索拉非尼逆转肝癌细胞多药耐药的实验研究
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摘要
一、研究背景
原发性肝癌(HCC)是世界常见的恶性肿瘤之一,全世界半数左右的肝癌患者集中在中国,我国每年约有11万人死于肝癌,居恶性肿瘤病死率的第二位。目前临床肝癌诊疗中还存在着早期诊断难、复发转移率高、治疗缺乏针对性、源头创新药物和治疗手段少等问题。在肝癌的综合治疗中,化疗是其中重要方法之一,而肿瘤多药耐药(MDR)是严重影响化疗效果及患者生存的主要原因之一。肿瘤多药耐药在临床肿瘤治疗中已普遍存在,成为目前肿瘤化疗的主要障碍之一。
MDR是指肿瘤细胞对一种抗肿瘤药物出现耐药的同时,对其他结构及作用机制不同的抗肿瘤药物也产生交叉耐药的现象。MDR的产生机制目前并未阐明,目前认为肿瘤化疗MDR的主要分子生物学机制为:①跨膜转运蛋白将化疗药物从肿瘤细胞中泵出,从而减少抗肿瘤药物在细胞中聚集,主要包括MDR1编码的P-糖蛋白(P-gp)、MDR相关蛋白(MRP)以及肺耐药相关蛋白(LRP);②细胞内与氧化还原和解毒有关的酶系统发生改变,如还原型谷胱苷肽(GSH)和谷胱苷肽巯基转移酶(GST);③药物靶分子的改变,如蛋白激酶C(PKC)和DNA拓扑异构酶Ⅱ(TOPOⅡ);④控制凋亡的基因和蛋白的改变,如Ras、Bcl-2、P53、c-fos和c-jun等。
随着人们对肿瘤生物学行为的日益深入,肿瘤分子靶向治疗是继传统的三大治疗方法——手术、化疗和放疗后又一有力的肿瘤治疗手段而得到广泛的关注。肿瘤分子靶向治疗是针对可能导致细胞癌变的环节,如细胞信号传导通路、原癌基因和抑癌基因、细胞因子及受体、抗肿瘤血管形成、自杀基因等,从分子水平来逆转这种恶性生物学行为,从而抑制肿瘤细胞生长,甚至使其完全消退的一种全新的生物治疗模式。由于该治疗手段是特异性针对于在肿瘤的发生发展过程中起关键作用的靶分子及其调控的信号转导通路,因此,不但具有了抗肿瘤治疗的特异性和选择性,而且避免了常规化疗药物的无选择性所致的毒副作用和耐药性。
分子靶向药物是肿瘤治疗研究的一个新领域,由于这类药物作用的高选择性以及较小的副作用,成为近年来抗癌药物研究的热点和发展趋势,也是提高肝癌治疗效果的研究方向之一。索拉非尼(sorafenib,BAY 43—9006)是一种新型口服多靶点的抗肿瘤药,它是一种双芳基尿素类口服多激酶抑制剂,具有双重抗肿瘤作用,一方面通过抑制RAF/MEK/ERK信号传导通路,直接抑制肿瘤生长,另一方面通过抑制几种与新生血管生成和肿瘤发展有关的酪氨酸激酶受体的活性,包括血管内皮生长因子-2(VEGFR-2)、血管内皮生长因子-3(VEGFR-3)、血小板源生长因子-β(PDGFR-β)和c-kit原癌基因,阻断肿瘤新生血管生成,间接抑制肿瘤细胞的生长,从而起到抗肿瘤作用。
细胞内和细胞间的信号转导是生物体各种生物学行为的基础,信号通路的异常与肿瘤的关系十分密切。肝癌发生MDR过程中所涉及的各种细胞生理活动,需要依赖于细胞内复杂的信号转导系统与各种激酶的激活。丝裂原活化蛋白激酶(MAPK)信号转导通路是介导细胞外刺激到细胞内反应的重要信号转导系统,调节着细胞的增殖、分化、凋亡和细胞间相互作用。MAPK是哺乳动物细胞内广泛存在的一类丝氨酸/苏氨酸蛋白激酶,目前,发现了4条平行的MAPK信号转导通路,分别为细胞外信号调节激酶(ERK)通路、c-Jun氨基末端激酶(JNK)通路、p38通路及ERK5/BMK1通路。资料显示,MAPK通路在肿瘤化疗耐药中发挥着重要的作用,抗肿瘤药物能够引起MAPK信号转导系统的激活及MDR的产生。目前大多数研究都认为,ERK的过度激活与许多肿瘤的化疗耐药性存在明显正相关,其作用机制可能是通过调控耐药相关基因和蛋白的表达。通过调节肿瘤细胞MARK激酶系统的表达有可能逆转MDR,成为逆转MDR的新方法。
肿瘤靶向治疗的进展随着分子生物学技术的发展和对发病机制从细胞、分子水平的进一步认识而进入一个全新的时代。国内尚未见有关分子靶向药物对肿瘤耐药相关蛋白的影响的研究,而国外研究也只多见于体外研究,有报道,单克隆抗体-西妥昔单抗能逆转大肠癌对化疗药物的耐药性,但是多靶向药物对耐药蛋白的影响尚未见报道。因此,对索拉非尼逆转肝癌耐药的分子机制进行深入研究,可以有计划地联合应用化疗药物和索拉非尼进行治疗,以取得最好的治疗效果,达到最大限度地改善病人生存质量的目的,为肝癌的综合治疗、提高疗效提供理论基础。
二、研究目的
多靶点药物索拉非尼分别在生长因子与其膜受体结合和细胞内信号转导两个水平上阻断肿瘤血管生成和肿瘤细胞的增殖,能有效的抑制肝癌细胞的生长和血管形成,本研究旨在确定索拉非尼对肝癌细胞多药耐药的逆转作用,寻找出索拉非尼对多药耐药蛋白的影响,并从分子水平和细胞信号转导两个方面探讨索拉非尼对化疗药物多药耐药的可能逆转机制。
三、研究方法
1.选择肝癌细胞系BEL-7402细胞、肝癌耐药细胞系BEL-7402/5-FU细胞做为实验细胞,测量两种细胞的生长和死亡率曲线、细胞倍增时间、细胞核分裂指数,并以流式细胞仪检测细胞表面P-gp蛋白的表达率。
2.以MTT比色法测定(1、2、4、8、16)/μmol/L索拉非尼对BEL-7402/5-FU细胞的剂量效应曲线,并以流式细胞仪检测上述剂量的索拉非尼对BEL-7402/5-FU细胞内罗丹明123(Rho123)浓度的影响,并根据上述实验结果选取合适的索拉非尼实验剂量。
3.实验分为三组:BEL-7402细胞组、BEL-7402/5-FU细胞组、BEL-7402/5-FU细胞+sorafenib(4μmol/L)组。
3.1以阿霉素(ADM)、健择(GEM)、顺铂(DDP)、氟尿嘧啶(5-FU)四种化疗药物作用上述三组细胞,通过MTT比色法检测细胞存活率的变化,评价上述四种化疗药物对三组细胞的生长抑制作用,并得出细胞的半数抑制率(IC_(50)),以判断化疗药物敏感性,并计算出索拉非尼对肝癌耐药细胞的逆转倍数;
3.2流式细胞仪测定三组细胞表面P-gp蛋白的表达率、Rho123的蓄积作用及细胞的周期分布;
3.3免疫细胞化学法检测三组细胞表面P-gp、MRP蛋白的表达;
3.4半定量PCR及实时荧光定量PCR法检测三组细胞MDR1、MRP基因的mRNA水平的变化;
3.5 Western-blot法检测三组细胞P-gp、MRP、ERK、pERK、pJNK蛋白水平并分析相关性。
4.统计方法
实验数据均采用SPSS 13.0软件进行统计学分析:两样本比较采用独立样本t检验分析。多组样本比较采用One-way ANOVA比较各组细胞的差异,用LSD法进行多重比较;如果方差不齐,采用近似F检验(Welch方法)及多重比较的Tamhane法分析。结果以均数±标准差((?)±SD)表示,P＜0.05认为有统计学意义。
四、结果
1.BEL-7402细胞及BEL-7402/5-FU细胞形态均呈上皮样单层排列,每3d～4 d传代一次,对数生长期两种细胞的存活率均＞95%,BEL-7402/5-FU细胞的多核巨细胞较BEL-7402细胞明显增多(P=0.000)。BEL-7402细胞倍增时间为23 h,BEL-7402/5-FU细胞倍增时间为32 h,明显高于BEL-7402细胞(P=0.007)。
2.BEL-7402细胞表面P-gp蛋白的表达率为11.64%±1.03%,BEL-7402/5-FU细胞为68.94%±1.45%,显著高于亲本细胞(P=0.000)。传代后BEL-7402细胞分裂指数逐渐上升,到第4 d达到高峰,占69.33‰±0.96‰,培养5 d后,分裂指数开始下降,到第8 d降为最低。传代后BEL-7402/5-FU细胞分裂指数也呈上升趋势,到第5 d达到高峰,占69.83‰±1.82‰,培养6 d后,分裂指数开始下降,到第8 d降为最低。
3.索拉非尼在4μmol/L以下对BEL-7402/5-FU细胞无明显毒性,超过此浓度,其毒性呈剂量效应,IC_(50)为(30.69±0.93)μmol/L。当索拉非尼在(1～8)μmol/L浓度时,细胞内Rho123荧光强度随索拉非尼的浓度的增加而升高;当索拉非尼浓度超过8μmol/L时,细胞内Rho123荧光强度不再增加,显示索拉非尼浓度在4μmol/L时逆转效率较高且毒副作用较小。
4.对ADM、GEM、DDP、5-FU这四种化疗药物,BEL-7402/5-FU细胞均显示耐药性,其IC_(50)分别为:(16.74±1.12)μmol/L、(253.56±2.41)μmol/L、(11.48±5.45)μmol/L、(893.02±2.55)μmol/L,而BEL-7402细胞相对较敏感,其IC_(50)明显下降(P=0.000),分别为:(3.56±0.03)μmol/L、(17.82±0.13)μmol/L、(3.35±0.05)μmol/L、(38.31±0.09)μmol/L,耐药细胞较亲本细胞对四种化疗药物的耐药倍数分别为:4.70、14.23、3.30、23.36,经4μmol/L的索拉非尼作用后,耐药细胞BEL-7402/5-FU对各抗癌药的敏感性增强,其IC_(50)明显下降(P≤0.001),分别为:(5.57±0.57)μmol/L、(35.63±1.30)μmol/L、(5.45±0.88)μmol/L、(88.31±1.60)μmol/L,其逆转倍数分别为:2.98、7.16、1.99、10.08。
5.BEL-7402/5-FU细胞经索拉非尼4μmol/L作用24 h后,P-gp表达率为36.34%±1.12%,BEL-7402/5-FU细胞的P-gp表达率为67.44%±1.69%,BEL-7402细胞的表达率为30.66%±1.17%,肝癌耐药细胞BEL-7402/5-FU经索拉非尼作用后,P-gp表达率较用药前明显下降(P=0.002)。
6.在Rho123蓄积实验中,BEL-7402细胞内Rho123荧光很强,表达率为76.01%±3.47%,而BEL-7402/5-FU细胞内Rho123荧光很弱,表达率仅为19.83%±1.44%,加索拉非尼4μmol/L后,波峰右移,细胞内荧光明显增强,表达率为49.80%±1.35%,提示索拉非尼可明显增加耐药细胞对Rho123的蓄积(P=0.000)。
7.流式细胞仪检测细胞周期显示,索拉非尼可使细胞周期阻滞于G_0/G_1期,经4μmol/L索拉非尼作用24 h后,BEL-7402细胞、BEL-7402/5-FU细胞、BEL-7402/5-FU+sorafenib组G_0/G_1期细胞比率分别为:76.48%±1.23%、38.29%±1.40%、56.45%±2.08%,S期细胞比率分别为:15.69%±1.13%、48.15%±1.37%、34.70%±2.79%,加药后BEL-7402/5-FU细胞的G_0/G_1期细胞比率明显高于用药前(P=0.000),S期细胞比率明显低于用药前(P=0.000)。
8.光学显微镜下观察,P-gp、MRP蛋白阳性反应呈棕黄色,主要位于胞质中,呈细颗粒状均匀分布,耐药细胞BEL-7402/5-FU组表达增加,颜色加深,广泛分布于胞核及胞质中;经4μmol/L索拉非尼作用后的耐药细胞P-gp、MRP蛋白表达明显下降,颜色变浅,胞质内棕黄色颗粒明显减少。
9.半定量PCR结果显示:索拉非尼4μmol/L作用BEL-7402/5-FU细胞24 h后,BEL-7402、BEL-7402/5-FU、BEL-7402/5-FU+sorafenib组MDR1/β-actin比值分别为:0.484±0.065、0.776±0.069、0.503±0.082,BEL-7402/5-FU细胞经索拉非尼作用后,MDR1基因表达较用药前下降了27.3%(P=0.004)。MRP/β-actin比值分别为:0.350±0.026、0.698±0.123、0.493±0.024,BEL-7402/5-FU细胞经索拉非尼作用后,MRP基因表达较用药前下降了20.5%(P=0.000)。
10.实时荧光定量PCR结果显示:BEL-7402组MDR1基因的CT值为23.65±0.21,β-actin的CT值为20.87±0.16;BEL-7402/5-FU组MDR1基因的CT值为23.85±0.23,β—actin的CT值为19.02±0.13;BEL-7402/5-FU+sorafenib组MDR1基因的CT值为23.33±0.22,β-actin的CT值为19.43±0.15。根据公式计算2~(-△△CT)为2~(-0.63),故BEL-7402/5-FU+sorafenib组MDR1基因的含量约为BEL-7402/5-FU组的64.62%,逆转率约为35.38%。BEL-7402组MRP基因的CT值为26.83±0.21,β-actin的CT值为20.87±0.16;BEL-7402/5-FU组MRP基因的CT值为26.87±0.23,β-actin的CT值为19.32±0.13;BEL-7402/5-FU+sorafenib组MRP基因的CT值为26.51±0.22,β-actin的CT值为19.43±0.15。根据公式计算2~(-△△CT)为2~(-0.47),故BEL-7402/5-FU+sorafenib组MRP基因的含量约为BEL-7402/5-FU组的72.20%,逆转率约为27.80%。
11.Western-blot结果显示,经4μmol/L索拉非尼作用后,BEL-7402/5-FU细胞P-gp蛋白(0.257±0.014)表达较用药前(0.400±0.014)明显下降(P=0.000);MRP蛋白(0.253±0.024)表达较用药前(0.396±0.007)明显下降(P=0.000);ERK蛋白(0.913±0.006)表达较用药前(0.917±0.018)无明显变化(P=0.683);pERK蛋白(0.663±0.009)表达较用药前(0.934±0.006)明显下降(P=0.000);pJNK蛋白(0.536±0.007)表达较用药前(0.305±0.009)明显升高(P=0.000)。
五、结论
1.索拉非尼在体外可明显提高肝癌耐药细胞对化疗药物的敏感性,并可以使细胞周期阻滞于G_0/G_1期,同时减少S期细胞比率,使肿瘤细胞增殖受阻。
2.通过免疫细胞化学、半定量及实时荧光定量PCR、蛋白印迹结果显示,索拉非尼在体外逆转肝癌多药耐药的机制可能是下调MDR1、MRP基因的表达,从而抑制了跨膜转运蛋白的功能,使进入细胞内的化疗药物泵出减少,增加了细胞内化疗药物的浓度,使其对耐药细胞的杀伤作用增强。
3.索拉非尼还可以使ERK信号转导通路受抑制,JNK信号转导通路激活,从而使肿瘤细胞增殖受阻,说明索拉非尼逆转肝癌多药耐药的机制还可能与影响MAPK信号转导通路的有关分子相关。
4.通过本实验证明索拉非尼在体外表现出较强的逆转肝癌细胞多药耐药性的作用,为进一步深入进行动物体内实验逆转MDR和临床MDR逆转提供了重要的参考价值。
Background
Primary liver cancer is one of the most common malignant tumors in the world, and about half of the patients with liver cancer focused in China,where about 110,000 people die from liver cancer each year,ranking the second in malignant tumor mortality.Presently,there still existed some problems in clinical diagnosis and treatment of liver cancer,such as a difficult early diagnosis to make,high rate of recurrence and metastasis,and lack of relevance for treatment,original innovative drugs and various treatment means.Chemotherapy is one of the important ways in the comprehensive treatment for liver cancer,while the tumor multidrug resistance (MDR) is one of the main reasons for significant effects on chemotherapy and patients'survival.The tumor MDR has been widespread in clinical tumor therapy, becoming one of the main obstacles in chemotherapy.
MDR refers to the drug resistance of tumor cells to a kind of antitumor drug, simultaneously,cross resistance also occurs to other antitumor drugs with different structure and mechanism.At present,the production mechanism of MDR remains unclear,the main MDR mechanisms of molecular biology in tumor chemotherapy are considered as:①transmembrane proteins,reduce the antitumor drug accumulation in cells by pumping chemotherapy drugs from tumor cells,mainly including MDR1 encoded P-glycoprotein(P-gp),MDR-related protein(MRP) and lung resistance related protein(LRP);②enzyme systems which relate to oxidation reduction and detoxification in cells change,such as reduced glutathione(GSH) and glutathione S-transferase(GST);③target molecule of drug change,like protein kinase C(PKC) and DNA topoisomeraseⅡ(TOPOⅡ);④genes and proteins which control apoptosis change,such as Ras,Bcl-2,P53,c-fos and c-jun.
With people go increased deep into the tumor biological behavior,molecular target therapy--another powerful mean for tumor treatment after the three major traditional methods(surgery,chemotherapy and radiotherapy)--has attracted wide attention.Molecular target therapy for tumor aim at the links which may lead to cell carcinogenesis,such as cell signaling pathway,proto-oncogene,tumor suppressor gene,cytokine,receptor,anti-tumor angiogenesis and suicide gene.It is a new biotherapy mode for inhibiting,even dissipating the tumor cell by reversing the malignant biological behavior from molecular level.Because the treatment is specific for the target molecule and signal transduction pathway which play a key role in the development of tumor,it not only possess specificity and selectivity of anti-tumor-treatment, but also avoid side effects and drug resistance caused by the no-choice conventional chemotherapy.
Molecular targeted drug is a new research field on tumor therapy.For the high selectivity and small side-effect of this kind of drugs,it has become a hot research and a development trend on anticancer drugs in recent years,as well as a research direction to enhance the curative effect of liver cancer.Sorafenib(BAY 43-9006) is a new type of oral multi-target antitumor drug.It is a type of oral multikinase inhibitor of dual-aryl ure with dual anti-tumor effect.One hand,it inhibits tumor growth directly by inhibiting the RAF/MEK/ERK signal transduction pathway;the other hand,by inhibiting the activity of several tyrosine kinase receptors related to tumor angiogenesis and development,including vascular endothelial growth factor -2 (VEGFR-2),vascular endothelial growth factor -3(VEGFR-3),platelet-derived growth factor-β(PDGFR-β) and c-kit proto-oncogene,it blocks tumor angiogenesis, indirectly inhibiting tumor cell growth,thus playing the effect on anti-tumor.
Intracellular and intercellular signal transduction is the basis of various biological behaviors of organisms,and the abnormal signal pathway is closely related to tumor.The physiological activities of various cells involved in the development of MDR depend on the intracellular complex signal transduction system and the activation of various kinase.Mitogen-activated protein kinase(MAPK) signal transduction pathway is the important signal transduction system to mediate extracellular stimulation to intracellular response,regulating the cell proliferation, differentiation,apoptosis and interaction.MAPK is a class of serine / threonine protein kinase widely distributed in mammalian cells.At present,4 parallel MAPK signal transduction pathway have been found,namely,extracellular signal-regulated kinase(ERK) pathway,c-Jun N-terminal kinase(JNK) pathway,p38 pathway and ERK5/BMK1 pathway.It is showed that MAPK pathway played an important role in chemotherapy MDR,antitulnor drug could induce the activation of MAPK signal transduction system and the emergence of MDR.It is considered in most current studies that the excessive activation of ERK and chemotherapy MDR of many tumors showed significant positive difference,its possible mechanism might be the regulation of drug resistance gene and protein expression.It may become a new method to reverse MDR by regulating the expression of MARK kinase system in tumor cells.
With the development of molecular biological technique and the further understanding of the pathogenesis on cells and molecular level,the targeted tumor therapy goes into a new era.The relevant research of the effect of molecular targeted drug on tumor resistance-associated protein has not been found in China,and only most in vitro studies in other countries.It has been reported that monoclonal antibody - cetuximab could reverse colorectal cancer resistance to chemotherapeutic drugs, properties,but no impact of multi-targeted drugs on drug-resistant protein has been reported.Therefore,a further study of the molecular mechanism of Sorafenib reverse liver cancer resistance can help to the treatment by the designed combination of chemotherapy drugs and Sorafenib,obtaining the best curative effect,maximizing the improvement for patient survival quality,and providing a theoretical foundation for the comprehensive treatment and efficacy enhancement for liver cancer.
Objective
The multi-target drug Sorafenib blocks tumor angiogenesis and tumor cell proliferation on both the level of growth factor combining with its membrane receptor and intracellular signal transduction.It can effectively inhibit hepotoma cell growth and angiogenesis.This study was to determine the reversal effect of Sorafenib on liver cancer cell MDR,look for the effect of Sorafenib on MDR protein,and explore the possible reversal mechanism of Sorafenib to chemotherapy drug MDR from molecular level and cellular signal transduction.
Methods
1.BEL-7402 cell of hepatoma cell line and BEL-7402/5-FU cell of hepatoma cell line resistant were selected as experimental cell.Their growth curve,cell doubling time and cell mitotic index were measured,and expression rate of P-gp protein in cell surface was checked by flow cytometry.
2.By MTT colorimetric assay,the dose-response curve of(1,2,4,8,16)μmol/L Sorafenib on BEL-7402/5-FU cell was drawn,and with the flow cytometry to detect the effect of Sorafenib with above-mentioned dose on Rho123 concentration in BEL-7402/5-FU cell,then,the appropriate dose of Sorafenib was selected according to the above experimental results.
3.It was divided into three groups:BEL-7402 cell,BEL-7402/5-FU cell,and BEL-7402/5-FU cell + sorafenib(4μmol/L) group.
3.1 The effect of the four kind of chemotherapy drugs,including adriamycin (ADM),gemcitabine(GEM),cisplatin(DDP),fluorouracil(5-FU),was taken on the above three groups of cells.Changes in cell survival rate were detected by MTT colorimetric assay,and the growth inhibition effect of these drugs on the three groups of cells was evaluated,coming to the half cell inhibition rate(IC50),thus,the sensitivity of chemotherapeutic drugs was determined and the reversal multiple of Sorafenib to hepatoma drug resistance cells.
3.2 P-gp protein expression rate in cell surface,Rho123 accumulation and cell cycle distribution of the cells in three groups were determined by flow cytometry;
3.3 Immunocytochemistry method was applied to detect P-gp and MRP protein expression;
3.4 Semi-quantitative PCR and fluorescent real-time quantitative PCR were adopted to detect the mRNA level of MDR1 and MRP gene;
3.5 With the western-blot method,protein level of P-gp,MRP,ERK,pERK and pJNK was determined and the relevance was analyzed.
4.Statistical Methods
All data were analyzed with software SPSS 13.0,the independent sample t was adopted to check the two-sample compare.Multi-group compare was analyzed with One-way ANOVA to detect the differences,LSD analysis to detect homogeneity of variance;while Pseudo F test(Welch) and Tamhane analysis used if uneven variance. The results were expressed as mean±standard deviation((?)±SD),P＜0.05 was treated as statistical significance.
Results
1.BEL-7402 cell and BEL-7402/5-FU cell showed epithelioid single-layer arrange,with passage once every 3 to 4 days,the survival rates of the two kind of cells in logarithmic growth phase were＞95%.The multinucleated giant cells in BEL-7402/5-FU cell increased significantly compared with which in BEL-7402 cell (P=0.000).The doubling time of BEL-7402 cell was 23 h,and BEL-7402/5-FU cell 32 h,significantly higher than that of BEL-7402 cell(P=0.007).
2.The expression rate of P-gp protein in BEL-7402 cell surface was 11.64%±1.03%,while which in BEL-7402/5-FU cell was 68.94%±1.45%,significantly higher than which of the parental cell(P=0.000).The mitotic index of BEL-7402 cell gradually increased after passage,reached the peak in the 4th day,accounting for 69.33‰±0.96‰.After 5 d cultivation,the mitotic index began to decline,was the lowest in the 8th day.The mitotic index of BEL-7402/5-FU cell was also on the rise after passage,reached the peak in the 5th day,accounting for 69.83‰±1.82‰.After 6 d cultivation,the mitotic index began to decline,and was the lowest in the 8th day.
3.Sorafenib showed no obvious toxicity to BEL-7402/5-FU cell under the dose of 4μmol/L,and its toxicity showed dose effect once over this concentration,IC_(50) was(30.69±0.93)μmol/L.At(1～8)μmol/L concentration,intracellular Rho123 fluorescence intensity increased with the Sorafenib concentration;While over the concentration of 8μmol/L,the intracellular Rho 123 fluorescence intensity would no longer increase,indicating higher reverse efficiency and less side-effects of Sorafenib at the concentration of 4μmol/L.
4.To all these four chemotherapy drugs - ADM,GEM,DDP and 5-FU, BEL-7402/5-FU cell showed resistance,and their IC_(50) were(16.74±1.12)μmol/L, (253.56±2.41)μmol/L,(11.48±5.45)μmol/L and(893.02±2.55)μmol/L, respectively.While BEL-7402 cell are relatively sensitive,the IC_(50) were respectively (3.56±0.03)μmol/L,(17.82±0.13)μmol/L,(3.35±0.05)μmol/L and(38.31±0.09)μmol/L.Compared with parental cells,the resistance multiple of drug-resistant cells to the four chemotherapy drugs were 4.70,14.23,3.30 and 23.36,respectively.Under the action of 4μmol/L Sorafenib,the sensitivity of the drug-resistant cell BEL-7402/5-FU enhanced to all the anticancer drugs,the IC50 decreased significantly(P(?)0.001),were(5.57±0.57)μmol/L,(35.63±1.30)μmol/L,(5.45±0.88)μmol/L and(88.31±1.60)μmol/L,respectively.The reversal multiple were 2.98,7.16,1.99 and 10.08.
5.After the 24 h effect of 4μmol/L Sorafenib on BEL-7402/5-FU cell,P-gp expression rate was 36.34%±1.12%,P-gp expression rate of BEL-7402/5-FU cell was 67.44%±1.69%,and the expression rate of BEL-7402 cell was 30.66%±1.17%. P-gp expression rate of hepatoma cell drug-resistant cell BEL-7402/5-FU significantly decreased after Sorafenib effect(P=0.002).
6.In Rho123 accumulation experiment,the Rho123 fluorescence was very strong in BEL-7402 cell,and the expression rate was 76.01%±3.47%;while which in BEL-7402/5-FU cell was weak,the expression rate was only 19.83%±1.44%. After 4μmol/L Sorafenib was added,the peak shifted to right,the intracellular fluorescence markedly increased,and the expression rate was 49.80%±1.35%.It indicated that Sorafenib could markedly increase the accumulation of drug-resistant cells to Rho123(P=0.000).
7.The cell cycle detected with flow cytometry showed that Sorafenib could block cell cycle in G0/G1 phase.After 24 h effect of 4μmol/L Sorafenib,the cell ratio of BEL-7402 cell,BEL-7402/5-FU cell and BEL-7402/5-FU + sorafenib group in G0/G1 phase,were respectively:76.48%±1.23%,38.29%±1.40%and 56.45%±2.08%.While which in S phase were 15.69%±1.13%,48.15%±1.37%and 34.70%±2.79%.The cell ratio of BEL-7402/5-FU cell in G0/G1 phase after adding with Sorafenib was significantly higher than that before treatment(P=0.000),the cell ratio of S phase was significantly lower than that before treatment(P=0.000).
8.With optical microscopy,the positive response of P-gp and MRP protein showed brown-yellow,mainly located in cytoplasm,showing uniform fine granular distribution.The expression of drug-resistant cell BEL-7402/5-FU group increased, the color deepened,widely distributed in the nucleus and cytoplasm;After the application of 4μmol/L Sorafenib,P-gp and MRP protein expression of drug-resistant cells decreased significantly,the color became shallow,the brown-yellow granules in cytoplasm reduced significantly.
9.Semi-quantitative PCR results showed that:after 24 h effect of 4μmol/L Sorafenib on BEL-7402/5-FU cell,the MDR1/β-actin ratio of BEL-7402, BEL-7402/5-FU and BEL-7402/5-FU + sorafenib group were respectively 0.484±0.065,0.776±0.069 and 0.503±0.082.After Sorafenib treatment,MDR1 gene expression of BEL-7402/5-FU cell decreased 27.3%compared with that before the treatment(P=0.004).The MRP/β-actin ratio were:0.350±0.026,0.698±0.123 and 0.493±0.024,respectively.After the effect of Sorafenib on BEL-7402/5-FU cell, MRP gene expression decreased 20.5%compare with that before the treatment(P= 0.000).
10.Real-time quantitative PCR results showed that:the CT value of MDR1 gene in BEL-7402 group was 23.65±0.21,and the CT value ofβ-actin was 20.87±0.16; The CT value of MDR1 gene in BEL-7402/5-FU group was 23.85±0.23,and the CT value ofβ-actin was 19.32±0.13;The CT value of MDR1 gene in BEL-7402/5-FU + sorafenib group was 23.33±0.22,and the CT value ofβ-actin was 19.43±0.15.2~(-△△CT) was calculated as 2~(-0.63) according to formula,so the content of MDR1 gene in BEL-7402/5-FU + sorafenib group was about 64.62%of which in BEL-7402/5-FU group,the reversal rate was about 35.38%.The CT value of MRP gene in BEL-7402 group was 26.83±0.21,and the CT value ofβ-actin was 20.87±0.16;The CT value of MRP gene in BEL-7402/5-FU group was 26.87±0.23,and the CT value ofβ-actin was 19.32±0.13;The CT value of MRP gene in BEL-7402/5-FU + sorafenib group was 26.51±0.22,and the CT value ofβ-actin was 19.43±0.15.2~(-△△CT) was calculated as 2~(-0.47) according to formula,so the content of MRP gene in BEL-7402/5-FU + sorafenib group was about 72.20%of which in BEL-7402/5-FU group,the reversal rate was about 27.80%.
11.Western-blot results showed that after the application of 4μmol/L Sorafenib, P-gp protein(0.257±0.014) expression in BEL-7402/5-FU cell was decreased significantly compared with that before the treatment(0.400±0.014)(P=0.000); MRP protein(0.253±0.024) expression decreased significantly compared with that of before treatment(0.396±0.007)(P=0.000);ERK protein expression (0.913±0.006) showed no significant change compared with that before treatment (0.917±0.018)(P=0.683);pERK protein(0.663±0.009) expression decreased significantly compared with that before treatment(0.934±0.006)(P=0.000); pJNK protein(0.536±0.007) expression significantly increased compared with that before treatment(0.305±0.009)(P=0.000).
Conclusion
1.Sorafenib could markedly enhance the sensitivity of drug-resistant hepatocellular carcinoma cells to chemotherapeutic drug in vitro,and block cell cycle in G0/G1 phase,reduce the cell ratio of S phase,thus blocking the tumor cell proliferation.
2.The results of immunocytochemistry,semi-quantitative and real-time fluorescence quantitative PCR and western blot showed that the possible mechanisms of Sorafenib reverse hepatoma MDR in vitro were the down-regulated MDR1 and MRP gene expression inhibited the function of the transmembrane transporter protein, decreased the chemotherapy drugs pump out of the cells,increased concentration of chemotherapeutic drugs in cells,thus enhancing the killing effect to drug-resistant cells.
3.Sorafenib could also inhibit the ERK signal transduction pathway;activate the JNK signal transduction pathway,thus blocking the tumor cell proliferation.It indicated that the mechanism of Sorafenib reverse hepatoma MDR might also relate to the molecule which impacted the MAPK signal transduction pathway.
4.It was proved in this experiment that Sorafenib showed strong effect of reversing hepatoma cell MDR in vitro,reversal of multidrug resistance of hepatocellular carcinoma cell function,providing an important clue for the further experimental and clinical MDR reversing in vivo of animal.
原发性肝癌(HCC)是世界常见的恶性肿瘤之一,全世界半数左右的肝癌患者集中在中国,我国每年约有11万人死于肝癌,居恶性肿瘤病死率的第二位。目前临床肝癌诊疗中还存在着早期诊断难、复发转移率高、治疗缺乏针对性、源头创新药物和治疗手段少等问题。在肝癌的综合治疗中,化疗是其中重要方法之一,而肿瘤多药耐药(MDR)是严重影响化疗效果及患者生存的主要原因之一。肿瘤多药耐药在临床肿瘤治疗中已普遍存在,成为目前肿瘤化疗的主要障碍之一。
MDR是指肿瘤细胞对一种抗肿瘤药物出现耐药的同时,对其他结构及作用机制不同的抗肿瘤药物也产生交叉耐药的现象。MDR的产生机制目前并未阐明,目前认为肿瘤化疗MDR的主要分子生物学机制为:①跨膜转运蛋白将化疗药物从肿瘤细胞中泵出,从而减少抗肿瘤药物在细胞中聚集,主要包括MDR1编码的P-糖蛋白(P-gp)、MDR相关蛋白(MRP)以及肺耐药相关蛋白(LRP);②细胞内与氧化还原和解毒有关的酶系统发生改变,如还原型谷胱苷肽(GSH)和谷胱苷肽巯基转移酶(GST);③药物靶分子的改变,如蛋白激酶C(PKC)和DNA拓扑异构酶Ⅱ(TOPOⅡ);④控制凋亡的基因和蛋白的改变,如Ras、Bcl-2、P53、c-fos和c-jun等。
随着人们对肿瘤生物学行为的日益深入,肿瘤分子靶向治疗是继传统的三大治疗方法——手术、化疗和放疗后又一有力的肿瘤治疗手段而得到广泛的关注。肿瘤分子靶向治疗是针对可能导致细胞癌变的环节,如细胞信号传导通路、原癌基因和抑癌基因、细胞因子及受体、抗肿瘤血管形成、自杀基因等,从分子水平来逆转这种恶性生物学行为,从而抑制肿瘤细胞生长,甚至使其完全消退的一种全新的生物治疗模式。由于该治疗手段是特异性针对于在肿瘤的发生发展过程中起关键作用的靶分子及其调控的信号转导通路,因此,不但具有了抗肿瘤治疗的特异性和选择性,而且避免了常规化疗药物的无选择性所致的毒副作用和耐药性。
分子靶向药物是肿瘤治疗研究的一个新领域,由于这类药物作用的高选择性以及较小的副作用,成为近年来抗癌药物研究的热点和发展趋势,也是提高肝癌治疗效果的研究方向之一。索拉非尼(sorafenib,BAY 43—9006)是一种新型口服多靶点的抗肿瘤药,它是一种双芳基尿素类口服多激酶抑制剂,具有双重抗肿瘤作用,一方面通过抑制RAF/MEK/ERK信号传导通路,直接抑制肿瘤生长,另一方面通过抑制几种与新生血管生成和肿瘤发展有关的酪氨酸激酶受体的活性,包括血管内皮生长因子-2(VEGFR-2)、血管内皮生长因子-3(VEGFR-3)、血小板源生长因子-β(PDGFR-β)和c-kit原癌基因,阻断肿瘤新生血管生成,间接抑制肿瘤细胞的生长,从而起到抗肿瘤作用。
细胞内和细胞间的信号转导是生物体各种生物学行为的基础,信号通路的异常与肿瘤的关系十分密切。肝癌发生MDR过程中所涉及的各种细胞生理活动,需要依赖于细胞内复杂的信号转导系统与各种激酶的激活。丝裂原活化蛋白激酶(MAPK)信号转导通路是介导细胞外刺激到细胞内反应的重要信号转导系统,调节着细胞的增殖、分化、凋亡和细胞间相互作用。MAPK是哺乳动物细胞内广泛存在的一类丝氨酸/苏氨酸蛋白激酶,目前,发现了4条平行的MAPK信号转导通路,分别为细胞外信号调节激酶(ERK)通路、c-Jun氨基末端激酶(JNK)通路、p38通路及ERK5/BMK1通路。资料显示,MAPK通路在肿瘤化疗耐药中发挥着重要的作用,抗肿瘤药物能够引起MAPK信号转导系统的激活及MDR的产生。目前大多数研究都认为,ERK的过度激活与许多肿瘤的化疗耐药性存在明显正相关,其作用机制可能是通过调控耐药相关基因和蛋白的表达。通过调节肿瘤细胞MARK激酶系统的表达有可能逆转MDR,成为逆转MDR的新方法。
肿瘤靶向治疗的进展随着分子生物学技术的发展和对发病机制从细胞、分子水平的进一步认识而进入一个全新的时代。国内尚未见有关分子靶向药物对肿瘤耐药相关蛋白的影响的研究,而国外研究也只多见于体外研究,有报道,单克隆抗体-西妥昔单抗能逆转大肠癌对化疗药物的耐药性,但是多靶向药物对耐药蛋白的影响尚未见报道。因此,对索拉非尼逆转肝癌耐药的分子机制进行深入研究,可以有计划地联合应用化疗药物和索拉非尼进行治疗,以取得最好的治疗效果,达到最大限度地改善病人生存质量的目的,为肝癌的综合治疗、提高疗效提供理论基础。
二、研究目的
多靶点药物索拉非尼分别在生长因子与其膜受体结合和细胞内信号转导两个水平上阻断肿瘤血管生成和肿瘤细胞的增殖,能有效的抑制肝癌细胞的生长和血管形成,本研究旨在确定索拉非尼对肝癌细胞多药耐药的逆转作用,寻找出索拉非尼对多药耐药蛋白的影响,并从分子水平和细胞信号转导两个方面探讨索拉非尼对化疗药物多药耐药的可能逆转机制。
三、研究方法
1.选择肝癌细胞系BEL-7402细胞、肝癌耐药细胞系BEL-7402/5-FU细胞做为实验细胞,测量两种细胞的生长和死亡率曲线、细胞倍增时间、细胞核分裂指数,并以流式细胞仪检测细胞表面P-gp蛋白的表达率。
2.以MTT比色法测定(1、2、4、8、16)/μmol/L索拉非尼对BEL-7402/5-FU细胞的剂量效应曲线,并以流式细胞仪检测上述剂量的索拉非尼对BEL-7402/5-FU细胞内罗丹明123(Rho123)浓度的影响,并根据上述实验结果选取合适的索拉非尼实验剂量。
3.实验分为三组:BEL-7402细胞组、BEL-7402/5-FU细胞组、BEL-7402/5-FU细胞+sorafenib(4μmol/L)组。
3.1以阿霉素(ADM)、健择(GEM)、顺铂(DDP)、氟尿嘧啶(5-FU)四种化疗药物作用上述三组细胞,通过MTT比色法检测细胞存活率的变化,评价上述四种化疗药物对三组细胞的生长抑制作用,并得出细胞的半数抑制率(IC_(50)),以判断化疗药物敏感性,并计算出索拉非尼对肝癌耐药细胞的逆转倍数;
3.2流式细胞仪测定三组细胞表面P-gp蛋白的表达率、Rho123的蓄积作用及细胞的周期分布;
3.3免疫细胞化学法检测三组细胞表面P-gp、MRP蛋白的表达;
3.4半定量PCR及实时荧光定量PCR法检测三组细胞MDR1、MRP基因的mRNA水平的变化;
3.5 Western-blot法检测三组细胞P-gp、MRP、ERK、pERK、pJNK蛋白水平并分析相关性。
4.统计方法
实验数据均采用SPSS 13.0软件进行统计学分析:两样本比较采用独立样本t检验分析。多组样本比较采用One-way ANOVA比较各组细胞的差异,用LSD法进行多重比较;如果方差不齐,采用近似F检验(Welch方法)及多重比较的Tamhane法分析。结果以均数±标准差((?)±SD)表示,P＜0.05认为有统计学意义。
四、结果
1.BEL-7402细胞及BEL-7402/5-FU细胞形态均呈上皮样单层排列,每3d～4 d传代一次,对数生长期两种细胞的存活率均＞95%,BEL-7402/5-FU细胞的多核巨细胞较BEL-7402细胞明显增多(P=0.000)。BEL-7402细胞倍增时间为23 h,BEL-7402/5-FU细胞倍增时间为32 h,明显高于BEL-7402细胞(P=0.007)。
2.BEL-7402细胞表面P-gp蛋白的表达率为11.64%±1.03%,BEL-7402/5-FU细胞为68.94%±1.45%,显著高于亲本细胞(P=0.000)。传代后BEL-7402细胞分裂指数逐渐上升,到第4 d达到高峰,占69.33‰±0.96‰,培养5 d后,分裂指数开始下降,到第8 d降为最低。传代后BEL-7402/5-FU细胞分裂指数也呈上升趋势,到第5 d达到高峰,占69.83‰±1.82‰,培养6 d后,分裂指数开始下降,到第8 d降为最低。
3.索拉非尼在4μmol/L以下对BEL-7402/5-FU细胞无明显毒性,超过此浓度,其毒性呈剂量效应,IC_(50)为(30.69±0.93)μmol/L。当索拉非尼在(1～8)μmol/L浓度时,细胞内Rho123荧光强度随索拉非尼的浓度的增加而升高;当索拉非尼浓度超过8μmol/L时,细胞内Rho123荧光强度不再增加,显示索拉非尼浓度在4μmol/L时逆转效率较高且毒副作用较小。
4.对ADM、GEM、DDP、5-FU这四种化疗药物,BEL-7402/5-FU细胞均显示耐药性,其IC_(50)分别为:(16.74±1.12)μmol/L、(253.56±2.41)μmol/L、(11.48±5.45)μmol/L、(893.02±2.55)μmol/L,而BEL-7402细胞相对较敏感,其IC_(50)明显下降(P=0.000),分别为:(3.56±0.03)μmol/L、(17.82±0.13)μmol/L、(3.35±0.05)μmol/L、(38.31±0.09)μmol/L,耐药细胞较亲本细胞对四种化疗药物的耐药倍数分别为:4.70、14.23、3.30、23.36,经4μmol/L的索拉非尼作用后,耐药细胞BEL-7402/5-FU对各抗癌药的敏感性增强,其IC_(50)明显下降(P≤0.001),分别为:(5.57±0.57)μmol/L、(35.63±1.30)μmol/L、(5.45±0.88)μmol/L、(88.31±1.60)μmol/L,其逆转倍数分别为:2.98、7.16、1.99、10.08。
5.BEL-7402/5-FU细胞经索拉非尼4μmol/L作用24 h后,P-gp表达率为36.34%±1.12%,BEL-7402/5-FU细胞的P-gp表达率为67.44%±1.69%,BEL-7402细胞的表达率为30.66%±1.17%,肝癌耐药细胞BEL-7402/5-FU经索拉非尼作用后,P-gp表达率较用药前明显下降(P=0.002)。
6.在Rho123蓄积实验中,BEL-7402细胞内Rho123荧光很强,表达率为76.01%±3.47%,而BEL-7402/5-FU细胞内Rho123荧光很弱,表达率仅为19.83%±1.44%,加索拉非尼4μmol/L后,波峰右移,细胞内荧光明显增强,表达率为49.80%±1.35%,提示索拉非尼可明显增加耐药细胞对Rho123的蓄积(P=0.000)。
7.流式细胞仪检测细胞周期显示,索拉非尼可使细胞周期阻滞于G_0/G_1期,经4μmol/L索拉非尼作用24 h后,BEL-7402细胞、BEL-7402/5-FU细胞、BEL-7402/5-FU+sorafenib组G_0/G_1期细胞比率分别为:76.48%±1.23%、38.29%±1.40%、56.45%±2.08%,S期细胞比率分别为:15.69%±1.13%、48.15%±1.37%、34.70%±2.79%,加药后BEL-7402/5-FU细胞的G_0/G_1期细胞比率明显高于用药前(P=0.000),S期细胞比率明显低于用药前(P=0.000)。
8.光学显微镜下观察,P-gp、MRP蛋白阳性反应呈棕黄色,主要位于胞质中,呈细颗粒状均匀分布,耐药细胞BEL-7402/5-FU组表达增加,颜色加深,广泛分布于胞核及胞质中;经4μmol/L索拉非尼作用后的耐药细胞P-gp、MRP蛋白表达明显下降,颜色变浅,胞质内棕黄色颗粒明显减少。
9.半定量PCR结果显示:索拉非尼4μmol/L作用BEL-7402/5-FU细胞24 h后,BEL-7402、BEL-7402/5-FU、BEL-7402/5-FU+sorafenib组MDR1/β-actin比值分别为:0.484±0.065、0.776±0.069、0.503±0.082,BEL-7402/5-FU细胞经索拉非尼作用后,MDR1基因表达较用药前下降了27.3%(P=0.004)。MRP/β-actin比值分别为:0.350±0.026、0.698±0.123、0.493±0.024,BEL-7402/5-FU细胞经索拉非尼作用后,MRP基因表达较用药前下降了20.5%(P=0.000)。
10.实时荧光定量PCR结果显示:BEL-7402组MDR1基因的CT值为23.65±0.21,β-actin的CT值为20.87±0.16;BEL-7402/5-FU组MDR1基因的CT值为23.85±0.23,β—actin的CT值为19.02±0.13;BEL-7402/5-FU+sorafenib组MDR1基因的CT值为23.33±0.22,β-actin的CT值为19.43±0.15。根据公式计算2~(-△△CT)为2~(-0.63),故BEL-7402/5-FU+sorafenib组MDR1基因的含量约为BEL-7402/5-FU组的64.62%,逆转率约为35.38%。BEL-7402组MRP基因的CT值为26.83±0.21,β-actin的CT值为20.87±0.16;BEL-7402/5-FU组MRP基因的CT值为26.87±0.23,β-actin的CT值为19.32±0.13;BEL-7402/5-FU+sorafenib组MRP基因的CT值为26.51±0.22,β-actin的CT值为19.43±0.15。根据公式计算2~(-△△CT)为2~(-0.47),故BEL-7402/5-FU+sorafenib组MRP基因的含量约为BEL-7402/5-FU组的72.20%,逆转率约为27.80%。
11.Western-blot结果显示,经4μmol/L索拉非尼作用后,BEL-7402/5-FU细胞P-gp蛋白(0.257±0.014)表达较用药前(0.400±0.014)明显下降(P=0.000);MRP蛋白(0.253±0.024)表达较用药前(0.396±0.007)明显下降(P=0.000);ERK蛋白(0.913±0.006)表达较用药前(0.917±0.018)无明显变化(P=0.683);pERK蛋白(0.663±0.009)表达较用药前(0.934±0.006)明显下降(P=0.000);pJNK蛋白(0.536±0.007)表达较用药前(0.305±0.009)明显升高(P=0.000)。
五、结论
1.索拉非尼在体外可明显提高肝癌耐药细胞对化疗药物的敏感性,并可以使细胞周期阻滞于G_0/G_1期,同时减少S期细胞比率,使肿瘤细胞增殖受阻。
2.通过免疫细胞化学、半定量及实时荧光定量PCR、蛋白印迹结果显示,索拉非尼在体外逆转肝癌多药耐药的机制可能是下调MDR1、MRP基因的表达,从而抑制了跨膜转运蛋白的功能,使进入细胞内的化疗药物泵出减少,增加了细胞内化疗药物的浓度,使其对耐药细胞的杀伤作用增强。
3.索拉非尼还可以使ERK信号转导通路受抑制,JNK信号转导通路激活,从而使肿瘤细胞增殖受阻,说明索拉非尼逆转肝癌多药耐药的机制还可能与影响MAPK信号转导通路的有关分子相关。
4.通过本实验证明索拉非尼在体外表现出较强的逆转肝癌细胞多药耐药性的作用,为进一步深入进行动物体内实验逆转MDR和临床MDR逆转提供了重要的参考价值。
Background
Primary liver cancer is one of the most common malignant tumors in the world, and about half of the patients with liver cancer focused in China,where about 110,000 people die from liver cancer each year,ranking the second in malignant tumor mortality.Presently,there still existed some problems in clinical diagnosis and treatment of liver cancer,such as a difficult early diagnosis to make,high rate of recurrence and metastasis,and lack of relevance for treatment,original innovative drugs and various treatment means.Chemotherapy is one of the important ways in the comprehensive treatment for liver cancer,while the tumor multidrug resistance (MDR) is one of the main reasons for significant effects on chemotherapy and patients'survival.The tumor MDR has been widespread in clinical tumor therapy, becoming one of the main obstacles in chemotherapy.
MDR refers to the drug resistance of tumor cells to a kind of antitumor drug, simultaneously,cross resistance also occurs to other antitumor drugs with different structure and mechanism.At present,the production mechanism of MDR remains unclear,the main MDR mechanisms of molecular biology in tumor chemotherapy are considered as:①transmembrane proteins,reduce the antitumor drug accumulation in cells by pumping chemotherapy drugs from tumor cells,mainly including MDR1 encoded P-glycoprotein(P-gp),MDR-related protein(MRP) and lung resistance related protein(LRP);②enzyme systems which relate to oxidation reduction and detoxification in cells change,such as reduced glutathione(GSH) and glutathione S-transferase(GST);③target molecule of drug change,like protein kinase C(PKC) and DNA topoisomeraseⅡ(TOPOⅡ);④genes and proteins which control apoptosis change,such as Ras,Bcl-2,P53,c-fos and c-jun.
With people go increased deep into the tumor biological behavior,molecular target therapy--another powerful mean for tumor treatment after the three major traditional methods(surgery,chemotherapy and radiotherapy)--has attracted wide attention.Molecular target therapy for tumor aim at the links which may lead to cell carcinogenesis,such as cell signaling pathway,proto-oncogene,tumor suppressor gene,cytokine,receptor,anti-tumor angiogenesis and suicide gene.It is a new biotherapy mode for inhibiting,even dissipating the tumor cell by reversing the malignant biological behavior from molecular level.Because the treatment is specific for the target molecule and signal transduction pathway which play a key role in the development of tumor,it not only possess specificity and selectivity of anti-tumor-treatment, but also avoid side effects and drug resistance caused by the no-choice conventional chemotherapy.
Molecular targeted drug is a new research field on tumor therapy.For the high selectivity and small side-effect of this kind of drugs,it has become a hot research and a development trend on anticancer drugs in recent years,as well as a research direction to enhance the curative effect of liver cancer.Sorafenib(BAY 43-9006) is a new type of oral multi-target antitumor drug.It is a type of oral multikinase inhibitor of dual-aryl ure with dual anti-tumor effect.One hand,it inhibits tumor growth directly by inhibiting the RAF/MEK/ERK signal transduction pathway;the other hand,by inhibiting the activity of several tyrosine kinase receptors related to tumor angiogenesis and development,including vascular endothelial growth factor -2 (VEGFR-2),vascular endothelial growth factor -3(VEGFR-3),platelet-derived growth factor-β(PDGFR-β) and c-kit proto-oncogene,it blocks tumor angiogenesis, indirectly inhibiting tumor cell growth,thus playing the effect on anti-tumor.
Intracellular and intercellular signal transduction is the basis of various biological behaviors of organisms,and the abnormal signal pathway is closely related to tumor.The physiological activities of various cells involved in the development of MDR depend on the intracellular complex signal transduction system and the activation of various kinase.Mitogen-activated protein kinase(MAPK) signal transduction pathway is the important signal transduction system to mediate extracellular stimulation to intracellular response,regulating the cell proliferation, differentiation,apoptosis and interaction.MAPK is a class of serine / threonine protein kinase widely distributed in mammalian cells.At present,4 parallel MAPK signal transduction pathway have been found,namely,extracellular signal-regulated kinase(ERK) pathway,c-Jun N-terminal kinase(JNK) pathway,p38 pathway and ERK5/BMK1 pathway.It is showed that MAPK pathway played an important role in chemotherapy MDR,antitulnor drug could induce the activation of MAPK signal transduction system and the emergence of MDR.It is considered in most current studies that the excessive activation of ERK and chemotherapy MDR of many tumors showed significant positive difference,its possible mechanism might be the regulation of drug resistance gene and protein expression.It may become a new method to reverse MDR by regulating the expression of MARK kinase system in tumor cells.
With the development of molecular biological technique and the further understanding of the pathogenesis on cells and molecular level,the targeted tumor therapy goes into a new era.The relevant research of the effect of molecular targeted drug on tumor resistance-associated protein has not been found in China,and only most in vitro studies in other countries.It has been reported that monoclonal antibody - cetuximab could reverse colorectal cancer resistance to chemotherapeutic drugs, properties,but no impact of multi-targeted drugs on drug-resistant protein has been reported.Therefore,a further study of the molecular mechanism of Sorafenib reverse liver cancer resistance can help to the treatment by the designed combination of chemotherapy drugs and Sorafenib,obtaining the best curative effect,maximizing the improvement for patient survival quality,and providing a theoretical foundation for the comprehensive treatment and efficacy enhancement for liver cancer.
Objective
The multi-target drug Sorafenib blocks tumor angiogenesis and tumor cell proliferation on both the level of growth factor combining with its membrane receptor and intracellular signal transduction.It can effectively inhibit hepotoma cell growth and angiogenesis.This study was to determine the reversal effect of Sorafenib on liver cancer cell MDR,look for the effect of Sorafenib on MDR protein,and explore the possible reversal mechanism of Sorafenib to chemotherapy drug MDR from molecular level and cellular signal transduction.
Methods
1.BEL-7402 cell of hepatoma cell line and BEL-7402/5-FU cell of hepatoma cell line resistant were selected as experimental cell.Their growth curve,cell doubling time and cell mitotic index were measured,and expression rate of P-gp protein in cell surface was checked by flow cytometry.
2.By MTT colorimetric assay,the dose-response curve of(1,2,4,8,16)μmol/L Sorafenib on BEL-7402/5-FU cell was drawn,and with the flow cytometry to detect the effect of Sorafenib with above-mentioned dose on Rho123 concentration in BEL-7402/5-FU cell,then,the appropriate dose of Sorafenib was selected according to the above experimental results.
3.It was divided into three groups:BEL-7402 cell,BEL-7402/5-FU cell,and BEL-7402/5-FU cell + sorafenib(4μmol/L) group.
3.1 The effect of the four kind of chemotherapy drugs,including adriamycin (ADM),gemcitabine(GEM),cisplatin(DDP),fluorouracil(5-FU),was taken on the above three groups of cells.Changes in cell survival rate were detected by MTT colorimetric assay,and the growth inhibition effect of these drugs on the three groups of cells was evaluated,coming to the half cell inhibition rate(IC50),thus,the sensitivity of chemotherapeutic drugs was determined and the reversal multiple of Sorafenib to hepatoma drug resistance cells.
3.2 P-gp protein expression rate in cell surface,Rho123 accumulation and cell cycle distribution of the cells in three groups were determined by flow cytometry;
3.3 Immunocytochemistry method was applied to detect P-gp and MRP protein expression;
3.4 Semi-quantitative PCR and fluorescent real-time quantitative PCR were adopted to detect the mRNA level of MDR1 and MRP gene;
3.5 With the western-blot method,protein level of P-gp,MRP,ERK,pERK and pJNK was determined and the relevance was analyzed.
4.Statistical Methods
All data were analyzed with software SPSS 13.0,the independent sample t was adopted to check the two-sample compare.Multi-group compare was analyzed with One-way ANOVA to detect the differences,LSD analysis to detect homogeneity of variance;while Pseudo F test(Welch) and Tamhane analysis used if uneven variance. The results were expressed as mean±standard deviation((?)±SD),P＜0.05 was treated as statistical significance.
Results
1.BEL-7402 cell and BEL-7402/5-FU cell showed epithelioid single-layer arrange,with passage once every 3 to 4 days,the survival rates of the two kind of cells in logarithmic growth phase were＞95%.The multinucleated giant cells in BEL-7402/5-FU cell increased significantly compared with which in BEL-7402 cell (P=0.000).The doubling time of BEL-7402 cell was 23 h,and BEL-7402/5-FU cell 32 h,significantly higher than that of BEL-7402 cell(P=0.007).
2.The expression rate of P-gp protein in BEL-7402 cell surface was 11.64%±1.03%,while which in BEL-7402/5-FU cell was 68.94%±1.45%,significantly higher than which of the parental cell(P=0.000).The mitotic index of BEL-7402 cell gradually increased after passage,reached the peak in the 4th day,accounting for 69.33‰±0.96‰.After 5 d cultivation,the mitotic index began to decline,was the lowest in the 8th day.The mitotic index of BEL-7402/5-FU cell was also on the rise after passage,reached the peak in the 5th day,accounting for 69.83‰±1.82‰.After 6 d cultivation,the mitotic index began to decline,and was the lowest in the 8th day.
3.Sorafenib showed no obvious toxicity to BEL-7402/5-FU cell under the dose of 4μmol/L,and its toxicity showed dose effect once over this concentration,IC_(50) was(30.69±0.93)μmol/L.At(1～8)μmol/L concentration,intracellular Rho123 fluorescence intensity increased with the Sorafenib concentration;While over the concentration of 8μmol/L,the intracellular Rho 123 fluorescence intensity would no longer increase,indicating higher reverse efficiency and less side-effects of Sorafenib at the concentration of 4μmol/L.
4.To all these four chemotherapy drugs - ADM,GEM,DDP and 5-FU, BEL-7402/5-FU cell showed resistance,and their IC_(50) were(16.74±1.12)μmol/L, (253.56±2.41)μmol/L,(11.48±5.45)μmol/L and(893.02±2.55)μmol/L, respectively.While BEL-7402 cell are relatively sensitive,the IC_(50) were respectively (3.56±0.03)μmol/L,(17.82±0.13)μmol/L,(3.35±0.05)μmol/L and(38.31±0.09)μmol/L.Compared with parental cells,the resistance multiple of drug-resistant cells to the four chemotherapy drugs were 4.70,14.23,3.30 and 23.36,respectively.Under the action of 4μmol/L Sorafenib,the sensitivity of the drug-resistant cell BEL-7402/5-FU enhanced to all the anticancer drugs,the IC50 decreased significantly(P(?)0.001),were(5.57±0.57)μmol/L,(35.63±1.30)μmol/L,(5.45±0.88)μmol/L and(88.31±1.60)μmol/L,respectively.The reversal multiple were 2.98,7.16,1.99 and 10.08.
5.After the 24 h effect of 4μmol/L Sorafenib on BEL-7402/5-FU cell,P-gp expression rate was 36.34%±1.12%,P-gp expression rate of BEL-7402/5-FU cell was 67.44%±1.69%,and the expression rate of BEL-7402 cell was 30.66%±1.17%. P-gp expression rate of hepatoma cell drug-resistant cell BEL-7402/5-FU significantly decreased after Sorafenib effect(P=0.002).
6.In Rho123 accumulation experiment,the Rho123 fluorescence was very strong in BEL-7402 cell,and the expression rate was 76.01%±3.47%;while which in BEL-7402/5-FU cell was weak,the expression rate was only 19.83%±1.44%. After 4μmol/L Sorafenib was added,the peak shifted to right,the intracellular fluorescence markedly increased,and the expression rate was 49.80%±1.35%.It indicated that Sorafenib could markedly increase the accumulation of drug-resistant cells to Rho123(P=0.000).
7.The cell cycle detected with flow cytometry showed that Sorafenib could block cell cycle in G0/G1 phase.After 24 h effect of 4μmol/L Sorafenib,the cell ratio of BEL-7402 cell,BEL-7402/5-FU cell and BEL-7402/5-FU + sorafenib group in G0/G1 phase,were respectively:76.48%±1.23%,38.29%±1.40%and 56.45%±2.08%.While which in S phase were 15.69%±1.13%,48.15%±1.37%and 34.70%±2.79%.The cell ratio of BEL-7402/5-FU cell in G0/G1 phase after adding with Sorafenib was significantly higher than that before treatment(P=0.000),the cell ratio of S phase was significantly lower than that before treatment(P=0.000).
8.With optical microscopy,the positive response of P-gp and MRP protein showed brown-yellow,mainly located in cytoplasm,showing uniform fine granular distribution.The expression of drug-resistant cell BEL-7402/5-FU group increased, the color deepened,widely distributed in the nucleus and cytoplasm;After the application of 4μmol/L Sorafenib,P-gp and MRP protein expression of drug-resistant cells decreased significantly,the color became shallow,the brown-yellow granules in cytoplasm reduced significantly.
9.Semi-quantitative PCR results showed that:after 24 h effect of 4μmol/L Sorafenib on BEL-7402/5-FU cell,the MDR1/β-actin ratio of BEL-7402, BEL-7402/5-FU and BEL-7402/5-FU + sorafenib group were respectively 0.484±0.065,0.776±0.069 and 0.503±0.082.After Sorafenib treatment,MDR1 gene expression of BEL-7402/5-FU cell decreased 27.3%compared with that before the treatment(P=0.004).The MRP/β-actin ratio were:0.350±0.026,0.698±0.123 and 0.493±0.024,respectively.After the effect of Sorafenib on BEL-7402/5-FU cell, MRP gene expression decreased 20.5%compare with that before the treatment(P= 0.000).
10.Real-time quantitative PCR results showed that:the CT value of MDR1 gene in BEL-7402 group was 23.65±0.21,and the CT value ofβ-actin was 20.87±0.16; The CT value of MDR1 gene in BEL-7402/5-FU group was 23.85±0.23,and the CT value ofβ-actin was 19.32±0.13;The CT value of MDR1 gene in BEL-7402/5-FU + sorafenib group was 23.33±0.22,and the CT value ofβ-actin was 19.43±0.15.2~(-△△CT) was calculated as 2~(-0.63) according to formula,so the content of MDR1 gene in BEL-7402/5-FU + sorafenib group was about 64.62%of which in BEL-7402/5-FU group,the reversal rate was about 35.38%.The CT value of MRP gene in BEL-7402 group was 26.83±0.21,and the CT value ofβ-actin was 20.87±0.16;The CT value of MRP gene in BEL-7402/5-FU group was 26.87±0.23,and the CT value ofβ-actin was 19.32±0.13;The CT value of MRP gene in BEL-7402/5-FU + sorafenib group was 26.51±0.22,and the CT value ofβ-actin was 19.43±0.15.2~(-△△CT) was calculated as 2~(-0.47) according to formula,so the content of MRP gene in BEL-7402/5-FU + sorafenib group was about 72.20%of which in BEL-7402/5-FU group,the reversal rate was about 27.80%.
11.Western-blot results showed that after the application of 4μmol/L Sorafenib, P-gp protein(0.257±0.014) expression in BEL-7402/5-FU cell was decreased significantly compared with that before the treatment(0.400±0.014)(P=0.000); MRP protein(0.253±0.024) expression decreased significantly compared with that of before treatment(0.396±0.007)(P=0.000);ERK protein expression (0.913±0.006) showed no significant change compared with that before treatment (0.917±0.018)(P=0.683);pERK protein(0.663±0.009) expression decreased significantly compared with that before treatment(0.934±0.006)(P=0.000); pJNK protein(0.536±0.007) expression significantly increased compared with that before treatment(0.305±0.009)(P=0.000).
Conclusion
1.Sorafenib could markedly enhance the sensitivity of drug-resistant hepatocellular carcinoma cells to chemotherapeutic drug in vitro,and block cell cycle in G0/G1 phase,reduce the cell ratio of S phase,thus blocking the tumor cell proliferation.
2.The results of immunocytochemistry,semi-quantitative and real-time fluorescence quantitative PCR and western blot showed that the possible mechanisms of Sorafenib reverse hepatoma MDR in vitro were the down-regulated MDR1 and MRP gene expression inhibited the function of the transmembrane transporter protein, decreased the chemotherapy drugs pump out of the cells,increased concentration of chemotherapeutic drugs in cells,thus enhancing the killing effect to drug-resistant cells.
3.Sorafenib could also inhibit the ERK signal transduction pathway;activate the JNK signal transduction pathway,thus blocking the tumor cell proliferation.It indicated that the mechanism of Sorafenib reverse hepatoma MDR might also relate to the molecule which impacted the MAPK signal transduction pathway.
4.It was proved in this experiment that Sorafenib showed strong effect of reversing hepatoma cell MDR in vitro,reversal of multidrug resistance of hepatocellular carcinoma cell function,providing an important clue for the further experimental and clinical MDR reversing in vivo of animal.
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