索拉非尼联合三氧化二砷对肝癌细胞株和肝癌裸鼠移植瘤模型作用的研究
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摘要
研究背景
肝细胞癌是常见的恶性肿瘤,在世界范围内排在第5位,每年死于肝细胞癌的患者超过六百万例。其中大部分患者死于诊断后1年内。由于肝癌起病隐匿,大多数患者在确诊时已是晚期,缺乏有效的治疗药物和手段。对于这类病人,全身化疗、介入栓塞化疗以及射频消融等各治疗手段疗效均不满意。迫切需要一种新的治疗方法。
研究表明新生血管的生成和Raf/MEK/ERK级联的信号传导在肝细胞癌进展中发挥重要作用。细胞外信号调节激酶(ERK)通路是丝裂原活化蛋白激酶(MAPK)信号传导通路的一种。活化的Ras能激活Raf-1,而活化的Raf-1优先通过MEK将信号传导至ERK_1/ERK_2,从而促进细胞生物增殖和抑制细胞凋亡。其中Raf丝/苏氨酸激酶[A-Raf,B-Raf及Raf-1(或C-Raf)]是MAPK级联的第1类激酶,也是细胞增殖及存活的关键调节因子。另外,近来已证明野生型Raf-1能够不依赖于MAPK信号系统,而通过与细胞凋亡及抗细胞凋亡蛋白的直接相互作用来延长细胞存活时间。已有研究发现Raf激酶在大部分肝细胞癌中过度表达,并且Raf/MEK/ERK通路能被HBV、HCV感染和有丝分裂生长因子所激活。此外,肝细胞癌是一种富含血管的肿瘤,肿瘤血管的生成与VEGF相关。血管内皮生长因子(VEGF)是目前所知道的最强直接作用于血管内皮细胞的生长因子,在肿瘤血管生成过程中发挥关键作用。Li等应用RT-PCR技术分析肝细胞癌中VEGF的mRNA表达,显示肝癌组织中VEGF的mRNA表达阳性率明显高于非癌肝组织。因而通过阻断Raf/MEK/ERK通路和抑制血管生成使治疗肝细胞癌成为可能。此外,肝细胞癌形成过程中,不仅癌细胞异常增生,而且突变的细胞凋亡减少,肝癌细胞对凋亡诱导反应明显缺陷。因此,采取促进肝癌细胞凋亡的措施,将有可能成为未来治疗肝癌的有效手段。
索拉非尼(Sorafenib)是第一种口服多激酶抑制剂,一方面可靶向作用于肿瘤细胞及肿瘤血管上的丝氨酸/苏氨酸激酶及受体酪氨酸激酶,通过抑制受体酪氨酸激酶KIT和FLT-3,以及Raf/MEK/ERK途径中丝氨酸/苏氨酸激酶,抑制肿瘤细胞增生;另一方面,通过上游抑制受体酪氨酸激酶VEGFR和PDGFR,及下游抑制Raf/MEK/ERK途径中丝氨酸/苏氨酸激酶,抑制肿瘤血管生成。Wilhelm等已证明,在大批带有致癌的K-ras和/或b-raf突变体的肿瘤细胞内,索拉非尼能够抑制MAPK信号传导系统,并且还能有效抑制分别由VEGF-和PDGFβ-激发的VEGFR2和PDGFRβPTK_S酸化。Li Liu等亦证实了索拉非尼在肝癌细胞株PLC/PRF/5中可阻断Raf/MEK/ERK信号传导通路,抑制血管形成并通过下调抗凋亡蛋白Mcl-1的表达和降低elF4E的磷酸化水平诱导细胞的凋亡。在索拉非尼治疗晚期肝细胞癌的Ⅱ期临床试验和Ⅲ期双盲随机(SHARP)研究中,索拉非尼可显著延长晚期HCC患者的总体生存期。索拉非尼成为第一个可改善晚期HCC患者生存期的靶向治疗药物。而如何提高其靶向抗血管,抑制增殖的作用,则是本研究的重点。
三氧化二砷(Arsenic trioxide,As_2O_3)作为凋亡诱导剂,在HCC的研究中发现对体内外肝癌细胞有抑制增殖和诱导凋亡作用。此外,陈惠英、华海清等的研究认为,三氧化二砷有显著抑制血管内皮细胞作用,提示三氧化二砷同时还具有抗肿瘤血管形成的作用。
根据索拉非尼和三氧化二砷在肝细胞癌中促进凋亡,抑制增殖和肿瘤血管的生成的机制各有不同,将两药联合多靶点,多途径治疗肝细胞癌具有可行性。本试验主要从体外和体内分别研究索拉非尼和三氧化二砷联合对肝细胞癌的抑制作用,证实两药联合可以具有协同作用,为进一步的临床应用提供实验依据。
研究目的
本研究旨在观察索拉非尼与三氧化二砷联合对体内、外肝癌细胞生长的抑制作用,并探讨其可能的效应机制。通过对细胞凋亡相关分子、细胞信号转导相关分子的检测探讨二者联合的机制;并应用荷瘤裸鼠动物模型研究了二者对肿瘤血管生成的抑制作用。
研究方法
1.HepG2肝癌细胞体外传代培养,细胞培养至对数生长期后实验细胞分为4组:分别为空白对照组、索拉非尼单药组、三氧化二砷单药组、索拉非尼联合三氧化二砷组。检测细胞增殖抑制率时索拉非尼药物剂量分别为1.5、3μmol/L;三氧化二砷剂量分别为2、4μmol/l;索拉非尼联合三氧化二砷组的药物剂量是1.5+2、3+4μmol/L。其它实验中药物剂量索拉非尼均为3μmol/L,三氧化二砷均为4μmol/L,联合组为索拉非尼+三氧化二砷3+4μmol/L。
2.以MTT法检测24、48、72h各时间点各组药物对人肝癌细胞HepG2增殖的OD值。根据OD值计算细胞生长抑制率。采用协同作用q值判断索拉非尼和三氧化二砷联用的性质:q＞1.15为协同作用,0.85≤q≤1.15为相加作用,q＜0.85为拮抗作用。
3.细胞培养及分组同上,用流式细胞仪于各组药物作用后48h检测细胞周期的分布与细胞凋亡率。
4.细胞培养及分组同上,用流式细胞仪检测各组药物作用48h后细胞线粒体膜电位变化。
5.细胞培养及分组同上,各组药物作用后48h提取蛋白,Western blot法检测各组细胞凋亡相关分子Bcl-2、Mcl-1蛋白表达以及细胞信号转导相关分子pERK1/2、ERK1/2的表达水平。
6.制备肝癌细胞裸鼠皮下移植瘤模型,将40只荷瘤裸鼠随机分成4组,空白对照组、索拉非尼单药组、三氧化二砷单药组、索拉非尼联合三氧化二砷组。观察治疗前后移植瘤的瘤积变化。于用药后第23天处死荷瘤裸鼠,称重并收集皮下移植瘤,肿瘤组织免疫组织化学HE染色观察病理学变化情况,免疫组织化学法检测各组移植瘤组织的微血管密度(MVD)及VEGF表达情况。
7.统计分析:所有数据采用SPSS 13.0统计软件进行统计处理,实验数据用均数±标准差((?)±s)表示,两样本均数的比较采用Independent-Samples T Test;多组间均数比较采用单因素方差分析,组间两两比较采用LSD法,方差不齐的采用Dunnett'T3法;等级资料采用非参数检验(2 Independent Samples Test)。显著性水准为α=0.05。
研究结果
1.MTT比色法检测显示,索拉非尼和三氧化二砷单独抑制HepG2细胞增殖的程度均呈时间和剂量依赖效应(P＜0.05);各浓度联合组与单药组抑制率均有显著性差异(P＜0.05),联合疗效优于单药。除索拉非尼3μmol/L和As_2O_34μmol/L联合用药组在24小时q值为1.121,表现为相加作用外,其余各浓度两药联合在24、48和72小时均表现为协同作用(q＞1.15),以索拉非尼3μmol/L和As_2O_34μmol/L联合用药组在48小时的协同作用最大(q=1.416)。
2.流式细胞仪检测细胞周期分布显示与对照组相比,三氧化二砷可使HepG2细胞生长阻滞于G_2/M期(P=0.000),而索拉非尼主要使S期细胞增加,与对照组相比有显著性差异(P=0.005)。联合用药后细胞与对照组相比主要阻滞在S期和G_2/M期(P值分别为0.001和0.015)。
3.在凋亡率方面,三氧化二砷组和索拉非尼组与对照组相比均有显著升高(P值分别为0.041和0.000)。联合用药组较三氧化二砷组和索拉非尼组均升高(P值分别为0.000和0.001)。
4.线粒体跨膜电位显示,药物处理组的相对荧光强度较对照组明显降低(P＜0.05),且联合用药使相对荧光强度降低更为明显,与另外三个组相比差异具有统计学意义(P＜0.05),说明单独使用三氧化二砷和索拉非尼能使细胞线粒体跨膜电位降低,联合作用强于单药。
5.Western blot结果显示,
(1)单独应用索拉非尼和三氧化二砷能抑制ERK磷酸化,索拉非尼和三氧化二砷联合可以进一步减少pERK的水平。药物作用后对总的ERK蛋白水平无显著性影响。
(2)索拉非尼对Bcl-2蛋白水平无明显影响,三氧化二砷显著下调Bcl-2蛋白的表达,联合应用索拉非尼后,对三氧化二砷下调Bcl-2蛋白有促进作用。
(3)Mcl-1蛋白的表达结果显示,索拉非尼组和联合组Mcl-1的表达低于三氧化二砷组和对照组,三氧化二砷组和对照组未见显著差异,索拉非尼组和联合组无显著差异。
6.动物试验结果显示,与对照组比较,单药索拉非尼组及三氧化二砷组、联合组的瘤积和瘤重均显著减少,差异有统计学意义(P＜0.05)。联合组的瘤积和瘤重较单药索拉非尼组及三氧化二砷组显著减少,差异有统计学意义(P＜0.05)。
7.肿瘤组织切片免疫组化结果显示,三氧化二砷组和索拉非尼组均较对照组的血管密度减少,差异有显著性(P值均为0.000)。并且联合用药组较三氧化二砷组和索拉非尼组的血管密度减少,差异有显著性(P值均为0.000)。与对照组相比,索拉非尼、三氧化二砷单药组和联合组均可使VEGF表达明显减少,差异有统计学意义(P＜0.05)。与索拉非尼和三氧化二砷单药组相比,联合组VEGF的表达下降更明显,差异有统计学意义(P＜0.05)。
初步结论
索拉非尼及三氧化二砷对HepG2肝癌细胞均有抑制增殖作用,呈时间和剂量依赖性。两药联合的作用更显著。两药阻滞细胞在不同的周期,联合用药可明显增加肝癌细胞的凋亡。两药联合的效应机制与ERK细胞信号转导通路有关。索拉非尼和三氧化二砷作用于凋亡途径的不同靶点,或协同作用于某些相同的靶点,分别增加了对凋亡相关分子影响的广度和强度,从而增强了联合诱导肝癌细胞凋亡的作用。此外,索拉非尼及三氧化二砷在体内还协同抑制肝癌细胞移植瘤的生长和血管生成,增强了抗肿瘤效果。
Background
Hepatocellular carcinoma(HCC),is the fifth most common cancer in the world and is responsible for＞6000,000 deaths annual.Patients with HCC die withinl year after the diagnosis were conformed.Unfortunately,the disease is often diagnosed at a late stage when curative therapies are least effective.For these patients, medical treatments,including chemotherapy,chemoembolization,ablation,and proton beam therapy,remain disappointing.There is an urgent need for new therapies for this aggressive disease.
Both angiogenesis and signaling through the Raf/mitogen-avtivated protein (MAP)/extracellular-signal-regulate-kinase(ERK)-kinase(MEK)/ERK(RAF/MEK/ER K)cascade play critical roles in the development of HCC.Activation of Ras signaling pathways is an important mechanism by which human cancers develop.Raf-1 kinases are serine/threonine protein kinases that function in this path way as downstream effectual molecules of Ras.Raf-1 can transmit the signal to ERK_1/ERK_2 through MEK,initiates a mitogenic kinase cascade that ultimately modulates gene expression via the phosphorylation of transcription factors,which can have profound effects on cellular proliferation and anti-apoptosis.Recent evidence suggests that Raf-1 can prolong the life of cell through the action with apoptosis protein and anti-apoptosis protein.Studies have found Raf kinase is over-expressed in a high percentage of HCC patient tumors,and the RAF/MEK/ERK pathway can be activated by major etiologic factors such as HBV and HCV infection and mitogenic grown factors.On the other hand,anti-angiogenesis therapies,which inhibit blood vessel formation,may have treatment effect of HCC tumors depend on a rich blood supply.Vascular-endothelial growth factors(VEGF) are major factors for cancer angiogenesis.Professor Li's study found mRNA of VEGF is over-expressed in tumors of HCC patients through RT-PCR.Therefore,inhibition of both angiogenesis and RAF/MEK/ERK signaling may represent an attractive approach for the treatment of HCC.One the other hand, studies found inhibition of apoptosis was important for HCC development.So inducing apoptosis may be a good treatment for HCC.
Sorafenib is a multikinase inhibitor that has shown to block proliferation of tumor cells and angiogenesis by inhibiting serine/threonine kinases(c-Raf mutant and wild-type B-Raf),as well as the receptor tyrosine kinases vascular endothelial growth factor receptor 2(VEGFR_2),VEGFR_3,platelet-derived growth factor receptor (PDGFR),FLT3 and C-KIT.A report by Wilhelm et al showed that Sorafenib exhibited the RAF/MEK/ERK pathway and VEGFR_2 and PDGFRβ-PTKs phosphorylation which were acted by VEGF and PDGFRβin the cancer cells with mutation of k-ras and/or b-raf.Li Liu et al also found Sorafenib inhibited RAF/MEK/ERK pathway and the phosphorylation of eIF4E and down-regulated antiapoptotic protein Mcl-1,induced tumor cell apoptosis in PLC/PRF/5 xenografts. The phaseⅡandⅢclinical studies(SHARP trial) reported that Sorafenib improved overall survival time in 44%HCC patients.Sorafenib was the first drug improving overall survival time of the HCC patients.
Arsenic trioxide(As_2O_3) has shown remarkable curative effect inducing tumor cell apoptosis in HCC.On the other hand,Hui-Ying Cheng,Hai-qing Hua et al also found As_2O_3 could inhibit the vascularization and electron microscopy showed that As_2O_3 could damage primitive mesenchymal cells and vascular endothelial cells and inhibit neovascular formation.It is considered that As_2O_3 can inhibit the tumor neovascularization.
As Sorafenib and As_2O_3 has different mechanisms of inducing tumor cell apoptosis,inhibiting tumor cell proliferation and inhibiting angiogenesis,it is possible that using two drugs to treat HCC through different ways and targets.
This study is designed for using two drugs in vitro and in vivo to confirm that they have synergistic effect for the inhibition of HCC.It also can supply evidence for the clinical study.
Objective
To investigate the inhibitory effect and molecular mechanisms of sorafenib combined with Arsenic trioxide(As_2O_3) on hepatocellular carcinoma cells and neoplasms.
Methods
1.HepG2(p53 wild type) human HCC tumor cells were cultured in RPMI 1640 containing 10%fetal calf serum.Exponentially growing cells were chosen for experiment.HepG2 cells were divided into 4 groups:control group,Sorafenib-treated group,As_2O_3-treated group and combination treatment group.MTT assay included cells treated with Sorafenib(1.5、3μmol/L),As_2O_3(2、4μmol/L) alone and combination(1.5+2and 3+4μmol/L) for 24,48 and 72 hours respectively.In other tests cells were treated with Sorafenib(3μmol/L) and As_2O_3(4μmol/L) alone and combination.
2.Groups as the above mentioned,the inhibitory of HepG2 cells were detected by MTT assay after treatment.Calculate the inhibitory rates of cells according OD value. Interaction between Sorafenib and As_2O_3 was assessed using the q value,where q＞1.15,0.85≤q≤1.15,and q＜0.85 indicated synergistic,additive,and antagonistic effects respectively.
3.Groups as the above mentioned,collected cells after 48h and analyzed cell cycle and apoptosis by flow cytometry.
4.Groups as the above mentioned,collected cells after 48h and analyzed the changes of mitochondrial membrane potential(Δψm) labeled by Rhodamine123 and examined by flow cytometry.
5.Groups as the above mentioned,collected cells after 48h and the proteins were extracted from cells.Western Blot was performed for protein expression of Bcl-2、Mcl-1、pERK 1/2、ERK1/2.
6.Construct nude mouse tumor animal model.The nude mouse with HepG_2 cell xenografts were randomized into control group,As_2O_3-treated group, Sorafenib-treated group and combination-treated group.Every group had 10 mice. Evaluate volume of tumor in four groups before and after treatments.Execute the nude mouse at the twenty-three day after treatments,to strip and weigh the neoplasm for evaluating the inhibition of the anti-tumor.Determine tumor tissue VEGF protein expressions and micro-vessel density(MVD,assessed by CD34 immunohistochemistry).
7.Statistical analysis:All data were analysed by SPSS 13.0 statistical software.Data were expressed as mean±S.D.Two independent samples were analyzed by T test. The others data were analyzed by One-way ANOVA followed by LSD multiple comparison tests.Rank data were analyzed by 2 Independent Samples Test.P-values were considered to be significant at＜0.05.
Results
1.The MTT result indicated:Combination of Sorafenib and As_2O_3 enhanced inhibition of proliferation in HepG2 cells(P＜0.05).They have synergistic effect for the inhibition of HepG2 cells(q＞1.15) except Sorafenib(3μmol/L) together with As_2O_3(4μmol/L) showed additive effect at 24h(q=1.121).When Sorafenib(3μmol/L) together with As_2O_3(4μmol/L) effected at 48h,q-value was the biggest(p=1.416). 2 Sorafenib and As_2O_3 alone and together affected cell cycle of HepG2 cells. Sorafenib alone can induce accumulation of cells in S phase compared with untreated control group(P=0.005).As_2O_3 alone can induce accumulation of cells in G_2/M phase compared with untreated control group(P=0.000).Combinaton group showed greater increase of S and G_2/M phases than control group(P-value was 0.001and 0.015 individually).
3 Sorafenib and As_2O_3 alone induced more HepG2 cells apoptosis compared with untreated control(P-value was 0.000and 0.041 individually).Cells apoptosis was increased induced by combinaton of Sorafenib with As_2O_3 compared with each agent (P-value was 0.001 and 0.000 individually).
4 The mitochondrial membrane potential was down-regulated in As_2O_3,Sorafenib or combination group compared with untreated control group(P＜0.05).The affection by the combination was stronger than that alone(P＜0.05).
5.There was no difference of the expression of ERK among four groups.The decrease of pERK expression was seen after treated with sorafenib and As_2O_3 alone and the combination,and the latter was more obvious.Sorafenib alone and in combination with As_2O_3 could decrease Mcl-1 expressions,while As_2O_3 alone and in combination with Sorafenib can decrease Bcl-2 expressions and the one of combination was more obvious.
6.The tumor volume and weight of Sorafenib and As_2O_3 alone groups were lower than the control groups after treatments(P＜0.05).The group of combination was the lowest among the four groups(P＜0.05).
7.The immunohistochemistry results showed:the MVD were decreased in the three groups compared with control group(P=0.000).The one of combination was more obvious compared with Sorafenib and As_2O_3 alone groups(P=0.000).The expressions of VEGF were obviously decreased in Sorafenib and As_2O_3 alone and in combination groups(P＜0.05),the one of combination was more obvious compared with Sorafenib and As_2O_3 alone groups(P＜0.05).
Conclusion
Sorafenib and As_2O_3 alone or combination induced cell cycle arrest.Sorafenib combined with As_2O_3 showed synergistic effect on inhibition proliferation of cells and induction apoptosis and they had a synergistic effect.The mechanisms of synergistic effect were closely related to the blockage of cell-cycle progress and the promotion of apoptosis as well as affecting signaling pathway.Sorafenib and As_2O_3 affected different targets or the same target in deeper degree resulting in enhancing the anti-tumor effect.The inhibition mechanisms were also attributed to inhibition of tumor angiogenesis in vivo.
肝细胞癌是常见的恶性肿瘤,在世界范围内排在第5位,每年死于肝细胞癌的患者超过六百万例。其中大部分患者死于诊断后1年内。由于肝癌起病隐匿,大多数患者在确诊时已是晚期,缺乏有效的治疗药物和手段。对于这类病人,全身化疗、介入栓塞化疗以及射频消融等各治疗手段疗效均不满意。迫切需要一种新的治疗方法。
研究表明新生血管的生成和Raf/MEK/ERK级联的信号传导在肝细胞癌进展中发挥重要作用。细胞外信号调节激酶(ERK)通路是丝裂原活化蛋白激酶(MAPK)信号传导通路的一种。活化的Ras能激活Raf-1,而活化的Raf-1优先通过MEK将信号传导至ERK_1/ERK_2,从而促进细胞生物增殖和抑制细胞凋亡。其中Raf丝/苏氨酸激酶[A-Raf,B-Raf及Raf-1(或C-Raf)]是MAPK级联的第1类激酶,也是细胞增殖及存活的关键调节因子。另外,近来已证明野生型Raf-1能够不依赖于MAPK信号系统,而通过与细胞凋亡及抗细胞凋亡蛋白的直接相互作用来延长细胞存活时间。已有研究发现Raf激酶在大部分肝细胞癌中过度表达,并且Raf/MEK/ERK通路能被HBV、HCV感染和有丝分裂生长因子所激活。此外,肝细胞癌是一种富含血管的肿瘤,肿瘤血管的生成与VEGF相关。血管内皮生长因子(VEGF)是目前所知道的最强直接作用于血管内皮细胞的生长因子,在肿瘤血管生成过程中发挥关键作用。Li等应用RT-PCR技术分析肝细胞癌中VEGF的mRNA表达,显示肝癌组织中VEGF的mRNA表达阳性率明显高于非癌肝组织。因而通过阻断Raf/MEK/ERK通路和抑制血管生成使治疗肝细胞癌成为可能。此外,肝细胞癌形成过程中,不仅癌细胞异常增生,而且突变的细胞凋亡减少,肝癌细胞对凋亡诱导反应明显缺陷。因此,采取促进肝癌细胞凋亡的措施,将有可能成为未来治疗肝癌的有效手段。
索拉非尼(Sorafenib)是第一种口服多激酶抑制剂,一方面可靶向作用于肿瘤细胞及肿瘤血管上的丝氨酸/苏氨酸激酶及受体酪氨酸激酶,通过抑制受体酪氨酸激酶KIT和FLT-3,以及Raf/MEK/ERK途径中丝氨酸/苏氨酸激酶,抑制肿瘤细胞增生;另一方面,通过上游抑制受体酪氨酸激酶VEGFR和PDGFR,及下游抑制Raf/MEK/ERK途径中丝氨酸/苏氨酸激酶,抑制肿瘤血管生成。Wilhelm等已证明,在大批带有致癌的K-ras和/或b-raf突变体的肿瘤细胞内,索拉非尼能够抑制MAPK信号传导系统,并且还能有效抑制分别由VEGF-和PDGFβ-激发的VEGFR2和PDGFRβPTK_S酸化。Li Liu等亦证实了索拉非尼在肝癌细胞株PLC/PRF/5中可阻断Raf/MEK/ERK信号传导通路,抑制血管形成并通过下调抗凋亡蛋白Mcl-1的表达和降低elF4E的磷酸化水平诱导细胞的凋亡。在索拉非尼治疗晚期肝细胞癌的Ⅱ期临床试验和Ⅲ期双盲随机(SHARP)研究中,索拉非尼可显著延长晚期HCC患者的总体生存期。索拉非尼成为第一个可改善晚期HCC患者生存期的靶向治疗药物。而如何提高其靶向抗血管,抑制增殖的作用,则是本研究的重点。
三氧化二砷(Arsenic trioxide,As_2O_3)作为凋亡诱导剂,在HCC的研究中发现对体内外肝癌细胞有抑制增殖和诱导凋亡作用。此外,陈惠英、华海清等的研究认为,三氧化二砷有显著抑制血管内皮细胞作用,提示三氧化二砷同时还具有抗肿瘤血管形成的作用。
根据索拉非尼和三氧化二砷在肝细胞癌中促进凋亡,抑制增殖和肿瘤血管的生成的机制各有不同,将两药联合多靶点,多途径治疗肝细胞癌具有可行性。本试验主要从体外和体内分别研究索拉非尼和三氧化二砷联合对肝细胞癌的抑制作用,证实两药联合可以具有协同作用,为进一步的临床应用提供实验依据。
研究目的
本研究旨在观察索拉非尼与三氧化二砷联合对体内、外肝癌细胞生长的抑制作用,并探讨其可能的效应机制。通过对细胞凋亡相关分子、细胞信号转导相关分子的检测探讨二者联合的机制;并应用荷瘤裸鼠动物模型研究了二者对肿瘤血管生成的抑制作用。
研究方法
1.HepG2肝癌细胞体外传代培养,细胞培养至对数生长期后实验细胞分为4组:分别为空白对照组、索拉非尼单药组、三氧化二砷单药组、索拉非尼联合三氧化二砷组。检测细胞增殖抑制率时索拉非尼药物剂量分别为1.5、3μmol/L;三氧化二砷剂量分别为2、4μmol/l;索拉非尼联合三氧化二砷组的药物剂量是1.5+2、3+4μmol/L。其它实验中药物剂量索拉非尼均为3μmol/L,三氧化二砷均为4μmol/L,联合组为索拉非尼+三氧化二砷3+4μmol/L。
2.以MTT法检测24、48、72h各时间点各组药物对人肝癌细胞HepG2增殖的OD值。根据OD值计算细胞生长抑制率。采用协同作用q值判断索拉非尼和三氧化二砷联用的性质:q＞1.15为协同作用,0.85≤q≤1.15为相加作用,q＜0.85为拮抗作用。
3.细胞培养及分组同上,用流式细胞仪于各组药物作用后48h检测细胞周期的分布与细胞凋亡率。
4.细胞培养及分组同上,用流式细胞仪检测各组药物作用48h后细胞线粒体膜电位变化。
5.细胞培养及分组同上,各组药物作用后48h提取蛋白,Western blot法检测各组细胞凋亡相关分子Bcl-2、Mcl-1蛋白表达以及细胞信号转导相关分子pERK1/2、ERK1/2的表达水平。
6.制备肝癌细胞裸鼠皮下移植瘤模型,将40只荷瘤裸鼠随机分成4组,空白对照组、索拉非尼单药组、三氧化二砷单药组、索拉非尼联合三氧化二砷组。观察治疗前后移植瘤的瘤积变化。于用药后第23天处死荷瘤裸鼠,称重并收集皮下移植瘤,肿瘤组织免疫组织化学HE染色观察病理学变化情况,免疫组织化学法检测各组移植瘤组织的微血管密度(MVD)及VEGF表达情况。
7.统计分析:所有数据采用SPSS 13.0统计软件进行统计处理,实验数据用均数±标准差((?)±s)表示,两样本均数的比较采用Independent-Samples T Test;多组间均数比较采用单因素方差分析,组间两两比较采用LSD法,方差不齐的采用Dunnett'T3法;等级资料采用非参数检验(2 Independent Samples Test)。显著性水准为α=0.05。
研究结果
1.MTT比色法检测显示,索拉非尼和三氧化二砷单独抑制HepG2细胞增殖的程度均呈时间和剂量依赖效应(P＜0.05);各浓度联合组与单药组抑制率均有显著性差异(P＜0.05),联合疗效优于单药。除索拉非尼3μmol/L和As_2O_34μmol/L联合用药组在24小时q值为1.121,表现为相加作用外,其余各浓度两药联合在24、48和72小时均表现为协同作用(q＞1.15),以索拉非尼3μmol/L和As_2O_34μmol/L联合用药组在48小时的协同作用最大(q=1.416)。
2.流式细胞仪检测细胞周期分布显示与对照组相比,三氧化二砷可使HepG2细胞生长阻滞于G_2/M期(P=0.000),而索拉非尼主要使S期细胞增加,与对照组相比有显著性差异(P=0.005)。联合用药后细胞与对照组相比主要阻滞在S期和G_2/M期(P值分别为0.001和0.015)。
3.在凋亡率方面,三氧化二砷组和索拉非尼组与对照组相比均有显著升高(P值分别为0.041和0.000)。联合用药组较三氧化二砷组和索拉非尼组均升高(P值分别为0.000和0.001)。
4.线粒体跨膜电位显示,药物处理组的相对荧光强度较对照组明显降低(P＜0.05),且联合用药使相对荧光强度降低更为明显,与另外三个组相比差异具有统计学意义(P＜0.05),说明单独使用三氧化二砷和索拉非尼能使细胞线粒体跨膜电位降低,联合作用强于单药。
5.Western blot结果显示,
(1)单独应用索拉非尼和三氧化二砷能抑制ERK磷酸化,索拉非尼和三氧化二砷联合可以进一步减少pERK的水平。药物作用后对总的ERK蛋白水平无显著性影响。
(2)索拉非尼对Bcl-2蛋白水平无明显影响,三氧化二砷显著下调Bcl-2蛋白的表达,联合应用索拉非尼后,对三氧化二砷下调Bcl-2蛋白有促进作用。
(3)Mcl-1蛋白的表达结果显示,索拉非尼组和联合组Mcl-1的表达低于三氧化二砷组和对照组,三氧化二砷组和对照组未见显著差异,索拉非尼组和联合组无显著差异。
6.动物试验结果显示,与对照组比较,单药索拉非尼组及三氧化二砷组、联合组的瘤积和瘤重均显著减少,差异有统计学意义(P＜0.05)。联合组的瘤积和瘤重较单药索拉非尼组及三氧化二砷组显著减少,差异有统计学意义(P＜0.05)。
7.肿瘤组织切片免疫组化结果显示,三氧化二砷组和索拉非尼组均较对照组的血管密度减少,差异有显著性(P值均为0.000)。并且联合用药组较三氧化二砷组和索拉非尼组的血管密度减少,差异有显著性(P值均为0.000)。与对照组相比,索拉非尼、三氧化二砷单药组和联合组均可使VEGF表达明显减少,差异有统计学意义(P＜0.05)。与索拉非尼和三氧化二砷单药组相比,联合组VEGF的表达下降更明显,差异有统计学意义(P＜0.05)。
初步结论
索拉非尼及三氧化二砷对HepG2肝癌细胞均有抑制增殖作用,呈时间和剂量依赖性。两药联合的作用更显著。两药阻滞细胞在不同的周期,联合用药可明显增加肝癌细胞的凋亡。两药联合的效应机制与ERK细胞信号转导通路有关。索拉非尼和三氧化二砷作用于凋亡途径的不同靶点,或协同作用于某些相同的靶点,分别增加了对凋亡相关分子影响的广度和强度,从而增强了联合诱导肝癌细胞凋亡的作用。此外,索拉非尼及三氧化二砷在体内还协同抑制肝癌细胞移植瘤的生长和血管生成,增强了抗肿瘤效果。
Background
Hepatocellular carcinoma(HCC),is the fifth most common cancer in the world and is responsible for＞6000,000 deaths annual.Patients with HCC die withinl year after the diagnosis were conformed.Unfortunately,the disease is often diagnosed at a late stage when curative therapies are least effective.For these patients, medical treatments,including chemotherapy,chemoembolization,ablation,and proton beam therapy,remain disappointing.There is an urgent need for new therapies for this aggressive disease.
Both angiogenesis and signaling through the Raf/mitogen-avtivated protein (MAP)/extracellular-signal-regulate-kinase(ERK)-kinase(MEK)/ERK(RAF/MEK/ER K)cascade play critical roles in the development of HCC.Activation of Ras signaling pathways is an important mechanism by which human cancers develop.Raf-1 kinases are serine/threonine protein kinases that function in this path way as downstream effectual molecules of Ras.Raf-1 can transmit the signal to ERK_1/ERK_2 through MEK,initiates a mitogenic kinase cascade that ultimately modulates gene expression via the phosphorylation of transcription factors,which can have profound effects on cellular proliferation and anti-apoptosis.Recent evidence suggests that Raf-1 can prolong the life of cell through the action with apoptosis protein and anti-apoptosis protein.Studies have found Raf kinase is over-expressed in a high percentage of HCC patient tumors,and the RAF/MEK/ERK pathway can be activated by major etiologic factors such as HBV and HCV infection and mitogenic grown factors.On the other hand,anti-angiogenesis therapies,which inhibit blood vessel formation,may have treatment effect of HCC tumors depend on a rich blood supply.Vascular-endothelial growth factors(VEGF) are major factors for cancer angiogenesis.Professor Li's study found mRNA of VEGF is over-expressed in tumors of HCC patients through RT-PCR.Therefore,inhibition of both angiogenesis and RAF/MEK/ERK signaling may represent an attractive approach for the treatment of HCC.One the other hand, studies found inhibition of apoptosis was important for HCC development.So inducing apoptosis may be a good treatment for HCC.
Sorafenib is a multikinase inhibitor that has shown to block proliferation of tumor cells and angiogenesis by inhibiting serine/threonine kinases(c-Raf mutant and wild-type B-Raf),as well as the receptor tyrosine kinases vascular endothelial growth factor receptor 2(VEGFR_2),VEGFR_3,platelet-derived growth factor receptor (PDGFR),FLT3 and C-KIT.A report by Wilhelm et al showed that Sorafenib exhibited the RAF/MEK/ERK pathway and VEGFR_2 and PDGFRβ-PTKs phosphorylation which were acted by VEGF and PDGFRβin the cancer cells with mutation of k-ras and/or b-raf.Li Liu et al also found Sorafenib inhibited RAF/MEK/ERK pathway and the phosphorylation of eIF4E and down-regulated antiapoptotic protein Mcl-1,induced tumor cell apoptosis in PLC/PRF/5 xenografts. The phaseⅡandⅢclinical studies(SHARP trial) reported that Sorafenib improved overall survival time in 44%HCC patients.Sorafenib was the first drug improving overall survival time of the HCC patients.
Arsenic trioxide(As_2O_3) has shown remarkable curative effect inducing tumor cell apoptosis in HCC.On the other hand,Hui-Ying Cheng,Hai-qing Hua et al also found As_2O_3 could inhibit the vascularization and electron microscopy showed that As_2O_3 could damage primitive mesenchymal cells and vascular endothelial cells and inhibit neovascular formation.It is considered that As_2O_3 can inhibit the tumor neovascularization.
As Sorafenib and As_2O_3 has different mechanisms of inducing tumor cell apoptosis,inhibiting tumor cell proliferation and inhibiting angiogenesis,it is possible that using two drugs to treat HCC through different ways and targets.
This study is designed for using two drugs in vitro and in vivo to confirm that they have synergistic effect for the inhibition of HCC.It also can supply evidence for the clinical study.
Objective
To investigate the inhibitory effect and molecular mechanisms of sorafenib combined with Arsenic trioxide(As_2O_3) on hepatocellular carcinoma cells and neoplasms.
Methods
1.HepG2(p53 wild type) human HCC tumor cells were cultured in RPMI 1640 containing 10%fetal calf serum.Exponentially growing cells were chosen for experiment.HepG2 cells were divided into 4 groups:control group,Sorafenib-treated group,As_2O_3-treated group and combination treatment group.MTT assay included cells treated with Sorafenib(1.5、3μmol/L),As_2O_3(2、4μmol/L) alone and combination(1.5+2and 3+4μmol/L) for 24,48 and 72 hours respectively.In other tests cells were treated with Sorafenib(3μmol/L) and As_2O_3(4μmol/L) alone and combination.
2.Groups as the above mentioned,the inhibitory of HepG2 cells were detected by MTT assay after treatment.Calculate the inhibitory rates of cells according OD value. Interaction between Sorafenib and As_2O_3 was assessed using the q value,where q＞1.15,0.85≤q≤1.15,and q＜0.85 indicated synergistic,additive,and antagonistic effects respectively.
3.Groups as the above mentioned,collected cells after 48h and analyzed cell cycle and apoptosis by flow cytometry.
4.Groups as the above mentioned,collected cells after 48h and analyzed the changes of mitochondrial membrane potential(Δψm) labeled by Rhodamine123 and examined by flow cytometry.
5.Groups as the above mentioned,collected cells after 48h and the proteins were extracted from cells.Western Blot was performed for protein expression of Bcl-2、Mcl-1、pERK 1/2、ERK1/2.
6.Construct nude mouse tumor animal model.The nude mouse with HepG_2 cell xenografts were randomized into control group,As_2O_3-treated group, Sorafenib-treated group and combination-treated group.Every group had 10 mice. Evaluate volume of tumor in four groups before and after treatments.Execute the nude mouse at the twenty-three day after treatments,to strip and weigh the neoplasm for evaluating the inhibition of the anti-tumor.Determine tumor tissue VEGF protein expressions and micro-vessel density(MVD,assessed by CD34 immunohistochemistry).
7.Statistical analysis:All data were analysed by SPSS 13.0 statistical software.Data were expressed as mean±S.D.Two independent samples were analyzed by T test. The others data were analyzed by One-way ANOVA followed by LSD multiple comparison tests.Rank data were analyzed by 2 Independent Samples Test.P-values were considered to be significant at＜0.05.
Results
1.The MTT result indicated:Combination of Sorafenib and As_2O_3 enhanced inhibition of proliferation in HepG2 cells(P＜0.05).They have synergistic effect for the inhibition of HepG2 cells(q＞1.15) except Sorafenib(3μmol/L) together with As_2O_3(4μmol/L) showed additive effect at 24h(q=1.121).When Sorafenib(3μmol/L) together with As_2O_3(4μmol/L) effected at 48h,q-value was the biggest(p=1.416). 2 Sorafenib and As_2O_3 alone and together affected cell cycle of HepG2 cells. Sorafenib alone can induce accumulation of cells in S phase compared with untreated control group(P=0.005).As_2O_3 alone can induce accumulation of cells in G_2/M phase compared with untreated control group(P=0.000).Combinaton group showed greater increase of S and G_2/M phases than control group(P-value was 0.001and 0.015 individually).
3 Sorafenib and As_2O_3 alone induced more HepG2 cells apoptosis compared with untreated control(P-value was 0.000and 0.041 individually).Cells apoptosis was increased induced by combinaton of Sorafenib with As_2O_3 compared with each agent (P-value was 0.001 and 0.000 individually).
4 The mitochondrial membrane potential was down-regulated in As_2O_3,Sorafenib or combination group compared with untreated control group(P＜0.05).The affection by the combination was stronger than that alone(P＜0.05).
5.There was no difference of the expression of ERK among four groups.The decrease of pERK expression was seen after treated with sorafenib and As_2O_3 alone and the combination,and the latter was more obvious.Sorafenib alone and in combination with As_2O_3 could decrease Mcl-1 expressions,while As_2O_3 alone and in combination with Sorafenib can decrease Bcl-2 expressions and the one of combination was more obvious.
6.The tumor volume and weight of Sorafenib and As_2O_3 alone groups were lower than the control groups after treatments(P＜0.05).The group of combination was the lowest among the four groups(P＜0.05).
7.The immunohistochemistry results showed:the MVD were decreased in the three groups compared with control group(P=0.000).The one of combination was more obvious compared with Sorafenib and As_2O_3 alone groups(P=0.000).The expressions of VEGF were obviously decreased in Sorafenib and As_2O_3 alone and in combination groups(P＜0.05),the one of combination was more obvious compared with Sorafenib and As_2O_3 alone groups(P＜0.05).
Conclusion
Sorafenib and As_2O_3 alone or combination induced cell cycle arrest.Sorafenib combined with As_2O_3 showed synergistic effect on inhibition proliferation of cells and induction apoptosis and they had a synergistic effect.The mechanisms of synergistic effect were closely related to the blockage of cell-cycle progress and the promotion of apoptosis as well as affecting signaling pathway.Sorafenib and As_2O_3 affected different targets or the same target in deeper degree resulting in enhancing the anti-tumor effect.The inhibition mechanisms were also attributed to inhibition of tumor angiogenesis in vivo.
引文
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