-
宫颈癌是女性第4大最常见的癌症,虽然它是一种可预防的癌症,但全球每年约有52.8万名妇女被诊断患有宫颈癌,死亡26.6万例,其中85%的死亡病例发生在发展中国家[1]。宫颈癌的发展是一个历时数年的缓慢过程,因为宫颈癌在早期没有明显症状,所以当检测出时大多已经到了晚期。人乳头瘤病毒(HPV),尤其是16和18 HPV的感染是发展为宫颈癌的主要危险因素[2]。宫颈癌细胞HeLa是研究宫颈癌的典型细胞系,它不同于人类的一般细胞,HeLa细胞具有无限增殖的能力,而且增殖速度异常迅速[3]。细胞凋亡是一种程序性死亡,与癌症的发生发展密切相关,促进癌细胞发生凋亡是治疗癌症的主要方法之一[4]。细胞死亡受体通路和线粒体通路是现阶段比较认同的2条引起细胞凋亡的途径[5],在线粒体凋亡通路中,Caspase-9为凋亡的启动者,其活化后产生Cleaved Caspase-9活化片段[6],Caspase-3是执行凋亡的因子[7],当其发生活化后会产生一个活性片段即Cleaved Caspase-3,它能够反映Caspase-3的活化程度以及细胞发生凋亡的程度[8]。线粒体通路引起的凋亡首先是使线粒体膜电位丧失从而增加它的通透性,这样细胞色素C等使细胞发生凋亡的物质就会从线粒体中释放出来。细胞色素C会和Caspase-9等因子相互作用,使Caspase-3激活,从而启动Caspase依赖的凋亡通路[9]。
FINDLAY等[10]首次在海葵中分离得到WP-1[(22E, 24R)-5, 8-epidioxy-5α, 8α-ergosta-6, 9(11), 22E-trien-3β-ol],之后又先后在海南粗榧内生真菌S26[11]、可食用蘑菇[12]等中分离出该化合物。研究表明,WP-1对肝癌细胞Hep3B有明显的细胞毒性,可以使细胞DNA断裂和染色质浓缩从而诱导肝癌细胞Hep3B发生凋亡,但是对正常细胞的毒性较低,说明其具有一定的特异性[13]。它还可以抑制白血病细胞HL60活力,并通过诱导CDKN1A表达来抑制结直肠癌细胞HT29的生长,从而引起细胞周期停滞和凋亡[14]。上述研究结果均表明WP-1具有一定的抗肿瘤活性,但其对宫颈癌的作用目前尚不清楚。本实验所用的WP-1是本课题组从红树林植物角果木(Ceriops tagal)内生真菌Cytospora sp.中分离鉴定的麦角甾醇类化合物[15],笔者采用MTT比色法和克隆形成实验等来研究WP-1对HeLa细胞增殖、凋亡的影响,旨在为其临床应用及新药开发提供依据。
Effects of Bio-active Molecules Isolated from Endophytic Fungi of Ceriops tagal on the Proliferation and Apoptosis of HeLa Cells
-
摘要: 为了解红树林植物角果木(Ceriops tagal)内生壳囊孢属真菌Cytospora sp.提取物WP-1[(22E, 24R)-5, 8-epidioxy-5α, 8α-ergosta-6, 9(11), 22E-trien-3β-ol]抑制人宫颈癌HeLa细胞的效果,笔者采用MTT比色法、克隆形成实验、Hoechst 33258染色和蛋白免疫印迹法来研究WP-1对HeLa细胞增殖和凋亡的影响。结果表明,随着WP-1浓度增加,HeLa细胞的存活率显著降低,而且该提取物能明显抑制HeLa细胞的克隆形成,当其浓度达到10 μmol·L−1时,HeLa细胞出现染色质浓缩以及核小体碎裂的凋亡现象。蛋白免疫印迹法结果显示,随着WP-1剂量的增加,与凋亡相关的蛋白Caspase-3及Caspase-9的表达下调,Cleaved Caspase-3,Cleaved Caspase-9的表达明显上调。本实验结果显示,WP-1可以抑制HeLa细胞的增殖,并促进HeLa细胞发生凋亡。Abstract: In order to understand the effect of WP-1 [(22E, 24R)-5, 8-epidioxy-5α, 8α-ergosta-6, 9(11), 22E-trien-3β-ol] isolated from Cytospora sp., an endophytic fungus from the mangrove plant Ceriops tagal on human cervical cancer HeLa cells, the proliferation and apoptosis of HeLa cells treated with WP-1 was determined and analyzed by using MTT colorimetry, clone formation, Hoechst 33258 staining and Western blot. It was found that with the increase of WP-1 concentration the survival rate of the HeLa cells decreased significantly, and that the WP-1 significantly inhibited the colony formation of cells. When treated with WP-1 at a concentration of 10 μmol·L−1 the HeLa cells showed chromatin condensation and nucleosome fragmentation, which were characteristic of apoptosis. Western blot analysis showed that with the increase of WP-1 dose, the expressions of apoptosis-related proteins Caspase-3 and Caspase-9 were down-regulated in the HeLa cells, while the expressions of Cleaved Caspase-3 and Cleaved Caspase-9 were significantly up-regulated. The results show that WP-1 can inhibit the proliferation of HeLa cells and promote apoptosis of HeLa cells.
-
-
[1] SCHWALBE N. Human papillomavirus (HPV) and related cancers in the global alliance for vaccines and immunization (GAVI) countries. A WHO/ICO HPV information centre report foreword [J]. Vaccine, 2012, 30(4): 1 − 83. [2] COMPARETTO C, BORRUTO F. Cervical cancer screening: a never-ending developing program [J]. World Journal of Clinical Cases, 2015, 3(7): 82 − 92. [3] 裘伟廷. 海里埃塔·拉克丝:缔造“永生”传奇[J]. 世界文化, 2017(6): 54 − 56. doi: 10.3969/j.issn.1005-9172.2017.06.025 [4] DILLON C P, GREEN D R. Molecular cell biology of apoptosis and necroptosis in cancer [J]. Advances in Experimental Medicine and Biology, 2016, 930: 1 − 23. [5] XIONG Y, LU Q J, ZHAO J, et al. Metformin inhibits growth of hepatocellular carcinoma cells by inducing apoptosis via mitochondrion-mediated pathway [J]. Asian Pacific Journal of Cancer Prevention, 2012, 13(7): 3275 − 3279. doi: 10.7314/APJCP.2012.13.7.3275 [6] 尤良震, 吴袁元, 尹昀东, 等. 丹蛭降糖胶囊对高糖诱导的人脐静脉内皮细胞凋亡及线粒体途径与死亡受体途径的影响[J]. 中华中医药杂志, 2019(8): 3675 − 3680. [7] TSAI Y H, LIN J J, MA Y S, et al. Fisetin inhibits cell proliferation through the induction of G0/G1 phase arrest and Caspase-3-mediated apoptosis in mouse leukemia cells [J]. American Journal of Chinese Medicine, 2019, 47(4): 1 − 23. [8] JOHANNA B, BROCK H, KATHRYN L, et al. A Caspase-3 reporter for fluorescence lifetime imaging of single-cell apoptosis [J]. Cells, 2018, 7(6): 1 − 20. [9] YAN S L, HUANG C Y, WU S T, et al. Oleanolic acid and ursolic acid induce apoptosis in four human liver cancer cell lines [J]. Toxicology in Vitro, 2010, 24(3): 842 − 848. doi: 10.1016/j.tiv.2009.12.008 [10] FINDLAY J A,PATIL A D. A novel sterol peroxide from the sea anenome Metridium senile [J]. Steroids, 1984, 44(3): 261 − 265. [11] 陈苹, 吴娇, 戴好富, 等. 海南粗榧内生真菌S26化学成分研究[J]. 中国药物化学杂志, 2008, 18(4): 279 − 283. doi: 10.3969/j.issn.1005-0108.2008.04.010 [12] NIUE A W, OMOLO J O, CHEPLOGOI P K, et al. Cytotoxic ergostane derivatives from the edible mushroom Termitomyces microcarpus (Lyophyllaceae) [J]. Biochemical Systematics & Ecology, 2018, 76: 12 − 14. [13] CHEN Y K, KUO Y H, CHIANG B H, et al. Cytotoxic activities of 9,11-dehydroergosterol peroxide and ergosterol peroxide from the fermentation mycelia of Ganoderma lucidum cultivated in the medium containing leguminous plants on Hep3B cells [J]. Journal of Agricultural & Food Chemistry, 2009, 57(13): 5713 − 5719. [14] MASUKO K, MITSURU Y, MAYUMI O K, et al. 5α,8α-Epidioxy-22E-ergosta-6,9(11),22-trien-3β-ol from an edible mushroom suppresses growth of HL60 leukemia and HT29 colon adenocarcinoma cells [J]. Biological & Pharmaceutical Bulletin, 2006, 29(4): 755 − 759. [15] DENG Q, LI G, SUN M Y, et al. A new antimicrobial sesquiterpene isolated from endophytic fungus Cytospora sp. from the Chinese mangrove plant Ceriops tagal [J]. Natural Product Research, 2020, 34(10): 1404 − 1408. doi: 10.1080/14786419.2018.1512993 [16] YANG H C, WANG J Y, BU X Y, et al. Resveratrol restores sensitivity of glioma cells to temozolamide through inhibiting the activation of Wnt signaling pathway [J]. Journal of Cellular Physiology, 2019, 234(5): 6783 − 6800. doi: 10.1002/jcp.27409 [17] 宁晓燕. RNA干扰抑制h POT1表达对人胃癌细胞生物学特性的影响[D]. 上海: 第三军医大学, 2007. [18] 罗凤仙, 解静, 钱颖艳, 等. 辣木生物碱抑制宫颈癌Hela细胞增殖和诱导其凋亡的作用[J]. 现代食品科技, 2020, 36(2): 18 − 24. [19] YANG Y, JIA G, WANG Q, et al. Tubeimoside-1 induces glioma apoptosis through regulation of Bax/Bcl-2 and the ROS/Cytochrome C/Caspase-3 pathway [J]. Oncotargets & Therapy, 2015, 8: 303 − 311. [20] 郭玮, 罗琼, 刘帅, 等. 麦角甾醇对S180荷瘤小鼠抑瘤作用及机制[J]. 中国公共卫生, 2015, 31(12): 1606 − 1608. doi: 10.11847/zgggws2015-31-12-24 [21] LIN M Z, LI H J, ZHAO Y, et al. 2-Naphthoic acid ergosterol ester, an ergosterol derivative, exhibits anti-tumor activity by promoting apoptosis and inhibiting angiogenesis [J]. Steroids, 2017, 122: 9 − 15. doi: 10.1016/j.steroids.2017.03.007 [22] LIN C N, KUO S H, WON S J. Steroids of formosan Ganoderma amboinense [J]. Phytochemistry, 1993, 32(6): 1549 − 1551. doi: 10.1016/0031-9422(93)85177-S [23] BATES M L, REID W W, WHITE J D. Duality of pathways in the oxidation of ergosterol to its peroxide in vivo [J]. Journal of the Chemical Society Chemical Communications, 1976(2): 44 − 45. doi: 10.1039/c39760000044 [24] TRIGOS A, ORTEGA-REGULES A. Selective destruction of microscopic fungi through photo-oxidation of ergosterol [J]. Mycologia, 2002, 94(4): 563 − 568. doi: 10.1080/15572536.2003.11833184 [25] KOBORI M, YOSHIDA M, OHNISHI-KAMEYAMA M, et al. Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cells [J]. British Journal of Pharmacology, 2007, 150(2): 209 − 219. doi: 10.1038/sj.bjp.0706972 [26] TAKEI T, YOSHIDA M, OHNISHI-KAMEYAMA M, et al. Ergosterol peroxide, an apoptosis-inducing component isolated from Sarcodon aspratus (Berk.) S. Ito [J]. Bioscience Biotechnology & Biochemistry, 2005, 69(1): 212 − 215.