耐药基因ABCG2在食管癌中的表达及青蒿琥酯逆转耐药的作用研究
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摘要
目的:食管癌(esophageal carcinoma, EC)是最常见的消化道恶性肿瘤之一,我国是食管癌高发区,发病率仅次于胃癌而居消化道肿瘤发病率第二位,严重威胁着人们的生命健康。化疗是目前食管癌综合治疗的主要方法之一,但是在化疗过程中出现的多药耐药(Multidrug resistance,MDR)是导致临床化疗失败的重要原因,尤其对于一些复发性肿瘤多药耐药的现象就更为普遍。化疗过程中出现的多药耐药现象,是指一种药物作用于肿瘤细胞产生耐药性后,该肿瘤对未接触过的、结构无关、机制不同的抗肿瘤药物也具有了交叉耐药。由于多药耐药决定了临床上化疗的成败及疗效,目前对多药耐药的研究非常广泛。研究发现,肿瘤细胞主动将化疗药物排出细胞外是引起多药耐药产生的机制之一,而此种机制常与一类跨膜蛋白有关,进一步研究发现,这类跨膜蛋白多属于三磷酸腺苷结合盒(ATP-binding cassette, ABC)转运蛋白家族,目前在人类至少有48种,其中研究最多的ABC蛋白为P-糖蛋白(P-gp)又称三磷酸腺苷结合转运蛋白B1(ATP-binding cassette B1,ABCB1)和乳腺癌耐药蛋白(breast cancer resistance protein, BCRP)又称三磷酸腺苷结合转运蛋白G2(ATP-binding cassette G2, ABCG2)等。ABCB1和ABCG2均属ABC超家族成员,具有药物泵的作用,可以利用ATP释放的能量逆浓度梯度,将细胞内抗肿瘤药物泵出细胞外,减少细胞内药物的有效浓度,从而产生耐药。其中P-gp与肿瘤多药耐药的关系研究最为广泛,已证实P-gp与多种肿瘤的多药耐药产生有关,并称由P-gp引起的多药耐药为经典的多药耐药途径。ABCG2是近年来发现的与多药耐药有关的因子,已有多篇文献报导ABCG2在多种肿瘤组织和多种肿瘤耐药细胞中高表达,参与肿瘤多药耐药的形成。
本实验利用了多项实验方法检测ABCB1及ABCG2基因、蛋白在食管癌组织、非典型增生及食管正常粘膜中的表达,观察其是否参与食管癌发生及耐药的形成。实验中利用阿霉素(Adriamycin, ADM)诱导食管癌细胞产生耐药的方法建立食管癌耐药细胞株(Eca109/ADM),并观察耐药细胞株中ABCB1和ABCG2表达情况,初探ABCB1及ABCG2与食管癌多药耐药的关系。利用基因转染的方法将ABCG2基因转染至食管癌细胞中观察转染细胞是否能引起耐药形成,探讨ABCG2与食管癌多药耐药关系,为研究ABCG2特性提供细胞模型。同时还利用裸鼠皮下种植瘤模型在体研究ABCG2与食管癌耐药关系。
青蒿琥酯(artesunate, Art)是我国常用的抗疟药物,对一些重症疟疾和耐药性疟疾患者具有较好的抗疟作用,随着研究的深入,发现青蒿琥酯除了具有抗疟作用外,还具有抗肿瘤等活性,青蒿琥酯对多种肿瘤细胞具有生长抑制作用,本课题小组以前的研究结果也提示青蒿琥酯对食管癌细胞有生长抑制作用。同时研究发现,青蒿琥酯抗肿瘤具有不产生交叉耐药的特点,提示青蒿琥酯可能具有逆转肿瘤耐药的作用。青蒿琥酯具有低毒副作用的特点,如果能将其开发为肿瘤多药耐药的逆转剂具有广泛的临床应用前景。实验中利用多项技术,应用细胞及动物模型研究青蒿琥酯是否具有逆转食管癌耐药的作用。
为此,本实验利用了多项实验技术研究ABCB1及ABCG2表达与食管癌多药耐药的关系及青蒿琥酯逆转食管癌耐药作用及机制,为临床上食管癌的化疗提供一些实验依据。
方法:
1 ABCB1及ABCG2在食管癌中的表达及其生物学意义收集食管鳞状细胞癌手术切除标本150例,每例标本分别取癌组织、癌旁组织(距癌边缘2-5cm)及手术切缘正常食管粘膜(距癌边缘5cm以上)。从中选取80例食管鳞状细胞癌、非典型增生及正常食管粘膜组织。经病理组织学诊断,80例食管鳞状细胞癌中59例为高中分化鳞癌,21例为低分化鳞癌;54例侵及纤维膜,26例未达到纤维膜;27例淋巴结转移, 53例未转移。采用逆转录聚合酶链反应( reverse transcription-polymerase chain reaction, RT-PCR)、流式细胞术方法(flow cytometry, FCM)和免疫组织化学方法(immunohistochemistry, IHC)检测80例食管癌组织、非典型增生、正常粘膜组织中ABCB1及ABCG2基因和蛋白的表达,分析ABCB1及ABCG2表达与食管癌临床病理特征的关系。
2阿霉素诱导食管癌耐药细胞中ABCG2的表达及其意义
采用药物持续接触浓度递增诱导法建立食管癌耐药细胞,将处于对数生长期Eca109细胞接种于含低浓度阿霉素(0.002μg/ml)的培养基中,根据细胞的生长情况不断提高药物浓度,直到细胞能在含阿霉素浓度为0.02μg/ml的培养基中稳定生长,历时8个月,细胞命名为Eca109/ADM细胞。
采用MTT法检测ADM作用Eca109、Eca109/ADM细胞后细胞生长抑制率。计算半数抑制浓度(IC50),从而计算耐药指数(RI)=耐药细胞IC50/亲本细胞IC50。
RT-PCR、Western-blot、FCM方法检测Eca109、Eca109/ADM细胞中ABCG2基因及蛋白的表达情况,FCM检测ABCB1蛋白表达。激光共聚焦荧光显微镜技术检测Eca109/ADM细胞中ABCG2蛋白定位及表达。ADM药物干预Eca109、Eca109/ADM细胞生长,利用FCM检测Eca109、Eca109/ADM细胞中ADM含量,从而反映细胞对药物外排作用。Art、ADM药物干预Eca109/ADM细胞,FCM检测细胞凋亡及细胞中ABCB1、ABCG2表达。
3食管癌耐药细胞系Eca109/ABCG2的建立及其生物学特征的研究利用分子生物学技术构建pCDNA3.1(+)-ABCG2重组质粒。采用脂质体转染方法将pCDNA3.1(+)-ABCG2重组质粒转染Eca109细胞,同时也设置空载体pCDNA3.1转染组。转染72 h后用G418进行稳定转染细胞的筛选,筛选出来的阳性细胞分别命名为Eca109/ABCG2、Eca109/PCDNA3.1细胞。
应用FCM方法测定转染效率,MTT方法检测ADM、DNR、MIT对Eca109/ABCG2、Eca109/PCDNA3.1、Eca109细胞生长抑制作用。ADM干预细胞生长,FCM方法检测细胞中ADM的含量。RT-PCR、FCM、Western-blot方法检测Eca109/ABCG2、Eca109/PCDNA3.1、Eca109细胞中ABCG2基因及蛋白的表达,免疫细胞化学方法检测ABCG2蛋白在细胞中表达及定位。
4青蒿琥酯逆转Eca109/ABCG2细胞对阿霉素耐药作用及机制应用MTT方法检测Art、ADM及Art与ADM联合作用对Eca109/ABCG2细胞的生长抑制率。用FCM方法检测Art、ADM及Art与ADM联合作用对Eca109/ABCG2细胞凋亡及细胞内ADM含量的影响。应用RT-PCR、FCM方法检测Art、ADM及Art与ADM联合作用Eca109/ABCG2细胞,细胞中ABCG2基因及蛋白表达量的变化。
5青蒿琥酯逆转裸鼠种植食管癌耐药作用研究
将60只BALB/c nu/nu裸鼠(4周龄,雌雄各半,17~20g)按体重随机分为10组,每组6只(雌雄各半)。其中注射Eca109/ABCG2细胞裸鼠皮下种植瘤共7组,注射Eca109细胞裸鼠皮下种植瘤共3组。裸鼠皮下注射细胞一周后成瘤,开始用药。
药物注射方式及时间:Art,腹腔注射,1次/d,连用7d,停药一周,再继续连用7d;ADM,腹腔注射,1次/3d,共注射7次。
注射Eca109/ABCG2细胞皮下种植瘤裸鼠模型分组:实验组分别给予25和50 mg/kg Art;1和4 mg/kg ADM;ADM联合Art用药组,1 mg/kg ADM与25和50 mg/kg Art联合用药。对照组给予生理盐水。
注射Eca109细胞皮下种植瘤裸鼠模型分组:实验组分别给予1和4 mg/kg ADM。对照组给予生理盐水。
实验期间隔日测量一次裸鼠的体重及皮下种植瘤的长、短径。药物停用一天后采用拉颈法统一无痛苦处死裸鼠,剥离皮下肿瘤称重。计算肿瘤体积及重量抑制率。取出瘤组织分别用于HE、IHC、RT-PCR、FCM方法检测。FCM检测皮下种植瘤组织细胞凋亡、细胞内ADM含量、ABCG2蛋白的表达,IHC方法检测种植瘤细胞ABCG2蛋白表达及定位。RT-PCR方法检测皮下种植瘤细胞中ABCG2 mRNA表达量。
结果:
1 ABCB1及ABCG2在食管癌中的表达及其生物学意义
RT-PCR及FCM结果显示,ABCB1和ABCG2 mRNA及蛋白在食管癌组织中表达均显著高于食管正常粘膜(P <0.01),在食管正常粘膜、非典型增生、癌组织之间呈现逐渐增高趋势(P <0.05)。食管癌组织中,ABCB1及ABCG2 mRNA和蛋白与分化程度、浸润深度及有无淋巴结转移密切相关(P <0.05),与性别和年龄无明显相关(P >0.05)。食管癌组织中ABCG2 mRNA及蛋白表达与ABCB1 mRNA及蛋白表达无显著相关性(rs=0.077, P=0.499; rs=-0.087, P=0.444)。
IHC结果显示:ABCB1和ABCG2阳性产物定位于细胞膜和胞浆,呈棕黄色颗粒状,弥漫或散在分布。食管鳞癌组织中ABCB1和ABCG2蛋白阳性表达率(77.50%和86.25%)明显高于不典型增生组织(31.25%和26.25%)和正常粘膜(6.25%和3.75%)(P <0.01)。
2阿霉素诱导食管癌耐药细胞中ABCG2的表达及其意义
历时8个月,成功培养了耐药细胞株Eca109/ADM,与Eca109细胞相比,Eca109/ADM细胞体积变大,形态更不规则。MTT方法检测,阿霉素(ADM)药物作用Eca109/ADM和Eca109细胞24h,IC50值分别为15.45±1.15,4.69±0.88,Eca109/ADM细胞耐药系数为3.29。
RT-PCR、FCM、Western-blot方法检测到Eca109/ADM细胞中ABCG2 mRNA和蛋白的表达量较Eca109细胞显著增高(P<0.05)。激光共聚焦荧光显微镜检测显示,Eca109/ADM细胞中ABCG2蛋白主要表达在细胞膜及胞浆,与Eca109细胞相比,ABCG2蛋白表达增高。FCM检测ABCB1蛋白在Eca109/ADM细胞表达量显著高于Eca109细胞(P<0.05)。
药物外排试验,FCM检测Eca109/ADM细胞外排阿霉素的作用强于Eca109细胞。Art与ADM联合用药于Eca109/ADM细胞,细胞凋亡率显著高于ADM、Art单独应用(P <0.05)。Art作用Eca109/ADM细胞48h,细胞中ABCG2蛋白表达量显著降低(P<0.05),ABCB1蛋白表达无显著差异(P>0.05)。
3食管癌耐药细胞系Eca109/ABCG2的建立及其生物学特征的研究
应用脂质体转染方法,将pCDNA3.1(+)-ABCG2重组质粒及空载体PCDNA3.1成功转染入Eca109细胞中,并应用G418成功筛选出稳定转染细胞,转染PCDNA3.1-ABCG2重组质粒与空载体PCDNA3.1的阳性克隆细胞分别记为Eca109/ABCG2、Eca109/PCDNA3.1细胞。
MTT方法检测结果显示,阿霉素作用Eca109/ABCG2、Eca109/PCDNA3.1、Eca109细胞24h的IC50值分别为18.61±3.94、4.18±0.14、4.69±0.88,Eca109/ABCG2细胞耐药性增加,相对于Eca109细胞其耐药指数为3.97。阿霉素作用Eca109/ABCG2细胞的IC50值与Eca109细胞比较,显著增高(P<0.01),阿霉素作用Eca109/PCDNA3.1细胞的IC50值与Eca109细胞比较无差别(P>0.05)。DNR和MIT对Eca109/ABCG2细胞IC50值也分别增加,耐药指数分别为3.50和3.15。
Eca109/ABCG2细胞药物外排实验结果显示,Eca109/ABCG2细胞外排阿霉素的作用较Eca109、Eca109/PCDNA3.1细胞显著增加(P <0.01)。
RT-PCR、Western-blot和FCM检测结果显示,Eca109/ABCG2细胞中ABCG2 mRNA和蛋白表达水平较Eca109/PCDNA3.1、Eca109细胞显著升高(P <0.05)。
免疫细胞化学方法检测结果显示,Eca109/ ABCG2细胞中ABCG2蛋白主要表达在细胞膜及胞浆,与Eca109细胞相比,ABCG2蛋白表达增高。4青蒿琥酯逆转Eca109/ABCG2细胞对阿霉素耐药作用及机制
MTT结果显示,Art与ADM联合作用组与单独用Art及ADM组相比,对Eca109/ ABCG2细胞生长抑制率均显著增高(P <0.05)。
Art与ADM联合作用组与单独应用Art及ADM组相比,Eca109/ ABCG2细胞凋亡率均显著增高(P <0.05)。
RT-PCR及FCM结果显示,Art与ADM联合作用组与单独用ADM组相比,Eca109/ ABCG2细胞中ABCG2 mRNA及蛋白表达量显著降低(P <0.05)。
5青蒿琥酯逆转裸鼠种植食管癌耐药作用研究
成功构建人食管癌裸鼠种植瘤模型,成瘤率达100%。相同浓度的ADM组,接种Eca109/ABCG2细胞皮下种植瘤体积和重量与接种Eca109细胞皮下种植瘤相比均显著增高(P<0.05)。Art与ADM联合作用组与单独应用Art或ADM组相比,Eca109/ABCG2细胞皮下种植瘤的体积及重量均显著降低(P <0.05)。
Art与ADM联合作用组与单独应用ADM组相比,Eca109/ABCG2细胞皮下种植瘤中ABCG2 mRNA及蛋白表达量均显著降低(P <0.05)。
结论:
1在食管上皮癌变过程中,食管鳞癌组织中ABCB1及ABCG2表达明显增高,且在食管不典型增生Ⅰ级至Ⅲ级之间呈逐渐增高趋势,提示ABCB1和ABCG2过表达可能参与食管鳞癌发生以及耐药性的产生。ABCB1和ABCG2表达无相关性,提示ABCB1与ABCG2引起的耐药可能具有不同的耐药谱。ABCG2可能成为食管癌耐药逆转新的靶点。
2应用ADM持续接触浓度递增诱导法,成功建立食管癌耐药细胞Eca109/ADM。食管癌耐药细胞Eca109/ADM高表达ABCB1和ABCG2,提示ABCB1和ABCG2参与了Eca109/ADM细胞耐药的形成。青蒿琥酯可以提高ADM对Eca109/ADM细胞的杀伤作用。青蒿琥酯可以降低Eca109/ADM细胞ABCG2表达而ABCB1表达无显著差异,提示青蒿琥酯可以降低Eca109/ADM细胞ABCG2表达,从而逆转其耐药。
3首次应用脂质体转染方法建立多药耐药食管癌细胞株Eca109/ABCG2,目前国内外文献尚未见相关报道。Eca109/ABCG2细胞具有ABCG2耐药表型,能够稳定表达ABCG2蛋白,是研究ABCG2生物学特征良好的多药耐药细胞模型。
4首次初探青蒿琥酯逆转食管癌耐药机制,国内外尚未见相关报道。青蒿琥酯与ADM联合应用可以降低Eca109/ABCG2细胞中ABCG2的表达,抑制ABCG2外排药物的作用,增加细胞内ADM含量,提高ADM对Eca109/ABCG2细胞杀伤作用,从而逆转Eca109/ABCG2细胞对ADM的耐药。
5建立了食管癌耐药细胞皮下种植瘤裸鼠模型,为研究食管癌耐药提供理想的动物模型。青蒿琥酯与ADM联合应用,能增强ADM在体内抑制人食管癌耐药细胞裸鼠皮下种植瘤的生长,且无明显的毒副作用,为将青蒿琥酯应用为食管癌耐药逆转剂提供了实验依据。青蒿琥酯有望成为高效、低毒的耐药逆转剂。
Objectives:Esophageal carcinoma is one of the most common malignant gastrointestinal tumors. The incidence of esophageal carcinoma is high in China, which is the second in gastrointestinal cancers, and it is a great threaten to human health. In the present, chemotherapy is one of the esophageal carcinoma combined modality therapies, but the mutidrug resistance (MDR) in the course of chemotherapy is a major barrier to the success of chemotherapy, especially to recurrent tumors. Multidrug resistance is a kind of resistance of cancer cells to multiple classes of chemotherapic drugs that can be structurally and mechanistically unrelated. When tumor cells develop drug resistance, they become resistant not only to the treated drug, but also to a variety of structurally and functionally unrelated drugs. Because the therapeutic effect is determined by multidrug resistance, multidrug resistance is generally studied lately. There is accumulating evidence that active export of anticancer drugs from cells is one of the major mechanisms of mutidrug resistance. Tumor cells often gain drug resistance through the overexpression of membrane trasport proteins that effectively efflux anticancer drugs. It has been convincingly documented that several ATP-dependent drug transporters can cause drug resistance in cancer cells by actively extruding the clinically administered chemotherapeutic drugs. Increased transmembrane efflux of antitumor drugs is one of the best-characterized mechanisms of MDR and is mediated through the overexpression of ATP-binding cassette (ABC) transporter superfamily members. By far the well-known major drug transporters, i.e., ABCB1 (P-glycoprotein or MDR1) and ABCG2 (BCRP/MXR/ABCP), have been characterized in details with respect to their structure and function. These drug trasporters belong to the human ABC transporter gene family that consists of 48 gene members at least. The best-characerized mechanism of MDR involves P-gp. P-gp overexpression in tumor cells results in broad resistance to a variety of anticancer drugs with different chemical structures and mechanism of action, so we call it to“typical multidrug resistance”. Recently, it’s reported that overexpression of ABCG2 confered drug resistance upon malignant cells to various chemotherapeutic drugs. Expression of ABCG2 in various tumors was high and participated in the development of multidrug resistance.
In our study, the gene and protein expression of ABCB1 and ABCG2 was detected by various experimental methods, to study the relationship between the expression of ABCB1, ABCG2 and the resistance of esophageal carcinoma. Multidrug resistance cell Eca109/ADM induced by ADM was established. Expression of ABCB1 and ABCG2 in Eca109/ADM was studied. In order to study the relationship between the ABCG2 and esophageal carcinoma MDR, the ABCG2 gene was transfected into Eca109 cells.
Artesunate is a remarkable antimalarial agent, espacially to severe and drug-resistant cases. In the present investigation, artesunate is not only an antimalarial agent but also an anticancer agent. Artesunate could inhibit the growth of varietal tumor cells. We have studied that artesunate could inhibit the growth of esophageal cancer cells. Artesunate could also have anticancer effect to drug-resistant cells which hint that artesunate could reverse the drug resistance of cancer cells. Artesunate has low adverse effect, so it can be developed to multidrug resistance reversing drugs. In our research, the drug reversal effect of artesunate to esophageal carcinoma was studied in cellular and animal levels.
Therefore, the relationship between ABCB1, ABCG2 expression in esophageal carcinoma and the esophageal carcinoma multidrug resistance, and drug reversal effect of artesunate were investigated in this study, which could be beneficial to the chemotherapy of esophageal carcinaoma in clinic. Methods:
1 Expression of ABCB1 and ABCG2 in esophageal carcinoma and related biology significance
Esophageal carcinoma tissues, paired adjacent mucosa (2~5cm from margin of esophageal carcinoma), and paired normal mucosa (at least 5cm from margin of esophageal carcinoma) were obtained from 150 resected surgical specimens of esophageal squamous cell carcinoma (ESCC). All the specimens were verified by pathologic diagnosis, 80 cases of esophageal squamous cell carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were selected from 150 specimens. 80 ESCC tissues included well and moderately differentiated ESCC(n=59), poorly differentiated ESCC(n=21) ; fibrous membrane invasion(n=54), fibrous membrane untouched(n=26); lymph node metastasis positive(n=27), lymph node metastasis negative(n=53). ABCB1, ABCG2 gene and protein expression in 80 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry(IHC) and flow cytometry(FCM). Relationship between their expressions and clinical pathological features was analyzed.
2 Expression of ABCG2 in adriamycin-resistant human esophageal cancer cells
Drug-resistant cells of esophageal cancer was established by gradually increasing the ADM concentration in the culture medium of human esophageal cancer cell line (Eca109) from 0.002μg/ml ADM to 0.02μg/ml ADM for 8 months. The drug-resistant cell was named Eca109/ADM cell.
Cell inhibitory rate of ADM on the growth of Eca109 and Eca109/ADM cells was investigated by MTT and IC50, resistance index (RI) (RI=Eca109/ADM IC50/ Eca109 IC50) was calculated. ABCG2 gene and protein expression in Eca109 and Eca109/ADM cells were investigated by RT-PCR, Western-blot as well as FCM. ABCB1 protein was detected by FCM. Location and expression of ABCG2 in Eca109/ADM cells were investigated by Laser scan confocal microscope (LSCM). Drug excretion of Eca109/ADM cells was detected by FCM. The apoptosis, ABCB1 and ABCG2 expression of Eca109, Eca109/ADM cells after treatment of Art and ADM were investigated by FCM.
3 Establishment of Eca109/ABCG2, a drug resistant esophageal cancer cell line, and its biological profile
We clone the whole length of ABCG2 gene from gene bank and ligate the whole length of ABCG2 gene to the PCDNA3.1 vector to construct the custom-crafted plasmid PCDNA3.1-ABCG2. The positive clones PCDNA3.1-ABCG2 was transfected to Eca109 cells and positive cell clones were selected with G418 after transfected 72h. Eca109 cell transfected with PCDNA3.1 as control group. Eca109 cell transfected with PCDNA3.1-ABCG2 and PCDNA3.1 was named Eca109/ABCG2 and Eca109/PCDNA3.1 cell respectively.
Transfected rate was investigated by FCM and the inhibitory effect of ADM, DNR and MIT on the growth of Eca109/ABCG2, Eca109/PCDNA3.1 as well as Eca109 cells was detected by MTT. The content of ADM in cells after treatment of ADM was investigated by FCM. Gene and protein expression of ABCG2 were investigated by RT-PCR, FCM as well as Western-blot. Location of ABCG2 in cells was measured by immunocytochemistry (ICH).
4 The ADM resistance reversal effect of Art on Eca109/ABCG2 cells and related mechanism
The inhibitory effect of Art, ADM and Art+ADM on the growth of Eca109/ABCG2 cells was investigated by MTT. Apoptosis of Eca109/ABCG2 cells and the content of ADM in Eca109/ABCG2 cells after treatment of Art, ADM as well as Art+ADM were detected by FCM. Gene and protein expression of ABCG2 in Eca109/ABCG2 cells after Art, ADM and Art+ADM interference were detected by RT-PCR and FCM.
5 The resistance-reversal effect of Art on human esophageal cacer transplanted in nude mice
60 (30 of male and 30 of female) nude mice (BALB/c, nu/nu, 4 wk old, 17~20g) were randomly divided into 10 groups (6 in each group) by weight. Each of nude mice in 7 groups was inoculated with 200μl Eca109/ABCG2 cells (6×106 each mouse) subcutaneously on the left subscapularis. Each of nude mice in 3 groups was inoculated with 200μl Eca109 cells (6×106 each mouse) subcutaneously on the left subscapularis. On the seventh day after inoculation, all of the nude mice were formed tumor, starting to use drugs.
Modality and time of drug injection: Art, peritoneal injection per a day for 2 courses, each course contained 7 days and there was a seven-day interval between two courses. ADM was treated with peritoneal injection per three days for 7 times.
Nude mice inoculated with Eca109/ABCG2 cells were divided into 7 groups as follows: 2 groups received Art (25, 50mg/kg/d, respectively). 2 groups received ADM (1, 4mg/kg/3d, respectively). 2 groups received ADM (1mg/kg/3d) + Art (25, 50mg/kg/d) respectively. The negative control group received saline.
Nude mice inoculated with Eca109 cells were divided into 3 groups as follows: the experimental groups received ADM (1, 4mg/kg/3d, respectively). The negative control group received saline.
The weight and the shortest, longest diameters of the tumors were measured with vernier caliper each 2 days.
Mice were sacrificed after experiments course and all tumor tissues were weighed. Cell apoptosis, the ADM content and ABCG2 protein of tumor cells were examined by FCM. Expression and location of ABCG2 protein of tumor cells were detected by IHC. ABCG2 mRNA expression of tumor cells was investigated by RT-PCR.
Results:
1 Expression of ABCB1 and ABCG2 in esophageal carcinoma and related biology significance
RT-PCR and FCM results show: mRNA and protein expression of ABCB1, ABCG2 in esophageal cancer tissues were significantly higher than normal tissues (P<0.01). In esophageal cancer tissues, mRNA and protein expression of ABCB1, ABCG2 were no difference between age and gender (P>0.05), but the expression of ABCB1, ABCG2 in poorly differentiated, fibrous membrane invasion and lymph node metastasis positive ESCC were significantly higher than the expression in well and moderately differentiated, fibrous membrane untouched and lymph node metastasis negative (P<0.05). There was no significant association between ABCG2 mRNA, protein expression and ABCB1 mRNA, protein expression (rs =0.077,P=0.499; rs =-0.087,P=0.444, respectively).
IHC results show: ABCB1 and ABCG2 positive protein were located in cell membrane and cytolymph as buffy granular and diffused distribution. The positive rates of ABCB1 and ABCG2 protein were significantly higher in ESCC (77.50% and 86.25%) than those in squamous dysplasia (31.25% and 26.25%) and normal squamous epithelium (6.25% and 3.75%) (P<0.01).
2 Expression of ABCG2 in adriamycin-resistant human esophageal cancer cells
Drug resistant cell Eca109/ADM was established successfully for 8 monthes. Comparing Eca109/ADM cell with Eca109 cell, Eca109/ADM cell became bigger volume and more irregularity morphology. The IC50 of Eca109/ADM and Eca109 cells to ADM was 15.45±1.15,4.69±0.88 after the treatment of ADM for 24h. The RI of Eca109/ADM was 3.29.
ABCG2 mRNA and protein expression of Eca109/ADM cells were significantly higher than Eca109 cells (P<0.05). ABCG2 protein of Eca109/ADM was located in cell membrane and cytolymph and the protein expression was higher than Eca109 cells. ABCB1 protein expression of Eca109/ADM cells was significantly higher than the expression of Eca109 cells (P<0.05). Drug excretion of Eca109/ADM cells was higher than Eca109 cells.
The cell apoptosis rate of Art+ADM group on Eca109/ADM cells was significantly higher than ADM group (P<0.05). ABCG2 protein expression of Eca109/ADM cells was significantly decreased after the treatment of Art for 48h (P<0.05), but ABCB1 protein expression of Eca109/ADM cells was no difference (P>0.05).
3 Establishment of Eca109/ABCG2, a drug resistant esophageal cancer cell line, and its biological profile
Eca109/ABCG2 and Eca109/PCDNA3.1 cell were established successfully. The IC50 of Eca109/ABCG2, Eca109/PCDNA3.1 and Eca109 cells was 18.61±3.94, 4.18±0.14, 4.69±0.88 after treatment of ADM for 24h. The RI of Eca109/ABCG2 was 3.97. The IC50 of Eca109/ABCG2 cells was significantly higher compared with Eca109 cells (P<0.01). The IC50 of Eca109/ PCDNA3.1 cells was no difference compared with Eca109 cells (P>0.05). The IC50 of Eca109/ABCG2 cells to DNR and MIT was higher than Eca109 cells, The RI was 3.50 and 3.15 respectively.
Drug excretion of Eca109/ABCG2 cells was significantly higher than Eca109/ PCDNA3.1 and Eca109 cells (P<0.01).
ABCG2 mRNA and protein expression of Eca109/ABCG2 cells was significantly higher than Eca109/PCDNA3.1 and Eca109 cells (P<0.05). ICC results show: ABCG2 protein of Eca109/ABCG2 was located in cell membrane and cytolymph, and the protein expression was higher than the expression of Eca109/PCDNA3.1 and Eca109 cells.
4 The ADM resistance reversal effect of Art on Eca109/ABCG2 cell and related mechanism.
MTT results show: The inhibitory effect of Art+ADM on the growth of Eca109/ABCG2 cells was significantly higer than Art or ADM (P<0.05). The Eca109/ABCG2 cells apoptosis rate of Art+ADM group was significantly higher than Art or ADM group (P<0.05). ABCG2 mRNA and protein expression of Eca109/ABCG2 cells after the treatment of Art+ADM were significantly lower than ADM (P<0.05).
5 The resistance-reversal effect of Art on human esophageal cancer transplanted in nude mice
The model of esophageal cancer xenograft in nude mice was successfully established, the tumorigenic rate was 100%. In the same ADM concentration group, volume and weight of transplanted tumor inoculated with Eca109/ABCG2 cells was significantly higher than transplanted tumor inoculated with Eca109 cells (P<0.05). In the transplanted tumor inoculated with Eca109/ABCG2 cells groups, tumor volume and weight of Art + ADM group was significantly lower than Art or ADM group (P<0.05), and ABCG2 mRNA, protein expression in tumor cells of Art + ADM group were significantly lower than ADM group (P<0.05).
Conclusions:
1 In the process of carcinogenesis of the esophagus, the expression of ABCB1 and ABCG2 in ESCC were significantly higher than those in squamous dysplasia and normal squamous epithelium. The expression of ABCB1 and ABCG2 in squamous dysplasia were gradually high from gradeⅠto gradeⅢ. High expression of ABCB1 and ABCG2 might be correlated with tumor progression and drug resistance. There was no correlation between ABCB1 expression and ABCG2 expression in ESCC, which suggest that the drug resistance induced by ABCB1 or ABCG2 might have different mechanism. ABCG2 might be new therapy target of esophageal carcinoma drug resistance.
2 Drug resistant cell Eca109/ADM was established successfully by gradually increasing the ADM concentration in the culture medium of human esophageal cancer cell line (Eca109). ABCB1 and ABCG2 expression were higher in Eca109/ADM cells than those in Eca109 cells, which suggested that ABCB1 and ABCG2 might participate in the drug resistance of Eca109/ADM cells. Artesunate could significantly increase the inhibitory effect of ADM on the growth of Eca109/ADM cells and decrease the ABCG2 expression of Eca109/ADM cells but not ABCB1, which suggested that artesunate could reverse the drug resistance of Eca109/ADM cells by decreasing ABCG2 expression of Eca109/ADM cells.
3 For the first time, multidrug resistance cell (Eca109/ABCG2) of esophageal cancer was successfully established by transfection of liposomes. Eca109/ABCG2 cell possessed ABCG2-resistant biological profile and could stably express the ABCG2 protein, so the Eca109/ABCG2 cell was ideally multidrug resistant cell model which had ABCG2 biological profile.
4 For the first time, resistance-reversal effect of artesunate on esophageal cancer was explored in our study. The cell growth inhibitory effect of ADM on Eca109/ABCG2 cells was significantly increased by combining Art and ADM. Art could significantly reduce the ABCG2 expression of Eca109/ABCG2 cells which could inhibit the drugs excretion and increase the ADM content in Eca109/ABCG2 cells, so artesunate could reverse the ADM drug resistance of Eca109/ABCG2 cells.
5 The model of esophageal cancer xenograft in nude mice was successfully established, which could provide an ideal model for the research of esophageal cancer drug-resistance. The growth inhibitory effect of ADM on esophageal cancer xenograft in vivo was enhanced by combining artesunate without causing obvious side effects in treated mice, which coule provide some experimental reference for artesunate as resistance-reversal agent. Artesunate could become high-performance and harmfulless resistance-reversal agent in clinic.
本实验利用了多项实验方法检测ABCB1及ABCG2基因、蛋白在食管癌组织、非典型增生及食管正常粘膜中的表达,观察其是否参与食管癌发生及耐药的形成。实验中利用阿霉素(Adriamycin, ADM)诱导食管癌细胞产生耐药的方法建立食管癌耐药细胞株(Eca109/ADM),并观察耐药细胞株中ABCB1和ABCG2表达情况,初探ABCB1及ABCG2与食管癌多药耐药的关系。利用基因转染的方法将ABCG2基因转染至食管癌细胞中观察转染细胞是否能引起耐药形成,探讨ABCG2与食管癌多药耐药关系,为研究ABCG2特性提供细胞模型。同时还利用裸鼠皮下种植瘤模型在体研究ABCG2与食管癌耐药关系。
青蒿琥酯(artesunate, Art)是我国常用的抗疟药物,对一些重症疟疾和耐药性疟疾患者具有较好的抗疟作用,随着研究的深入,发现青蒿琥酯除了具有抗疟作用外,还具有抗肿瘤等活性,青蒿琥酯对多种肿瘤细胞具有生长抑制作用,本课题小组以前的研究结果也提示青蒿琥酯对食管癌细胞有生长抑制作用。同时研究发现,青蒿琥酯抗肿瘤具有不产生交叉耐药的特点,提示青蒿琥酯可能具有逆转肿瘤耐药的作用。青蒿琥酯具有低毒副作用的特点,如果能将其开发为肿瘤多药耐药的逆转剂具有广泛的临床应用前景。实验中利用多项技术,应用细胞及动物模型研究青蒿琥酯是否具有逆转食管癌耐药的作用。
为此,本实验利用了多项实验技术研究ABCB1及ABCG2表达与食管癌多药耐药的关系及青蒿琥酯逆转食管癌耐药作用及机制,为临床上食管癌的化疗提供一些实验依据。
方法:
1 ABCB1及ABCG2在食管癌中的表达及其生物学意义收集食管鳞状细胞癌手术切除标本150例,每例标本分别取癌组织、癌旁组织(距癌边缘2-5cm)及手术切缘正常食管粘膜(距癌边缘5cm以上)。从中选取80例食管鳞状细胞癌、非典型增生及正常食管粘膜组织。经病理组织学诊断,80例食管鳞状细胞癌中59例为高中分化鳞癌,21例为低分化鳞癌;54例侵及纤维膜,26例未达到纤维膜;27例淋巴结转移, 53例未转移。采用逆转录聚合酶链反应( reverse transcription-polymerase chain reaction, RT-PCR)、流式细胞术方法(flow cytometry, FCM)和免疫组织化学方法(immunohistochemistry, IHC)检测80例食管癌组织、非典型增生、正常粘膜组织中ABCB1及ABCG2基因和蛋白的表达,分析ABCB1及ABCG2表达与食管癌临床病理特征的关系。
2阿霉素诱导食管癌耐药细胞中ABCG2的表达及其意义
采用药物持续接触浓度递增诱导法建立食管癌耐药细胞,将处于对数生长期Eca109细胞接种于含低浓度阿霉素(0.002μg/ml)的培养基中,根据细胞的生长情况不断提高药物浓度,直到细胞能在含阿霉素浓度为0.02μg/ml的培养基中稳定生长,历时8个月,细胞命名为Eca109/ADM细胞。
采用MTT法检测ADM作用Eca109、Eca109/ADM细胞后细胞生长抑制率。计算半数抑制浓度(IC50),从而计算耐药指数(RI)=耐药细胞IC50/亲本细胞IC50。
RT-PCR、Western-blot、FCM方法检测Eca109、Eca109/ADM细胞中ABCG2基因及蛋白的表达情况,FCM检测ABCB1蛋白表达。激光共聚焦荧光显微镜技术检测Eca109/ADM细胞中ABCG2蛋白定位及表达。ADM药物干预Eca109、Eca109/ADM细胞生长,利用FCM检测Eca109、Eca109/ADM细胞中ADM含量,从而反映细胞对药物外排作用。Art、ADM药物干预Eca109/ADM细胞,FCM检测细胞凋亡及细胞中ABCB1、ABCG2表达。
3食管癌耐药细胞系Eca109/ABCG2的建立及其生物学特征的研究利用分子生物学技术构建pCDNA3.1(+)-ABCG2重组质粒。采用脂质体转染方法将pCDNA3.1(+)-ABCG2重组质粒转染Eca109细胞,同时也设置空载体pCDNA3.1转染组。转染72 h后用G418进行稳定转染细胞的筛选,筛选出来的阳性细胞分别命名为Eca109/ABCG2、Eca109/PCDNA3.1细胞。
应用FCM方法测定转染效率,MTT方法检测ADM、DNR、MIT对Eca109/ABCG2、Eca109/PCDNA3.1、Eca109细胞生长抑制作用。ADM干预细胞生长,FCM方法检测细胞中ADM的含量。RT-PCR、FCM、Western-blot方法检测Eca109/ABCG2、Eca109/PCDNA3.1、Eca109细胞中ABCG2基因及蛋白的表达,免疫细胞化学方法检测ABCG2蛋白在细胞中表达及定位。
4青蒿琥酯逆转Eca109/ABCG2细胞对阿霉素耐药作用及机制应用MTT方法检测Art、ADM及Art与ADM联合作用对Eca109/ABCG2细胞的生长抑制率。用FCM方法检测Art、ADM及Art与ADM联合作用对Eca109/ABCG2细胞凋亡及细胞内ADM含量的影响。应用RT-PCR、FCM方法检测Art、ADM及Art与ADM联合作用Eca109/ABCG2细胞,细胞中ABCG2基因及蛋白表达量的变化。
5青蒿琥酯逆转裸鼠种植食管癌耐药作用研究
将60只BALB/c nu/nu裸鼠(4周龄,雌雄各半,17~20g)按体重随机分为10组,每组6只(雌雄各半)。其中注射Eca109/ABCG2细胞裸鼠皮下种植瘤共7组,注射Eca109细胞裸鼠皮下种植瘤共3组。裸鼠皮下注射细胞一周后成瘤,开始用药。
药物注射方式及时间:Art,腹腔注射,1次/d,连用7d,停药一周,再继续连用7d;ADM,腹腔注射,1次/3d,共注射7次。
注射Eca109/ABCG2细胞皮下种植瘤裸鼠模型分组:实验组分别给予25和50 mg/kg Art;1和4 mg/kg ADM;ADM联合Art用药组,1 mg/kg ADM与25和50 mg/kg Art联合用药。对照组给予生理盐水。
注射Eca109细胞皮下种植瘤裸鼠模型分组:实验组分别给予1和4 mg/kg ADM。对照组给予生理盐水。
实验期间隔日测量一次裸鼠的体重及皮下种植瘤的长、短径。药物停用一天后采用拉颈法统一无痛苦处死裸鼠,剥离皮下肿瘤称重。计算肿瘤体积及重量抑制率。取出瘤组织分别用于HE、IHC、RT-PCR、FCM方法检测。FCM检测皮下种植瘤组织细胞凋亡、细胞内ADM含量、ABCG2蛋白的表达,IHC方法检测种植瘤细胞ABCG2蛋白表达及定位。RT-PCR方法检测皮下种植瘤细胞中ABCG2 mRNA表达量。
结果:
1 ABCB1及ABCG2在食管癌中的表达及其生物学意义
RT-PCR及FCM结果显示,ABCB1和ABCG2 mRNA及蛋白在食管癌组织中表达均显著高于食管正常粘膜(P <0.01),在食管正常粘膜、非典型增生、癌组织之间呈现逐渐增高趋势(P <0.05)。食管癌组织中,ABCB1及ABCG2 mRNA和蛋白与分化程度、浸润深度及有无淋巴结转移密切相关(P <0.05),与性别和年龄无明显相关(P >0.05)。食管癌组织中ABCG2 mRNA及蛋白表达与ABCB1 mRNA及蛋白表达无显著相关性(rs=0.077, P=0.499; rs=-0.087, P=0.444)。
IHC结果显示:ABCB1和ABCG2阳性产物定位于细胞膜和胞浆,呈棕黄色颗粒状,弥漫或散在分布。食管鳞癌组织中ABCB1和ABCG2蛋白阳性表达率(77.50%和86.25%)明显高于不典型增生组织(31.25%和26.25%)和正常粘膜(6.25%和3.75%)(P <0.01)。
2阿霉素诱导食管癌耐药细胞中ABCG2的表达及其意义
历时8个月,成功培养了耐药细胞株Eca109/ADM,与Eca109细胞相比,Eca109/ADM细胞体积变大,形态更不规则。MTT方法检测,阿霉素(ADM)药物作用Eca109/ADM和Eca109细胞24h,IC50值分别为15.45±1.15,4.69±0.88,Eca109/ADM细胞耐药系数为3.29。
RT-PCR、FCM、Western-blot方法检测到Eca109/ADM细胞中ABCG2 mRNA和蛋白的表达量较Eca109细胞显著增高(P<0.05)。激光共聚焦荧光显微镜检测显示,Eca109/ADM细胞中ABCG2蛋白主要表达在细胞膜及胞浆,与Eca109细胞相比,ABCG2蛋白表达增高。FCM检测ABCB1蛋白在Eca109/ADM细胞表达量显著高于Eca109细胞(P<0.05)。
药物外排试验,FCM检测Eca109/ADM细胞外排阿霉素的作用强于Eca109细胞。Art与ADM联合用药于Eca109/ADM细胞,细胞凋亡率显著高于ADM、Art单独应用(P <0.05)。Art作用Eca109/ADM细胞48h,细胞中ABCG2蛋白表达量显著降低(P<0.05),ABCB1蛋白表达无显著差异(P>0.05)。
3食管癌耐药细胞系Eca109/ABCG2的建立及其生物学特征的研究
应用脂质体转染方法,将pCDNA3.1(+)-ABCG2重组质粒及空载体PCDNA3.1成功转染入Eca109细胞中,并应用G418成功筛选出稳定转染细胞,转染PCDNA3.1-ABCG2重组质粒与空载体PCDNA3.1的阳性克隆细胞分别记为Eca109/ABCG2、Eca109/PCDNA3.1细胞。
MTT方法检测结果显示,阿霉素作用Eca109/ABCG2、Eca109/PCDNA3.1、Eca109细胞24h的IC50值分别为18.61±3.94、4.18±0.14、4.69±0.88,Eca109/ABCG2细胞耐药性增加,相对于Eca109细胞其耐药指数为3.97。阿霉素作用Eca109/ABCG2细胞的IC50值与Eca109细胞比较,显著增高(P<0.01),阿霉素作用Eca109/PCDNA3.1细胞的IC50值与Eca109细胞比较无差别(P>0.05)。DNR和MIT对Eca109/ABCG2细胞IC50值也分别增加,耐药指数分别为3.50和3.15。
Eca109/ABCG2细胞药物外排实验结果显示,Eca109/ABCG2细胞外排阿霉素的作用较Eca109、Eca109/PCDNA3.1细胞显著增加(P <0.01)。
RT-PCR、Western-blot和FCM检测结果显示,Eca109/ABCG2细胞中ABCG2 mRNA和蛋白表达水平较Eca109/PCDNA3.1、Eca109细胞显著升高(P <0.05)。
免疫细胞化学方法检测结果显示,Eca109/ ABCG2细胞中ABCG2蛋白主要表达在细胞膜及胞浆,与Eca109细胞相比,ABCG2蛋白表达增高。4青蒿琥酯逆转Eca109/ABCG2细胞对阿霉素耐药作用及机制
MTT结果显示,Art与ADM联合作用组与单独用Art及ADM组相比,对Eca109/ ABCG2细胞生长抑制率均显著增高(P <0.05)。
Art与ADM联合作用组与单独应用Art及ADM组相比,Eca109/ ABCG2细胞凋亡率均显著增高(P <0.05)。
RT-PCR及FCM结果显示,Art与ADM联合作用组与单独用ADM组相比,Eca109/ ABCG2细胞中ABCG2 mRNA及蛋白表达量显著降低(P <0.05)。
5青蒿琥酯逆转裸鼠种植食管癌耐药作用研究
成功构建人食管癌裸鼠种植瘤模型,成瘤率达100%。相同浓度的ADM组,接种Eca109/ABCG2细胞皮下种植瘤体积和重量与接种Eca109细胞皮下种植瘤相比均显著增高(P<0.05)。Art与ADM联合作用组与单独应用Art或ADM组相比,Eca109/ABCG2细胞皮下种植瘤的体积及重量均显著降低(P <0.05)。
Art与ADM联合作用组与单独应用ADM组相比,Eca109/ABCG2细胞皮下种植瘤中ABCG2 mRNA及蛋白表达量均显著降低(P <0.05)。
结论:
1在食管上皮癌变过程中,食管鳞癌组织中ABCB1及ABCG2表达明显增高,且在食管不典型增生Ⅰ级至Ⅲ级之间呈逐渐增高趋势,提示ABCB1和ABCG2过表达可能参与食管鳞癌发生以及耐药性的产生。ABCB1和ABCG2表达无相关性,提示ABCB1与ABCG2引起的耐药可能具有不同的耐药谱。ABCG2可能成为食管癌耐药逆转新的靶点。
2应用ADM持续接触浓度递增诱导法,成功建立食管癌耐药细胞Eca109/ADM。食管癌耐药细胞Eca109/ADM高表达ABCB1和ABCG2,提示ABCB1和ABCG2参与了Eca109/ADM细胞耐药的形成。青蒿琥酯可以提高ADM对Eca109/ADM细胞的杀伤作用。青蒿琥酯可以降低Eca109/ADM细胞ABCG2表达而ABCB1表达无显著差异,提示青蒿琥酯可以降低Eca109/ADM细胞ABCG2表达,从而逆转其耐药。
3首次应用脂质体转染方法建立多药耐药食管癌细胞株Eca109/ABCG2,目前国内外文献尚未见相关报道。Eca109/ABCG2细胞具有ABCG2耐药表型,能够稳定表达ABCG2蛋白,是研究ABCG2生物学特征良好的多药耐药细胞模型。
4首次初探青蒿琥酯逆转食管癌耐药机制,国内外尚未见相关报道。青蒿琥酯与ADM联合应用可以降低Eca109/ABCG2细胞中ABCG2的表达,抑制ABCG2外排药物的作用,增加细胞内ADM含量,提高ADM对Eca109/ABCG2细胞杀伤作用,从而逆转Eca109/ABCG2细胞对ADM的耐药。
5建立了食管癌耐药细胞皮下种植瘤裸鼠模型,为研究食管癌耐药提供理想的动物模型。青蒿琥酯与ADM联合应用,能增强ADM在体内抑制人食管癌耐药细胞裸鼠皮下种植瘤的生长,且无明显的毒副作用,为将青蒿琥酯应用为食管癌耐药逆转剂提供了实验依据。青蒿琥酯有望成为高效、低毒的耐药逆转剂。
Objectives:Esophageal carcinoma is one of the most common malignant gastrointestinal tumors. The incidence of esophageal carcinoma is high in China, which is the second in gastrointestinal cancers, and it is a great threaten to human health. In the present, chemotherapy is one of the esophageal carcinoma combined modality therapies, but the mutidrug resistance (MDR) in the course of chemotherapy is a major barrier to the success of chemotherapy, especially to recurrent tumors. Multidrug resistance is a kind of resistance of cancer cells to multiple classes of chemotherapic drugs that can be structurally and mechanistically unrelated. When tumor cells develop drug resistance, they become resistant not only to the treated drug, but also to a variety of structurally and functionally unrelated drugs. Because the therapeutic effect is determined by multidrug resistance, multidrug resistance is generally studied lately. There is accumulating evidence that active export of anticancer drugs from cells is one of the major mechanisms of mutidrug resistance. Tumor cells often gain drug resistance through the overexpression of membrane trasport proteins that effectively efflux anticancer drugs. It has been convincingly documented that several ATP-dependent drug transporters can cause drug resistance in cancer cells by actively extruding the clinically administered chemotherapeutic drugs. Increased transmembrane efflux of antitumor drugs is one of the best-characterized mechanisms of MDR and is mediated through the overexpression of ATP-binding cassette (ABC) transporter superfamily members. By far the well-known major drug transporters, i.e., ABCB1 (P-glycoprotein or MDR1) and ABCG2 (BCRP/MXR/ABCP), have been characterized in details with respect to their structure and function. These drug trasporters belong to the human ABC transporter gene family that consists of 48 gene members at least. The best-characerized mechanism of MDR involves P-gp. P-gp overexpression in tumor cells results in broad resistance to a variety of anticancer drugs with different chemical structures and mechanism of action, so we call it to“typical multidrug resistance”. Recently, it’s reported that overexpression of ABCG2 confered drug resistance upon malignant cells to various chemotherapeutic drugs. Expression of ABCG2 in various tumors was high and participated in the development of multidrug resistance.
In our study, the gene and protein expression of ABCB1 and ABCG2 was detected by various experimental methods, to study the relationship between the expression of ABCB1, ABCG2 and the resistance of esophageal carcinoma. Multidrug resistance cell Eca109/ADM induced by ADM was established. Expression of ABCB1 and ABCG2 in Eca109/ADM was studied. In order to study the relationship between the ABCG2 and esophageal carcinoma MDR, the ABCG2 gene was transfected into Eca109 cells.
Artesunate is a remarkable antimalarial agent, espacially to severe and drug-resistant cases. In the present investigation, artesunate is not only an antimalarial agent but also an anticancer agent. Artesunate could inhibit the growth of varietal tumor cells. We have studied that artesunate could inhibit the growth of esophageal cancer cells. Artesunate could also have anticancer effect to drug-resistant cells which hint that artesunate could reverse the drug resistance of cancer cells. Artesunate has low adverse effect, so it can be developed to multidrug resistance reversing drugs. In our research, the drug reversal effect of artesunate to esophageal carcinoma was studied in cellular and animal levels.
Therefore, the relationship between ABCB1, ABCG2 expression in esophageal carcinoma and the esophageal carcinoma multidrug resistance, and drug reversal effect of artesunate were investigated in this study, which could be beneficial to the chemotherapy of esophageal carcinaoma in clinic. Methods:
1 Expression of ABCB1 and ABCG2 in esophageal carcinoma and related biology significance
Esophageal carcinoma tissues, paired adjacent mucosa (2~5cm from margin of esophageal carcinoma), and paired normal mucosa (at least 5cm from margin of esophageal carcinoma) were obtained from 150 resected surgical specimens of esophageal squamous cell carcinoma (ESCC). All the specimens were verified by pathologic diagnosis, 80 cases of esophageal squamous cell carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were selected from 150 specimens. 80 ESCC tissues included well and moderately differentiated ESCC(n=59), poorly differentiated ESCC(n=21) ; fibrous membrane invasion(n=54), fibrous membrane untouched(n=26); lymph node metastasis positive(n=27), lymph node metastasis negative(n=53). ABCB1, ABCG2 gene and protein expression in 80 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry(IHC) and flow cytometry(FCM). Relationship between their expressions and clinical pathological features was analyzed.
2 Expression of ABCG2 in adriamycin-resistant human esophageal cancer cells
Drug-resistant cells of esophageal cancer was established by gradually increasing the ADM concentration in the culture medium of human esophageal cancer cell line (Eca109) from 0.002μg/ml ADM to 0.02μg/ml ADM for 8 months. The drug-resistant cell was named Eca109/ADM cell.
Cell inhibitory rate of ADM on the growth of Eca109 and Eca109/ADM cells was investigated by MTT and IC50, resistance index (RI) (RI=Eca109/ADM IC50/ Eca109 IC50) was calculated. ABCG2 gene and protein expression in Eca109 and Eca109/ADM cells were investigated by RT-PCR, Western-blot as well as FCM. ABCB1 protein was detected by FCM. Location and expression of ABCG2 in Eca109/ADM cells were investigated by Laser scan confocal microscope (LSCM). Drug excretion of Eca109/ADM cells was detected by FCM. The apoptosis, ABCB1 and ABCG2 expression of Eca109, Eca109/ADM cells after treatment of Art and ADM were investigated by FCM.
3 Establishment of Eca109/ABCG2, a drug resistant esophageal cancer cell line, and its biological profile
We clone the whole length of ABCG2 gene from gene bank and ligate the whole length of ABCG2 gene to the PCDNA3.1 vector to construct the custom-crafted plasmid PCDNA3.1-ABCG2. The positive clones PCDNA3.1-ABCG2 was transfected to Eca109 cells and positive cell clones were selected with G418 after transfected 72h. Eca109 cell transfected with PCDNA3.1 as control group. Eca109 cell transfected with PCDNA3.1-ABCG2 and PCDNA3.1 was named Eca109/ABCG2 and Eca109/PCDNA3.1 cell respectively.
Transfected rate was investigated by FCM and the inhibitory effect of ADM, DNR and MIT on the growth of Eca109/ABCG2, Eca109/PCDNA3.1 as well as Eca109 cells was detected by MTT. The content of ADM in cells after treatment of ADM was investigated by FCM. Gene and protein expression of ABCG2 were investigated by RT-PCR, FCM as well as Western-blot. Location of ABCG2 in cells was measured by immunocytochemistry (ICH).
4 The ADM resistance reversal effect of Art on Eca109/ABCG2 cells and related mechanism
The inhibitory effect of Art, ADM and Art+ADM on the growth of Eca109/ABCG2 cells was investigated by MTT. Apoptosis of Eca109/ABCG2 cells and the content of ADM in Eca109/ABCG2 cells after treatment of Art, ADM as well as Art+ADM were detected by FCM. Gene and protein expression of ABCG2 in Eca109/ABCG2 cells after Art, ADM and Art+ADM interference were detected by RT-PCR and FCM.
5 The resistance-reversal effect of Art on human esophageal cacer transplanted in nude mice
60 (30 of male and 30 of female) nude mice (BALB/c, nu/nu, 4 wk old, 17~20g) were randomly divided into 10 groups (6 in each group) by weight. Each of nude mice in 7 groups was inoculated with 200μl Eca109/ABCG2 cells (6×106 each mouse) subcutaneously on the left subscapularis. Each of nude mice in 3 groups was inoculated with 200μl Eca109 cells (6×106 each mouse) subcutaneously on the left subscapularis. On the seventh day after inoculation, all of the nude mice were formed tumor, starting to use drugs.
Modality and time of drug injection: Art, peritoneal injection per a day for 2 courses, each course contained 7 days and there was a seven-day interval between two courses. ADM was treated with peritoneal injection per three days for 7 times.
Nude mice inoculated with Eca109/ABCG2 cells were divided into 7 groups as follows: 2 groups received Art (25, 50mg/kg/d, respectively). 2 groups received ADM (1, 4mg/kg/3d, respectively). 2 groups received ADM (1mg/kg/3d) + Art (25, 50mg/kg/d) respectively. The negative control group received saline.
Nude mice inoculated with Eca109 cells were divided into 3 groups as follows: the experimental groups received ADM (1, 4mg/kg/3d, respectively). The negative control group received saline.
The weight and the shortest, longest diameters of the tumors were measured with vernier caliper each 2 days.
Mice were sacrificed after experiments course and all tumor tissues were weighed. Cell apoptosis, the ADM content and ABCG2 protein of tumor cells were examined by FCM. Expression and location of ABCG2 protein of tumor cells were detected by IHC. ABCG2 mRNA expression of tumor cells was investigated by RT-PCR.
Results:
1 Expression of ABCB1 and ABCG2 in esophageal carcinoma and related biology significance
RT-PCR and FCM results show: mRNA and protein expression of ABCB1, ABCG2 in esophageal cancer tissues were significantly higher than normal tissues (P<0.01). In esophageal cancer tissues, mRNA and protein expression of ABCB1, ABCG2 were no difference between age and gender (P>0.05), but the expression of ABCB1, ABCG2 in poorly differentiated, fibrous membrane invasion and lymph node metastasis positive ESCC were significantly higher than the expression in well and moderately differentiated, fibrous membrane untouched and lymph node metastasis negative (P<0.05). There was no significant association between ABCG2 mRNA, protein expression and ABCB1 mRNA, protein expression (rs =0.077,P=0.499; rs =-0.087,P=0.444, respectively).
IHC results show: ABCB1 and ABCG2 positive protein were located in cell membrane and cytolymph as buffy granular and diffused distribution. The positive rates of ABCB1 and ABCG2 protein were significantly higher in ESCC (77.50% and 86.25%) than those in squamous dysplasia (31.25% and 26.25%) and normal squamous epithelium (6.25% and 3.75%) (P<0.01).
2 Expression of ABCG2 in adriamycin-resistant human esophageal cancer cells
Drug resistant cell Eca109/ADM was established successfully for 8 monthes. Comparing Eca109/ADM cell with Eca109 cell, Eca109/ADM cell became bigger volume and more irregularity morphology. The IC50 of Eca109/ADM and Eca109 cells to ADM was 15.45±1.15,4.69±0.88 after the treatment of ADM for 24h. The RI of Eca109/ADM was 3.29.
ABCG2 mRNA and protein expression of Eca109/ADM cells were significantly higher than Eca109 cells (P<0.05). ABCG2 protein of Eca109/ADM was located in cell membrane and cytolymph and the protein expression was higher than Eca109 cells. ABCB1 protein expression of Eca109/ADM cells was significantly higher than the expression of Eca109 cells (P<0.05). Drug excretion of Eca109/ADM cells was higher than Eca109 cells.
The cell apoptosis rate of Art+ADM group on Eca109/ADM cells was significantly higher than ADM group (P<0.05). ABCG2 protein expression of Eca109/ADM cells was significantly decreased after the treatment of Art for 48h (P<0.05), but ABCB1 protein expression of Eca109/ADM cells was no difference (P>0.05).
3 Establishment of Eca109/ABCG2, a drug resistant esophageal cancer cell line, and its biological profile
Eca109/ABCG2 and Eca109/PCDNA3.1 cell were established successfully. The IC50 of Eca109/ABCG2, Eca109/PCDNA3.1 and Eca109 cells was 18.61±3.94, 4.18±0.14, 4.69±0.88 after treatment of ADM for 24h. The RI of Eca109/ABCG2 was 3.97. The IC50 of Eca109/ABCG2 cells was significantly higher compared with Eca109 cells (P<0.01). The IC50 of Eca109/ PCDNA3.1 cells was no difference compared with Eca109 cells (P>0.05). The IC50 of Eca109/ABCG2 cells to DNR and MIT was higher than Eca109 cells, The RI was 3.50 and 3.15 respectively.
Drug excretion of Eca109/ABCG2 cells was significantly higher than Eca109/ PCDNA3.1 and Eca109 cells (P<0.01).
ABCG2 mRNA and protein expression of Eca109/ABCG2 cells was significantly higher than Eca109/PCDNA3.1 and Eca109 cells (P<0.05). ICC results show: ABCG2 protein of Eca109/ABCG2 was located in cell membrane and cytolymph, and the protein expression was higher than the expression of Eca109/PCDNA3.1 and Eca109 cells.
4 The ADM resistance reversal effect of Art on Eca109/ABCG2 cell and related mechanism.
MTT results show: The inhibitory effect of Art+ADM on the growth of Eca109/ABCG2 cells was significantly higer than Art or ADM (P<0.05). The Eca109/ABCG2 cells apoptosis rate of Art+ADM group was significantly higher than Art or ADM group (P<0.05). ABCG2 mRNA and protein expression of Eca109/ABCG2 cells after the treatment of Art+ADM were significantly lower than ADM (P<0.05).
5 The resistance-reversal effect of Art on human esophageal cancer transplanted in nude mice
The model of esophageal cancer xenograft in nude mice was successfully established, the tumorigenic rate was 100%. In the same ADM concentration group, volume and weight of transplanted tumor inoculated with Eca109/ABCG2 cells was significantly higher than transplanted tumor inoculated with Eca109 cells (P<0.05). In the transplanted tumor inoculated with Eca109/ABCG2 cells groups, tumor volume and weight of Art + ADM group was significantly lower than Art or ADM group (P<0.05), and ABCG2 mRNA, protein expression in tumor cells of Art + ADM group were significantly lower than ADM group (P<0.05).
Conclusions:
1 In the process of carcinogenesis of the esophagus, the expression of ABCB1 and ABCG2 in ESCC were significantly higher than those in squamous dysplasia and normal squamous epithelium. The expression of ABCB1 and ABCG2 in squamous dysplasia were gradually high from gradeⅠto gradeⅢ. High expression of ABCB1 and ABCG2 might be correlated with tumor progression and drug resistance. There was no correlation between ABCB1 expression and ABCG2 expression in ESCC, which suggest that the drug resistance induced by ABCB1 or ABCG2 might have different mechanism. ABCG2 might be new therapy target of esophageal carcinoma drug resistance.
2 Drug resistant cell Eca109/ADM was established successfully by gradually increasing the ADM concentration in the culture medium of human esophageal cancer cell line (Eca109). ABCB1 and ABCG2 expression were higher in Eca109/ADM cells than those in Eca109 cells, which suggested that ABCB1 and ABCG2 might participate in the drug resistance of Eca109/ADM cells. Artesunate could significantly increase the inhibitory effect of ADM on the growth of Eca109/ADM cells and decrease the ABCG2 expression of Eca109/ADM cells but not ABCB1, which suggested that artesunate could reverse the drug resistance of Eca109/ADM cells by decreasing ABCG2 expression of Eca109/ADM cells.
3 For the first time, multidrug resistance cell (Eca109/ABCG2) of esophageal cancer was successfully established by transfection of liposomes. Eca109/ABCG2 cell possessed ABCG2-resistant biological profile and could stably express the ABCG2 protein, so the Eca109/ABCG2 cell was ideally multidrug resistant cell model which had ABCG2 biological profile.
4 For the first time, resistance-reversal effect of artesunate on esophageal cancer was explored in our study. The cell growth inhibitory effect of ADM on Eca109/ABCG2 cells was significantly increased by combining Art and ADM. Art could significantly reduce the ABCG2 expression of Eca109/ABCG2 cells which could inhibit the drugs excretion and increase the ADM content in Eca109/ABCG2 cells, so artesunate could reverse the ADM drug resistance of Eca109/ABCG2 cells.
5 The model of esophageal cancer xenograft in nude mice was successfully established, which could provide an ideal model for the research of esophageal cancer drug-resistance. The growth inhibitory effect of ADM on esophageal cancer xenograft in vivo was enhanced by combining artesunate without causing obvious side effects in treated mice, which coule provide some experimental reference for artesunate as resistance-reversal agent. Artesunate could become high-performance and harmfulless resistance-reversal agent in clinic.
引文
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2 Ceckova M, Libra A, Pavek P, et al. Expression and functional activity of breast cancer resistance protein (BCRP, ABCG2) transporter in the human choriocarcinoma cell line BeWo. Clin Exp Pharmacol Physiol, 2006, 33(1-2):58-65
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