喜树碱诱导SMMC-7721肝癌细胞的蛋白质组学研究
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摘要
研究目的
喜树碱是从喜树(珙桐科,喜树属)中提取出的五环生物碱,是拓扑异构酶Ⅰ抑制剂,能可逆性地与拓扑异构酶Ⅰ-DNA可裂解复合物结合。喜树碱衍生物伊利替康和拓扑替康已广泛应用于各种肿瘤的治疗,尤其是肝癌。可裂解的DNA-拓扑异构酶-喜树碱三元复合物一旦转变成不可裂解复合物,将导致DNA损伤、DNA修复机制的激活,细胞周期调控蛋白的表达变化和细胞凋亡等一系列信号通路的改变。这些通路中关键调控分子的失活极大地影响了癌变过程,并与拓扑异构酶Ⅰ抑制剂的作用密切相关。以多聚(ADP-核糖)聚合酶(PARP)为例,该蛋白参与了DNA修复机制,对维持基因稳定性有重要作用。喜树碱可激活PARP的表达,PARP缺失或基因敲除的细胞很容易发生癌变,并且对喜树碱的细胞毒作用高度敏感,同时PARP抑制剂亦可以增强喜树碱衍生物的抗肿瘤作用。此外,喜树碱诱导的细胞信号通路中,CHK1,CHK2和NF-κB等细胞周期阻滞和细胞凋亡相关的信号分子均发挥着重要的调控作用,这些功能蛋白的抑制剂同样可以强化喜树碱的细胞毒性,并成为国内外肿瘤研究的热点和难点。因此,在模式体系中进一步研究喜树碱作用后的关键调控因子,探索可靠、敏感的生物靶标,有助于完善肿瘤应对细胞毒药物作用的特异性调控网络,为肿瘤治疗奠定坚实的理论基础。本实验采用了双向凝胶电泳的方法对喜树碱诱导的SMMC-7721肝癌细胞蛋白质组的变化进行分离和分析,并对喜树碱诱导半乳糖凝集素-1(Galectin-1)表达变化的调控机制作出了初步的探索。
半乳糖凝集素-1是凝集素家族成员之一,广泛参与了细胞生命活动,如调节细胞粘附和增殖、肿瘤转移、细胞凋亡、免疫逃逸和血管再生等。正常肝脏组织中,Galectin-1几乎不表达,但是在肝癌细胞中往往过量表达。此外利用RNA干扰技术抑制Galectin-1的表达后,肿瘤细胞对细胞毒药物SN-38(喜树碱的衍生物)的敏感性得到大幅度增加,而加入重组的Galectin-1后,这种敏感性可以被部分抵消。有报道称Galectin-1组织特异性表达与DNA甲基化模式有关,而且在去甲基化作用下,其表达可得到提高。而DNA甲基转移酶(DNMTs)负责DNA复制过程中维持DNA的甲基化模式。因此,DNMTs是否参与了喜树碱对Galectin-1的表达调控,并影响喜树碱诱导的增殖抑制和细胞凋亡值得进一步研究。空白对照组的细胞总蛋白质,行双向凝胶电泳(2-D)。电泳图谱经图像扫描分析后,选取了若干个表达量大,并且有差异的蛋白质点进行MALDI-TOF质谱分析。采用荧光定量RT-PCR和Western Blot方法对2-D结果进行验证,并检测了喜树碱对SMMC-7721细胞Galectin-1 mRNA和蛋白表达的影响。随后应用ELISA法,检测了喜树碱对SMMC-7721肝癌细胞DNMTs表达的影响。接着通过MTT实验和流式细胞术分析,检测了DNMTs抑制剂5-脱氧氮杂胞苷、喜树碱单独用药或者联合用药对SMMC-7721细胞增殖和凋亡的影响。最后检测了低浓度5-脱氧氮杂胞苷对SMMC-7721细胞周期分布的影响情况。
结果
Galectin-1的分离和鉴定
喜树碱处理SMMC-7721细胞6小时后,收集细胞总蛋白行双向凝胶电泳(2-D),分析喜树碱处理组和空白对照组的总蛋白质的差异。在相同实验条件下进行3次2-D,银染显色后得到喜树碱处理组(实验组)和对照组SMMC-7721细胞蛋白质的2-D图谱。PDQuest软件分析显示,实验组蛋白质点数为761±56个,对照组为731±80个,两块平均胶的匹配率分别为70%,61%。比较两组细胞平均胶的差异(差异2倍以上认为有差异),有317个蛋白仅在实验组细胞中出现,有30个蛋白仅在对照组细胞中出现;有386个蛋白点在两组细胞中均存在,其中在实验组中表达量显著高于对照组的蛋白质点有37个;在对照组中表达量高于实验组的蛋白质点有29个。质谱结果显示,喜树碱诱导的某个表达下降的蛋白为Galectin-1。
Galectin-1表达的检测
本实验运用了实时荧光定量反转录PCR和Western免疫杂交技术,测定了喜树碱刺激后Galectin-1在mRNA和蛋白水平上的表达变化。结果显示喜树碱(55μg/ml)刺激SMMC-7721细胞6 h后,Galectin-1的mRNA和蛋白表达显著下降(P＜0.05),与比较蛋白质组学结果一致。
喜树碱下调DNMTs的表达
蛋白质组学研究发现,喜树碱可调控SMMC-7721肝癌细胞Galectin-1的表达变化,但是其具体调控机制尚不明确。据报道,Galectin-1基因启动子附近区域富含GC区,DNA甲基化是该基因表达重要的调控机制。因此DNA甲基化机制是否参与了喜树碱诱导的信号通路,并影响Galectin-1的表达值得进一步研究。ELISA法检测了喜树碱(55μg/ml)对SMMC-7721肝癌细胞DNMTs表达的影响。结果显示,喜树碱显著性地抑制DNMTs的表达(P＜0.05),抑制程度随着时间延长而增加,并且呈时间依赖性。
DNA甲基转移酶抑制剂增强了喜树碱的抑制细胞增殖和促凋亡作用
5-脱氧氮杂胞苷(DAC)是一种DNA甲基转移酶抑制剂。体外研究证实,它通过去甲基化作用能使多种CpG岛过甲基化的抑癌基因重新表达,而恢复抑癌功能。喜树碱显著性地抑制DNMTs的表达,提示喜树碱的作用机制与DNA甲基化有关,因此本部分实验检测了5-脱氧氮杂胞苷、喜树碱单独用药或者联合用药对SMMC-7721细胞增殖和凋亡的影响。结果表明,低浓度(0.0228μg/ml)5-脱氧氮杂胞苷并不影响SMMC-7721细胞增殖和凋亡,而同样浓度喜树碱对低浓度5-脱氧氮杂胞苷预处理的SMMC-7721细胞的增殖抑制程度和促凋亡的作用明显高于喜树碱单独作用组(P＜0.05)。
5-脱氧氮杂胞苷增强喜树碱的抑制细胞增殖和促凋亡作用与细胞周期分布无关
喜树碱是细胞周期特异性药物,主要作用于细胞S期。大量研究表明,5-脱氧氮杂胞苷影响细胞周期分布,造成细胞周期的阻滞,S期和G2/M期的聚集。因此5-脱氧氮杂胞苷增强喜树碱细胞增殖抑制和促凋亡作用可能与细胞周期分布的改变有关。本实验检测了5-脱氧氮杂胞苷(0.0228μg/ml)对SMMC-7721细胞周期分布的影响。结果显示,低浓度5-脱氧氮杂胞苷并未影响SMMC-7721细胞的周期分布。
结论
本实验建立了一套分辨率高、重复性好的蛋白质组学方法,并证明了该方法在寻找喜树碱诱导的相关下游信号蛋白的有效性和可行性,提供了SMMC-7721肝癌细胞蛋白质的双向电泳图谱,比较和分析了喜树碱诱导SMMC-7721细胞后蛋白质点的差异,并对差异蛋白进行了质谱鉴定。
本实验发现喜树碱的分子作用机制中新的下游蛋白,DNA甲基转移酶,并且提示喜树碱抑制DNMTs的活性,是喜树碱的分子药理作用新的重要环节。在抑制细胞的增殖和促进细胞凋亡的作用方面,DNMTs抑制剂5-脱氧氮杂胞苷和喜树碱联合用药组的效果明显高于喜树碱单独用药组,提示5-脱氧氮杂胞苷预处理可以强化喜树碱对肝癌细胞的细胞毒作用,为肝癌的治疗提供了新的思路。
Objective
Camptothecin(CPT) was originally isolated from the bark of the Chinese tree, Camptotheca acuminate(Nyssaceae.family),and is a DNA Topoisomerase I inhibitor that reversibly binds to Top 1 cleavage complexes(Top lccs).Several CPT derivatives such as Irinotecan and Topotecan are now commonly used in the treatment of certain human cancers,especially hepatocellular carcinoma.The conversion of Top lccs into irreversible Top 1 covalent complexes results in DNA damage,the induction of DNA repair mechanisms,and changes in expression for cell-cycle proteins related to growth arrest and apoptosis,and defects in key factors of these pathways are essential to the oncogenic process,and are related to the effects of Top 1 inhibitors.For example,CPT can activate Poly(ADP-ribose) polymerase(PARP) which is involved in DNA-repair mechanisms and plays a crucial role in maintaining gene stability.PARP-deficient and knockout cells have been shown to be hypersensitive to CPT and more susceptible to oncogenic transformation. Furthermore,administration of a PARP inhibitor increased the anti-tumor activity of CPT derivatives in a mouse model.Inhibitors of CHK1,CHK2,and NF-κB,which are involved in CPT-induced cellular pathways,have also shown enhanced cytotoxicity when combined with CPT treatment.It is therefore essential to discover new regulation factor induced by CPT in the model system,in order to fulfill the specific regulation network induced by cytotoxic drugs in cancer cell and build theoretical foundation for cancer therapy.The goal of the present study was to use proteomics to identify proteins that change expression following treatment with CPT in the hepatocellular carcinoma cell line,SMMC-7721.
Galectin-1 is a member of the lectin family which has many receptors and is involved in mediating cell adhesion and proliferation,as well as tumor metastasis, apoptosis,immune escape,and angiogenesis.In normal liver tissue,Galectin-1 expression is barely detectable,however,it was found to be overexpressed in primary hepatocellular carcinoma cells.Moreover,administration of siRNA targeted to galectin-1 gene increased the sensitivity of siRNA-expressing cells to the cytotoxic effects of SN-38,a CPT derivative,in the glioma cell line,U87 MG.Expression of recombinant Galectin-1 partially abrogated the increase in sensitivity observed with siRNA expression.The proximal promoter region of the Galectin-1 gene is extremely GC-rich.Therefore,we hypothesized that DNA methyltransferases(DNMTs) may be involved in CPT-induced signaling and affect cell proliferation inhibition and apoptosis induced by CPT.
Methods
SMMC-7721 cells were treated with 55μg/ml CPT for 6 h,then total protein of CPT treated group and control group were collected and separated by two dimensional electrophoresis(2-D).Several spots differentially expressed in two groups were analyzed by a mass spectrometer MALDI-TOF-MS/MS(ABI 4700).Then Quantitative real-time PCR(RT-PCR) and Western blot analysis confirmed mRNA and protein expression changes in Galectin-1 respectively.DNMTs activity of SMMC-7721 cell line after CPT treatment was analyzed by ELISA assay.To evaluate whether regulation of DNMTs is associated with CPT-induced cell death,SMMC-7721 cells were pre-treated with the DNMT inhibitor,5-aza-2'deoxycytidine(DAC),and cell proliferation inhibition and apoptosis were evaluated after CPT treatment.Finally cell cycle distributions were analyzed after DAC treatment.
Results
Isolation and Identification of Galectin-1
Addition of CPT to SMMC-7221 cells resulted in concentration dependent growth inhibition.CPT treatment(55μg/ml) was used to generate samples for two-dimensional electrophoresis.The silver-stained gel provided a comprehensive view of the major proteins expressed in the hepatocellular carcinoma cell line,SMMC-7721. Although some horizontal streaks persisted,protein spots were generally well resolved. 2D-gels for both treated samples and control gels consistently provided good sensitivity and high reproducibility with detection of more than 700 silver stained proteins.High efficiency matching was achieved in each group.A specific protein that showed decreased expression following treatment with CPT,was subsequently identified as Galectin-1 using MALDI-TOF-MS/MS analysis.
Confirmation of Galectin-1 Identification
To confirm the changes in Galectin-1 protein expression observed by 2D-gel electrophoresis,both mRNA and protein expression levels were evaluated following CPT treatment using Quantitative Real-Time RT-PCR and Western blot analysis,respectively. Based on data from real-time RT-PCR analysis,6 h after treatment with CPT(55μg/ml) mRNA expression of Galectin-1 was significantly down regulated.However,24 h after treatment with CPT,Galectin-1 mRNA expression returned to its baseline level.Using the same time points,Western blot analysis of protein expression levels of Galectin-1 in SMMC-7721 cells following CPT treatment were consistent with the data from real-time RT-PCR analysis.Staining forβ-actin was used as a control.
CPT induces down-regulation of DNMTs
The proximal promoter region of the Galectin-1 gene is extremely GC-rich. Therefore,we hypothesized that DNA methyltransferases(DNMTs) may be involved in CPT-induced signaling.The activity of DNMTs decreased with incubation of SMMC-7721 cells with CPT.Significant differences in the activity of DNMTs were observed between: control samples and samples collected at the 3rd h timpoint,between the 3rd h and 9th h timepoint samples,and between the 9th h and 24th h timepoint samples.These results confirmed our hypothesis that DNMTs were involved in CPT-induced cellular responses.
Enhanced cell cytotoxicity after DAC and CPT co-treatment
To evaluate whether regulation of DNMTs is associated with CPT-induced cell death,SMMC-7721 cells were pre-treated with the DNMT inhibitor,5-aza-2'deoxycytidine (DAC),and cell viability and apoptosis were evaluated after CPT treatment.MTT and Flow Cytometry assays were used to detect differences in cell proliferation and apoptosis in SMMC-7721 cells with and without DAC pre-treatment followed by incubation with CPT for 48 h.DAC treatment alone could not affect cell proliferation and apoptosis in SMMC-7721 compared to blank control group,but inhibition of DNMTs by DAC followed by administration of CPT resulted in a significant inhibition of proliferation and enhancement of apoptosis in SMMC-7721 cells compared to CPT treatment alone.
DAC and cell cycle distributions
CPT is a cell cycle specific drug,mainly worked in S phase.DAC could lead cell cycle arrest in S phase and G2/M phase in some kinds of cell type.Therefore changes in cell cycle may play a role in enhanced cytotoxic effects of CPT by pretreatment of DAC. However DAC did not affect cell cycle distribution of SMMC-7721 cell line.
Conclusion
A 2-D method with high resolution and good reproductivity was built in this study, and was proved to be an effective and viable way to discover new regulation proteins induced by CPT.This study provided 2D map of SMMC-7721 cell line,and compared the differences in protein expressions between CPT treated group and control group.
Our study reported on the discovery of a novel downstream protein,DNA methyltransferase(DNMT),linking Topoisomerase I inhibition by CPT leading to the decrease expression of Galectin-1 and growth inhibition in hepatocellular carcinomas SMMC-7721 cells.Moreover,in combination with the DNMT inhibitor DAC,CPT markedly enhanced its antitumor activity.We also provided new evidence to indicate that CPT can modulate Galectin-1 gene and protein expression in the human hepatocellular carcinoma cell line,SMMC-7721.Our results provided new insight into the molecular mechanisms of CPT and the potential to optimize therapies for treatment of hepatocellular carcinomas.
喜树碱是从喜树(珙桐科,喜树属)中提取出的五环生物碱,是拓扑异构酶Ⅰ抑制剂,能可逆性地与拓扑异构酶Ⅰ-DNA可裂解复合物结合。喜树碱衍生物伊利替康和拓扑替康已广泛应用于各种肿瘤的治疗,尤其是肝癌。可裂解的DNA-拓扑异构酶-喜树碱三元复合物一旦转变成不可裂解复合物,将导致DNA损伤、DNA修复机制的激活,细胞周期调控蛋白的表达变化和细胞凋亡等一系列信号通路的改变。这些通路中关键调控分子的失活极大地影响了癌变过程,并与拓扑异构酶Ⅰ抑制剂的作用密切相关。以多聚(ADP-核糖)聚合酶(PARP)为例,该蛋白参与了DNA修复机制,对维持基因稳定性有重要作用。喜树碱可激活PARP的表达,PARP缺失或基因敲除的细胞很容易发生癌变,并且对喜树碱的细胞毒作用高度敏感,同时PARP抑制剂亦可以增强喜树碱衍生物的抗肿瘤作用。此外,喜树碱诱导的细胞信号通路中,CHK1,CHK2和NF-κB等细胞周期阻滞和细胞凋亡相关的信号分子均发挥着重要的调控作用,这些功能蛋白的抑制剂同样可以强化喜树碱的细胞毒性,并成为国内外肿瘤研究的热点和难点。因此,在模式体系中进一步研究喜树碱作用后的关键调控因子,探索可靠、敏感的生物靶标,有助于完善肿瘤应对细胞毒药物作用的特异性调控网络,为肿瘤治疗奠定坚实的理论基础。本实验采用了双向凝胶电泳的方法对喜树碱诱导的SMMC-7721肝癌细胞蛋白质组的变化进行分离和分析,并对喜树碱诱导半乳糖凝集素-1(Galectin-1)表达变化的调控机制作出了初步的探索。
半乳糖凝集素-1是凝集素家族成员之一,广泛参与了细胞生命活动,如调节细胞粘附和增殖、肿瘤转移、细胞凋亡、免疫逃逸和血管再生等。正常肝脏组织中,Galectin-1几乎不表达,但是在肝癌细胞中往往过量表达。此外利用RNA干扰技术抑制Galectin-1的表达后,肿瘤细胞对细胞毒药物SN-38(喜树碱的衍生物)的敏感性得到大幅度增加,而加入重组的Galectin-1后,这种敏感性可以被部分抵消。有报道称Galectin-1组织特异性表达与DNA甲基化模式有关,而且在去甲基化作用下,其表达可得到提高。而DNA甲基转移酶(DNMTs)负责DNA复制过程中维持DNA的甲基化模式。因此,DNMTs是否参与了喜树碱对Galectin-1的表达调控,并影响喜树碱诱导的增殖抑制和细胞凋亡值得进一步研究。空白对照组的细胞总蛋白质,行双向凝胶电泳(2-D)。电泳图谱经图像扫描分析后,选取了若干个表达量大,并且有差异的蛋白质点进行MALDI-TOF质谱分析。采用荧光定量RT-PCR和Western Blot方法对2-D结果进行验证,并检测了喜树碱对SMMC-7721细胞Galectin-1 mRNA和蛋白表达的影响。随后应用ELISA法,检测了喜树碱对SMMC-7721肝癌细胞DNMTs表达的影响。接着通过MTT实验和流式细胞术分析,检测了DNMTs抑制剂5-脱氧氮杂胞苷、喜树碱单独用药或者联合用药对SMMC-7721细胞增殖和凋亡的影响。最后检测了低浓度5-脱氧氮杂胞苷对SMMC-7721细胞周期分布的影响情况。
结果
Galectin-1的分离和鉴定
喜树碱处理SMMC-7721细胞6小时后,收集细胞总蛋白行双向凝胶电泳(2-D),分析喜树碱处理组和空白对照组的总蛋白质的差异。在相同实验条件下进行3次2-D,银染显色后得到喜树碱处理组(实验组)和对照组SMMC-7721细胞蛋白质的2-D图谱。PDQuest软件分析显示,实验组蛋白质点数为761±56个,对照组为731±80个,两块平均胶的匹配率分别为70%,61%。比较两组细胞平均胶的差异(差异2倍以上认为有差异),有317个蛋白仅在实验组细胞中出现,有30个蛋白仅在对照组细胞中出现;有386个蛋白点在两组细胞中均存在,其中在实验组中表达量显著高于对照组的蛋白质点有37个;在对照组中表达量高于实验组的蛋白质点有29个。质谱结果显示,喜树碱诱导的某个表达下降的蛋白为Galectin-1。
Galectin-1表达的检测
本实验运用了实时荧光定量反转录PCR和Western免疫杂交技术,测定了喜树碱刺激后Galectin-1在mRNA和蛋白水平上的表达变化。结果显示喜树碱(55μg/ml)刺激SMMC-7721细胞6 h后,Galectin-1的mRNA和蛋白表达显著下降(P＜0.05),与比较蛋白质组学结果一致。
喜树碱下调DNMTs的表达
蛋白质组学研究发现,喜树碱可调控SMMC-7721肝癌细胞Galectin-1的表达变化,但是其具体调控机制尚不明确。据报道,Galectin-1基因启动子附近区域富含GC区,DNA甲基化是该基因表达重要的调控机制。因此DNA甲基化机制是否参与了喜树碱诱导的信号通路,并影响Galectin-1的表达值得进一步研究。ELISA法检测了喜树碱(55μg/ml)对SMMC-7721肝癌细胞DNMTs表达的影响。结果显示,喜树碱显著性地抑制DNMTs的表达(P＜0.05),抑制程度随着时间延长而增加,并且呈时间依赖性。
DNA甲基转移酶抑制剂增强了喜树碱的抑制细胞增殖和促凋亡作用
5-脱氧氮杂胞苷(DAC)是一种DNA甲基转移酶抑制剂。体外研究证实,它通过去甲基化作用能使多种CpG岛过甲基化的抑癌基因重新表达,而恢复抑癌功能。喜树碱显著性地抑制DNMTs的表达,提示喜树碱的作用机制与DNA甲基化有关,因此本部分实验检测了5-脱氧氮杂胞苷、喜树碱单独用药或者联合用药对SMMC-7721细胞增殖和凋亡的影响。结果表明,低浓度(0.0228μg/ml)5-脱氧氮杂胞苷并不影响SMMC-7721细胞增殖和凋亡,而同样浓度喜树碱对低浓度5-脱氧氮杂胞苷预处理的SMMC-7721细胞的增殖抑制程度和促凋亡的作用明显高于喜树碱单独作用组(P＜0.05)。
5-脱氧氮杂胞苷增强喜树碱的抑制细胞增殖和促凋亡作用与细胞周期分布无关
喜树碱是细胞周期特异性药物,主要作用于细胞S期。大量研究表明,5-脱氧氮杂胞苷影响细胞周期分布,造成细胞周期的阻滞,S期和G2/M期的聚集。因此5-脱氧氮杂胞苷增强喜树碱细胞增殖抑制和促凋亡作用可能与细胞周期分布的改变有关。本实验检测了5-脱氧氮杂胞苷(0.0228μg/ml)对SMMC-7721细胞周期分布的影响。结果显示,低浓度5-脱氧氮杂胞苷并未影响SMMC-7721细胞的周期分布。
结论
本实验建立了一套分辨率高、重复性好的蛋白质组学方法,并证明了该方法在寻找喜树碱诱导的相关下游信号蛋白的有效性和可行性,提供了SMMC-7721肝癌细胞蛋白质的双向电泳图谱,比较和分析了喜树碱诱导SMMC-7721细胞后蛋白质点的差异,并对差异蛋白进行了质谱鉴定。
本实验发现喜树碱的分子作用机制中新的下游蛋白,DNA甲基转移酶,并且提示喜树碱抑制DNMTs的活性,是喜树碱的分子药理作用新的重要环节。在抑制细胞的增殖和促进细胞凋亡的作用方面,DNMTs抑制剂5-脱氧氮杂胞苷和喜树碱联合用药组的效果明显高于喜树碱单独用药组,提示5-脱氧氮杂胞苷预处理可以强化喜树碱对肝癌细胞的细胞毒作用,为肝癌的治疗提供了新的思路。
Objective
Camptothecin(CPT) was originally isolated from the bark of the Chinese tree, Camptotheca acuminate(Nyssaceae.family),and is a DNA Topoisomerase I inhibitor that reversibly binds to Top 1 cleavage complexes(Top lccs).Several CPT derivatives such as Irinotecan and Topotecan are now commonly used in the treatment of certain human cancers,especially hepatocellular carcinoma.The conversion of Top lccs into irreversible Top 1 covalent complexes results in DNA damage,the induction of DNA repair mechanisms,and changes in expression for cell-cycle proteins related to growth arrest and apoptosis,and defects in key factors of these pathways are essential to the oncogenic process,and are related to the effects of Top 1 inhibitors.For example,CPT can activate Poly(ADP-ribose) polymerase(PARP) which is involved in DNA-repair mechanisms and plays a crucial role in maintaining gene stability.PARP-deficient and knockout cells have been shown to be hypersensitive to CPT and more susceptible to oncogenic transformation. Furthermore,administration of a PARP inhibitor increased the anti-tumor activity of CPT derivatives in a mouse model.Inhibitors of CHK1,CHK2,and NF-κB,which are involved in CPT-induced cellular pathways,have also shown enhanced cytotoxicity when combined with CPT treatment.It is therefore essential to discover new regulation factor induced by CPT in the model system,in order to fulfill the specific regulation network induced by cytotoxic drugs in cancer cell and build theoretical foundation for cancer therapy.The goal of the present study was to use proteomics to identify proteins that change expression following treatment with CPT in the hepatocellular carcinoma cell line,SMMC-7721.
Galectin-1 is a member of the lectin family which has many receptors and is involved in mediating cell adhesion and proliferation,as well as tumor metastasis, apoptosis,immune escape,and angiogenesis.In normal liver tissue,Galectin-1 expression is barely detectable,however,it was found to be overexpressed in primary hepatocellular carcinoma cells.Moreover,administration of siRNA targeted to galectin-1 gene increased the sensitivity of siRNA-expressing cells to the cytotoxic effects of SN-38,a CPT derivative,in the glioma cell line,U87 MG.Expression of recombinant Galectin-1 partially abrogated the increase in sensitivity observed with siRNA expression.The proximal promoter region of the Galectin-1 gene is extremely GC-rich.Therefore,we hypothesized that DNA methyltransferases(DNMTs) may be involved in CPT-induced signaling and affect cell proliferation inhibition and apoptosis induced by CPT.
Methods
SMMC-7721 cells were treated with 55μg/ml CPT for 6 h,then total protein of CPT treated group and control group were collected and separated by two dimensional electrophoresis(2-D).Several spots differentially expressed in two groups were analyzed by a mass spectrometer MALDI-TOF-MS/MS(ABI 4700).Then Quantitative real-time PCR(RT-PCR) and Western blot analysis confirmed mRNA and protein expression changes in Galectin-1 respectively.DNMTs activity of SMMC-7721 cell line after CPT treatment was analyzed by ELISA assay.To evaluate whether regulation of DNMTs is associated with CPT-induced cell death,SMMC-7721 cells were pre-treated with the DNMT inhibitor,5-aza-2'deoxycytidine(DAC),and cell proliferation inhibition and apoptosis were evaluated after CPT treatment.Finally cell cycle distributions were analyzed after DAC treatment.
Results
Isolation and Identification of Galectin-1
Addition of CPT to SMMC-7221 cells resulted in concentration dependent growth inhibition.CPT treatment(55μg/ml) was used to generate samples for two-dimensional electrophoresis.The silver-stained gel provided a comprehensive view of the major proteins expressed in the hepatocellular carcinoma cell line,SMMC-7721. Although some horizontal streaks persisted,protein spots were generally well resolved. 2D-gels for both treated samples and control gels consistently provided good sensitivity and high reproducibility with detection of more than 700 silver stained proteins.High efficiency matching was achieved in each group.A specific protein that showed decreased expression following treatment with CPT,was subsequently identified as Galectin-1 using MALDI-TOF-MS/MS analysis.
Confirmation of Galectin-1 Identification
To confirm the changes in Galectin-1 protein expression observed by 2D-gel electrophoresis,both mRNA and protein expression levels were evaluated following CPT treatment using Quantitative Real-Time RT-PCR and Western blot analysis,respectively. Based on data from real-time RT-PCR analysis,6 h after treatment with CPT(55μg/ml) mRNA expression of Galectin-1 was significantly down regulated.However,24 h after treatment with CPT,Galectin-1 mRNA expression returned to its baseline level.Using the same time points,Western blot analysis of protein expression levels of Galectin-1 in SMMC-7721 cells following CPT treatment were consistent with the data from real-time RT-PCR analysis.Staining forβ-actin was used as a control.
CPT induces down-regulation of DNMTs
The proximal promoter region of the Galectin-1 gene is extremely GC-rich. Therefore,we hypothesized that DNA methyltransferases(DNMTs) may be involved in CPT-induced signaling.The activity of DNMTs decreased with incubation of SMMC-7721 cells with CPT.Significant differences in the activity of DNMTs were observed between: control samples and samples collected at the 3rd h timpoint,between the 3rd h and 9th h timepoint samples,and between the 9th h and 24th h timepoint samples.These results confirmed our hypothesis that DNMTs were involved in CPT-induced cellular responses.
Enhanced cell cytotoxicity after DAC and CPT co-treatment
To evaluate whether regulation of DNMTs is associated with CPT-induced cell death,SMMC-7721 cells were pre-treated with the DNMT inhibitor,5-aza-2'deoxycytidine (DAC),and cell viability and apoptosis were evaluated after CPT treatment.MTT and Flow Cytometry assays were used to detect differences in cell proliferation and apoptosis in SMMC-7721 cells with and without DAC pre-treatment followed by incubation with CPT for 48 h.DAC treatment alone could not affect cell proliferation and apoptosis in SMMC-7721 compared to blank control group,but inhibition of DNMTs by DAC followed by administration of CPT resulted in a significant inhibition of proliferation and enhancement of apoptosis in SMMC-7721 cells compared to CPT treatment alone.
DAC and cell cycle distributions
CPT is a cell cycle specific drug,mainly worked in S phase.DAC could lead cell cycle arrest in S phase and G2/M phase in some kinds of cell type.Therefore changes in cell cycle may play a role in enhanced cytotoxic effects of CPT by pretreatment of DAC. However DAC did not affect cell cycle distribution of SMMC-7721 cell line.
Conclusion
A 2-D method with high resolution and good reproductivity was built in this study, and was proved to be an effective and viable way to discover new regulation proteins induced by CPT.This study provided 2D map of SMMC-7721 cell line,and compared the differences in protein expressions between CPT treated group and control group.
Our study reported on the discovery of a novel downstream protein,DNA methyltransferase(DNMT),linking Topoisomerase I inhibition by CPT leading to the decrease expression of Galectin-1 and growth inhibition in hepatocellular carcinomas SMMC-7721 cells.Moreover,in combination with the DNMT inhibitor DAC,CPT markedly enhanced its antitumor activity.We also provided new evidence to indicate that CPT can modulate Galectin-1 gene and protein expression in the human hepatocellular carcinoma cell line,SMMC-7721.Our results provided new insight into the molecular mechanisms of CPT and the potential to optimize therapies for treatment of hepatocellular carcinomas.
引文
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