胃癌多药耐药异质性的临床与基础研究
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摘要
化疗是胃癌综合治疗的重要措施,尤其在手术切除原发肿瘤患者的进一步治疗中,由于胃癌对射线治疗不敏感,化疗已成为术后治疗的主要手段,但肿瘤多药耐药(Multidrug resistance, MDR)的存在常导致化疗失败。MDR是指肿瘤细胞对一种抗肿瘤药产生耐药性的同时,对其他结构不同、作用靶点相异的抗肿瘤药也产生交叉性耐药的现象。MDR包括原发性(先天性)耐药(natural MDR)和继发性耐药(acquired MDR):原发性耐药是肿瘤细胞固有的对化疗药物不敏感,故首次使用化疗药物时肿瘤细胞就产生耐药,胃癌即属于原发性MDR表型较强的肿瘤;继发性耐药指肿瘤初始对化疗药物敏感,但经过化疗药物作用后产生的MDR现象,在临床消化道肿瘤的化疗中也常发现这种情况,即病人初始对一线化疗方案敏感,但经过几个疗程化疗后机体内转移/残留病灶开始对药物产生耐受,迫使临床医师不得不改用二线化疗方案。胃癌的这种MDR特性导致化疗有效率不足50%,目前进展期患者5年生存率仅30%与之关系密切,如能对胃癌MDR进行逆转,则对提高化疗效果、改善患者预后意义重大。
迄今关于肿瘤MDR的研究已历30余年,在MDR中作用明确的途径有近10条之多:①MDR-1/P-gp的过度表达;②多药耐药相关蛋白(MRP)的过度表达;③肺耐药相关蛋白(LRP)对药物的胞吐作用;④细胞解毒系统和DNA损伤修复作用增强;⑤药物靶点在质和量上的改变,减弱药物的细胞毒性作用;⑥凋亡抑制基因(如Survivin、Bcl-2)表达提高及促凋亡基因(如Bax、Fas)表达的降低;⑦细胞外环境(pH、温度、氧浓度)的改变等,近来研究热点为HIF-1α;⑧其他与MDR相关的因子,如COX-2已成为逆转胃癌MDR的重要靶点并在研究中证实COX-2抑制剂具有可靠的MDR逆转效果。一般认为,这些因子/途径综合作用导致了胃癌MDR的产生。
但随着研究的深入,学者们发现目前这些MDR机制在解释MDR现象中存在很大不足:①以上机制并不能完全解释胃癌的MDR现象,有待进一步深入研究。这是由于目前研究大多是对一个/数个MDR相关基因及其蛋白质表达进行探讨,在细胞系或动物实验中往往结论明确,但在实体肿瘤研究中常无法解释所有MDR现象。②恶性肿瘤尽管由单克隆增殖而来,但在演进过程中多种因素的作用下可能出现不同的基因或大分子的改变,在抗癌药敏感性及MDR相关因子表达等方面可产生差异变化(即异质性)。从临床角度来看,我国常见胃癌多属进展期,淋巴结转移率达60%-80%,并成为影响预后的独立因素。胃癌患者术后多死于局部复发/或转移。目前化疗多为经验性方案,疗效不佳;虽然已有以原发灶肿瘤细胞体外药敏试验作为选择化疗药物依据的研究,但其结果既不等同于体内的多途径、多因素参与MDR时的结果,也不等同于已发生MDR异质化的转移灶的结果。这种情况在胃癌原发灶与转移灶二者间的差异如何变化,以及淋巴结转移灶中这种异质的MDR的机制如何,目前国内外文献报道很少。
本系列研究基于以上问题,准备在胃癌MDR方面进行下列研究:第一系列分为四部分,从目前研究最多的MDR途径入手,了解各MDR因子与肿瘤细胞化疗药敏性的关系,并对胃癌淋巴结转移灶的MDR特性进行研究并与原发灶比较,以确定胃癌在演进过程中,肿瘤细胞MDR异质性(Heterogeneity)变化情况;第二系列在已有研究结果的基础上,使用目前先进的蛋白质组学(Proteomics)技术对不同胃癌细胞株的差异表达蛋白质进行筛选鉴定,以期找到新的、在胃癌MDR中起关键作用的蛋白质,为今后胃癌MDR的机制探索及逆转奠定基础。
由于蛋白质是生命结构和生命活动的直接执行者,因此,对蛋白质的研究较基因研究更能体现生物功能,因而更为重要。蛋白质组是指由基因组编码的全部蛋白质,也可以说是指一个细胞或组织或有机体全部蛋白质的存在及活动方式。蛋白质组学(Proteomics)则是以及因编码的所有蛋白质组为研究对象,从整体的角度分析细胞内动态变化的蛋白质组成与活动规律。蛋白质组学研究主要依赖三大技术:蛋白质组分的分离技术、鉴定技术和蛋白质结构和功能预测技术。蛋白质组学技术为肿瘤MDR研究带来了新的思维方式和研究领域,它可以从组织或细胞蛋白质整体水平这一全新角度来研究肿瘤MDR机制。在方法学上,蛋白质组学技术与其他蛋白质研究技术不同,该技术于实验前无须确定明确的研究对象(某一种或几种蛋白质),也无须准备相应抗体,这大大拓宽了研究范围,可以更为准确的对生命现象进行研究分析。
本研究以临床胃癌组织及不同胃癌细胞株为研究对象,采用芬丹明B显色法(SRB法)原代细胞培养化疗药敏实验检测胃癌原发灶、淋巴结转移灶肿瘤细胞的体外化疗药敏性;以免疫组化法检测胃癌原发灶、淋巴结转移灶各种多药耐药因子表达;采用双向电泳及基质辅助激光解吸电离-飞行时间质谱鉴定法从不同胃癌细胞株分离鉴定目的蛋白质。对以上结果进行分析,旨在达到以下研究目标:①了解胃癌细胞的体外化疗药敏性与各种MDR因子的关系,确定MDR因子在不同机制药物耐药性形成中的作用;②探讨胃癌原发灶、淋巴结转移灶MDR表型的异质性变化及规律,为原发灶切除后针对转移灶化疗提供理论和实验依据;③从不同胃癌细胞株中确定新的影响胃癌MDR的蛋白质,为今后进一步研究胃癌MDR关键蛋白并实现胃癌MDR逆转奠定基础。
本课题六部分的具体内容如下:
第一部分胃癌原发灶、区域淋巴结转移灶体外化疗药敏性的变化及其意义
目的:检测胃癌原发灶、区域淋巴结转移灶肿瘤细胞对11种胃癌化疗药物的体外敏感性,对其变化进行了分析,并对其意义进行了探讨。
方法:对56例伴淋巴结转移的胃癌原发灶及淋巴结转移灶新鲜肿瘤组织进行芬丹明B显色法(SRB法)原代细胞培养化疗药敏实验,比较两种病灶对11种化疗药物(5-FU、VP-16、HCPT、PTX、L-OHP、CDDP、eADM、THP、MMC、VCR、MTX)敏感性的差异,对结果进行t检验及Spearman相关分析。
结果:11种化疗药物对胃癌原发灶和淋巴结转移灶的肿瘤细胞平均抑制率均低于50%,7种药物对原发灶和淋巴结转移灶的肿瘤细胞抑制率有差异(均P<0.05),其中HCPT,L-OHP,VCR对转移淋巴结细胞抑制率明显低于原发灶(t分别为2.22、2.14、3.60,均P<0.05), VP-16、eADM、THP、MMC对原发灶肿瘤细胞抑制率明显低于淋巴结转移灶(t分别为-2.17、-2.03、-2.02、-2.43,均P<0.05) ,5-FU,PTX,CDDP,MTX对两种病灶肿瘤细胞的抑制率无明显统计学差异(均P>0.05)。5-FU,VP-16,HCPT,L-OHP,PTX,THP,MTX对原发灶和转移淋巴结的肿瘤细胞抑制率呈正相关(r分别为0.3735、0.4080、0.4067、0.5177、0.4006、0.5164、0.3566,均P<0.05)。
小结:1.以SRB显色法行胃癌细胞肿瘤体外化疗敏感性实验具有灵敏度高、结果准确、操作简单、成本较低等优点,值得临床推广。2.11种化疗药物对胃癌原发灶、淋巴结转移灶肿瘤细胞抑制率均较低,提示胃癌细胞对化疗药物均存在原发性MDR,以单药化疗控制肿瘤细胞是不现实的。3.大部分(7/11种)胃癌常用化疗药物(eADM、VP-16、THP、MMC、HCPT、L-OHP、VCR)对肿瘤细胞的抑制率在原发灶、转移淋巴结存在异质性变化,提示以原发灶药敏性结果指导术后针对转移/残留病灶的化疗是不准确的。
第二部分胃癌原发灶、区域淋巴结转移灶经典耐药因子P-gp、GST-π、TopoⅡα异质性表达及与体外化疗药敏性关系的研究
目的:MDR1/P-gp的药泵作用和GST-π的解毒作用及TopoⅡα的药物靶点调控作用是目前研究最多的肿瘤MDR的经典途径,在目前研究中受关注最多,研究也最为深入、广泛。本部分研究检测了胃癌原发灶和转移淋巴结P-gp、GST-π、TopoⅡα表达及肿瘤细胞体外化疗药物敏感性,并对结果进行了分析,对其意义进行了探讨。
方法:对56例胃癌新鲜肿瘤组织及转移淋巴结进行肿瘤细胞培养芬丹明B显色法化疗药敏性实验,并对原发灶和区域淋巴结转移灶组织行P-gp、GST-π、TopoⅡα免疫组化染色。计量资料采用配对t检验,等级资料使用Wilcoxon配对秩和检验、双变量相关分析并计算Spearman等级相关系数。
结果:1.胃癌原发灶、淋巴结转移灶化疗药敏性结果同第一部分。2. P-gp、GST-π在转移灶中的表达程度明显高于原发灶(S分别为182、111,均P<0.01),而TopoⅡα在原发灶中表达强于转移灶(S=-234, P<0.01)。双变量相关分析显示,在原发灶与转移淋巴结间P-gp表达具有明显正相关性(r=0.3303, P<0.05);而GST-π、TopoⅡα在原发肿瘤与淋巴结转移灶间无明显相关性(均P>0.05)。3.在胃癌原发灶中,P-gp强表达时,肿瘤细胞对PTX、HCPT、CDDP的耐药性增强(t分别为3.40、3.01、3.73,均P<0.01);GST-π强表达时CDDP对肿瘤细胞的抑制率降低(t=2.02, P<0.05);而TopoⅡα则与VCR、L-OHP的耐药性有关(t分别为-2.82、-2.82,均P<0.01)。在胃癌淋巴结转移灶中P-gp强表达时肿瘤细胞对VP-16、MTX的敏感性降低(t分别为2.92、2.18,均P<0.05);GST-π强表达与肿瘤细胞对11种药物的耐药性变化无关(均P>0.05);转移灶中TopoⅡα强表达时肿瘤细胞对PTX的敏感性增高(t=-3.04, P<0.01)。4.在肿瘤原发灶,P-gp表达强度与HCPT、PTX、CDDP对肿瘤细胞的平均抑制率均呈负相关(r=-0.2856, r=-0.2967,P=0.0264;均P<0.05);转移灶中P-gp表达程度与VP-16、MTX对肿瘤细胞抑制率均具有负相关性(r=-0.2753, r=-0.2665;均P<0.05)。原发灶GST-π表达程度与5-FU、VP-16的抑制率呈负相关(r=-0.3475, r=-0.2913;均P<0.05),转移灶GST-π表达程度则与HCPT、VCR的抑制率均具有负相关性(r=-0.3703, r=-0.3218;均P<0.05)。原发、转移灶中P-gp、GST-π表达与药物抑制率均未发现具有统计学意义的正相关(均P>0.05)。在肿瘤原发灶,TopoⅡα表达程度与L-OHP对肿瘤细胞的平均抑制率均呈正相关(r=0.3243, P<0.05);在转移灶中,TopoⅡα表达程度则与PTX对肿瘤细胞抑制率具有正相关性(r=0.4085,均P<0.01)。
小结:1. P-gp、GST-π、TopoⅡα在胃癌原发灶和区域淋巴结转移灶中均有较高的阳性表达率,且其表达在原发灶及淋巴结转移灶中出现了异质性改变,P-gp、GST-π在区域淋巴结转移灶中的表达程度明显高于原发灶,而TopoⅡα在转移灶中表达较原发灶减弱(S分别为182、111、-234,均P<0.01)。检测其在淋巴结转移灶中的表达情况对反映术后患者的MDR状态有重要意义。2.在胃癌原发灶和转移淋巴结P-gp、GST-π、TopoⅡα对化疗药物敏感性的影响也存在异质性改变,这些因子均参与了胃癌MDR,且与部分化疗药物的体外药敏性有关,但其表达并不能反映所有药物的耐药情况,以原发灶MDR因子表达来预测转移/残留病灶的化疗敏感性也是不准确的。3. P-gp、GST-π、TopoⅡα在临床组织中的表达情况不能解释全部MDR现象,进一步研究寻找新的主要MDR因子对胃癌MDR研究意义重大。
第三部分胃癌原发灶、区域淋巴结转移灶凋亡相关因子p53、Survivin、Bcl-2、Bax异质性表达及与体外化疗药敏性关系的研究
目的:近来研究显示肿瘤细胞凋亡调控机制在胃癌MDR中发挥着重要作用,故本部分研究对胃癌原发灶、淋巴结转移灶的凋亡相关因子p53、Survivin、Bcl-2、Bax表达情况进行了检测,并对其异质性变化情况及其与体外化疗药敏性的关系进行了分析。
方法:对56例胃癌新鲜肿瘤组织及区域淋巴结转移灶进行肿瘤细胞培养芬丹明B显色法化疗药敏性实验,并对原发灶和转移灶行凋亡相关蛋白p53、Survivin、Bcl-2、Bax以免疫组化染色检测其表达。计量资料采用配对t检验,等级资料使用Wilcoxon配对秩和检验、双变量相关分析并计算Spearman等级相关系数。
结果:1.胃癌原发灶、淋巴结转移灶化疗药敏性结果同第一部分。2.经Wilcoxon配对秩和检验,Bcl-2、Bax在转移灶中的表达程度明显高于原发灶(S分别为117、111,均P<0.05),而p53、Survivin在转移灶中表达与原发灶无明显差异(均P>0.05)。双变量相关分析显示,在原发灶与转移淋巴结间p53、Bcl-2、Bax表达具有明显正相关性(r分别为0.7255、0.4262、0.2901;均P<0.05)。3.在胃癌原发灶中,p53强表达时,肿瘤细胞对PTX、CDDP的耐药性增强(t分别为2.18、3.64,均P<0.05);Survivin强表达时VCR、PTX、eADM对肿瘤细胞的抑制率降低(t分别为2.10、3.02、2.07,均P<0.05),但L-OHP对肿瘤细胞的抑制率反而升高(t=-2.10,P<0.05);而Bcl-2强表达则与5-FU、PTX、eADM和HCPT的耐药性增强有关(t分别为2.76、2.38、2.16、4.27,均P<0.05);Bax强表达时,5-FU、eADM、HCPT对肿瘤细胞的抑制率升高(t分别为-2.51、-3.32、-2.30,均P<0.05)。在胃癌淋巴结转移灶中p53强表达时肿瘤细胞对PTX的敏感性降低(t=2.03,P<0.05);转移灶中Survivin强表达时肿瘤细胞仅对MTX的敏感性降低(t=3.28,P<0.01);转移灶Bcl-2表达增强时肿瘤细胞对5-FU、VCR、VP-16、PTX的耐药性增强(t分别为2.43、2.65、6.78、3.19,均P<0.05);转移灶Bax表达增强则未发现与肿瘤细胞耐药性增强有关(均P>0.05)。4.经Spearman相关分析,在肿瘤原发灶和转移灶中,原发灶p53表达程度与CDDP的抑制率呈负相关(r=-0.4648, P<0.01),转移灶中未发现p53与11种药物对肿瘤细胞抑制率均具有相关性(均P>0.05)。原发灶Survivin表达程度与5-FU、PTX、eADM的抑制率呈负相关(r分别为-0.4805、-0.2979、-0.2861;均P<0.05),而与L-OHP的抑制率呈正相关(r=0.3329, P<0.05);转移灶Survivin表达程度则与MTX的抑制率均具有负相关性(r=-0.3611, P<0.01)。在肿瘤原发灶,Bcl-2表达程度与5-FU、VP-16、HCPT、PTX、eADM对肿瘤细胞的平均抑制率均呈负相关(r分别为-0.3722、-0.3032、-0.5035、-0.3973、-0.2712;均P<0.05);在转移灶中,Bcl-2表达程度则与5-FU、VP-16、PTX、VCR对肿瘤细胞抑制率具有负相关性(r分别为-0.2737、-0.6238、-0.3918、-0.3469,均P<0.05)。原发灶Bax表达程度与5-FU、HCPT、eADM的抑制率呈正相关(r分别为0.4377、0.3666、0.3517,均P<0.05),转移灶Bax表达程度则未发现与药物对肿瘤细胞的抑制率均具有相关性(均P>0.05)。
小结:1.凋亡相关因子在胃癌原发灶和转移淋巴结中表达情况及相互间的相关关系都出现了异质性改变,Bcl-2、Bax在区域淋巴结转移灶中的表达程度明显高于原发灶(均P<0.05),而p53、Survivin在转移灶中表达与原发灶无明显差异(均P>0.05);检测其在淋巴结转移灶中的表达情况对反映术后患者的MDR状态有重要意义。2.在胃癌原发灶和转移淋巴结中各凋亡相关因子对化疗药物敏感性的影响也存在异质性改变,研究证实这些因子均参与了胃癌MDR,且与部分化疗药物的体外药敏性有关,但其表达并不能反映所有药物的耐药情况,以原发灶凋亡相关因子表达来预测转移/残留病灶的化疗敏感性也是不准确的。3.本实验虽证实凋亡相关因子与胃癌MDR有关,但尚未发现任何一种凋亡相关因子与胃癌细胞全部/大部分MDR现象有关,寻找胃癌MDR关键因子、实现MDR逆转尚待进一步深入研究。
第四部分胃癌原发灶、区域淋巴结转移灶COX-2表达与多药耐药因子及与体外化疗敏感性关系的研究
目的:研究表明环氧合酶-2(COX-2)与胃癌MDR关系密切,可通过调节其他MDR因子表达参与胃癌MDR。故本研究对胃癌原发灶、淋巴结转移灶的COX-2表达情况进行了检测,并对其异质性变化情况及其与其他MDR因子、体外化疗药敏性的关系进行了分析。
方法:对56例胃癌新鲜肿瘤组织及转移淋巴结进行肿瘤细胞培养芬丹明B显色法化疗药敏性实验,并对原发灶和转移灶行COX-2及其他MDR因子免疫组化染色。统计方法同第二部分。
结果:1.胃癌原发灶、淋巴结转移灶化疗药敏性结果同第一部分。2. COX-2在转移灶中的表达程度明显高于原发灶(S=190, P<0.01);双变量相关分析显示,在原发灶与转移淋巴结间COX-2表达具有明显正相关性(r=0.3511, P<0.01)。3.在胃癌原发灶中,COX-2表达与GST-π、Survivin、Bcl-2表达呈正相关(r分别为0.2741、0.7024、0.5925,均P <0.05),与TopoⅡα、Bax表达呈负相关(r分别为-0.3488、-0.3292,均P<0.05)。在淋巴结转移灶中,COX-2表达与P-gp、Survivin、Bcl-2表达呈正相关(r分别为0.3490、0.2836、0.5368,均P<0.05)。4.在胃癌原发灶中,COX-2强表达时,5-FU、VCR、PTX、eADM、HCPT对肿瘤细胞的抑制率降低(t分别为2.38、2.50、3.15、2.39、2.55,均P<0.05);在胃癌淋巴结转移灶中,COX-2表达增强时肿瘤细胞对5-FU、VCR、VP-16、MTX的耐药性增强(t分别为3.33、4.11、2.90、2.63,均P<0.05)。5.在肿瘤原发灶,COX-2表达程度与5-FU、HCPT、PTX、eADM、THP、VCR对肿瘤细胞的平均抑制率均呈负相关(r分别为-0.5385、-0.3077、-0.3850、-0.2991、-0.3466、-0.4158,均P<0.05);在转移灶中,COX-2表达程度则与5-FU、VP-16、PTX、VCR、MTX对肿瘤细胞抑制率具有负相关性(r分别为-0.4120、-0.4421、-0.3300、-0.3597、-0.2836,均P <0.05)。
小结:1. COX-2在转移过程中发生了异质性变化,且转移灶中COX-2与原发灶表达存在正相关关系,说明COX-2对肿瘤的转移起促进作用。2.原发灶中COX-2与P-gp、GST-π、TopoⅡα等经典MDR因子的关系与转移灶情况不同。3. COX-2与凋亡相关因子p53、Survivin、Bcl-2、Bax的关系在原发灶和淋巴结转移灶中也不相同。4.与原发灶COX-2表达有关的化疗药物和转移灶中不同,提示COX-2逆转剂在术后化疗中的意义尚不能确定,进一步深入研究其机制并寻找新MDR相关因子有重要意义。第五部分不同分化程度胃癌细胞株差异表达蛋白质的筛选鉴定及与胃癌多药耐药关系的研究
目的:研究发现消化道肿瘤的MDR与肿瘤分化程度有关,故本研究以不同分化的胃癌细胞为研究对象,应用先进的蛋白质组学技术对胃癌分化相关蛋白质进行了筛选鉴定,并对其与胃癌MDR的关系进行了分析。
方法:采用双向凝胶电泳及基质辅助激光解吸电离-飞行时间质谱鉴定法对三种分化程度不同的胃癌细胞株MKN28(高分化)、SGC7901(中分化)和BGC823(低分化)进行了研究,并应用蛋白印迹试验法对鉴定出的部分蛋白质在胃癌细胞株及胃癌组织中的表达进行了验证。
结果:三种胃癌细胞株的双向电泳凝胶所得蛋白质点均在1000个左右,表达差异明显的蛋白质点14个,经质谱仪分析,8种蛋白质得到鉴定,为类硫氧还蛋白过氧化物酶、甘油醛-3-磷酸脱氢酶、β-微管蛋白多肽、假定蛋白、锌指蛋白139、蛋白酪氨酸激酶、钙网蛋白前体、原肌球蛋白。这些蛋白质与胃癌细胞信号转导、维持细胞稳态、参与糖酵解及肿瘤药物代谢、抗氧化损伤等功能有关。经蛋白印迹试验法检测,证实这些蛋白质在胃癌细胞株及胃癌组织中的表达与蛋白质组学所得结果一致。
小结:1.不同分化的胃癌细胞蛋白质表达存在差异。本研究中双向电泳分离出的蛋白质点较多,鉴定的蛋白质点经Western blot对肿瘤组织及细胞株进行验证,所得结果均与蛋白质组学结果相符合,说明本研究有较高的可信性及意义。2.对8种差异蛋白质的分析显示,这些蛋白质涉及肿瘤药物代谢、细胞信号转导、维持细胞稳态、参与糖酵解及抗氧化损伤等多个方面,这些蛋白质的表达与胃癌细胞分化程度密切相关,其表达差异造成了胃癌生物学活性的不同,以其为靶标进行干预有可能逆转胃癌细胞的某些恶性生物学行为。
第六部分胃癌细胞株SGC7901及耐药细胞株SGC7901/VCR差异点筛选鉴定的蛋白质组学研究
目的:为获得与胃癌MDR直接相关的新蛋白质,本研究应用蛋白质组学技术对胃癌细胞株SGC7901和耐药细胞株SGC7901/VCR的差异蛋白质进行了筛选鉴定,并对结果及意义进行了分析。
方法:同第五部分。
结果:在两种胃癌细胞株中所得蛋白质点均超过1000个,找到表达差异明显的蛋白质点9个,经质谱分析,7种蛋白质得到鉴定,为S60核糖体蛋白L23、电压依赖性阴离子选择性通道蛋白1、锌指蛋白394、角蛋白I型细胞骨架9、核糖核酸3'-端磷酸化酶、matrin型锌指蛋白2、Sideroflexin-1。这些蛋白质与肿瘤药物代谢、胃癌细胞信号转导、维持细胞稳态、参与增殖凋亡、促进细胞成熟等功能有关。经蛋白印迹试验法检测,证实这些蛋白质在胃癌细胞株中的表达与蛋白质组学所得结果一致。
小结: 1.本研究中胃癌细胞株SGC7901及其耐药细胞株SGC7901/VCR的双向电泳凝胶所得蛋白质点较多,质谱鉴定的蛋白质经Western blot验证,所得结果与蛋白质组学结果符合,说明本研究的可信性较高,所得蛋白质点与胃癌细胞MDR有直接相关性。2.对差异蛋白质的分析显示,这些蛋白质大多涉及肿瘤药物代谢,同时涉及胃癌细胞的其他生物学特性,这些蛋白质的表达与胃癌细胞MDR密切相关,其表达差异造成了胃癌细胞MDR表型的不同,进一步进行研究有可能找到关键/重要MDR因子,以其为靶标进行干预有可能逆转胃癌细胞的MDR特性,提高术后化疗效果并改善预后。
综合上述六部分内容,本研究得出如下结论:
1.胃癌细胞对大部分化疗药物的体外药敏性在原发灶、转移淋巴结存在异质性变化,以原发灶药敏性结果指导术后针对转移/残留病灶的化疗是不准确的,术后化疗应针对转移灶的特点进行。
2.经典MDR因子(P-gp、GST-π、TopoⅡα)和凋亡相关因子(p53、Survivin、Bcl-2、Bax)表达在原发灶及淋巴结转移灶中出现了异质性改变;在胃癌原发灶和转移淋巴结经典MDR因子和凋亡相关因子对化疗药物敏感性的影响也存在异质性改变,这些因子的表达只与部分化疗药物的体外药敏性有关,并不能反映所有药物的耐药情况;这些因子在临床组织中不能解释全部MDR现象,研究寻找新的主要MDR因子对胃癌MDR研究意义重大。
3. COX-2对胃癌的转移起促进作用,且本身在转移中发生异质性改变。原发灶中COX-2与经典MDR因子和凋亡相关因子的关系与转移灶情况不同。与原发灶COX-2表达有关的化疗药物和转移灶不同。
4.应用蛋白质组学技术对筛选鉴定了8种胃癌分化相关蛋白质,并在胃癌细胞株和组织中得到了验证。经功能分析,发现其中β-微管蛋白多肽、蛋白酪氨酸激酶等与胃癌耐药有关。
5.以胃癌细胞株SGC7901和耐药细胞株SGC7901/VCR为研究对象,应用蛋白质组学技术鉴定出7种与胃癌MDR直接相关的蛋白质,并在胃癌细胞株中进行了验证。
Chemotherapy is important in comprehensive treatments of gastric carcinoma, especially in the patients with primary tumors resected, which is the most important therapeutic means, for this kind of cancer is insensitive to radiotherapy. But multidrug resistance (MDR) of gastric carcinoma often causes the failure of chemotherapy. MDR means that when tumor cells are resistant to a chemotherapeutic drug, they are often resistant to other drugs of different structure and mechanism. MDR contains natural MDR and acquired MDR. Natural MDR means that inherent insensitivity to chemotherapeutic drugs exist in tumor cells, so MDR will occur when a kind of chemotherapeutic drug is first used, and gastric carcinoma cells are tumor cells with obvious MDR phenotypes; acquired MDR means that tumor cells are sensitive to chemotherapeutic drugs in the beginning, but after several courses of treatment, MDR would occur, and it is common during clinical therapy for gastrointestinal tract carcinomas. MDR of gastric carcinoma causes the percentage of effectiveness is less than 50%, and it is related to low survival rate(less than 30%) of advanced gastric carcinoma. It will have great realistic meaning if the reversion of MDR for gastric carcinoma comes true.
Research about MDR of tumors has been on for over 30 years. Now nearly 10 kinds of mechanism have been proven to have explicit relationship with MDR, and they are as follows:①Overexpression of MDR1/P-gp;②Overexpression of MRP;③Enhancement of exocytosis by LRP;④Reinforcement of cell’s detoxification and repair effects of DNA damage;⑤The drug targets’sufficient either in quality or quantity;⑥Reinforcement of inhibitor of apoptosis proteins and reduction of apoptosis proteins;⑦Change of extracellular environment(such as pH, temperature or oxygenconcentration), hypoxia inducible factors-1alpha(HIF-1α) is research focus recently;⑧Other factors related to MDR, such as COX-2. It is generally recognized that MDR of gastric carcinoma is caused by all these factors’synergy.
But with detailed study, scholars have found that many defects exist in the explanation about MDR of gastric carcinoma with these known mechanisms. The problems exist as follows:①These mechanisms can not explain all phenomena of gastric cancer MDR. The reason is most studies’objects are one/several MDR-related genes and proteins, and they often can not be used to explain all MDR phenomena in clinical studies though there often has clear conclusion in the cell lines or animal experiments.②Malignant tumors come from monoclonal proliferation, but changes in the genes or molecules often appear in the evolution of tumors. Changes (Heterogeneity) may arise in the anti-cancer drug sensitivity and expression of MDR-related factors. From a clinical point of view, most gastric carcinomas in China belong to advanced stage, and metastasis rate of lymph node reaches 60%-80%, which has been thought as independent prognostic factor, and most patients died of gastric cancer recurrence / or metastasis after primary tumors were resected. Current chemotherapy regimens often are experiential programs, and the efficacy is poor. Some reports have shown that chemosensitivity test in vitro of tumor cells can be used as evidence in the selection of chemotherapeutics, but these results cannot reflect condition of multi-channel and multi-factors in vivo, also they cannot be on behalf of the results of lymph node metastases (LNMs). MDR in LNMs of gastric carcinoma has been rarely reported in the literature both at home and abroad.
Our study of these series is based on the problems above, and we prepare to conduct the following study of gastric cancer MDR. Our study is divided into two series as follows:①Series 1 is divided into 4 four parts, the relationship between MDR related factors and chemosensitivity in vitro has been investigated from the most established ways that lead to MDR of gastric carcinoma; characters of MDR in LNMs are also studied, and heterogeneity is analyzed compared with results of PT.②Series 2 is to find new key factor of MDR. Advanced techniques of proteomics are used to screen and identificate differentially expressed proteins in different gastric carcinoma cell lines. This study may be helpful in mechanism and reversal in MDR of gastric carcinoma.
Protein is the direct executor of the life’s structure and activities, so it’s more important to investigate protein than gene. Proteome means all the proteins encoded by genome and proteomics take all proteome encoded by genome as object, then it analysis dynamic changes of intracellular protein composition and activities of law as a whole. Research of proteomics depends on three techniques: technique of protein components’separation, identification technology of protein components and technique of function prediction. Proteomics brings new mode of thinking in the research of MDR about malignant tumors. Proteomics studies mechanism of MDR about malignant tumors from the overall level of tissue or cell protein. In the methodological, it’s unnecessary to determine the definite object of study, and also it’s unnecessary to prepare antibody in advance. This greatly broadens the scope of the study, and we can be more accurate analysis of life phenomena.
The main contents of this article are as follows: Part one: Chemosensitivity of regional lymph node metastases compared to the primary tumors with SRB assay in vitro for patients with advanced gastric carcinoma
Objective: To investigate the different chemosensitivity of lymph node metastases cultures compared to the primary tumor in gastric carcinoma gastric carcinoma by sulphorhodamine B (SRB) assay.
Methods: In vitro SRB assay was performed on 56 paired surgical specimens of primary tumors (PT) and lymph node metastases (LNMs) from 56 gastric carcinoma patients. The results were then compared with response to 11 antitumor drugs(5-FU, VP-16, HCPT, PTX, L-OHP, CDDP, eADM, THP, MMC, VCR, MTX). Data was dealt with t test and Spearman correlation analysis.
Results: 1.There was significantly different chemosensitivity of individuals to antitumor drugs between PT and LNMs in 7/11 drugs(all P<0.05). The inhibition rates of tumor cells in LNMs for HCPT, L-OHP and VCR were lower than those in PT(all P<0.05), whereas the inhibition rates of VP-16, eADM, THP, MMC were higher in LNMs than those in PT(all P<0.05). No significantly difference of inhibition ratio between PT and LNMs was found in 5-FU,PTX,CDDP,MTX(all P>0.05). 2.There was a significantly positive correlation between the inhibition rates of PT and LNMs for 5-FU,VP-16, HCPT, L-OHP,PTX,THP,MTX (r=0.3735~0.5177, all P<0.05).
Summary: 1. SRB assay has many advantages such as high sensitivity, pin-point accuracy, more simple operation, lower cost, etc. So it’s worthy of promotion in clinic. 2. All 11 drugs have low inhibition rates for gastric carcinoma cells , and it shows that natural MDR exists in gastric carcinoma cells, so it’s unrealistic to control this cancer with one drug. 3. Heterogeneity of chemosensitivity to most antitumor drugs is shown in metastatic lymph nodes in gastric cancers. The chemosensitivity of gastric carcinoma primary tumor in the drugs response assay in votro may not give a good indication of the chemosensitivity in metastatic lesions. Chemotherapy after operation should be carried on according to characteristic of LNMs in patients with advanced gastric carcinoma.
Part two: Relationship between expression of P-gp, GST-π, TopoⅡαand chemosensitivities in primary tumors(PT) and regional lymph node metastases(LNMs) of gastric carcinoma
Objects: The most studied classical pathways of MDR include drug pump of MDR1/P-gp, detoxification of GST-πand drug targets regulation of TopoⅡα. These pathways are most viewed. In this part, expression of P-gp, GST-π, TopoⅡand chemosensitivities were detected in PT and LNMs, their relationship was analyzed, and the significance that may exist was discussed.
Methods: In vitro SRB assay was performed on 56 paired fresh surgical specimens of PT and LNMs from 56 gastric carcinoma patients. Expression of P-gp, GST-π, TopoⅡwas tested by immunohistochemical staining. Measurement data were analyzed with t test, ranked data with Wilcoxon rank test, and Spearman correlation analysis was also used to calculate correlation coefficient.
Results: 1.The results of chemosensitivity in vitro in tumor cells of PT and LNMs were seen in Part one. 2. The expression of P-gp and GST-πwas higher in LNMs than in PT(both P<0.01), and expression of TopoⅡαwas higher in PT(P<0.01). There was positive correlativity of P-gp between PT and LNMs(r=0.3303, P<0.05), and no significant correlativity was found about GST-πand TopoⅡαbetween PT and LNMs(both P>0.05). 3. In PT the inhibition rates for PTX, HCPT, CDDP in P-gp strong expression group were lower than those in weak group (all P<0.05); The inhibition rate to CDDP was significantly lower for the GST-πstrong expression group in PT (P<0.05); and for strong expression of TopoⅡα, the inhibition rates for VCR, L-OHP were higher(both P<0.05). In LNMs, there were lower inhibition rates for VP-16, MTX in P-gp strong expression group (both P<0.05), and the inhibition rate to PTX was significantly lower for the TopoⅡαweak expression group in LNMs(P<0.05); no significant change was found between inhibition rates for these drugs and expression of GST-π(all P>0.05).4. The inhibition rates to HCPT, PTX, CDDP were negatively correlated with the expression of P-gp in PT (r=-0.2856, r=-0.2967,P=0.0264; all P<0.05), and negative correlation was found between the inhibition rates to VP-16, MTX and the expression of P-gp in LNMs(r=-0.2753, r=-0.2665; both P<0.05). The expression of GST-πwas negatively correlated with the inhibition rates to 5-Fu, VP-16 in PT( r=-0.3475, r=-0.2913; both P<0.05), and negatively correlated with HCPT, VCR(r=-0.3703, r=-0.3218;both P<0.05). The expression of TopoⅡαwas positively correlated with inhibition rates to L-OHP in PT(r=0.3243, P<0.05), and positive correlation was found between the expression of TopoⅡαand inhibition rates to PTX in LNMs(r=0.4085, P<0.05).
Summary: 1. There was higher positive expression rate of P-gp, GST-π, TopoⅡαbetween PT and LNMs of gastric carcinoma, and expression of these proteins showed heterogeneity between PT and LNMs. It’s of great significance to test expression of P-gp, GST-π, TopoⅡαin LNMs, and it can reflect the MDR status in patients after PT resected. 2. There was heterogeneity in the relationship about expression of P-gp, GST-π, TopoⅡαand chemosensitivity in 11 chemotherapeutics between PT and LNMs. The results showed that these factors all participate in MDR of gastric carcinoma, and there existed relationship between expression of them and chemosensitivity in part of chemotherapeutics, but expression of them could not reflect chemosensitivity of all drugs. So it was inaccurate to predict MDR of LNMs with results of PT. 3. P-gp, GST-π, TopoⅡαcould not explain all phenomena of MDR, so it’s of great significance to search new MDR related factors to explore the mechanism of MDR of gastric carcinoma.
Part three: Relationship between expression of apoptosis-related proteins (p53, Survivin, Bcl-2, Bax) and chemosensitivities in primary tumors (PT) and regional lymph node metastases (LNMs) of gastric carcinoma
Objective: Many recent researches have shown that regulatory mechanisms of apoptosis of tumor cells play an important role in MDR of gastric carcinoma.So in this part, expression of apoptosis-related proteins (p53, Survivin, Bcl-2, Bax) and chemosensitivities in vitro were detected in PT and LNMs, their relationship was analyzed, and the significance that may exist were discussed.
Methods: In vitro SRB assay was performed on 56 paired fresh surgical specimens of PT and LNMs from 56 gastric carcinoma patients. Expression of p53, Survivin, Bcl-2, Bax was tested by immunohistochemical staining. Measurement data were analyzed with t test, ranked data with Wilcoxon rank test, and Spearman correlation analysis was also used, and correlation coefficient was calculated.
Results: 1.The results of chemosensitivity in vitro in tumor cells of PT and LNMs were seen in Part one. 2. The expression of Bcl-2, Bax was higher in LNMs than in PT(both P<0.01), and there was no significant difference in expression of p53, Survivin between PT and LNMs(both P>0.05). There was positive correlativity of p53, Bcl-2, Bax between PT and LNMs(r=0.7255, r=0.4262, r=0.2901; P<0.05). 3. In PT the inhibition rates for PTX, CDDP in p53 strong expression group were lower than those in weak group (both P<0.05); the inhibition rates to VCR, PTX, eADM were significantly lower for the Survivin strong expression group in PT (all P<0.05), but for L-OHP, the inhibition rate was higher when the tumor with stronger expression of Survivin(P<0.05); and for strong expression of Bcl-2, the inhibition rates for 5-FU, PTX, eADM, HCPT were lower(all P<0.05); for strong expression of Bax, the inhibition rates for 5-FU, eADM, HCPT were higher(all P<0.05). In LNMs, there was lower inhibition rate for PTX in p53 strong expression group (P<0.05), the inhibition rate to MTX was significantly lower for the Survivin strong expression group in LNMs(P<0.01); and for strong expression of Bcl-2, the inhibition rates for 5-FU, VCR, VP-16, PTX were lower(all P<0.05); no significant change was found between inhibition rates for these drugs and expression of Bax(all P>0.05).4. There was negatively correlation between inhibition rate to CDDP and the expression of p53 in PT(P<0.01) , and no significant relationship was found between expression of p53 and inhibition rates for these 11 drugs(all P>0.05). The inhibition rates to 5-FU, PTX, eADM were negatively correlated with the expression of Survivin in PT (r=-0.4805, r=-0.2979, r=-0.2861; all P<0.05), and there was positive correlation was found between inhibition rate to L-OHP and expression of Survivin(r=0.3329, P<0.05); negative correlation was found between the inhibition rates to MTX and the expression of Survivin in LNMs(r=-0.3611; P<0.01). The expression of Bcl-2 was negatively correlated with the inhibition rates to 5-FU, VP-16, HCPT, PTX, eADM in PT(r=-0.3722, r=-0.3032, r=-0.5035, r=-0.3973, r=-0.2712;all P<0.05), and negatively correlated with 5-FU, VP-16, PTX, VCR(r=-0.2737, r=-0.6238, r=-0.3918, r=-0.3469;all P<0.05)in LNMs. The expression of Bax was positively correlated with inhibition rates to 5-FU, HCPT, eADM in PT(r=0.4377, r=0.3666, r=0.3517;all P<0.05), and no significant correlation was found between the expression of Bax and inhibition rates to these drugs in LNMs(all P>0.05).
Summary: 1. There was heterogeneity in expression of apoptosis-related proteins (p53, Survivin, Bcl-2, Bax) between PT and LNMs. The results showed that it’s valuable to test these factors in LNMs to reflect situation of MDR in patients with PT resected. 2. There was heterogeneity in the relationship between expression of p53, Survivin, Bcl-2, Bax and chemosensitivity in 11 chemotherapeutics between PT and LNMs. The results showed that these factors all participate in MDR of gastric carcinoma, and there existed relationship between expression of them and chemosensitivity in part of chemotherapeutics, but expression of them could not reflect chemosensitivity of all drugs. So it was inaccurate to predict MDR of LNMs with results of PT. 3. Results showed that p53, Survivin, Bcl-2, Bax all participate in MDR, but none of them could explain all phenomena of MDR, so it’s of great significance to search new key MDR related factors to explore the mechanism of MDR of gastric carcinoma.
Part four: Relationship between expressions of cyclooxygenase-2(COX-2), MDR related factors and chemosensitivities in vitro in primary tumors (PT) and regional lymph node metastases (LNMs) of gastric carcinoma
Objective: Many researches in recent years have shown that cyclooxygenase-2(COX-2) has close relationship with MDR of gastric carcinoma, and it can participate in MDR gastric carcinoma by regulating expression of other of MDR related factors. So in this part, expression of COX-2, some other MDR related factors and chemosensitivities in vitro were detected in PT and LNMs, their relationship was analyzed, and the significance that may exist was investigated.
Methods: In vitro SRB assay was performed on 56 paired fresh surgical specimens of PT and LNMs from 56 gastric carcinoma patients. Expression of COX-2, some other MDR related factors were tested by immunohistochemical staining. Statistical analysis was the same as Part three.
Results: 1.The results of chemosensitivity in vitro in tumor cells of PT and LNMs were seen in Part one. 2. The expression of COX-2 was higher in LNMs than in PT(P<0.01), and there was positive correlativity of COX-2 between PT and LNMs(r=0.3511, P<0.01). 3. In PT, there was positive correlation between the expression of COX-2 and other MDR related factors as following: GST-π, Survivin, Bcl-2(r=0.2741,r=0.7024,r=0.5925;all P <0.05), and negative correlation was found between expression of COX-2 and TopoⅡα, Bax(r=-0.3488, r=-0.3292,both P <0.05). In LNMs, positive correlation was found between expression of COX-2 and P-gp, Survivin, Bcl-2(r=0.3490,r=0.2836,r=0.5368;all P <0.05). 4.In PT the inhibition rates for 5-FU, VCR, PTX, eADM, HCPT in COX-2 strong expression group were lower than those in weak group (all P<0.01). In LNMs, there were lower inhibition rates for 5-FU, VCR, VP-16, MTX in COX-2 strong expression group (all P<0.05).5. The inhibition rates to 5-FU, HCPT, PTX, eADM, THP, VCR were negatively correlated with the expression of COX-2 in PT (r=-0.5385,r=-0.3077,r=-0.3850,r=-0.2991,r=-0.3466,r=-0.4158;all P <0.05), and negatively correlated with 5-FU, VP-16, PTX, VCR, MTX(r=-0.4120, r=-0.4421, r=-0.3300,r=-0.3597, r=-0.2836;all P <0.05)in LNMs.
Summary: 1. There was heterogeneity in expression of COX-2 between PT and LNMs, and positive correlativity was found between expression of COX-2 in PT and in LNMs, which showed that COX-2 played an important role in metastasis of gastric carcinoma. 2. There was heterogeneity in the relationship about COX-2 and P-gp, GST-π, TopoⅡαbetween PT and LNMs. 3. There was heterogeneity in the relationship of COX-2 and p53, Survivin, Bcl-2, Bax between PT and LNMs. 3. Results showed that COX-2 participates in MDR of gastric carcinoma, but it could not explain all phenomena of MDR, and it is uncertain about the effect of inhibitor of COX-2 in the clinical therapy. So it’s of great significance to search new key MDR related factors to explore the mechanism of MDR of gastric carcinoma.
Part five: Identification of differentiation-related proteins in gastric carcinoma cell lines by comparative proteomics and their relationship with MDR of gastric carcinoma
Objective: Researches have shown that MDR of digestive tract was in related to differentiation of tumor cells. So in this part, differentiation of gastric cancer cell lines were used as object to investigate differentiation-related proteins in human gastric carcinoma cell lines by comparative proteomics, and then their relationship with MDR of gastric carcinoma was studied.
Methods: 1 The holoproteins of human gastric carcinoma cell lines MKN28(well differentiated), SGC7901(moderately differentiated) and BGC823(poorly differentiated) were measured by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).Some proteins obtained through proteomics were tested by Western blot in the cell strain and tissues of gastric carcinoma.
Results: There were different proteins in human gastric carcinoma cell lines. All 14 different protein spots were found in 3 gastric carcinoma cell lines, and 8 spots were identified by MALDI-TOF-MS, and these proteins were probable thioredoxin peroxidase, glyceraldehyde-3-phosphate dehydrogenase (GAPD),β-tubulin polypeptide, hypothetical protein, zinc finger protein (ZNF)139, protein-tyrosine kinase, calreticulin precursor, tropomyosin. These proteins have relationship with the biological behavior of gastric carcinoma, such as signal transduction, cellular homeostasis, glycolysis, antioxidation action, multidrug resistance (MDR), etc. Result of Western blot about protein expression was in consonance with situation of proteomics.
Summary: 1. Proteins spots got from 3 gastric cells were all 1000 more or less, and 8 spots of 14 were identificated, part of them verificated by Western blot, which showed it was credible of the results. 2. These 8 proteins were involved in many biological behaviors of gastric carcinoma, such as signal transduction, cellular homeostasis, glycolysis, antioxidation action, multidrug resistance (MDR), etc. The results showed that intervention for these proteins could reverse malignant some biological behaviors of gastric carcinoma.
Part six: Identification of gastric carcinoma cell line SGC7901 and tumor MDR cell strain SGC7901/VCR by comparative proteomics
Objective: To obtain new proteins directly related to the MDR of gastric carcinoma, comparative proteomics was used in this part, and gastric carcinoma cell line SGC7901 and tumor MDR cell strain SGC7901/VCR were used as objects to identificate new differently expressed proteins.
Methods: Methods were the same as Part five.
Results: There were different proteins in these 2 gastric carcinoma cell lines. All 9 different protein spots were found, and 7 spots were identified by MALDI-TOF-MS. These proteins were 60S ribosomal protein L23, voltage-dependent anion-selective channel protein (VDAC1), zinc finger protein (ZNF)394, keratin, type I cytoskeletal 9(KⅠC9), RNA 3'-terminal phosphate cyclase(RTC1), zinc finger matrin-type protein 2, Sideroflexin-1. These proteins have relationship with the biological behavior of gastric carcinoma, such as multidrug resistance (MDR), cellular signal transduction, maintenance of cellular homeostasis, proliferation and apoptosis, maturation of cells, etc. Result of Western blot about protein expression was in consonance with situation of proteomics.
Summary: 1. Proteins spots got from 3 gastric cells were all over 1000, and 7 spots of 9 were identificated, parts of which were certificated by Western blot assay, which showed it was credible of the results, and these proteins were directly related to the MDR of gastric carcinoma. 2. These 7 proteins were involved in many biological behaviors of gastric carcinoma, such as multidrug resistance (MDR), cellular signal transduction, maintenance of cellular homeostasis, proliferation and apoptosis, maturation of cells, etc. The results showed that intervention for these proteins could reverse MDR of gastric carcinoma, improve the effect of chemotherapy, and improve the prognosis.
Conclusions:
1. There was heterogeneity in chemosensitivity in vitro related between PT and LNMs, so it’s inaccurate to design chemotherapy for patients with PT resected according to results of chemosensitivity test for PT, it should be designed to aim at MDR features of LNMs.
2. There was heterogeneity in expression of MDR related factors(P-gp, GST-π, TopoⅡα, p53, Survivin, Bcl-2, Bax) between PT and LNMs. Heterogeneity was also found in the relationship about expression of MDR related factors and chemosensitivity in chemotherapeutics between PT and LNMs. The results showed that these factors all participate in MDR of gastric carcinoma, and there existed relationship between expression of them and chemosensitivity in part of chemotherapeutics, but expression of them could not reflect chemosensitivity of all drugs. It’s of great significance to search new key MDR related factors to explore the mechanism of MDR of gastric carcinoma.
3. Results showed that COX-2 participates in MDR of gastric carcinoma, and there was heterogeneity in expression of COX-2 between PT and LNMs. Relationship between COX-2 and MDR related factors was different from that in LNMs, and heterogeneity was also found in the relationship about expression of COX-2 and chemosensitivity in chemotherapeutics between PT and LNMs.
4. 8 spots of 14 differentiation-related proteins were identificated, which were probable thioredoxin peroxidase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH),β-tubulin polypeptide, hypothetical protein, zinc finger protein (ZNF)139, protein-tyrosine kinase, calreticulin precursor, tropomyosin; part of them were certificated by Western blot. In these proteins,β-tubulin polypeptide and protein-tyrosine kinase were in related to MDR of gastric carcinoma.
5. 7 proteins directly related to the MDR of gastric carcinoma were identificated by comparative proteomics in gastric carcinoma cell line SGC7901 and tumor MDR cell strain SGC7901/VCR, which were 60S ribosomal protein L23, voltage-dependent anion-selective channel protein (VDAC1), zinc finger protein (ZNF)394, keratin, type I cytoskeletal 9(KⅠC9), RNA 3'-terminal phosphate cyclase(RTC1), zinc finger matrin-type protein 2, Sideroflexin-1; part of them were certificated by Western blot assay.
迄今关于肿瘤MDR的研究已历30余年,在MDR中作用明确的途径有近10条之多:①MDR-1/P-gp的过度表达;②多药耐药相关蛋白(MRP)的过度表达;③肺耐药相关蛋白(LRP)对药物的胞吐作用;④细胞解毒系统和DNA损伤修复作用增强;⑤药物靶点在质和量上的改变,减弱药物的细胞毒性作用;⑥凋亡抑制基因(如Survivin、Bcl-2)表达提高及促凋亡基因(如Bax、Fas)表达的降低;⑦细胞外环境(pH、温度、氧浓度)的改变等,近来研究热点为HIF-1α;⑧其他与MDR相关的因子,如COX-2已成为逆转胃癌MDR的重要靶点并在研究中证实COX-2抑制剂具有可靠的MDR逆转效果。一般认为,这些因子/途径综合作用导致了胃癌MDR的产生。
但随着研究的深入,学者们发现目前这些MDR机制在解释MDR现象中存在很大不足:①以上机制并不能完全解释胃癌的MDR现象,有待进一步深入研究。这是由于目前研究大多是对一个/数个MDR相关基因及其蛋白质表达进行探讨,在细胞系或动物实验中往往结论明确,但在实体肿瘤研究中常无法解释所有MDR现象。②恶性肿瘤尽管由单克隆增殖而来,但在演进过程中多种因素的作用下可能出现不同的基因或大分子的改变,在抗癌药敏感性及MDR相关因子表达等方面可产生差异变化(即异质性)。从临床角度来看,我国常见胃癌多属进展期,淋巴结转移率达60%-80%,并成为影响预后的独立因素。胃癌患者术后多死于局部复发/或转移。目前化疗多为经验性方案,疗效不佳;虽然已有以原发灶肿瘤细胞体外药敏试验作为选择化疗药物依据的研究,但其结果既不等同于体内的多途径、多因素参与MDR时的结果,也不等同于已发生MDR异质化的转移灶的结果。这种情况在胃癌原发灶与转移灶二者间的差异如何变化,以及淋巴结转移灶中这种异质的MDR的机制如何,目前国内外文献报道很少。
本系列研究基于以上问题,准备在胃癌MDR方面进行下列研究:第一系列分为四部分,从目前研究最多的MDR途径入手,了解各MDR因子与肿瘤细胞化疗药敏性的关系,并对胃癌淋巴结转移灶的MDR特性进行研究并与原发灶比较,以确定胃癌在演进过程中,肿瘤细胞MDR异质性(Heterogeneity)变化情况;第二系列在已有研究结果的基础上,使用目前先进的蛋白质组学(Proteomics)技术对不同胃癌细胞株的差异表达蛋白质进行筛选鉴定,以期找到新的、在胃癌MDR中起关键作用的蛋白质,为今后胃癌MDR的机制探索及逆转奠定基础。
由于蛋白质是生命结构和生命活动的直接执行者,因此,对蛋白质的研究较基因研究更能体现生物功能,因而更为重要。蛋白质组是指由基因组编码的全部蛋白质,也可以说是指一个细胞或组织或有机体全部蛋白质的存在及活动方式。蛋白质组学(Proteomics)则是以及因编码的所有蛋白质组为研究对象,从整体的角度分析细胞内动态变化的蛋白质组成与活动规律。蛋白质组学研究主要依赖三大技术:蛋白质组分的分离技术、鉴定技术和蛋白质结构和功能预测技术。蛋白质组学技术为肿瘤MDR研究带来了新的思维方式和研究领域,它可以从组织或细胞蛋白质整体水平这一全新角度来研究肿瘤MDR机制。在方法学上,蛋白质组学技术与其他蛋白质研究技术不同,该技术于实验前无须确定明确的研究对象(某一种或几种蛋白质),也无须准备相应抗体,这大大拓宽了研究范围,可以更为准确的对生命现象进行研究分析。
本研究以临床胃癌组织及不同胃癌细胞株为研究对象,采用芬丹明B显色法(SRB法)原代细胞培养化疗药敏实验检测胃癌原发灶、淋巴结转移灶肿瘤细胞的体外化疗药敏性;以免疫组化法检测胃癌原发灶、淋巴结转移灶各种多药耐药因子表达;采用双向电泳及基质辅助激光解吸电离-飞行时间质谱鉴定法从不同胃癌细胞株分离鉴定目的蛋白质。对以上结果进行分析,旨在达到以下研究目标:①了解胃癌细胞的体外化疗药敏性与各种MDR因子的关系,确定MDR因子在不同机制药物耐药性形成中的作用;②探讨胃癌原发灶、淋巴结转移灶MDR表型的异质性变化及规律,为原发灶切除后针对转移灶化疗提供理论和实验依据;③从不同胃癌细胞株中确定新的影响胃癌MDR的蛋白质,为今后进一步研究胃癌MDR关键蛋白并实现胃癌MDR逆转奠定基础。
本课题六部分的具体内容如下:
第一部分胃癌原发灶、区域淋巴结转移灶体外化疗药敏性的变化及其意义
目的:检测胃癌原发灶、区域淋巴结转移灶肿瘤细胞对11种胃癌化疗药物的体外敏感性,对其变化进行了分析,并对其意义进行了探讨。
方法:对56例伴淋巴结转移的胃癌原发灶及淋巴结转移灶新鲜肿瘤组织进行芬丹明B显色法(SRB法)原代细胞培养化疗药敏实验,比较两种病灶对11种化疗药物(5-FU、VP-16、HCPT、PTX、L-OHP、CDDP、eADM、THP、MMC、VCR、MTX)敏感性的差异,对结果进行t检验及Spearman相关分析。
结果:11种化疗药物对胃癌原发灶和淋巴结转移灶的肿瘤细胞平均抑制率均低于50%,7种药物对原发灶和淋巴结转移灶的肿瘤细胞抑制率有差异(均P<0.05),其中HCPT,L-OHP,VCR对转移淋巴结细胞抑制率明显低于原发灶(t分别为2.22、2.14、3.60,均P<0.05), VP-16、eADM、THP、MMC对原发灶肿瘤细胞抑制率明显低于淋巴结转移灶(t分别为-2.17、-2.03、-2.02、-2.43,均P<0.05) ,5-FU,PTX,CDDP,MTX对两种病灶肿瘤细胞的抑制率无明显统计学差异(均P>0.05)。5-FU,VP-16,HCPT,L-OHP,PTX,THP,MTX对原发灶和转移淋巴结的肿瘤细胞抑制率呈正相关(r分别为0.3735、0.4080、0.4067、0.5177、0.4006、0.5164、0.3566,均P<0.05)。
小结:1.以SRB显色法行胃癌细胞肿瘤体外化疗敏感性实验具有灵敏度高、结果准确、操作简单、成本较低等优点,值得临床推广。2.11种化疗药物对胃癌原发灶、淋巴结转移灶肿瘤细胞抑制率均较低,提示胃癌细胞对化疗药物均存在原发性MDR,以单药化疗控制肿瘤细胞是不现实的。3.大部分(7/11种)胃癌常用化疗药物(eADM、VP-16、THP、MMC、HCPT、L-OHP、VCR)对肿瘤细胞的抑制率在原发灶、转移淋巴结存在异质性变化,提示以原发灶药敏性结果指导术后针对转移/残留病灶的化疗是不准确的。
第二部分胃癌原发灶、区域淋巴结转移灶经典耐药因子P-gp、GST-π、TopoⅡα异质性表达及与体外化疗药敏性关系的研究
目的:MDR1/P-gp的药泵作用和GST-π的解毒作用及TopoⅡα的药物靶点调控作用是目前研究最多的肿瘤MDR的经典途径,在目前研究中受关注最多,研究也最为深入、广泛。本部分研究检测了胃癌原发灶和转移淋巴结P-gp、GST-π、TopoⅡα表达及肿瘤细胞体外化疗药物敏感性,并对结果进行了分析,对其意义进行了探讨。
方法:对56例胃癌新鲜肿瘤组织及转移淋巴结进行肿瘤细胞培养芬丹明B显色法化疗药敏性实验,并对原发灶和区域淋巴结转移灶组织行P-gp、GST-π、TopoⅡα免疫组化染色。计量资料采用配对t检验,等级资料使用Wilcoxon配对秩和检验、双变量相关分析并计算Spearman等级相关系数。
结果:1.胃癌原发灶、淋巴结转移灶化疗药敏性结果同第一部分。2. P-gp、GST-π在转移灶中的表达程度明显高于原发灶(S分别为182、111,均P<0.01),而TopoⅡα在原发灶中表达强于转移灶(S=-234, P<0.01)。双变量相关分析显示,在原发灶与转移淋巴结间P-gp表达具有明显正相关性(r=0.3303, P<0.05);而GST-π、TopoⅡα在原发肿瘤与淋巴结转移灶间无明显相关性(均P>0.05)。3.在胃癌原发灶中,P-gp强表达时,肿瘤细胞对PTX、HCPT、CDDP的耐药性增强(t分别为3.40、3.01、3.73,均P<0.01);GST-π强表达时CDDP对肿瘤细胞的抑制率降低(t=2.02, P<0.05);而TopoⅡα则与VCR、L-OHP的耐药性有关(t分别为-2.82、-2.82,均P<0.01)。在胃癌淋巴结转移灶中P-gp强表达时肿瘤细胞对VP-16、MTX的敏感性降低(t分别为2.92、2.18,均P<0.05);GST-π强表达与肿瘤细胞对11种药物的耐药性变化无关(均P>0.05);转移灶中TopoⅡα强表达时肿瘤细胞对PTX的敏感性增高(t=-3.04, P<0.01)。4.在肿瘤原发灶,P-gp表达强度与HCPT、PTX、CDDP对肿瘤细胞的平均抑制率均呈负相关(r=-0.2856, r=-0.2967,P=0.0264;均P<0.05);转移灶中P-gp表达程度与VP-16、MTX对肿瘤细胞抑制率均具有负相关性(r=-0.2753, r=-0.2665;均P<0.05)。原发灶GST-π表达程度与5-FU、VP-16的抑制率呈负相关(r=-0.3475, r=-0.2913;均P<0.05),转移灶GST-π表达程度则与HCPT、VCR的抑制率均具有负相关性(r=-0.3703, r=-0.3218;均P<0.05)。原发、转移灶中P-gp、GST-π表达与药物抑制率均未发现具有统计学意义的正相关(均P>0.05)。在肿瘤原发灶,TopoⅡα表达程度与L-OHP对肿瘤细胞的平均抑制率均呈正相关(r=0.3243, P<0.05);在转移灶中,TopoⅡα表达程度则与PTX对肿瘤细胞抑制率具有正相关性(r=0.4085,均P<0.01)。
小结:1. P-gp、GST-π、TopoⅡα在胃癌原发灶和区域淋巴结转移灶中均有较高的阳性表达率,且其表达在原发灶及淋巴结转移灶中出现了异质性改变,P-gp、GST-π在区域淋巴结转移灶中的表达程度明显高于原发灶,而TopoⅡα在转移灶中表达较原发灶减弱(S分别为182、111、-234,均P<0.01)。检测其在淋巴结转移灶中的表达情况对反映术后患者的MDR状态有重要意义。2.在胃癌原发灶和转移淋巴结P-gp、GST-π、TopoⅡα对化疗药物敏感性的影响也存在异质性改变,这些因子均参与了胃癌MDR,且与部分化疗药物的体外药敏性有关,但其表达并不能反映所有药物的耐药情况,以原发灶MDR因子表达来预测转移/残留病灶的化疗敏感性也是不准确的。3. P-gp、GST-π、TopoⅡα在临床组织中的表达情况不能解释全部MDR现象,进一步研究寻找新的主要MDR因子对胃癌MDR研究意义重大。
第三部分胃癌原发灶、区域淋巴结转移灶凋亡相关因子p53、Survivin、Bcl-2、Bax异质性表达及与体外化疗药敏性关系的研究
目的:近来研究显示肿瘤细胞凋亡调控机制在胃癌MDR中发挥着重要作用,故本部分研究对胃癌原发灶、淋巴结转移灶的凋亡相关因子p53、Survivin、Bcl-2、Bax表达情况进行了检测,并对其异质性变化情况及其与体外化疗药敏性的关系进行了分析。
方法:对56例胃癌新鲜肿瘤组织及区域淋巴结转移灶进行肿瘤细胞培养芬丹明B显色法化疗药敏性实验,并对原发灶和转移灶行凋亡相关蛋白p53、Survivin、Bcl-2、Bax以免疫组化染色检测其表达。计量资料采用配对t检验,等级资料使用Wilcoxon配对秩和检验、双变量相关分析并计算Spearman等级相关系数。
结果:1.胃癌原发灶、淋巴结转移灶化疗药敏性结果同第一部分。2.经Wilcoxon配对秩和检验,Bcl-2、Bax在转移灶中的表达程度明显高于原发灶(S分别为117、111,均P<0.05),而p53、Survivin在转移灶中表达与原发灶无明显差异(均P>0.05)。双变量相关分析显示,在原发灶与转移淋巴结间p53、Bcl-2、Bax表达具有明显正相关性(r分别为0.7255、0.4262、0.2901;均P<0.05)。3.在胃癌原发灶中,p53强表达时,肿瘤细胞对PTX、CDDP的耐药性增强(t分别为2.18、3.64,均P<0.05);Survivin强表达时VCR、PTX、eADM对肿瘤细胞的抑制率降低(t分别为2.10、3.02、2.07,均P<0.05),但L-OHP对肿瘤细胞的抑制率反而升高(t=-2.10,P<0.05);而Bcl-2强表达则与5-FU、PTX、eADM和HCPT的耐药性增强有关(t分别为2.76、2.38、2.16、4.27,均P<0.05);Bax强表达时,5-FU、eADM、HCPT对肿瘤细胞的抑制率升高(t分别为-2.51、-3.32、-2.30,均P<0.05)。在胃癌淋巴结转移灶中p53强表达时肿瘤细胞对PTX的敏感性降低(t=2.03,P<0.05);转移灶中Survivin强表达时肿瘤细胞仅对MTX的敏感性降低(t=3.28,P<0.01);转移灶Bcl-2表达增强时肿瘤细胞对5-FU、VCR、VP-16、PTX的耐药性增强(t分别为2.43、2.65、6.78、3.19,均P<0.05);转移灶Bax表达增强则未发现与肿瘤细胞耐药性增强有关(均P>0.05)。4.经Spearman相关分析,在肿瘤原发灶和转移灶中,原发灶p53表达程度与CDDP的抑制率呈负相关(r=-0.4648, P<0.01),转移灶中未发现p53与11种药物对肿瘤细胞抑制率均具有相关性(均P>0.05)。原发灶Survivin表达程度与5-FU、PTX、eADM的抑制率呈负相关(r分别为-0.4805、-0.2979、-0.2861;均P<0.05),而与L-OHP的抑制率呈正相关(r=0.3329, P<0.05);转移灶Survivin表达程度则与MTX的抑制率均具有负相关性(r=-0.3611, P<0.01)。在肿瘤原发灶,Bcl-2表达程度与5-FU、VP-16、HCPT、PTX、eADM对肿瘤细胞的平均抑制率均呈负相关(r分别为-0.3722、-0.3032、-0.5035、-0.3973、-0.2712;均P<0.05);在转移灶中,Bcl-2表达程度则与5-FU、VP-16、PTX、VCR对肿瘤细胞抑制率具有负相关性(r分别为-0.2737、-0.6238、-0.3918、-0.3469,均P<0.05)。原发灶Bax表达程度与5-FU、HCPT、eADM的抑制率呈正相关(r分别为0.4377、0.3666、0.3517,均P<0.05),转移灶Bax表达程度则未发现与药物对肿瘤细胞的抑制率均具有相关性(均P>0.05)。
小结:1.凋亡相关因子在胃癌原发灶和转移淋巴结中表达情况及相互间的相关关系都出现了异质性改变,Bcl-2、Bax在区域淋巴结转移灶中的表达程度明显高于原发灶(均P<0.05),而p53、Survivin在转移灶中表达与原发灶无明显差异(均P>0.05);检测其在淋巴结转移灶中的表达情况对反映术后患者的MDR状态有重要意义。2.在胃癌原发灶和转移淋巴结中各凋亡相关因子对化疗药物敏感性的影响也存在异质性改变,研究证实这些因子均参与了胃癌MDR,且与部分化疗药物的体外药敏性有关,但其表达并不能反映所有药物的耐药情况,以原发灶凋亡相关因子表达来预测转移/残留病灶的化疗敏感性也是不准确的。3.本实验虽证实凋亡相关因子与胃癌MDR有关,但尚未发现任何一种凋亡相关因子与胃癌细胞全部/大部分MDR现象有关,寻找胃癌MDR关键因子、实现MDR逆转尚待进一步深入研究。
第四部分胃癌原发灶、区域淋巴结转移灶COX-2表达与多药耐药因子及与体外化疗敏感性关系的研究
目的:研究表明环氧合酶-2(COX-2)与胃癌MDR关系密切,可通过调节其他MDR因子表达参与胃癌MDR。故本研究对胃癌原发灶、淋巴结转移灶的COX-2表达情况进行了检测,并对其异质性变化情况及其与其他MDR因子、体外化疗药敏性的关系进行了分析。
方法:对56例胃癌新鲜肿瘤组织及转移淋巴结进行肿瘤细胞培养芬丹明B显色法化疗药敏性实验,并对原发灶和转移灶行COX-2及其他MDR因子免疫组化染色。统计方法同第二部分。
结果:1.胃癌原发灶、淋巴结转移灶化疗药敏性结果同第一部分。2. COX-2在转移灶中的表达程度明显高于原发灶(S=190, P<0.01);双变量相关分析显示,在原发灶与转移淋巴结间COX-2表达具有明显正相关性(r=0.3511, P<0.01)。3.在胃癌原发灶中,COX-2表达与GST-π、Survivin、Bcl-2表达呈正相关(r分别为0.2741、0.7024、0.5925,均P <0.05),与TopoⅡα、Bax表达呈负相关(r分别为-0.3488、-0.3292,均P<0.05)。在淋巴结转移灶中,COX-2表达与P-gp、Survivin、Bcl-2表达呈正相关(r分别为0.3490、0.2836、0.5368,均P<0.05)。4.在胃癌原发灶中,COX-2强表达时,5-FU、VCR、PTX、eADM、HCPT对肿瘤细胞的抑制率降低(t分别为2.38、2.50、3.15、2.39、2.55,均P<0.05);在胃癌淋巴结转移灶中,COX-2表达增强时肿瘤细胞对5-FU、VCR、VP-16、MTX的耐药性增强(t分别为3.33、4.11、2.90、2.63,均P<0.05)。5.在肿瘤原发灶,COX-2表达程度与5-FU、HCPT、PTX、eADM、THP、VCR对肿瘤细胞的平均抑制率均呈负相关(r分别为-0.5385、-0.3077、-0.3850、-0.2991、-0.3466、-0.4158,均P<0.05);在转移灶中,COX-2表达程度则与5-FU、VP-16、PTX、VCR、MTX对肿瘤细胞抑制率具有负相关性(r分别为-0.4120、-0.4421、-0.3300、-0.3597、-0.2836,均P <0.05)。
小结:1. COX-2在转移过程中发生了异质性变化,且转移灶中COX-2与原发灶表达存在正相关关系,说明COX-2对肿瘤的转移起促进作用。2.原发灶中COX-2与P-gp、GST-π、TopoⅡα等经典MDR因子的关系与转移灶情况不同。3. COX-2与凋亡相关因子p53、Survivin、Bcl-2、Bax的关系在原发灶和淋巴结转移灶中也不相同。4.与原发灶COX-2表达有关的化疗药物和转移灶中不同,提示COX-2逆转剂在术后化疗中的意义尚不能确定,进一步深入研究其机制并寻找新MDR相关因子有重要意义。第五部分不同分化程度胃癌细胞株差异表达蛋白质的筛选鉴定及与胃癌多药耐药关系的研究
目的:研究发现消化道肿瘤的MDR与肿瘤分化程度有关,故本研究以不同分化的胃癌细胞为研究对象,应用先进的蛋白质组学技术对胃癌分化相关蛋白质进行了筛选鉴定,并对其与胃癌MDR的关系进行了分析。
方法:采用双向凝胶电泳及基质辅助激光解吸电离-飞行时间质谱鉴定法对三种分化程度不同的胃癌细胞株MKN28(高分化)、SGC7901(中分化)和BGC823(低分化)进行了研究,并应用蛋白印迹试验法对鉴定出的部分蛋白质在胃癌细胞株及胃癌组织中的表达进行了验证。
结果:三种胃癌细胞株的双向电泳凝胶所得蛋白质点均在1000个左右,表达差异明显的蛋白质点14个,经质谱仪分析,8种蛋白质得到鉴定,为类硫氧还蛋白过氧化物酶、甘油醛-3-磷酸脱氢酶、β-微管蛋白多肽、假定蛋白、锌指蛋白139、蛋白酪氨酸激酶、钙网蛋白前体、原肌球蛋白。这些蛋白质与胃癌细胞信号转导、维持细胞稳态、参与糖酵解及肿瘤药物代谢、抗氧化损伤等功能有关。经蛋白印迹试验法检测,证实这些蛋白质在胃癌细胞株及胃癌组织中的表达与蛋白质组学所得结果一致。
小结:1.不同分化的胃癌细胞蛋白质表达存在差异。本研究中双向电泳分离出的蛋白质点较多,鉴定的蛋白质点经Western blot对肿瘤组织及细胞株进行验证,所得结果均与蛋白质组学结果相符合,说明本研究有较高的可信性及意义。2.对8种差异蛋白质的分析显示,这些蛋白质涉及肿瘤药物代谢、细胞信号转导、维持细胞稳态、参与糖酵解及抗氧化损伤等多个方面,这些蛋白质的表达与胃癌细胞分化程度密切相关,其表达差异造成了胃癌生物学活性的不同,以其为靶标进行干预有可能逆转胃癌细胞的某些恶性生物学行为。
第六部分胃癌细胞株SGC7901及耐药细胞株SGC7901/VCR差异点筛选鉴定的蛋白质组学研究
目的:为获得与胃癌MDR直接相关的新蛋白质,本研究应用蛋白质组学技术对胃癌细胞株SGC7901和耐药细胞株SGC7901/VCR的差异蛋白质进行了筛选鉴定,并对结果及意义进行了分析。
方法:同第五部分。
结果:在两种胃癌细胞株中所得蛋白质点均超过1000个,找到表达差异明显的蛋白质点9个,经质谱分析,7种蛋白质得到鉴定,为S60核糖体蛋白L23、电压依赖性阴离子选择性通道蛋白1、锌指蛋白394、角蛋白I型细胞骨架9、核糖核酸3'-端磷酸化酶、matrin型锌指蛋白2、Sideroflexin-1。这些蛋白质与肿瘤药物代谢、胃癌细胞信号转导、维持细胞稳态、参与增殖凋亡、促进细胞成熟等功能有关。经蛋白印迹试验法检测,证实这些蛋白质在胃癌细胞株中的表达与蛋白质组学所得结果一致。
小结: 1.本研究中胃癌细胞株SGC7901及其耐药细胞株SGC7901/VCR的双向电泳凝胶所得蛋白质点较多,质谱鉴定的蛋白质经Western blot验证,所得结果与蛋白质组学结果符合,说明本研究的可信性较高,所得蛋白质点与胃癌细胞MDR有直接相关性。2.对差异蛋白质的分析显示,这些蛋白质大多涉及肿瘤药物代谢,同时涉及胃癌细胞的其他生物学特性,这些蛋白质的表达与胃癌细胞MDR密切相关,其表达差异造成了胃癌细胞MDR表型的不同,进一步进行研究有可能找到关键/重要MDR因子,以其为靶标进行干预有可能逆转胃癌细胞的MDR特性,提高术后化疗效果并改善预后。
综合上述六部分内容,本研究得出如下结论:
1.胃癌细胞对大部分化疗药物的体外药敏性在原发灶、转移淋巴结存在异质性变化,以原发灶药敏性结果指导术后针对转移/残留病灶的化疗是不准确的,术后化疗应针对转移灶的特点进行。
2.经典MDR因子(P-gp、GST-π、TopoⅡα)和凋亡相关因子(p53、Survivin、Bcl-2、Bax)表达在原发灶及淋巴结转移灶中出现了异质性改变;在胃癌原发灶和转移淋巴结经典MDR因子和凋亡相关因子对化疗药物敏感性的影响也存在异质性改变,这些因子的表达只与部分化疗药物的体外药敏性有关,并不能反映所有药物的耐药情况;这些因子在临床组织中不能解释全部MDR现象,研究寻找新的主要MDR因子对胃癌MDR研究意义重大。
3. COX-2对胃癌的转移起促进作用,且本身在转移中发生异质性改变。原发灶中COX-2与经典MDR因子和凋亡相关因子的关系与转移灶情况不同。与原发灶COX-2表达有关的化疗药物和转移灶不同。
4.应用蛋白质组学技术对筛选鉴定了8种胃癌分化相关蛋白质,并在胃癌细胞株和组织中得到了验证。经功能分析,发现其中β-微管蛋白多肽、蛋白酪氨酸激酶等与胃癌耐药有关。
5.以胃癌细胞株SGC7901和耐药细胞株SGC7901/VCR为研究对象,应用蛋白质组学技术鉴定出7种与胃癌MDR直接相关的蛋白质,并在胃癌细胞株中进行了验证。
Chemotherapy is important in comprehensive treatments of gastric carcinoma, especially in the patients with primary tumors resected, which is the most important therapeutic means, for this kind of cancer is insensitive to radiotherapy. But multidrug resistance (MDR) of gastric carcinoma often causes the failure of chemotherapy. MDR means that when tumor cells are resistant to a chemotherapeutic drug, they are often resistant to other drugs of different structure and mechanism. MDR contains natural MDR and acquired MDR. Natural MDR means that inherent insensitivity to chemotherapeutic drugs exist in tumor cells, so MDR will occur when a kind of chemotherapeutic drug is first used, and gastric carcinoma cells are tumor cells with obvious MDR phenotypes; acquired MDR means that tumor cells are sensitive to chemotherapeutic drugs in the beginning, but after several courses of treatment, MDR would occur, and it is common during clinical therapy for gastrointestinal tract carcinomas. MDR of gastric carcinoma causes the percentage of effectiveness is less than 50%, and it is related to low survival rate(less than 30%) of advanced gastric carcinoma. It will have great realistic meaning if the reversion of MDR for gastric carcinoma comes true.
Research about MDR of tumors has been on for over 30 years. Now nearly 10 kinds of mechanism have been proven to have explicit relationship with MDR, and they are as follows:①Overexpression of MDR1/P-gp;②Overexpression of MRP;③Enhancement of exocytosis by LRP;④Reinforcement of cell’s detoxification and repair effects of DNA damage;⑤The drug targets’sufficient either in quality or quantity;⑥Reinforcement of inhibitor of apoptosis proteins and reduction of apoptosis proteins;⑦Change of extracellular environment(such as pH, temperature or oxygenconcentration), hypoxia inducible factors-1alpha(HIF-1α) is research focus recently;⑧Other factors related to MDR, such as COX-2. It is generally recognized that MDR of gastric carcinoma is caused by all these factors’synergy.
But with detailed study, scholars have found that many defects exist in the explanation about MDR of gastric carcinoma with these known mechanisms. The problems exist as follows:①These mechanisms can not explain all phenomena of gastric cancer MDR. The reason is most studies’objects are one/several MDR-related genes and proteins, and they often can not be used to explain all MDR phenomena in clinical studies though there often has clear conclusion in the cell lines or animal experiments.②Malignant tumors come from monoclonal proliferation, but changes in the genes or molecules often appear in the evolution of tumors. Changes (Heterogeneity) may arise in the anti-cancer drug sensitivity and expression of MDR-related factors. From a clinical point of view, most gastric carcinomas in China belong to advanced stage, and metastasis rate of lymph node reaches 60%-80%, which has been thought as independent prognostic factor, and most patients died of gastric cancer recurrence / or metastasis after primary tumors were resected. Current chemotherapy regimens often are experiential programs, and the efficacy is poor. Some reports have shown that chemosensitivity test in vitro of tumor cells can be used as evidence in the selection of chemotherapeutics, but these results cannot reflect condition of multi-channel and multi-factors in vivo, also they cannot be on behalf of the results of lymph node metastases (LNMs). MDR in LNMs of gastric carcinoma has been rarely reported in the literature both at home and abroad.
Our study of these series is based on the problems above, and we prepare to conduct the following study of gastric cancer MDR. Our study is divided into two series as follows:①Series 1 is divided into 4 four parts, the relationship between MDR related factors and chemosensitivity in vitro has been investigated from the most established ways that lead to MDR of gastric carcinoma; characters of MDR in LNMs are also studied, and heterogeneity is analyzed compared with results of PT.②Series 2 is to find new key factor of MDR. Advanced techniques of proteomics are used to screen and identificate differentially expressed proteins in different gastric carcinoma cell lines. This study may be helpful in mechanism and reversal in MDR of gastric carcinoma.
Protein is the direct executor of the life’s structure and activities, so it’s more important to investigate protein than gene. Proteome means all the proteins encoded by genome and proteomics take all proteome encoded by genome as object, then it analysis dynamic changes of intracellular protein composition and activities of law as a whole. Research of proteomics depends on three techniques: technique of protein components’separation, identification technology of protein components and technique of function prediction. Proteomics brings new mode of thinking in the research of MDR about malignant tumors. Proteomics studies mechanism of MDR about malignant tumors from the overall level of tissue or cell protein. In the methodological, it’s unnecessary to determine the definite object of study, and also it’s unnecessary to prepare antibody in advance. This greatly broadens the scope of the study, and we can be more accurate analysis of life phenomena.
The main contents of this article are as follows: Part one: Chemosensitivity of regional lymph node metastases compared to the primary tumors with SRB assay in vitro for patients with advanced gastric carcinoma
Objective: To investigate the different chemosensitivity of lymph node metastases cultures compared to the primary tumor in gastric carcinoma gastric carcinoma by sulphorhodamine B (SRB) assay.
Methods: In vitro SRB assay was performed on 56 paired surgical specimens of primary tumors (PT) and lymph node metastases (LNMs) from 56 gastric carcinoma patients. The results were then compared with response to 11 antitumor drugs(5-FU, VP-16, HCPT, PTX, L-OHP, CDDP, eADM, THP, MMC, VCR, MTX). Data was dealt with t test and Spearman correlation analysis.
Results: 1.There was significantly different chemosensitivity of individuals to antitumor drugs between PT and LNMs in 7/11 drugs(all P<0.05). The inhibition rates of tumor cells in LNMs for HCPT, L-OHP and VCR were lower than those in PT(all P<0.05), whereas the inhibition rates of VP-16, eADM, THP, MMC were higher in LNMs than those in PT(all P<0.05). No significantly difference of inhibition ratio between PT and LNMs was found in 5-FU,PTX,CDDP,MTX(all P>0.05). 2.There was a significantly positive correlation between the inhibition rates of PT and LNMs for 5-FU,VP-16, HCPT, L-OHP,PTX,THP,MTX (r=0.3735~0.5177, all P<0.05).
Summary: 1. SRB assay has many advantages such as high sensitivity, pin-point accuracy, more simple operation, lower cost, etc. So it’s worthy of promotion in clinic. 2. All 11 drugs have low inhibition rates for gastric carcinoma cells , and it shows that natural MDR exists in gastric carcinoma cells, so it’s unrealistic to control this cancer with one drug. 3. Heterogeneity of chemosensitivity to most antitumor drugs is shown in metastatic lymph nodes in gastric cancers. The chemosensitivity of gastric carcinoma primary tumor in the drugs response assay in votro may not give a good indication of the chemosensitivity in metastatic lesions. Chemotherapy after operation should be carried on according to characteristic of LNMs in patients with advanced gastric carcinoma.
Part two: Relationship between expression of P-gp, GST-π, TopoⅡαand chemosensitivities in primary tumors(PT) and regional lymph node metastases(LNMs) of gastric carcinoma
Objects: The most studied classical pathways of MDR include drug pump of MDR1/P-gp, detoxification of GST-πand drug targets regulation of TopoⅡα. These pathways are most viewed. In this part, expression of P-gp, GST-π, TopoⅡand chemosensitivities were detected in PT and LNMs, their relationship was analyzed, and the significance that may exist was discussed.
Methods: In vitro SRB assay was performed on 56 paired fresh surgical specimens of PT and LNMs from 56 gastric carcinoma patients. Expression of P-gp, GST-π, TopoⅡwas tested by immunohistochemical staining. Measurement data were analyzed with t test, ranked data with Wilcoxon rank test, and Spearman correlation analysis was also used to calculate correlation coefficient.
Results: 1.The results of chemosensitivity in vitro in tumor cells of PT and LNMs were seen in Part one. 2. The expression of P-gp and GST-πwas higher in LNMs than in PT(both P<0.01), and expression of TopoⅡαwas higher in PT(P<0.01). There was positive correlativity of P-gp between PT and LNMs(r=0.3303, P<0.05), and no significant correlativity was found about GST-πand TopoⅡαbetween PT and LNMs(both P>0.05). 3. In PT the inhibition rates for PTX, HCPT, CDDP in P-gp strong expression group were lower than those in weak group (all P<0.05); The inhibition rate to CDDP was significantly lower for the GST-πstrong expression group in PT (P<0.05); and for strong expression of TopoⅡα, the inhibition rates for VCR, L-OHP were higher(both P<0.05). In LNMs, there were lower inhibition rates for VP-16, MTX in P-gp strong expression group (both P<0.05), and the inhibition rate to PTX was significantly lower for the TopoⅡαweak expression group in LNMs(P<0.05); no significant change was found between inhibition rates for these drugs and expression of GST-π(all P>0.05).4. The inhibition rates to HCPT, PTX, CDDP were negatively correlated with the expression of P-gp in PT (r=-0.2856, r=-0.2967,P=0.0264; all P<0.05), and negative correlation was found between the inhibition rates to VP-16, MTX and the expression of P-gp in LNMs(r=-0.2753, r=-0.2665; both P<0.05). The expression of GST-πwas negatively correlated with the inhibition rates to 5-Fu, VP-16 in PT( r=-0.3475, r=-0.2913; both P<0.05), and negatively correlated with HCPT, VCR(r=-0.3703, r=-0.3218;both P<0.05). The expression of TopoⅡαwas positively correlated with inhibition rates to L-OHP in PT(r=0.3243, P<0.05), and positive correlation was found between the expression of TopoⅡαand inhibition rates to PTX in LNMs(r=0.4085, P<0.05).
Summary: 1. There was higher positive expression rate of P-gp, GST-π, TopoⅡαbetween PT and LNMs of gastric carcinoma, and expression of these proteins showed heterogeneity between PT and LNMs. It’s of great significance to test expression of P-gp, GST-π, TopoⅡαin LNMs, and it can reflect the MDR status in patients after PT resected. 2. There was heterogeneity in the relationship about expression of P-gp, GST-π, TopoⅡαand chemosensitivity in 11 chemotherapeutics between PT and LNMs. The results showed that these factors all participate in MDR of gastric carcinoma, and there existed relationship between expression of them and chemosensitivity in part of chemotherapeutics, but expression of them could not reflect chemosensitivity of all drugs. So it was inaccurate to predict MDR of LNMs with results of PT. 3. P-gp, GST-π, TopoⅡαcould not explain all phenomena of MDR, so it’s of great significance to search new MDR related factors to explore the mechanism of MDR of gastric carcinoma.
Part three: Relationship between expression of apoptosis-related proteins (p53, Survivin, Bcl-2, Bax) and chemosensitivities in primary tumors (PT) and regional lymph node metastases (LNMs) of gastric carcinoma
Objective: Many recent researches have shown that regulatory mechanisms of apoptosis of tumor cells play an important role in MDR of gastric carcinoma.So in this part, expression of apoptosis-related proteins (p53, Survivin, Bcl-2, Bax) and chemosensitivities in vitro were detected in PT and LNMs, their relationship was analyzed, and the significance that may exist were discussed.
Methods: In vitro SRB assay was performed on 56 paired fresh surgical specimens of PT and LNMs from 56 gastric carcinoma patients. Expression of p53, Survivin, Bcl-2, Bax was tested by immunohistochemical staining. Measurement data were analyzed with t test, ranked data with Wilcoxon rank test, and Spearman correlation analysis was also used, and correlation coefficient was calculated.
Results: 1.The results of chemosensitivity in vitro in tumor cells of PT and LNMs were seen in Part one. 2. The expression of Bcl-2, Bax was higher in LNMs than in PT(both P<0.01), and there was no significant difference in expression of p53, Survivin between PT and LNMs(both P>0.05). There was positive correlativity of p53, Bcl-2, Bax between PT and LNMs(r=0.7255, r=0.4262, r=0.2901; P<0.05). 3. In PT the inhibition rates for PTX, CDDP in p53 strong expression group were lower than those in weak group (both P<0.05); the inhibition rates to VCR, PTX, eADM were significantly lower for the Survivin strong expression group in PT (all P<0.05), but for L-OHP, the inhibition rate was higher when the tumor with stronger expression of Survivin(P<0.05); and for strong expression of Bcl-2, the inhibition rates for 5-FU, PTX, eADM, HCPT were lower(all P<0.05); for strong expression of Bax, the inhibition rates for 5-FU, eADM, HCPT were higher(all P<0.05). In LNMs, there was lower inhibition rate for PTX in p53 strong expression group (P<0.05), the inhibition rate to MTX was significantly lower for the Survivin strong expression group in LNMs(P<0.01); and for strong expression of Bcl-2, the inhibition rates for 5-FU, VCR, VP-16, PTX were lower(all P<0.05); no significant change was found between inhibition rates for these drugs and expression of Bax(all P>0.05).4. There was negatively correlation between inhibition rate to CDDP and the expression of p53 in PT(P<0.01) , and no significant relationship was found between expression of p53 and inhibition rates for these 11 drugs(all P>0.05). The inhibition rates to 5-FU, PTX, eADM were negatively correlated with the expression of Survivin in PT (r=-0.4805, r=-0.2979, r=-0.2861; all P<0.05), and there was positive correlation was found between inhibition rate to L-OHP and expression of Survivin(r=0.3329, P<0.05); negative correlation was found between the inhibition rates to MTX and the expression of Survivin in LNMs(r=-0.3611; P<0.01). The expression of Bcl-2 was negatively correlated with the inhibition rates to 5-FU, VP-16, HCPT, PTX, eADM in PT(r=-0.3722, r=-0.3032, r=-0.5035, r=-0.3973, r=-0.2712;all P<0.05), and negatively correlated with 5-FU, VP-16, PTX, VCR(r=-0.2737, r=-0.6238, r=-0.3918, r=-0.3469;all P<0.05)in LNMs. The expression of Bax was positively correlated with inhibition rates to 5-FU, HCPT, eADM in PT(r=0.4377, r=0.3666, r=0.3517;all P<0.05), and no significant correlation was found between the expression of Bax and inhibition rates to these drugs in LNMs(all P>0.05).
Summary: 1. There was heterogeneity in expression of apoptosis-related proteins (p53, Survivin, Bcl-2, Bax) between PT and LNMs. The results showed that it’s valuable to test these factors in LNMs to reflect situation of MDR in patients with PT resected. 2. There was heterogeneity in the relationship between expression of p53, Survivin, Bcl-2, Bax and chemosensitivity in 11 chemotherapeutics between PT and LNMs. The results showed that these factors all participate in MDR of gastric carcinoma, and there existed relationship between expression of them and chemosensitivity in part of chemotherapeutics, but expression of them could not reflect chemosensitivity of all drugs. So it was inaccurate to predict MDR of LNMs with results of PT. 3. Results showed that p53, Survivin, Bcl-2, Bax all participate in MDR, but none of them could explain all phenomena of MDR, so it’s of great significance to search new key MDR related factors to explore the mechanism of MDR of gastric carcinoma.
Part four: Relationship between expressions of cyclooxygenase-2(COX-2), MDR related factors and chemosensitivities in vitro in primary tumors (PT) and regional lymph node metastases (LNMs) of gastric carcinoma
Objective: Many researches in recent years have shown that cyclooxygenase-2(COX-2) has close relationship with MDR of gastric carcinoma, and it can participate in MDR gastric carcinoma by regulating expression of other of MDR related factors. So in this part, expression of COX-2, some other MDR related factors and chemosensitivities in vitro were detected in PT and LNMs, their relationship was analyzed, and the significance that may exist was investigated.
Methods: In vitro SRB assay was performed on 56 paired fresh surgical specimens of PT and LNMs from 56 gastric carcinoma patients. Expression of COX-2, some other MDR related factors were tested by immunohistochemical staining. Statistical analysis was the same as Part three.
Results: 1.The results of chemosensitivity in vitro in tumor cells of PT and LNMs were seen in Part one. 2. The expression of COX-2 was higher in LNMs than in PT(P<0.01), and there was positive correlativity of COX-2 between PT and LNMs(r=0.3511, P<0.01). 3. In PT, there was positive correlation between the expression of COX-2 and other MDR related factors as following: GST-π, Survivin, Bcl-2(r=0.2741,r=0.7024,r=0.5925;all P <0.05), and negative correlation was found between expression of COX-2 and TopoⅡα, Bax(r=-0.3488, r=-0.3292,both P <0.05). In LNMs, positive correlation was found between expression of COX-2 and P-gp, Survivin, Bcl-2(r=0.3490,r=0.2836,r=0.5368;all P <0.05). 4.In PT the inhibition rates for 5-FU, VCR, PTX, eADM, HCPT in COX-2 strong expression group were lower than those in weak group (all P<0.01). In LNMs, there were lower inhibition rates for 5-FU, VCR, VP-16, MTX in COX-2 strong expression group (all P<0.05).5. The inhibition rates to 5-FU, HCPT, PTX, eADM, THP, VCR were negatively correlated with the expression of COX-2 in PT (r=-0.5385,r=-0.3077,r=-0.3850,r=-0.2991,r=-0.3466,r=-0.4158;all P <0.05), and negatively correlated with 5-FU, VP-16, PTX, VCR, MTX(r=-0.4120, r=-0.4421, r=-0.3300,r=-0.3597, r=-0.2836;all P <0.05)in LNMs.
Summary: 1. There was heterogeneity in expression of COX-2 between PT and LNMs, and positive correlativity was found between expression of COX-2 in PT and in LNMs, which showed that COX-2 played an important role in metastasis of gastric carcinoma. 2. There was heterogeneity in the relationship about COX-2 and P-gp, GST-π, TopoⅡαbetween PT and LNMs. 3. There was heterogeneity in the relationship of COX-2 and p53, Survivin, Bcl-2, Bax between PT and LNMs. 3. Results showed that COX-2 participates in MDR of gastric carcinoma, but it could not explain all phenomena of MDR, and it is uncertain about the effect of inhibitor of COX-2 in the clinical therapy. So it’s of great significance to search new key MDR related factors to explore the mechanism of MDR of gastric carcinoma.
Part five: Identification of differentiation-related proteins in gastric carcinoma cell lines by comparative proteomics and their relationship with MDR of gastric carcinoma
Objective: Researches have shown that MDR of digestive tract was in related to differentiation of tumor cells. So in this part, differentiation of gastric cancer cell lines were used as object to investigate differentiation-related proteins in human gastric carcinoma cell lines by comparative proteomics, and then their relationship with MDR of gastric carcinoma was studied.
Methods: 1 The holoproteins of human gastric carcinoma cell lines MKN28(well differentiated), SGC7901(moderately differentiated) and BGC823(poorly differentiated) were measured by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).Some proteins obtained through proteomics were tested by Western blot in the cell strain and tissues of gastric carcinoma.
Results: There were different proteins in human gastric carcinoma cell lines. All 14 different protein spots were found in 3 gastric carcinoma cell lines, and 8 spots were identified by MALDI-TOF-MS, and these proteins were probable thioredoxin peroxidase, glyceraldehyde-3-phosphate dehydrogenase (GAPD),β-tubulin polypeptide, hypothetical protein, zinc finger protein (ZNF)139, protein-tyrosine kinase, calreticulin precursor, tropomyosin. These proteins have relationship with the biological behavior of gastric carcinoma, such as signal transduction, cellular homeostasis, glycolysis, antioxidation action, multidrug resistance (MDR), etc. Result of Western blot about protein expression was in consonance with situation of proteomics.
Summary: 1. Proteins spots got from 3 gastric cells were all 1000 more or less, and 8 spots of 14 were identificated, part of them verificated by Western blot, which showed it was credible of the results. 2. These 8 proteins were involved in many biological behaviors of gastric carcinoma, such as signal transduction, cellular homeostasis, glycolysis, antioxidation action, multidrug resistance (MDR), etc. The results showed that intervention for these proteins could reverse malignant some biological behaviors of gastric carcinoma.
Part six: Identification of gastric carcinoma cell line SGC7901 and tumor MDR cell strain SGC7901/VCR by comparative proteomics
Objective: To obtain new proteins directly related to the MDR of gastric carcinoma, comparative proteomics was used in this part, and gastric carcinoma cell line SGC7901 and tumor MDR cell strain SGC7901/VCR were used as objects to identificate new differently expressed proteins.
Methods: Methods were the same as Part five.
Results: There were different proteins in these 2 gastric carcinoma cell lines. All 9 different protein spots were found, and 7 spots were identified by MALDI-TOF-MS. These proteins were 60S ribosomal protein L23, voltage-dependent anion-selective channel protein (VDAC1), zinc finger protein (ZNF)394, keratin, type I cytoskeletal 9(KⅠC9), RNA 3'-terminal phosphate cyclase(RTC1), zinc finger matrin-type protein 2, Sideroflexin-1. These proteins have relationship with the biological behavior of gastric carcinoma, such as multidrug resistance (MDR), cellular signal transduction, maintenance of cellular homeostasis, proliferation and apoptosis, maturation of cells, etc. Result of Western blot about protein expression was in consonance with situation of proteomics.
Summary: 1. Proteins spots got from 3 gastric cells were all over 1000, and 7 spots of 9 were identificated, parts of which were certificated by Western blot assay, which showed it was credible of the results, and these proteins were directly related to the MDR of gastric carcinoma. 2. These 7 proteins were involved in many biological behaviors of gastric carcinoma, such as multidrug resistance (MDR), cellular signal transduction, maintenance of cellular homeostasis, proliferation and apoptosis, maturation of cells, etc. The results showed that intervention for these proteins could reverse MDR of gastric carcinoma, improve the effect of chemotherapy, and improve the prognosis.
Conclusions:
1. There was heterogeneity in chemosensitivity in vitro related between PT and LNMs, so it’s inaccurate to design chemotherapy for patients with PT resected according to results of chemosensitivity test for PT, it should be designed to aim at MDR features of LNMs.
2. There was heterogeneity in expression of MDR related factors(P-gp, GST-π, TopoⅡα, p53, Survivin, Bcl-2, Bax) between PT and LNMs. Heterogeneity was also found in the relationship about expression of MDR related factors and chemosensitivity in chemotherapeutics between PT and LNMs. The results showed that these factors all participate in MDR of gastric carcinoma, and there existed relationship between expression of them and chemosensitivity in part of chemotherapeutics, but expression of them could not reflect chemosensitivity of all drugs. It’s of great significance to search new key MDR related factors to explore the mechanism of MDR of gastric carcinoma.
3. Results showed that COX-2 participates in MDR of gastric carcinoma, and there was heterogeneity in expression of COX-2 between PT and LNMs. Relationship between COX-2 and MDR related factors was different from that in LNMs, and heterogeneity was also found in the relationship about expression of COX-2 and chemosensitivity in chemotherapeutics between PT and LNMs.
4. 8 spots of 14 differentiation-related proteins were identificated, which were probable thioredoxin peroxidase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH),β-tubulin polypeptide, hypothetical protein, zinc finger protein (ZNF)139, protein-tyrosine kinase, calreticulin precursor, tropomyosin; part of them were certificated by Western blot. In these proteins,β-tubulin polypeptide and protein-tyrosine kinase were in related to MDR of gastric carcinoma.
5. 7 proteins directly related to the MDR of gastric carcinoma were identificated by comparative proteomics in gastric carcinoma cell line SGC7901 and tumor MDR cell strain SGC7901/VCR, which were 60S ribosomal protein L23, voltage-dependent anion-selective channel protein (VDAC1), zinc finger protein (ZNF)394, keratin, type I cytoskeletal 9(KⅠC9), RNA 3'-terminal phosphate cyclase(RTC1), zinc finger matrin-type protein 2, Sideroflexin-1; part of them were certificated by Western blot assay.
引文
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