大蒜素抑制内质网自噬影响胃癌SGC-7901细胞增殖和凋亡的研究
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摘要
目的探讨大蒜素诱导胃癌SGC-7901细胞凋亡过程中对内质网自噬的影响。方法将胃癌SGC-7901细胞分为对照组(正常培养)、大蒜素低、中、高剂量组(SGC-7901细胞分别加入12、24、48μg·m L-1大蒜素进行培养);采用CCk-8法检测各组细胞培养24 h、48 h、72 h后细胞增殖抑制率;TUNEL检测各组细胞凋亡率;免疫荧光法检测各组细胞LC3与Sec61蛋白共定位;Western blot检测各组细胞GRP78、PERK、p-PERK、e IF2a、ATF-4、CHOP、Beclin-1、LC3Ⅰ及LC3Ⅱ蛋白的表达。结果与对照组相比,①大蒜素低、中、高剂量组细胞ATG8与Sec61蛋白共定位数量随剂量的升高而降低,增殖抑制率、凋亡率随剂量的升高而升高(P<0.01)。②低、中、高剂量组细胞表达GRP78、PERK-、p-PERK、e IF2a、ATF-4及CHOP表达,且呈剂量依赖性,Beclin-1表达随剂量的增加而减少,LC3Ⅰ/LC3Ⅱ比值随剂量的增加而升高(P<0.01)。结论大蒜素可通过抑制胃癌SGC-7901细胞内质网自噬,诱导细胞内质网应激凋亡,从而起到抑制胃癌SGC-7901细胞增殖能力的作用。
Objective To investigate the effect of allicin on autophagy of endoplasmic reticulum(ER) in apoptosis of gastric cancer SGC-7901 cells. Methods Gastric cancer SGC-7901 cells were divided into control group(normal culture), low, medium and high doses of allicin group(SGC-7901 cells were added with 12, 24, 48 μg·m L-1 allicin for culture). The cell proliferation rate of each group was detected by CCk-8 method at 24 h, 48 h and 72 h after culture. TUNEL was used to detect apoptosis, and Immunofluorescence assay was used to detect the colocalization of LC3 and Sec61 proteins in each group. Western blot was used to detect the expression of GRP78, PERK, p-PERK, eIF2 a, ATF-4, CHOP, Beclin-1,LC3 I and LC3 II proteins. Results Compared with the control group, the co-localizations of ATG8 and Sec61 protein in the allicin groups were decreased along with the increase of the dose of allicin, while the proliferation inhibition rate and the apoptotic rate were increased along with the increase of the dose(P<0.01). Moreover, the expression of GRP78, PERK, p-PERK, eIF2 a, ATF-4 and CHOP in low, medium and high dose groups were increased in a dose-dependent manner(P<0.01). The expression of Beclin-1 was decreased with the increasing dose, while the ratio of LC3 I and LC3 II rose along with the increasing dose(P<0.01). Conclusion Allicin could inhibit the proliferation of gastric cancer SGC-7901 cells by inhibiting autophagy in the endoplasmic reticulum of gastric cancer SGC-7901 cells and inducing endoplasmic reticulum stress.
Objective To investigate the effect of allicin on autophagy of endoplasmic reticulum(ER) in apoptosis of gastric cancer SGC-7901 cells. Methods Gastric cancer SGC-7901 cells were divided into control group(normal culture), low, medium and high doses of allicin group(SGC-7901 cells were added with 12, 24, 48 μg·m L-1 allicin for culture). The cell proliferation rate of each group was detected by CCk-8 method at 24 h, 48 h and 72 h after culture. TUNEL was used to detect apoptosis, and Immunofluorescence assay was used to detect the colocalization of LC3 and Sec61 proteins in each group. Western blot was used to detect the expression of GRP78, PERK, p-PERK, eIF2 a, ATF-4, CHOP, Beclin-1,LC3 I and LC3 II proteins. Results Compared with the control group, the co-localizations of ATG8 and Sec61 protein in the allicin groups were decreased along with the increase of the dose of allicin, while the proliferation inhibition rate and the apoptotic rate were increased along with the increase of the dose(P<0.01). Moreover, the expression of GRP78, PERK, p-PERK, eIF2 a, ATF-4 and CHOP in low, medium and high dose groups were increased in a dose-dependent manner(P<0.01). The expression of Beclin-1 was decreased with the increasing dose, while the ratio of LC3 I and LC3 II rose along with the increasing dose(P<0.01). Conclusion Allicin could inhibit the proliferation of gastric cancer SGC-7901 cells by inhibiting autophagy in the endoplasmic reticulum of gastric cancer SGC-7901 cells and inducing endoplasmic reticulum stress.
引文
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[16]宋吉宁,董锦忠,陈文礼.芦荟大黄素对胃癌SGC-7901细胞株钙联蛋白/钙网蛋白循环及内质网应激凋亡的影响[J].中国临床研究, 2018, 31(6):725-729. doi:10.13429/j. cnki. cjcr.2018.06.002.
[17]张晓海,张洪涛,胡孝定,等.柴胡皂苷D对胃癌SGC-7901细胞生长、迁移抑制作用和Norrin、Livin的影响[J].浙江医学, 2017, 39(15):1248-1252. doi:10.12056/j.issn.1006-2785.2017.39.15.2016-1308.
[18]于洋,刘师兵,李松岩,等.内质网应激反应与人卵巢癌细胞顺铂耐药性关系的实验研究[J].中国民康医学, 2015,27(14):121-124. doi:10.3969/j. issn.1672-0369.2015.14.076.
[19]Cheng X, Feng H, Wu H, et al. Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer[J]. Cancer Lett,2018, 431:105-114. doi:10.1016/j. canlet.2018.05.046.
[20]袁兆新,金笛,张宏宇.14-3-3和CLIC4蛋白在U251细胞自噬中的相互作用[J].中风和神经疾病杂志, 2015,32(5):449-451.
[21]徐路,曾林川,于洋,等.钙离子在顺铂诱导宫颈癌HeLe细胞自噬反应中的作用[J].吉林大学学报(医学版), 2016, 42(6):1045-1048. doi:10.13481/j.1671-587x.20160601.
[2]邹文斌,李兆申.中国胃癌发病率及死亡率研究进展[J].中国实用内科杂志, 2014, 34(4):408-415.
[3]Bernales S, Schuck S, Walter P. ER-phagy:selective autophagy of the endoplasmic reticulum[J]. Autophagy, 2007,3(3):285-287.
[4]于亦鸣,钟斌诚,何茜,等.大蒜素对小细胞肺癌A549细胞增殖、迁移能力及凋亡的影响[J].临床和实验医学杂志, 2017, 16(18):1776-1779. doi:10.3969/j. issn.1671-4695.2017.18.003.
[5]陈传军,于志坚,吕亚莉,等.大蒜素抗人肝癌HepG-2细胞的实验研究[J].中华中医药学刊, 2014, 32(7):1763-1767. doi:10.13193/j. issn.1673-7717.2014.07.074.
[6]邵淑丽,刘锐,隋文静,等.大蒜素诱导结肠癌HT-29细胞凋亡[J].基因组学与应用生物学, 2015, 34(2):227-233. doi:10.13417/j. gab.034.000227.
[7]董益明,巩平,林芷伊,等.大蒜素对胃癌细胞上皮间质转化的影响[J].中国现代医药杂志, 2015, 17(2):18-21.doi:10.3969/j. issn.1672-9463.2015.02.005.
[8]张丹,汪圣毅,陈志武.大蒜素对人胃癌SGC-7901细胞迁移、增殖及凋亡的影响[J].中药材, 2013, 36(4):615-618.
[9]Lemasters JJ. Selective mitochondrial autophagy, or mitophagy,as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging[J]. Rejuvenation Res, 2005, 8(1):3-5.
[10]Khaminets A, Heinrich T, Mari M, et al. Regulation of endoplasmic reticulum turnover by selective autophagy[J]. Nature,2015, 522(7556):354-358. doi:10.1038/nature14498.
[11]Gagliostro V, Casas J, Caretti A, et al. Dihydroceramide delays cell cycle G1/S transition via activation of ER stress and induction of autophagy[J]. Int J Biochem Cell Biol., 2012,44(12):2135-2143. doi:10.1016/j. biocel.2012.08.025.
[12]Zhuang J, Li Y, Chi Y. Role of p38 MAPK activation and mitochondrial cytochrome-c release in allicin-induced apoptosis in SK-N-SH cells[J]. Anticancer Drugs, 2016,27(4):312-317. doi:10.1097/CAD.0000000000000340.
[13]王宇娟,霍俊玲,李婧,等.宫颈癌中HPV16 E6蛋白诱导内质网应激-自噬反应的研究[J].国际妇产科学杂志,2014, 41(6):658-662.
[14]Morel JD, Paatero AO, Wei J, et al. Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature[J]. Mol Cell Proteomics, 2018,17(9):1750-1765. doi:10.1074/mcp. RA118.000824.
[15]彭娜,胡泽昆,胡大军.自噬相关分子Beclin1和LC3在糖尿病肾脏疾病患者血清中的表达及意义[J].临床肾脏病杂志, 2018, 18(7):417-420. doi:10.3969/j. issn.1671-2390.2018.07.007.
[16]宋吉宁,董锦忠,陈文礼.芦荟大黄素对胃癌SGC-7901细胞株钙联蛋白/钙网蛋白循环及内质网应激凋亡的影响[J].中国临床研究, 2018, 31(6):725-729. doi:10.13429/j. cnki. cjcr.2018.06.002.
[17]张晓海,张洪涛,胡孝定,等.柴胡皂苷D对胃癌SGC-7901细胞生长、迁移抑制作用和Norrin、Livin的影响[J].浙江医学, 2017, 39(15):1248-1252. doi:10.12056/j.issn.1006-2785.2017.39.15.2016-1308.
[18]于洋,刘师兵,李松岩,等.内质网应激反应与人卵巢癌细胞顺铂耐药性关系的实验研究[J].中国民康医学, 2015,27(14):121-124. doi:10.3969/j. issn.1672-0369.2015.14.076.
[19]Cheng X, Feng H, Wu H, et al. Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer[J]. Cancer Lett,2018, 431:105-114. doi:10.1016/j. canlet.2018.05.046.
[20]袁兆新,金笛,张宏宇.14-3-3和CLIC4蛋白在U251细胞自噬中的相互作用[J].中风和神经疾病杂志, 2015,32(5):449-451.
[21]徐路,曾林川,于洋,等.钙离子在顺铂诱导宫颈癌HeLe细胞自噬反应中的作用[J].吉林大学学报(医学版), 2016, 42(6):1045-1048. doi:10.13481/j.1671-587x.20160601.