白头翁皂苷B4对肝癌细胞Huh-7及其荷瘤裸鼠肿瘤的抑制作用及机制研究
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摘要
目的:研究白头翁皂苷B4(AB4)对肝癌细胞Huh-7及其荷瘤裸鼠肿瘤的抑制作用及机制。方法:将Huh-7细胞分为AB4给药组、阴性对照组(等体积培养液)、阳性对照组(5-氟尿嘧啶50μmol/L),运用MTT法检测0、3、6、12、25、50、100、200、400、800、1 600μmol/L AB4作用Huh-7细胞12、24、36、48 h的增殖抑制率,计算半数抑制浓度(IC50);克隆形成试验评估50μmol/L AB4作用Huh-7细胞24 h的克隆细胞数;膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶(AnnexinⅤ-FITC/PI)染色检测50μmol/L AB4作用Huh-7细胞24 h的细胞凋亡率;Western blot法检测50μmol/L AB4作用Huh-7细胞24 h后B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(Caspase-3)、活化胱天蛋白酶3(Cleaved Caspase-3)、活化多腺苷二磷酸核糖聚合酶(Cleaved PARP)等凋亡蛋白的表达。将荷瘤裸鼠随机分为阴性对照组(生理盐水)、阳性对照组(5-氟尿嘧啶50 mg/kg)和AB4低、中、高剂量组(25、50、100mg/kg),每组3只,每天腹腔注射相应药物1次,连续19 d,观察裸鼠肿瘤生长情况,计算抑瘤率;苏木精-伊红(HE)染色观察肿瘤细胞形态变化。结果:AB4对Huh-7细胞的增殖抑制率随给药浓度的增加而增加,但50μmol/L后增加不明显,随作用时间的延长而增加,但作用24 h后增加不明显,AB4的IC50为(56.76±1.756)μmol/L。与阴性对照组比较,50μmol/L AB4作用Huh-7细胞24 h的克隆细胞数明显减少、Bcl-2蛋白表达明显减弱(P<0.05),细胞凋亡率和Bax、Caspase-3、Cleaved Caspase-3、Cleaved PARP蛋白表达均明显增加(P<0.05或P<0.01),且与阳性对照组差异无统计学意义。与阴性对照组比较,AB4低、中、高剂量组和阳性对照组荷瘤裸鼠的瘤体积明显减小(P<0.05),肿瘤细胞轮廓逐渐模糊,出现核固缩、核质不清晰、核碎裂现象,其抑瘤率分别为25.93%、39.15%、46.26%、42.83%。结论:AB4对Huh-7细胞及其荷瘤裸鼠均有抑制作用,其机制可能与上调Bax/Bcl-2比例、活化Caspase-3、裂解PARP、诱导细胞发生凋亡有关。
OBJECTIVE:To study the anti-tumor effect of anemoside B4(AB4) on hepatocellular carcinoma Huh-7 and tumor-bearing nude mice and its mechanism. METHODS:Huh-7 cells were divided into AB4 treatment group,negative control group(constant volume of culture medium)and positive control group(5-fluorouracil 50 μ mol/L). The inhibitory rate of Huh-7 cell proliferation was tested by MTT after treated with 0,3,6,12,25,50,100,200,400,800,1 600 μmol/L AB4 for 12,24,36,48 h;IC50 were calculated. The number of clone cells was evaluated by clone formation tests after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. The apoptosis rate of Huh-7 cells was tested by Annexin Ⅴ-FITC/PI double staining after treated with 50 μ mol/L AB4 for 24 h. The expression of apoptotic proteins such as Bcl-2,Bax,Caspase-3,Cleaved Caspase-3 and Cleaved PARP were tested by Western blot after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. Tumor-bearing nude mice were randomly divided into negative control group(normal saline),positive control group(5-fluorouracil 50 mg/kg),AB4 lwo-dose,medium-dose and high-dose groups(25,50,100 mg/kg),with 3 mice in each group. They were given relevant medicine intraperitoneally,once a day,for consecutive 19 d. The growth of tumor was observed,and anti-tumor rate was also calculated. HE staining was used to observe the morphology of tumor cells. RESULTS:The inhibition rate of AB4 on Huh-7 cell proliferation increased with the increase of concentration of AB4,but it did not increase significantly after reaching 50 μmol/L;it increased with the increase of time,but it did not increase significantly after 24 h. The IC50 of AB4 was(56.76±1.756)μmol/L.Compared with negative control group,the number of clone cells was decreased significantly after Huh-7 cells were treated with 50μmol/L AB4 for 24 h,while the protein expression of Bcl-2 was decreased significantly(P<0.05);the apoptotic rate,the protein expression of Bax,Caspase-3,Cleaved Caspase-3 and Cleaved PARP were increased significantly(P<0.05 or P<0.01),there was no statistical significance,compared with positive control group. Compared with negative control group,the volume of tumor was decreased significantly in AB4 low-dose,medium-dose and high-dose groups,positive control group(P<0.05);the outline of tumor cells become more and more blurred;there were nuclear pyknosis,unclear nucleoplasm and nuclear fragmentation;its anti-tumor rates were 25.93%,39.15%,46.26%,42.83%,respectively. CONCLUSIONS:AB4 inhibits Huh-7 cells and tumor-bearing mice, the mechanism of which may be associated with up-regulating the proportion of Bax/Bcl-2, activating Caspase-3,cracking PARP and inducing apoptosis.
OBJECTIVE:To study the anti-tumor effect of anemoside B4(AB4) on hepatocellular carcinoma Huh-7 and tumor-bearing nude mice and its mechanism. METHODS:Huh-7 cells were divided into AB4 treatment group,negative control group(constant volume of culture medium)and positive control group(5-fluorouracil 50 μ mol/L). The inhibitory rate of Huh-7 cell proliferation was tested by MTT after treated with 0,3,6,12,25,50,100,200,400,800,1 600 μmol/L AB4 for 12,24,36,48 h;IC50 were calculated. The number of clone cells was evaluated by clone formation tests after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. The apoptosis rate of Huh-7 cells was tested by Annexin Ⅴ-FITC/PI double staining after treated with 50 μ mol/L AB4 for 24 h. The expression of apoptotic proteins such as Bcl-2,Bax,Caspase-3,Cleaved Caspase-3 and Cleaved PARP were tested by Western blot after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. Tumor-bearing nude mice were randomly divided into negative control group(normal saline),positive control group(5-fluorouracil 50 mg/kg),AB4 lwo-dose,medium-dose and high-dose groups(25,50,100 mg/kg),with 3 mice in each group. They were given relevant medicine intraperitoneally,once a day,for consecutive 19 d. The growth of tumor was observed,and anti-tumor rate was also calculated. HE staining was used to observe the morphology of tumor cells. RESULTS:The inhibition rate of AB4 on Huh-7 cell proliferation increased with the increase of concentration of AB4,but it did not increase significantly after reaching 50 μmol/L;it increased with the increase of time,but it did not increase significantly after 24 h. The IC50 of AB4 was(56.76±1.756)μmol/L.Compared with negative control group,the number of clone cells was decreased significantly after Huh-7 cells were treated with 50μmol/L AB4 for 24 h,while the protein expression of Bcl-2 was decreased significantly(P<0.05);the apoptotic rate,the protein expression of Bax,Caspase-3,Cleaved Caspase-3 and Cleaved PARP were increased significantly(P<0.05 or P<0.01),there was no statistical significance,compared with positive control group. Compared with negative control group,the volume of tumor was decreased significantly in AB4 low-dose,medium-dose and high-dose groups,positive control group(P<0.05);the outline of tumor cells become more and more blurred;there were nuclear pyknosis,unclear nucleoplasm and nuclear fragmentation;its anti-tumor rates were 25.93%,39.15%,46.26%,42.83%,respectively. CONCLUSIONS:AB4 inhibits Huh-7 cells and tumor-bearing mice, the mechanism of which may be associated with up-regulating the proportion of Bax/Bcl-2, activating Caspase-3,cracking PARP and inducing apoptosis.
引文
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[23]YAO N,LI YJ,ZHANG DM,et al.B4G2 induces mitochondrial apoptosis by the ROS-mediated opening of Ca2+-dependent permeability transition pores[J].Cell Physiol Biochem,2015,37(3):838-852.
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[25]ZHENG Y,ZHOU F,WU X,et al.23-Hydroxybetulinic acid from Pulsatilla chinensis(Bunge)Regel synergizes the antitumor activities of doxorubicin in vitro and in vivo[J].J Ethnopharmacol,2010,128(3):615-622.
[2]RAWLA P,SUNKARA T,MURALIDHARAN P,et al.Update in global trends and aetiology of hepatocellular carcinoma[J].Contemp Oncol(Pozn),2018,22(3):141-150.
[3]BRUIX J,RAOUL JL,SHERMAN M,et al.Efficacy and safety of sorafenib in patients with advanced hepatocellular carcinoma:subanalyses of a phaseⅢtrial[J].J Hepatol,2012,57(4):821-829.
[4]BAHMAN AA,ABAZA MSI,KHOUSHIASH SI,et al.Sequence-dependent effect of sorafenib in combination with natural phenolic compounds on hepatic cancer cells and the possible mechanism of action[J].Int J Mol Med,2018,42(3):1695-1715.
[5]国家药典委员会.中华人民共和国药典:一部[S].2015年版.北京:中国医药科技出版社,2015:104.
[6]LI W,SUN YN,YAN XT,et al.Isolation of nematicidal triterpenoid saponins from Pulsatilla koreana root and their activities against Meloidogyne incognita[J].Molecules,2013,18(5):5306-5316.
[7]YAO D,LI H,GOU Y,et al.Betulinic acid-mediated inhibitory effect on hepatitis B virus by suppression of manganese superoxide dismutase expression[J].Febs J,2009,276(9):2599-2614.
[8]SIA D,VILLANUEVA A,FRIEDMAN SL,et al.Liver cancer cell of origin,molecular class,and effects on patient prognosis[J].Gastroenterology,2017,152(4):745-761.
[9]LIU X,ZHOU J,ZHOU N,et al.SYNJ2BP inhibits tumor growth and metastasis by activating DLL4 pathway in hepatocellular carcinoma[J].J Exp Clin Cancer Res,2016.DOI:10.1186/s13046-016-0385-0.
[10]CUI J,GONG Z,SHEN HM.The role of autophagy in liver cancer:molecular mechanisms and potential therapeutic targets[J].Biochim Biophys Acta,2013,1836(1):15-26.
[11]KHAN M,LI T,AHMAD KHAN MK,et al.Alantolactone induces apoptosis in HepG2 cells through GSH depletion,inhibition of STAT3 activation,and mitochondrial dysfunction[J].Biomed Res Int,2013.DOI:10.1155/2013/719858.
[12]ALLAIRE M,NAULT JC.Advances in management of hepatocellular carcinoma[J].Curr Opin Oncol,2017,29(4):288-295.
[13]ZHU JN,JIANG L,JIANG JH,et al.Hepatocyte nuclear factor-1beta enhances the stemness of hepatocellular carcinoma cells through activation of the notch pathway[J].Sci Rep,2017.DOI:10.1038/s41598-017-04116-7.
[14]郝轩轩,王新陆,崔琳,等.加参方浸膏对H2O2诱导的H9c2心肌细胞凋亡的干预作用研究[J].中国药房,2018,29(14):1898-1903.
[15]MUKHOPADHYAY S,PANDA PK,SINHA N,et al.Autophagy and apoptosis:where do they meet?[J].Apoptosis,2014,19(4):555-566.
[16]POULIQUEN D,BELLOT G,GUIHARD G,et al.Mitochondrial membrane permeabilization produced by PTP,Bax and apoptosis:a1H-NMR relaxation study[J].Cell Death Differ,2006,13(2):301-310.
[17]WANG Y,WANG H,GE H,et al.AG-1031 induced autophagic cell death and apoptosis in C6 glioma cells associated with notch-1 signaling pathway[J].J Cell Biochem,2018,119(7):5893-5903.
[18]KIRAZ Y,ADAN A,KARTAL YM,et al.Major apoptotic mechanisms and genes involved in apoptosis[J].Tumour Biol,2016,37(7):8471-8486.
[19]王晓霞,刘天龙,刘晶,等.黄芪甲苷对D-半乳糖诱导原代培养心肌细胞凋亡的保护作用及机制研究[J].中国药房,2018,29(9):1189-1193.
[20]LIU Q,CHEN W,JIAO Y,et al.Pulsatilla saponin A,an active molecule from Pulsatilla chinensis,induces cancer cell death and inhibits tumor growth in mouse xenograft models[J].J Surg Res,2014,188(2):387-395.
[21]HONG SW,JUNG KH,LEE HS,et al.SB365 inhibits angiogenesis and induces apoptosis of hepatocellular carcinoma through modulation of PI3K/Akt/mTOR signaling pathway[J].Cancer Sci,2012,103(11):1929-1937.
[22]田龙夫,张琦,王波涛,等.血根碱通过调控PI3K/Akt信号通路诱导胰腺癌细胞凋亡的机制[J].中国实验方剂学杂志,2018,24(19):166-171.
[23]YAO N,LI YJ,ZHANG DM,et al.B4G2 induces mitochondrial apoptosis by the ROS-mediated opening of Ca2+-dependent permeability transition pores[J].Cell Physiol Biochem,2015,37(3):838-852.
[24]ZHANG Y,BAO J,WANG K,et al.Pulsatilla saponin Dinhibits autophagic flux and synergistically enhances the anticancer activity of chemotherapeutic agents against HeLa cells[J].Am J Chin Med,2015,43(8):1657-1670.
[25]ZHENG Y,ZHOU F,WU X,et al.23-Hydroxybetulinic acid from Pulsatilla chinensis(Bunge)Regel synergizes the antitumor activities of doxorubicin in vitro and in vivo[J].J Ethnopharmacol,2010,128(3):615-622.