Condurango-glycoside-A fraction of Gonolobus condurango induces DNA damage associated senescence and apoptosis via ROS-dependent p53 signalling pathway in HeLa cells
Condurango-glycoside-A fraction of Gonolobus condurango induces DNA damage associated senescence and apoptosis via ROS-dependent p53 signalling pathway in HeLa cells
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DOI：
10.1007/s11010-013-1732-5
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发表时间：
2013-10-01
影响因子：
4.3
通讯作者：
Khuda-Bukhsh, Anisur Rahman
中科院分区：
文献类型：
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作者：
Bishayee, Kausik;Paul, Avijit;Khuda-Bukhsh, Anisur Rahman
魔芋植物提取物在包括顺势疗法在内的一些传统医学系统中被用作抗癌药物，但它显然缺乏任何科学验证。此外，还没有详细的研究表明，苦参苷-A(CGA)是苦参果的主要成分，是否具有强大的抗癌化合物作用。因此，我们研究了CGA对宫颈癌细胞(HeLa)的诱导凋亡作用。在不同的时间点对β-半乳糖苷酶活性和DNA损伤进行了关键的研究，在9-12h观察到诱导的DNA损伤，在较晚的阶段(CGA处理后18h)细胞出现衰老，从而暗示DNA损伤可能在诱导早熟细胞衰老中起作用。同时，随着活性氧(ROS)生成量的增加，细胞发生凋亡的数量也随之增加。P53蛋白表达上调，提示细胞凋亡可能是通过P53途径介导的。DCHFDA(4‘，6-二氨基-2-苯基吲哚二盐酸盐)实验、吖啶橙/溴化乙锭染色和Annexin V/PI实验结果共同证实了细胞凋亡是通过增加ROS生成来诱导的。细胞周期停滞于G0/G1期进一步证明了细胞增殖的抑制。P53、Akt、Bcl2、Bax、细胞色素c和caspase3等相关基因和蛋白的表达谱也提供了ROS介导的P53上调和Bax表达进一步增强以及随后细胞色素c释放和caspase3激活的证据。总体结果表明，CGA启动ROS的产生，促进P53表达上调，从而导致与DNA损伤相关的细胞凋亡和早衰。
Gonolobus condurango plant extract is used as an anticancer drug in some traditional systems of medicine including homeopathy, but it apparently lacks any scientific validation. Further, no detailed study is available to suggest whether condurango-glycoside-A (CGA), a major ingredient of condurango serves as a potent anticancer compound. Therefore, we investigated apoptosis-inducing ability of CGA against cervix carcinoma cells (HeLa). beta-galactosidase-activity and DNA damage were critically studied at different time points; while induced DNA-damage was observed at 9-12th hours, senescence of cells appeared at a later stage (18th hour after CGA treatment), implicating thereby a possible role of DNA damage in inducing pre-mature cell senescence. Concurrently, the number of cells undergoing apoptosis increased along with increase in reactive oxygen species (ROS) generation. Expression of p53 was also up-regulated, indicating that apoptosis could have been mediated through p53 pathway. DCHFDA (4',6-Diamidino-2-phenylindole dihydrochloride) assay, acridine orange/ethidium bromide staining and annexin V/PI assay results collectively confirmed that apoptosis was induced by increased ROS generation. Reduction in proliferation of cells was further evidenced by the cell cycle arrest at G0/G1 stage. Expression profiles of certain relevant genes and proteins like p53, Akt, Bcl-2, Bax, cytochrome c and caspase 3 also provided evidence of ROS mediated p53 up-regulation and further boost in Bax expression and followed by cytochrome c release and activation of caspase 3. Overall results suggest that CGA initiates ROS generation, promoting up-regulation of p53 expression, thus resulting in apoptosis and pre-mature senescence associated with DNA damage.