腹腔注射剂量为7.5mg/kg/d时sxpl-3即可有效抑制KYSE-150与KYSE-450细胞移植瘤的生长,抑制率分别为62%与74%；病理免疫组化切片以及肝肾功血生化检测显示sxpl-3对裸鼠肝肾组织无毒性。
综上所述,我们的结果证实,旱生香茶菜中对映-贝壳杉烷二萜类化合物sxp1-3,即xerophilusins B可通过线粒体依赖的内源性途径诱导人食管鳞癌KYSE-150与KYSE-450细胞凋亡,并可导致G2/M期阻滞,在体内实验中可显著抑制移植瘤的生长,同时对正常组织无毒性作用。因此,xerophilusins B可以作为治疗食管鳞癌的新的前导化合物。
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.
引文
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