目的 研究一种萘酰亚胺-多胺缀合物对人肝癌HepG2细胞的杀伤作用,并探讨其作用机制。方法 以体外培养的人肝癌HepG2细胞为研究对象,噻唑蓝法检测细胞毒性,AO/EB/Hochest 33342染色观察细胞凋亡形态,DCFH-DA检测HepG2细胞内ROS变化,Rh123染色检测线粒体膜电位变化,免疫荧光法检测caspase-9,PARP-1表达,PI染色分析细胞周期变化,流式细胞术分析细胞凋亡率。结果 噻唑蓝法显示,该萘酰亚胺-多胺缀合物呈剂量依赖性抑制HepG2细胞增殖,48 h的IC50为17.28 μmol·L-1, 24 h的IC50为29.71 μmol·L-1,10 μmol·L-1该萘酰亚胺-多胺缀合物作用48 h后:细胞内活性氧簇、caspase-9荧光强度均显著升高(P<0.01,n=20),Rh123、PARP-1荧光强度显著减低(P<0.01,n=20);细胞增殖阻滞在S期;25 μmol·L-1萘酰亚胺-多胺缀合物作用细胞48 h后,高内涵活细胞成像系统可见细胞核固缩呈圆珠状凋亡特征,流式细胞术显示细胞早期凋亡率为33.13%,晚期凋亡率为30.99%。结论 该萘酰亚胺-多胺缀合物对HepG2细胞具有较强的促凋亡作用,其机制与升高细胞内ROS水平,降低线粒体膜电位,进而活化caspase-9,通过线粒体途径诱导细胞凋亡有关,另外,该萘酰亚胺-多胺缀合物还通过下调PARP-1表达而发挥促凋亡作用。
Abstract
OBJECTIVE To study the effect of a novel naphthalimide-polyamine conjugate on apoptosis of human hepatocellular carcinoma HepG2 cell line. METHODS MTT assay was used to evaluate the cell inhibition rate. The cellular morphous was detected with AO/EB/Hoechst staining, the ROS was detected with DCFH-DA staining, the mitochondrial membrane potential(ΔΨm)was detected with Rh123 staining,the expression of caspase-9 and PARP-1 was detected by immunofluorescence method . The cell cycle was detected by HCS. Apoptosis of HepG2 cells was quantified by flow cytometry using Annexin V/PI stain. RESULTS Proliferation of HepG2 cells was inhibited significantly by the naphthalimide-polyamine conjugate in a dosage dependant manner. Under HCS, some HepG2 cells underwent a typical apoptotic morphologic change and the expression of ROS and caspase-9 was increased. The expression of PARP-1 and the mitochondrial membrane potential(ΔΨm)was detected decreased. Flow cytometry indicates 64.12% HepG2 cells were induced apoptosis after 48 h incubation with 25 μmol·L-1 naphthalimide-polyamine conjugate. CONCLUSION The naphthalimide-polyamine conjugate could significantly induce the apoptosis of HepG2 cells by the mitochondrial pathway. The mechanism is concerned with increasing ROS, decreasing the mitochondrial membrane potential, upgrading caspase-9 and decreasing the expression of PARP-1.
关键词
萘酰亚胺-多胺缀合物 /
肝癌HepG2细胞 /
凋亡
{{custom_keyword}} /
Key words
naphthalimide-polyamine conjugate /
hepatocellular carcinoma HepG2 cell line /
apoptosis
{{custom_keyword}} /
中图分类号:
R965
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] CHEN W Q,ZHENG R S,ZENG H M, et al. Report of cancer incidence and mortality in China,2011 [J]. China Cancer(中国肿瘤),2015,24(1):1-10.
[2] BRAA M F, RAMOS A. Naphathalimides as anticancer agents:Synthesis and biological activity[J]. Curr Med Chem, 2001, 1(3):237-255.
[3] TIAN Z Y,SU L P,XIE S Q, et al. Synthesis, biological activitity and fluorescence spectroscopy of naphthalimide-polyamine conjugates [J]. Chin J Org Chem(有机化学), 2013, 33(7):1514-1521.
[4] BERNARDI P, KRAUSKOPF A, BASSO E, et al. The mitochondrial permeability transition from in vitro artifact to disease target [J]. FEBS J, 2006, 273(10):2077-2099.
[5] SCHWEIKL H, PETZEL C, BOLAY C, et al. 2- Hydroxyethyl methacrylate- induced apoptosis through the ATM- and p53- dependent intrinsic mitochondrial pathway[J]. Biomaterials,2014,35(9):2890-2904.
[6] SETSUKINAI K, URANO Y, KAKINUMA K, et al. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species[J]. J Biol Chem, 2003, 278(5):3170-3175.
[7] KO Y J, JEONG J W, CHOI Y H, et al. Soy soluble polysaccharide induces apoptosis in HCT116 human colon cancer cells via reactive oxygen species generation[J]. Mol Med Rep, 2013,8(6):1767-1772.
[8] NABBEN M,SHABALINA I G,MOONEN- KORNIPS E, et al. Uncoupled respiration,ROS production,acute lipotoxicity and oxidative damage in isolated skeletal muscle mitochondria from UCP3- ablated mice[J]. Biochim Biophys Acta, 2011, 1807(9):1095-1105.
[9] XIONG Y,ZHANG M,CHEN F, et al. Roles of mitochondrial dysfunction in cardiovascular diseases [J]. Chin J Pathophysiol(中国病理生理杂志),2013,29(2):364-370.
[10] NIE C,LUO Y,ZHAO X,et al. Caspase- 9 mediates Puma activation in UCN- 01- induced apoptosis[J/OL]. Cell Death Dis, 2014,5:e1495.
[11] CASTIEL A, VISOCHEK L, MITTELMAN L, et al. Aphenanthrene derived PARP inhibitor is an extra-cent rosomes declustering agent exclusively eradicating human cancer celle[J]. BMC Cancer, 2011,11(1):412-426.
[12] AHMAD M, TORKY A, GLAHN F, et al. PARP-1 Expression and activity in primary human lung cells[J]. Arch Toxicol, 2011,85(6):669-679.
[13] MICHELS J, VITALE I, SENOVILLA L, et al. Synergistic interaction between cisplatin and PARP inhibitors in nonsmall cell lung cancer[J]. Cell Cycle, 2013,12(6):877-883.
[14] ABD-ELMAGEED Z Y, NAURA A S, ERRAMI Y, et al. The poly(ADP-ribose) polymerases(PARPs):New roles in intracellular transport[J]. Cell Signal, 2012,24(1):1-8.
[15] WANG C J, DELCROS J G, BIGGERSTAFF J, et al. Synthesis and biological evaluation of N1-(anthracen-9-ylmethyl) triamines as molecular recognition elements for the polyamine transporter [J]. J Med Chem, 2003, 46(13):2663-2671.
[16] TIAN Z Y, XIE S Q, DU Y W, et al. Synthesis, cytotoxicity and apoptosis of naphthalimide polyamine conjugates as antitumor agents [J]. Eur J Med Chem, 2009, 44(1):393-399.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}