小白菊内酯对胃癌细胞BGC-823的抑制作用及其机制
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摘要
目的研究小白菊内酯(PN)对胃癌细胞BGC-823的抑制作用及其机制。方法将BGC-823细胞分为DMSO组和PN组,DMSO组常规进行细胞培养,PN组则采用不同浓度的PN处理细胞。两组细胞采用CCK-8技术检测不同时间点细胞活力的变化,采用克隆形成实验检测细胞增殖能力的变化,采用流式细胞术检测细胞不同周期比例及凋亡水平,采用荧光实验检测细胞内活性氧簇(ROS)生成水平,采用Western Blot方法检测细胞周期及凋亡相关蛋白水平的变化。结果与DMSO组比较,随着PN浓度的增加BGC-823细胞的活力及增殖能力均下降(F=283.80、312.60,P<0.01)。24h时,PN组的总凋亡比例与DMSO组相比明显升高(t=27.57,P<0.01),且随着处理时间的延长凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶(caspase)8、caspase9和caspase3剪切型的表达明显增加(F=228.10～2 024.00,P<0.01)。与DMSO组相比较,PN组细胞周期被阻滞在G1期(t=4.22、7.44,P<0.05);而且随着处理时间的延长,细胞周期蛋白(Cyclin)D1及CyclinE1水平明显下降(F=159.40、25.40,P<0.01),P53及P21蛋白表达水平明显升高(F=53.74、73.03,P<0.01)。24h时,PN组细胞内ROS生成水平较DMSO组明显升高(t=10.92,P<0.01);而且随着处理时间的延长,细胞核内c-Myc基因、转录因子E2F1、核因子κB(NF-κB)以及磷酸化的信号传导及转录激活因子3(p-STAT3)蛋白的表达水平明显下降(F=306.90、1 067.00,t=8.72、9.15,P<0.01)。结论 PN对BGC-823细胞增殖能力呈剂量依赖性抑制,对细胞周期产生阻滞并诱导细胞凋亡,这种抗肿瘤作用可能是通过抑制STAT3-c-Myc-E2F1发挥作用的。
Objective To investigate the inhibitory effect of parthenolide(PN)on gastric cancer BGC-823 cells and related and mechanism. Methods BGC-823 cells were divided into DMSO group and PN group.The cells in the DMSO group were cultured with the conventional method,and those in the PN group were treated with different concentrations of PN.CCK-8 assay was used to measure the change in cell viability at different time points,colony-forming assay was used to measure the change in the proliferative capacity of BGC-823 cells,flow cytometry was used to measure the proportion of cells in different cell cycles and the apoptosis of cells,fluorescence assay was used to measure the level of reactive oxygen species(ROS)in cells,and Western Blot was used to measure the changes in the levels of cell cycle-and apoptosis-related proteins. Results Compared with the DMSO group,the PN group had significant reductions in the viability and proliferative capacity of BGC-823 cells in a concentration-dependent manner(F=283.80,312.60,P<0.01).At 24 hof PN exposure,compared with the DMSO group,the PN group had a significant increase in the proportion of apoptotic cells(t=27.57,P<0.01),as well as significant increases in the expression of the apoptosis-related proteins cleaved caspase-8,cleaved caspase-9,and cleaved caspase-3 over the time of treatment(F =228.10-2 024.00,P<0.01).Compared with the DMSO group,the PN group had a significantly higher number of cells arrested in G1 phase(t=4.22,7.44,P<0.05),as well as significant reductions in the levels of CyclinD1 and CyclinE1(F=159.40,25.40,P<0.01)and significant increases in the protein expression of P53 and P21(F=53.74,73.03,P<0.01)over the time of treatment.Compared with the DMSO group at 24 hof PN exposure,the PN group had a significant increase in the level of ROS(t=10.92,P<0.01),and over the time of treatment,the PN group had significant reductions in the protein expression of c-Myc,the transcription factor E2 F1,nuclear factor-kappa B,and phosphorylated STAT3(F=306.90,1 067.00,t=8.72,9.15,P<0.01). Conclusion PN can inhibit the proliferation of BGC-823 cells,induce G1-phase cell cycle arrest,and thus exerts an antitumor effect in a dose-dependent manner,possibly by inhibiting the STAT3-c-Myc-E2 F1 axis.
Objective To investigate the inhibitory effect of parthenolide(PN)on gastric cancer BGC-823 cells and related and mechanism. Methods BGC-823 cells were divided into DMSO group and PN group.The cells in the DMSO group were cultured with the conventional method,and those in the PN group were treated with different concentrations of PN.CCK-8 assay was used to measure the change in cell viability at different time points,colony-forming assay was used to measure the change in the proliferative capacity of BGC-823 cells,flow cytometry was used to measure the proportion of cells in different cell cycles and the apoptosis of cells,fluorescence assay was used to measure the level of reactive oxygen species(ROS)in cells,and Western Blot was used to measure the changes in the levels of cell cycle-and apoptosis-related proteins. Results Compared with the DMSO group,the PN group had significant reductions in the viability and proliferative capacity of BGC-823 cells in a concentration-dependent manner(F=283.80,312.60,P<0.01).At 24 hof PN exposure,compared with the DMSO group,the PN group had a significant increase in the proportion of apoptotic cells(t=27.57,P<0.01),as well as significant increases in the expression of the apoptosis-related proteins cleaved caspase-8,cleaved caspase-9,and cleaved caspase-3 over the time of treatment(F =228.10-2 024.00,P<0.01).Compared with the DMSO group,the PN group had a significantly higher number of cells arrested in G1 phase(t=4.22,7.44,P<0.05),as well as significant reductions in the levels of CyclinD1 and CyclinE1(F=159.40,25.40,P<0.01)and significant increases in the protein expression of P53 and P21(F=53.74,73.03,P<0.01)over the time of treatment.Compared with the DMSO group at 24 hof PN exposure,the PN group had a significant increase in the level of ROS(t=10.92,P<0.01),and over the time of treatment,the PN group had significant reductions in the protein expression of c-Myc,the transcription factor E2 F1,nuclear factor-kappa B,and phosphorylated STAT3(F=306.90,1 067.00,t=8.72,9.15,P<0.01). Conclusion PN can inhibit the proliferation of BGC-823 cells,induce G1-phase cell cycle arrest,and thus exerts an antitumor effect in a dose-dependent manner,possibly by inhibiting the STAT3-c-Myc-E2 F1 axis.
引文
[1]REYNOLDS C,ESRAILIAN E,HOMMES D.Quality improvement in gastroenterology:A systematic review of practical interventions for clinicians[J].Dig Dis Sci,2018,63(10):2507-2518.
[2]FONG A J,FAULKNER G,JONES J M,et al.A qualitative analysis of oncology clinicians'perceptions and barriers for physical activity counseling in breast cancer survivors[J].Support Care Cancer,2018,26(9):3117-3126.
[3]JIN X,ZHOU J,ZHANG Z,et al.The combined administration of parthenolide and ginsenoside CK in long circulation liposomes with targeted tLyp-1ligand induce mitochondriamediated lung cancer apoptosis[J].Artif Cells Nanomed Biotechnol,2018,46(Suppl3):S931-942.
[4]WANG M,LI Q.Parthenolide could become a promising and stable drug with anti-inflammatory effects[J].Nat Prod Res,2015,29(12):1092-1101.
[5]KALIA M,YADAV V K,SINGH P K,et al.Exploring the impact of parthenolide as anti-quorum sensing and anti-biofilm agent against pseudomonas aeruginosa[J].Life Sci,2018,199:96-103.
[6]WIEDHOPF R M,YOUNG M,BIANCHI E,et al.Tumor inhibitory agent from magnolia grandiflora(Magnoliaceae).Ⅰ.Parthenolide[J].J Pharm Sci,1973,62(2):345.
[7]LI X H,XIAO T,YANG J H,et al.Parthenolide attenuated bleomycin-induced pulmonary fibrosis via the NF-kappaB/Snail signaling pathway[J].Respir Res,2018,19(1):111.
[8]LIU M,BI H,YAN Y,et al.Parthenolide inhibits STAT3signaling by covalently targeting janus kinases[J].Molecules,2018,23(6):E1478
[9]FLORES-LOPEZ G,MORENO-LORENZANA D,AYALA-SANCHEZ M,et al.Parthenolide and DMAPT induce cell death in primitive CML cells through reactive oxygen species[J].J Cell Mol Med,2018,22(10):4899-4912.
[10]GAO H E,SUN Y,DING Y H,et al.Antineoplastic effects of CPPTL via the ROS/JNK pathway in acute myeloid leukemia[J].Oncotarget,2017,8(24):38990-39000.
[11]YANG C,YANG Q O,KONG Q J,et al.Parthenolide induces reactive oxygen species-mediated autophagic cell death in human osteosarcoma cells[J].Cell Physiol Biochem,2016,40(1-2):146-154.
[12]DUAN D,ZHANG J,YAO J,et al.Targeting Thioredoxin reductase by parthenolide contributes to inducing apoptosis of HeLa cells[J].J Biol Chem,2016,291(19):10021-10031.
[13]WU C,CHEN F,RUSHING J W,et al.Antiproliferative activities of parthenolide and golden feverfew extract against three human cancer cell lines[J].J Med Food,2006,9(1):55-61.
[14]SAXENA N,YADAV P,KUMAR O.The Fas/Fas ligand apoptotic pathway is involved in abrin-induced apoptosis[J].Toxicol Sci,2013,135(1):103-118.
[15]GUO X X,LI Y,SUN C,et al.P53-dependent Fas expression is critical for Ginsenoside Rh2triggered caspase-8activation in HeLa cells[J].Protein Cell,2014,5(3):224-234.
[16]CHOUDHARY G S,AL-HARBI S,ALMASAN A.Caspase-3activation is a critical determinant of genotoxic stress-induced apoptosis[J].Methods Mol Biol,2015,1219:1-9.
[17]MAO W,ZHU Z.Parthenolide inhibits hydrogen peroxideinduced osteoblast apoptosis[J].Mol Med Rep,2018,17(6):8369-8376.
[18]LI X,YANG H,KE J,et al.Smad4re-expression increases the sensitivity to parthenolide in colorectal cancer[J].Oncol Rep,2017,38(4):2317-2324.
[19]TALIB W H,Al Kury L T.Parthenolide inhibits tumor-promoting effects of nicotine in lung cancer by inducing P53-dependent apoptosis and inhibiting VEGF expression[J].Biomed Pharmacother,2018,107:1488-1495.
[20]LI C,GE Q,LIU J,et al.Effects of miR-1236-3p and miR-370-5p on activation of P21in various tumors and its inhibition on the growth of lung cancer cells[J].Tumour Biol,2017,39(6):1010428317710824.
[21]LIU B,ZHOU Z,ZHOU W,et al.Resveratrol inhibits proliferation in human colorectal carcinoma cells by inducing G1/Sphase cell cycle arrest and apoptosis through caspase/cyclinCDK pathways[J].Mol Med Rep,2014,10(4):1697-1702.
[22]LIN M,BI H,YAN Y,et al.Parthenolide suppresses nonsmall cell lung cancer GLC-82cells growth via B-Raf/MAPK/Erk pathway[J].Oncotarget,2017,8(14):23436-23447.
[23]LEE C H,YANG J R,CHEN,C Y,et al.Novel STAT3inhibitor LDOC1targets phospho-JAK2for degradation by inte-racting with LNX1and regulates the aggressiveness of lung cancer[J].Cancers(Basel),2019,11(1):pii:E63.
[24]BLOOM M J,SAKSENA S D,SWAIN G P,et al.The effects of IKK-beta inhibition on early NF-kappa-B activation and transcription of downstream genes[J].Cell Signal,2018,55:17-25.
[25]LI J,LIU Q,LIU Z,et al.KPNA2promotes metabolic reprogramming in glioblastomas by regulation of c-myc[J].J Exp Clin Cancer Res,2018,37(1):194.
[26]MAYANK A K,SHARMA S,DESHWAL R K,et al.LIMD1antagonizes E2F1activity and cell cycle progression by enhancing Rb function in cancer cells[J].Cell Biol Int,2014,38(7):809-817.
[27]QIAO L,ZHANG Q,ZHANG W,et al.The lysine acetyltransferase GCN5contributes to human papillomavirus oncoprotein E7-induced cell proliferation via up-regulating E2F1[J].J Cell Mol Med,2018,22(11):5333-5345.
[28]GAO S,CHEN M,WEI W,et al.Crosstalk of mTOR/PKM2and STAT3/c-Myc signaling pathways regulate the energy metabolism and acidic microenvironment of gastric cancer[J].JCell Biochem,2019,120:1193-1202.
[29]SHU F,ZOU X,TUO H,et al.Stathmin gene silencing suppresses proliferation,migration and invasion of gastric cancer cells via AKT/sCLU and STAT3signaling[J].Int J Oncol,2019,54(3):1086-1098.
[30]CHEN W,LI P,LIU Y,et al.Isoalantolactone induces apop-tosis through ROS-mediated ER stress and inhibition of STAT 3in prostate cancer cells[J].J Exp Clin Cancer Res,2018,37(1):309.
[31]FENG C,XIA Y,ZOU P,et al.Curcumin analog L48H37induces apoptosis through ROS-mediated endoplasmic reticulum stress and STAT3pathways in human lung cancer cells[J].Mol Carcinog,2017,56(7):1765-1777.
[32]ZHANG Y,LUO Y H,PIAO X J,et al.The design of 1,4-naphthoquinone derivatives and mechanisms underlying apoptosis induction through ROS-dependent MAPK/Akt/STAT3pathways in human lung cancer cells[J].Bioorg Med Chem,2019,27(8):1577-1587.
[2]FONG A J,FAULKNER G,JONES J M,et al.A qualitative analysis of oncology clinicians'perceptions and barriers for physical activity counseling in breast cancer survivors[J].Support Care Cancer,2018,26(9):3117-3126.
[3]JIN X,ZHOU J,ZHANG Z,et al.The combined administration of parthenolide and ginsenoside CK in long circulation liposomes with targeted tLyp-1ligand induce mitochondriamediated lung cancer apoptosis[J].Artif Cells Nanomed Biotechnol,2018,46(Suppl3):S931-942.
[4]WANG M,LI Q.Parthenolide could become a promising and stable drug with anti-inflammatory effects[J].Nat Prod Res,2015,29(12):1092-1101.
[5]KALIA M,YADAV V K,SINGH P K,et al.Exploring the impact of parthenolide as anti-quorum sensing and anti-biofilm agent against pseudomonas aeruginosa[J].Life Sci,2018,199:96-103.
[6]WIEDHOPF R M,YOUNG M,BIANCHI E,et al.Tumor inhibitory agent from magnolia grandiflora(Magnoliaceae).Ⅰ.Parthenolide[J].J Pharm Sci,1973,62(2):345.
[7]LI X H,XIAO T,YANG J H,et al.Parthenolide attenuated bleomycin-induced pulmonary fibrosis via the NF-kappaB/Snail signaling pathway[J].Respir Res,2018,19(1):111.
[8]LIU M,BI H,YAN Y,et al.Parthenolide inhibits STAT3signaling by covalently targeting janus kinases[J].Molecules,2018,23(6):E1478
[9]FLORES-LOPEZ G,MORENO-LORENZANA D,AYALA-SANCHEZ M,et al.Parthenolide and DMAPT induce cell death in primitive CML cells through reactive oxygen species[J].J Cell Mol Med,2018,22(10):4899-4912.
[10]GAO H E,SUN Y,DING Y H,et al.Antineoplastic effects of CPPTL via the ROS/JNK pathway in acute myeloid leukemia[J].Oncotarget,2017,8(24):38990-39000.
[11]YANG C,YANG Q O,KONG Q J,et al.Parthenolide induces reactive oxygen species-mediated autophagic cell death in human osteosarcoma cells[J].Cell Physiol Biochem,2016,40(1-2):146-154.
[12]DUAN D,ZHANG J,YAO J,et al.Targeting Thioredoxin reductase by parthenolide contributes to inducing apoptosis of HeLa cells[J].J Biol Chem,2016,291(19):10021-10031.
[13]WU C,CHEN F,RUSHING J W,et al.Antiproliferative activities of parthenolide and golden feverfew extract against three human cancer cell lines[J].J Med Food,2006,9(1):55-61.
[14]SAXENA N,YADAV P,KUMAR O.The Fas/Fas ligand apoptotic pathway is involved in abrin-induced apoptosis[J].Toxicol Sci,2013,135(1):103-118.
[15]GUO X X,LI Y,SUN C,et al.P53-dependent Fas expression is critical for Ginsenoside Rh2triggered caspase-8activation in HeLa cells[J].Protein Cell,2014,5(3):224-234.
[16]CHOUDHARY G S,AL-HARBI S,ALMASAN A.Caspase-3activation is a critical determinant of genotoxic stress-induced apoptosis[J].Methods Mol Biol,2015,1219:1-9.
[17]MAO W,ZHU Z.Parthenolide inhibits hydrogen peroxideinduced osteoblast apoptosis[J].Mol Med Rep,2018,17(6):8369-8376.
[18]LI X,YANG H,KE J,et al.Smad4re-expression increases the sensitivity to parthenolide in colorectal cancer[J].Oncol Rep,2017,38(4):2317-2324.
[19]TALIB W H,Al Kury L T.Parthenolide inhibits tumor-promoting effects of nicotine in lung cancer by inducing P53-dependent apoptosis and inhibiting VEGF expression[J].Biomed Pharmacother,2018,107:1488-1495.
[20]LI C,GE Q,LIU J,et al.Effects of miR-1236-3p and miR-370-5p on activation of P21in various tumors and its inhibition on the growth of lung cancer cells[J].Tumour Biol,2017,39(6):1010428317710824.
[21]LIU B,ZHOU Z,ZHOU W,et al.Resveratrol inhibits proliferation in human colorectal carcinoma cells by inducing G1/Sphase cell cycle arrest and apoptosis through caspase/cyclinCDK pathways[J].Mol Med Rep,2014,10(4):1697-1702.
[22]LIN M,BI H,YAN Y,et al.Parthenolide suppresses nonsmall cell lung cancer GLC-82cells growth via B-Raf/MAPK/Erk pathway[J].Oncotarget,2017,8(14):23436-23447.
[23]LEE C H,YANG J R,CHEN,C Y,et al.Novel STAT3inhibitor LDOC1targets phospho-JAK2for degradation by inte-racting with LNX1and regulates the aggressiveness of lung cancer[J].Cancers(Basel),2019,11(1):pii:E63.
[24]BLOOM M J,SAKSENA S D,SWAIN G P,et al.The effects of IKK-beta inhibition on early NF-kappa-B activation and transcription of downstream genes[J].Cell Signal,2018,55:17-25.
[25]LI J,LIU Q,LIU Z,et al.KPNA2promotes metabolic reprogramming in glioblastomas by regulation of c-myc[J].J Exp Clin Cancer Res,2018,37(1):194.
[26]MAYANK A K,SHARMA S,DESHWAL R K,et al.LIMD1antagonizes E2F1activity and cell cycle progression by enhancing Rb function in cancer cells[J].Cell Biol Int,2014,38(7):809-817.
[27]QIAO L,ZHANG Q,ZHANG W,et al.The lysine acetyltransferase GCN5contributes to human papillomavirus oncoprotein E7-induced cell proliferation via up-regulating E2F1[J].J Cell Mol Med,2018,22(11):5333-5345.
[28]GAO S,CHEN M,WEI W,et al.Crosstalk of mTOR/PKM2and STAT3/c-Myc signaling pathways regulate the energy metabolism and acidic microenvironment of gastric cancer[J].JCell Biochem,2019,120:1193-1202.
[29]SHU F,ZOU X,TUO H,et al.Stathmin gene silencing suppresses proliferation,migration and invasion of gastric cancer cells via AKT/sCLU and STAT3signaling[J].Int J Oncol,2019,54(3):1086-1098.
[30]CHEN W,LI P,LIU Y,et al.Isoalantolactone induces apop-tosis through ROS-mediated ER stress and inhibition of STAT 3in prostate cancer cells[J].J Exp Clin Cancer Res,2018,37(1):309.
[31]FENG C,XIA Y,ZOU P,et al.Curcumin analog L48H37induces apoptosis through ROS-mediated endoplasmic reticulum stress and STAT3pathways in human lung cancer cells[J].Mol Carcinog,2017,56(7):1765-1777.
[32]ZHANG Y,LUO Y H,PIAO X J,et al.The design of 1,4-naphthoquinone derivatives and mechanisms underlying apoptosis induction through ROS-dependent MAPK/Akt/STAT3pathways in human lung cancer cells[J].Bioorg Med Chem,2019,27(8):1577-1587.