β-谷甾醇诱导人胃癌细胞自噬与凋亡的作用及机制研究
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摘要
目的从体内外两个方面研究β-谷甾醇诱导胃癌细胞自噬的作用及机制,为β-谷甾醇抗胃癌作用研究提供实验依据。方法体外培养人胃癌SGC-7901细胞,设对照组和实验组,对照组仅用磷酸盐缓冲液(PBS)处理,实验组以不同浓度β-谷甾醇处理。四唑盐比色法(MTT)检测β-谷甾醇对胃癌细胞的抑制作用,流式细胞术检测β-谷甾醇对胃癌细胞的促凋亡作用,绿色荧光蛋白标记法(GFP)检测β-谷甾醇对胃癌细胞自噬诱导作用,蛋白印迹法(Western-blot)检测信号通道蛋白磷酸化磷脂酰肌醇3激酶(p-PI3K)、磷酸化蛋白激酶B(p-AKT)及磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的表达。用SGC-7901细胞制造胃癌裸鼠模型,经腹胫注射β-谷甾醇,3 d给药1次,共42d,观察移植瘤重量体积变化,免疫组化法检测移植瘤组织LC3-Ⅱ、LC3-Ⅰ表达。结果实验组β-谷甾醇浓度达20μmol/L时可明显抑制SGC-7901细胞活力,促进SGC-7901凋亡,与对照组比较差异有统计学意义(P <0. 05或P <0. 01);β-谷甾醇诱导SGC-7901细胞发生自噬效应,(20、40、80、160μmol/L)实验组细胞自噬率(19. 32±2. 51、32. 25±3. 18、57. 45±4. 02、78. 93±4. 21)与对照组(7. 28±2. 83)比较,差异有统计学意义(P <0. 05,P <0. 01);实验组β-谷甾醇下调p-PI3K、p-AKT及mTOR表达,与对照组比较差异有统计学意义(P <0. 05);实验组抑瘤率为(69. 8±3. 6)%,移植瘤的重量和体积明显减轻变小,与对照组比较差异均有统计学意义(P <0. 05);实验组β-谷甾醇上调裸鼠移植瘤细胞LC3-Ⅱ表达,下调LC3-Ⅰ表达,使LC3-Ⅱ/LC3-Ⅰ(7. 76±0. 35)升高,与对照组(0. 32±0. 21)比较,差异有统计学意义(t=7. 28,P=0. 008)。结论β-谷甾醇能抑制胃癌细胞增殖并促进其凋亡,其抗癌机制可能与β-谷甾醇通过PI3K/AKT/m TOR通路诱导细胞自噬作用有关。
Objective To study the effect and mechanism of autophagy in gastric cancer cells induced by β-sitosterol in vivo and in vitro,providing experimental evidence for antitumor study of β-sitosterol on gastric cancer. Methods Human gastric cancer SGC-7901 cells were cultured in vitro. The control group was treated with phosphate buffer( PBS) only,and the experimental group was treated with different concentrations of β-sitosterol. Tetrazolium salt colorimetry( MTT) was used to detect the inhibitory effect ofβ-sitosterol on gastric cancer cells. Flow cytometry was used to detect the apoptotic effect of β-sitosterol on gastric cancer cells. Green fluorescent protein labeling( GFP) was used to detect the induction of autophagy by β-sitosterol. Western blot was used to detect the expression of signal pathway proteins phosphorylated phosphoinositid 3-kinase( p-PI3 K),phosphorylated protein kinase B( p-AKT) and phosphorylated mammalian target of rapamycin( p-mTOR). The nude mice model of gastric cancer was established by SGC-7901 cells. β-sitosterol was injected abdominally once every 3 days for 42 days. The weight and volume of transplanted tumors were observed. The expression of LC3-Ⅱ and LC3-Ⅰ in transplanted tumors was detected by immunohistochemistry. Results β-sitosterol could inhibite the viability and promote the apoptosis of SGC-7901 cells,inducing the autophagy of SGC-7901 cells when its concentration reached 20 μmol/L. The autophagy rate of experimental group was( 19. 32 ± 2. 51,32. 25 ± 3. 18,57. 45 ± 4. 02,78. 93 ± 4. 21),which was significantly higher than that in the control group( 7. 28 ± 2. 83,P < 0. 05). In the experimental group,the expression of p-PI3 K,p-AKT and m TOR was down-regulated by β-sitosterol,which was significantly different from that in the control group( P < 0. 05). The inhibition rate of the experimental group was( 69. 8 ± 3. 6) %,and the weight and volume of the transplanted tumors were significantly reduced compared with those in the control group( P < 0. 05). β-sitosterol increased the expression of LC3-Ⅱ,decreased expression of LC3-Ⅰ,leading the rise of ratio of LC3-Ⅱ to LC3-Ⅰ in nude mouse tumor( t = 7. 28,P =0. 008). Conclusions β-sitosterol can inhibite proliferation and promote apoptosis of SGC-7901 cells. The antitumor mechanism may related to the autophagy induced by beta-sitosterol through PI3 K/AKT/m TOR pathway.
Objective To study the effect and mechanism of autophagy in gastric cancer cells induced by β-sitosterol in vivo and in vitro,providing experimental evidence for antitumor study of β-sitosterol on gastric cancer. Methods Human gastric cancer SGC-7901 cells were cultured in vitro. The control group was treated with phosphate buffer( PBS) only,and the experimental group was treated with different concentrations of β-sitosterol. Tetrazolium salt colorimetry( MTT) was used to detect the inhibitory effect ofβ-sitosterol on gastric cancer cells. Flow cytometry was used to detect the apoptotic effect of β-sitosterol on gastric cancer cells. Green fluorescent protein labeling( GFP) was used to detect the induction of autophagy by β-sitosterol. Western blot was used to detect the expression of signal pathway proteins phosphorylated phosphoinositid 3-kinase( p-PI3 K),phosphorylated protein kinase B( p-AKT) and phosphorylated mammalian target of rapamycin( p-mTOR). The nude mice model of gastric cancer was established by SGC-7901 cells. β-sitosterol was injected abdominally once every 3 days for 42 days. The weight and volume of transplanted tumors were observed. The expression of LC3-Ⅱ and LC3-Ⅰ in transplanted tumors was detected by immunohistochemistry. Results β-sitosterol could inhibite the viability and promote the apoptosis of SGC-7901 cells,inducing the autophagy of SGC-7901 cells when its concentration reached 20 μmol/L. The autophagy rate of experimental group was( 19. 32 ± 2. 51,32. 25 ± 3. 18,57. 45 ± 4. 02,78. 93 ± 4. 21),which was significantly higher than that in the control group( 7. 28 ± 2. 83,P < 0. 05). In the experimental group,the expression of p-PI3 K,p-AKT and m TOR was down-regulated by β-sitosterol,which was significantly different from that in the control group( P < 0. 05). The inhibition rate of the experimental group was( 69. 8 ± 3. 6) %,and the weight and volume of the transplanted tumors were significantly reduced compared with those in the control group( P < 0. 05). β-sitosterol increased the expression of LC3-Ⅱ,decreased expression of LC3-Ⅰ,leading the rise of ratio of LC3-Ⅱ to LC3-Ⅰ in nude mouse tumor( t = 7. 28,P =0. 008). Conclusions β-sitosterol can inhibite proliferation and promote apoptosis of SGC-7901 cells. The antitumor mechanism may related to the autophagy induced by beta-sitosterol through PI3 K/AKT/m TOR pathway.
引文
[1]王小俊,刘友东,季承博,等.超氧化物歧化酶2基因在胃癌组织中的表达及其对胃癌细胞生物学行为的影响[J].中华实验外科杂志,2018,35(8):1545-1547. DOI:10. 3760/cma. j. issn.1001-9030. 2018. 08. 048.
[2]周玲玉,徐彩,王乐琴,等.β-谷甾醇对人肺癌A549细胞增殖及凋亡的影响[J].国际检验医学杂志,2016,37(7):865-867.DOI:10. 3969/j. issn. 1673-4130. 2016. 07. 001.
[3]林明珠,赵岩,蔡恩博,等.β-谷甾醇对H22荷瘤小鼠体内抗肿瘤作用[J].中国公共卫生,2017,33(12):1797-1800. DOI:10.11847/zgggws2017-33-12-31.
[4] Rajavel T,Packiyaraj P,Suryanarayanan V,et al.β-Sitosterol targets Trx/Trx1 reductase to induce apoptosis in A549 cells via ROS mediated mitochondrial dysregulation and p53 activation[J]. Sci Rep,2018,8(1):2071. DOI:10. 1038/s41598-018-20311-6.
[5] Sharmila R,Sindhu G. Modulation of Angiogenesis, Proliferative Response and Apoptosis by β-Sitosterol in Rat Model of Renal Carcinogenesis[J]. Indian J Clin Biochem,2017,32(2):142-152. DOI:10.1007/s12291-016-0583-8.
[6]邓军容,黄伟,易红,等.谷氨酰胺分解代谢促进鼻咽癌自噬和放疗抵抗性[J].中国医师杂志,2018,20(6):820-825. DOI:10.3760/cma. j. issn. 1008-1372.2018. 06. 006.
[7]张新菊,张笑天,马中良. miR-17-92、自噬在肿瘤中的研究进展[J].生命科学,2017,29(11):1149-1155. DOI:10. 13376/j.cbls/2017152.
[8]闫红,吴正升,江冬瑞,等.乳腺癌中自噬相关基因LC3与凋亡相关基因XIAP的表达及意义[J].临床与实验病理学杂志,2016,32(4):409-412. DOI:10. 13315/j. cnki. cjcep. 2016. 04. 012.
[9]周笑,杨鹏,于雁. PI3K/Akt/m TOR信号转导通路与肿瘤[J].国际免疫学杂志,2016,39(3):280-284. DOI:10. 3760/cma. j. issn. 1673-4394. 2016. 03. 018.
[10]董会月,路君,林灵婧,等. ULK1复合物在细胞自噬中的作用[J/CD].中华细胞与干细胞杂志:电子版,2016,6(4):240-242 . DOI:10. 3877/cma. j. issn. 2095-1221. 2016. 04. 008.
[2]周玲玉,徐彩,王乐琴,等.β-谷甾醇对人肺癌A549细胞增殖及凋亡的影响[J].国际检验医学杂志,2016,37(7):865-867.DOI:10. 3969/j. issn. 1673-4130. 2016. 07. 001.
[3]林明珠,赵岩,蔡恩博,等.β-谷甾醇对H22荷瘤小鼠体内抗肿瘤作用[J].中国公共卫生,2017,33(12):1797-1800. DOI:10.11847/zgggws2017-33-12-31.
[4] Rajavel T,Packiyaraj P,Suryanarayanan V,et al.β-Sitosterol targets Trx/Trx1 reductase to induce apoptosis in A549 cells via ROS mediated mitochondrial dysregulation and p53 activation[J]. Sci Rep,2018,8(1):2071. DOI:10. 1038/s41598-018-20311-6.
[5] Sharmila R,Sindhu G. Modulation of Angiogenesis, Proliferative Response and Apoptosis by β-Sitosterol in Rat Model of Renal Carcinogenesis[J]. Indian J Clin Biochem,2017,32(2):142-152. DOI:10.1007/s12291-016-0583-8.
[6]邓军容,黄伟,易红,等.谷氨酰胺分解代谢促进鼻咽癌自噬和放疗抵抗性[J].中国医师杂志,2018,20(6):820-825. DOI:10.3760/cma. j. issn. 1008-1372.2018. 06. 006.
[7]张新菊,张笑天,马中良. miR-17-92、自噬在肿瘤中的研究进展[J].生命科学,2017,29(11):1149-1155. DOI:10. 13376/j.cbls/2017152.
[8]闫红,吴正升,江冬瑞,等.乳腺癌中自噬相关基因LC3与凋亡相关基因XIAP的表达及意义[J].临床与实验病理学杂志,2016,32(4):409-412. DOI:10. 13315/j. cnki. cjcep. 2016. 04. 012.
[9]周笑,杨鹏,于雁. PI3K/Akt/m TOR信号转导通路与肿瘤[J].国际免疫学杂志,2016,39(3):280-284. DOI:10. 3760/cma. j. issn. 1673-4394. 2016. 03. 018.
[10]董会月,路君,林灵婧,等. ULK1复合物在细胞自噬中的作用[J/CD].中华细胞与干细胞杂志:电子版,2016,6(4):240-242 . DOI:10. 3877/cma. j. issn. 2095-1221. 2016. 04. 008.