石蒜碱对结肠癌HT29细胞增殖、凋亡的影响及凋亡机制的初步探讨
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摘要
目的研究石蒜碱(lycorine)对结肠癌HT29细胞增殖和凋亡的影响,探讨其可能的凋亡机制。方法选择人结肠癌HT29细胞株进行培养与处理,按石蒜碱干预浓度为0μmol/L(对照组)、1μmol/L、4μmol/L、16μmol/L分为四组,探讨不同浓度石蒜碱对HT29细胞增殖的抑制作用、对HT29细胞克隆形成率的影响、对HT29细胞凋亡的形态学及凋亡率的影响,以及对细胞凋亡相关蛋白、MAPK/ERK通路蛋白的影响。结果 MTT结果显示,与对照组比较,各浓度的石蒜碱诱导HT29细胞24 h、48h后HT29细胞的存活率均下降(均P <0.05)。HT29细胞接种7 d后肉眼可见细胞集落形成,以上四组细胞克隆形成率分别为(77.00±3.61)%、(64.17±2.02)%、(51.50±2.78)%、(26.67±1.61)%。Hoechst 33258染色结果示,对照组细胞呈均匀的淡蓝色荧光、细胞核无明显凋亡形态学特征,余各浓度的石蒜碱组呈现不同程度的核浓缩、核碎裂、核边集及细胞呈现蓝白色荧光等典型的凋亡改变。流式细胞术凋亡检测结果显示,HT29细胞凋亡率随着石蒜碱浓度的增加而升高(P <0.05)。与对照组相比,各浓度石蒜碱组的Bcl-2、p-ERK蛋白表达量均减少(均P <0.05),Bax、Caspase-3蛋白表达量均增加(均P <0.05)。结论石蒜碱可抑制结肠癌HT29细胞的增殖并诱导其凋亡,其凋亡机制可能与抑制MAPK/ERK信号通路有关。
Objectives To investigate the effect of lycorine on proliferation and apoptosis of colon cancer HT29 cells and to investigate the possible apoptosis mechanism. Methods Four human colon cancer HT29 cell lines were cultured and were treated with different concentration of lycorine with 0 μmol/L(control group), 1 μmol/L, 4 μmol/L and 16 μmol/L, respectively. The effect of different concentrations on the inhibition of cell proliferation, rate of clone formation, apoptotic morphology and apoptotic rate, apoptosis related proteins and MAPK/ERK pathway proteins was investigated. Results Methylthiazolyldiphenyl-tetrazolium(MTT) bromide showed that compared with control group, survival rate of HT29 cells decreased at 24 h and 48 h after treated with all three concentrations of lycorine(P < 0.05). Colony formation was observed at the 7 thday after inoculation. The rate of colony formation was(77.00±3.61)%,(64.17±2.02)%,(51.50±2.78)%,(26.67±1.61)%, respectively, in the four groups. Hoechst 33258 staining showed that the cells in control group had uniform light blue fluorescence and had no obvious morphological characteristics of apoptosis in the nucleus. Cells treated with different concentration of lycorine showed typical apoptotic changes such as nuclear concentration, fragmentation, edge collection and blue-white fluorescence. Flow cytometry showed that the apoptotic rate of HT29 cells increased with the increase of Lycorine concentration(P < 0.05). Compared with control group, the expression of Bcl-2 and p-ERK decreased(P < 0.05), while the expression of Bax and Caspase-3 increased in lycorine-treated cells(P <0.05). Conclusion Lycorine could inhibit proliferation and induce apoptosis of colon cancer HT29 cells. The mechanism was possibly related to suppression of MAPK/ERK pathway.
Objectives To investigate the effect of lycorine on proliferation and apoptosis of colon cancer HT29 cells and to investigate the possible apoptosis mechanism. Methods Four human colon cancer HT29 cell lines were cultured and were treated with different concentration of lycorine with 0 μmol/L(control group), 1 μmol/L, 4 μmol/L and 16 μmol/L, respectively. The effect of different concentrations on the inhibition of cell proliferation, rate of clone formation, apoptotic morphology and apoptotic rate, apoptosis related proteins and MAPK/ERK pathway proteins was investigated. Results Methylthiazolyldiphenyl-tetrazolium(MTT) bromide showed that compared with control group, survival rate of HT29 cells decreased at 24 h and 48 h after treated with all three concentrations of lycorine(P < 0.05). Colony formation was observed at the 7 thday after inoculation. The rate of colony formation was(77.00±3.61)%,(64.17±2.02)%,(51.50±2.78)%,(26.67±1.61)%, respectively, in the four groups. Hoechst 33258 staining showed that the cells in control group had uniform light blue fluorescence and had no obvious morphological characteristics of apoptosis in the nucleus. Cells treated with different concentration of lycorine showed typical apoptotic changes such as nuclear concentration, fragmentation, edge collection and blue-white fluorescence. Flow cytometry showed that the apoptotic rate of HT29 cells increased with the increase of Lycorine concentration(P < 0.05). Compared with control group, the expression of Bcl-2 and p-ERK decreased(P < 0.05), while the expression of Bax and Caspase-3 increased in lycorine-treated cells(P <0.05). Conclusion Lycorine could inhibit proliferation and induce apoptosis of colon cancer HT29 cells. The mechanism was possibly related to suppression of MAPK/ERK pathway.
引文
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[13]张军,谷翔,黄问银,等.GLP-1对AGEs诱导H9C2心肌细胞凋亡的保护作用研究[J].中国药理学通报,2017,33(1):120-126.
[14]GEEST C R,BUITENHUIS M,GROOT K M,et al.Tight control of MEK-ERK activation is essential in regulating proliferation,survival,and cytokine production of CD34+-derived neutrophil progenitors[J].Blood,2009,114(16):3402-3412.
[15]NOWAK G.Protein kinase C-alpha and ERK1/2 mediate mitochondrial dysfunction,decreases in active Na+transport,and cisplatin-induced apoptosis in renal cells[J].J Biol Chem,2002,277(45):43377-43388.
[16]CAGNOL S,CHAMBARD J C.ERK and cell death:mechanisms of ERK-induced cell death--apoptosis,autophagy and senescence[J].FEBS J,2010,277(1):2-21.
[2]REJHOVáA,OPATTOVA A,CUMOVA A,et al.Natural compounds and combination therapy in colorectal cancer treatment[J].Eur J Med Chem,2018,144:582-594.
[3]YU H,QIU Y,PANG X,et al.Lycorine promotes autophagy and apoptosis via TCRP1/Akt/mTOR axis inactivation in human hepatocellular carcinoma[J].Mol Cancer Ther,2017,16(12):2711-2723.
[4]WANG J,XU J,XING G.Lycorine inhibits the growth and metastasis of breast cancer through the blockage of STAT3 signaling pathway[J].Acta Biochim Biophys Sin(Shanghai),2017,49(9):771-779.
[5]黄义强,张一鸣,金中,等.石蒜碱对肾癌细胞ACHN具有明显的抗肿瘤作用[J].南方医科大学学报,2016,36(6):857-862.
[6]ROY M,LIANG L,XIAO X,et al.Lycorine:A prospective natural lead for anticancer drug discovery[J].Biomed Pharmacother,2018,107:615-624.
[7]CHLAPEK P,REDOVA M,ZITTERBART K,et al.Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors:an expression profiling study[J].JExp Clin Cancer Res,2010,29:45.
[8]朱燕,谢丽,杨觅,等.重楼抗肿瘤作用机制的研究进展[J].癌症进展,2015,13(2):164-166.
[9]WANG C,WANG Q,LI X,et al.Lycorine induces apoptosis of bladder cancer T24 cells by inhibiting phospho-Akt and activating the intrinsic apoptotic cascade[J].Biochem Biophys Res Commun,2017,483(1):197-202.
[10]ZENG H,FU R,YAN L,et al.Lycorine induces apoptosis of A549 cells via AMPK-mammalian target of rapamycin(mTOR)-S6K signaling pathway[J].Med Sci Monit,2017,23:2035-2041.
[11]GALLUZZI L,MAIURI M C,VITALE I,et al.Cell death modalities:classification and pathophysiological implications[J].Cell Death Differ,2007,14(7):1237-1243.
[12]GALLUZZI L,VITALE I,AARONSON S A,et al.Molecular mechanisms of cell death:recommendations of the Nomenclature Committee on Cell Death 2018[J].Cell Death Differ,2018,25(3):486-541.
[13]张军,谷翔,黄问银,等.GLP-1对AGEs诱导H9C2心肌细胞凋亡的保护作用研究[J].中国药理学通报,2017,33(1):120-126.
[14]GEEST C R,BUITENHUIS M,GROOT K M,et al.Tight control of MEK-ERK activation is essential in regulating proliferation,survival,and cytokine production of CD34+-derived neutrophil progenitors[J].Blood,2009,114(16):3402-3412.
[15]NOWAK G.Protein kinase C-alpha and ERK1/2 mediate mitochondrial dysfunction,decreases in active Na+transport,and cisplatin-induced apoptosis in renal cells[J].J Biol Chem,2002,277(45):43377-43388.
[16]CAGNOL S,CHAMBARD J C.ERK and cell death:mechanisms of ERK-induced cell death--apoptosis,autophagy and senescence[J].FEBS J,2010,277(1):2-21.
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