TRAIL对胃癌细胞株SGC7901/VCR耐药基因表达的影响及TRAIL联合顺铂杀伤SGC7901/VCR的研究
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摘要
研究背景
胃癌的发病率仍较高,胃癌的诊治仍是肿瘤诊治中面临的挑战之一。早期胃癌诊治效果良好,十年生存率近90%,但国内早期胃癌的检出率仍较低。大部分患者诊断时已经处于中晚期而不得不接受化疗。化疗多年来仍是胃癌治疗的一个比较重要的方法。但胃癌化疗中一个令人沮丧的问题是胃癌细胞对化疗药物的耐药问题,又称为多药耐药(multidrug resistance, MDR)。因此,将胃癌耐药现象作为研究方向,探索其可能的内在机制和寻找对抗肿瘤细胞耐药的干预物将具有很大的价值,为筛选出抗耐药或逆转耐药的药物打下基础。
我们在以前的应用免疫组化的方法的研究中观察到在人的胃癌细胞中耐药基因如LRP、GST-π高表达且与COX-2呈正相关。同样ELISA法在体外培养的胃癌耐药细胞株SGC7901/VCR中,也观察到上述基因对应的蛋白表达被抑制的现象。用COX-2抑制剂塞来昔布能下调这种表达,证实COX-2高表达与上述耐药基因的活性的关系,显示用外来物对耐药基因的干预可能是有效的。肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor related apoptosis-inducing ligand, TRAIL)能诱导某些种类的肿瘤细胞凋亡,还对一些肿瘤细胞有细胞毒作用。近来还发现TRAIL和一些抗肿瘤药物联合应用时表现出协调效应,使耐药或部分耐药的肿瘤细胞变得对药物有敏感性了。TRAIL增加化疗药物杀伤肿瘤细胞的效力的机制何在?是经典的对肿瘤细胞的促凋亡作用、与抗肿瘤药物的协同杀伤肿瘤细胞作用还是通过抑制了肿瘤细胞耐药基因进而逆转肿瘤细胞的耐药性而增强了对肿瘤细胞的杀伤力。通过我们既往的研究结果,我们假设可能是后者即通过抑制肿瘤细胞耐药基因而使肿瘤细胞的耐药性发生逆转。若果真如此,将对解决困扰胃恶性肿瘤化疗的耐药问题提供较好的切入点。因此,本课题分为两个研究部分：第一部分我们采用不同浓度的TRAIL与胃癌耐药细胞株SGC7901/VCR共同培养,观察TRAIL对耐药基因MDR1,LRP和GST-π有无作用效果,上调还是下调?目的是对TRAIL在耐药基因的调节中扮演的角色进行探索；第二部分我们将TRAIL联合化疗药物顺铂加入胃癌细胞株SGC-7901/VCRTRAIL中,探讨亚毒性剂量的肿瘤坏死因子相关凋亡配体(TRAIL)联合小剂量化疗药物顺铂(DDP)能否协同抑制胃癌细胞株SGC-7901/VCR的活性、提高SGC-7901/VCR的凋亡率和干预其多药耐药基因1(MDR1)勺表达,为进一步走向临床研究打下基础。
第一部分：TRAILI对胃癌耐药细胞株SGC7901/VCR中MDR1, LRP和GST-π耐药基因表达的影响
目的：
研究TRAIL对胃癌细胞株SGC7901/VCR多药耐药基因MDR1, LRP和GST-π的表达的影响,为最终在临床上使用TRAIL逆转胃癌耐药,解决胃癌细胞多药耐药问题提供初步实验依据。
方法：
胃癌耐药细胞株SGC7901/VCR与不同浓度的TRAIL(50,100,200,400ug/L)培养48h,没有任何干预措施的SGC7901/VCR细胞株作为对照,RT-PCR检测各组胃癌耐药细胞株耐药基因MDRl,LRP,GST-π mRNA的表达。同时用ELISA法检测各组胃癌耐药细胞株中三种耐药蛋白P-gp、LRP和GST-π的表达含量：用t检验及单因素方差分析的统计学方法进行分析,研究TRAIL对胃癌耐药细胞株多药耐药基因表达的影响。
结果：
1. RT-PCR结果：
对照组中胃癌耐药细胞株SGC7901/VCR中的三个耐药基因MDR1,LRP,GST-π均呈阳性,mRNA值分别为0.88±0.01,0.91±0.04和1.01±0.13。胃癌耐药细胞株被四种浓度的TRAIL作用,48h后,胃癌耐药细胞株中的MDR1,LRP,GST-πmRNA的表达被抑制,并随着浓度的增高,抑制效益愈强,但未显示出线性关系。TRAIL组的MDR1mRNA值在四个浓度的实验组均较对照组显著降低(分别为0.78±0.02；0.69±0.05；0.55±0.01和0.53±0.01,与对照组相比,四组均p<0.05)。随着TRAIL的浓度50μ g/L、100μ g/L、200μ g/L递增,MDR1mRNA被抑制幅度有逐渐递增的趋势,各个浓度组之间均显示出显著性差别(P<0.05)。但当浓度增加到400μg/L时,对MDR1mRNA的抑制程度与200μ g/L时对比,无显著性差异(p>0.05)。同样, TRAIL组的LRPmRNA值在50,100,200,400μ g/L四个浓度的实验组均较对照组显著降低,其数值分别是0.85±0.05；0.76±0.05；0.59±0.04和0.58±0.02,全部四种浓度均较对照组显著降低p<0.05)。随着TRAIL的浓度50μg/L、100μ g/L、200μ g/L递增,LRPmRNA被抑制程度有逐渐递增的趋势,各个浓度组之间均显示出显著性差别(P<0.05)。但当浓度增加到400μ g/L时,对LRP mRNA的抑制程度与200μ g/L时对比,亦无显著性差异(p>0.05)。与前两者表现相似的是：TRAIL组的GST-π mRNA值在四个浓度的实验组均较对照组显著降低(分别为0.89±0.04；0.77±0.08；0.65±0.06；0.61±0.03。与对照组相比,四种浓度均显著降低,p<0.05)。随着TRAIL的浓度50μg/L、100μg/L、200μg/L递增,GST-π mRNA被抑制程度有逐渐递增的趋势,各个浓度组之间均显示出显著性差别(P<0.05)。但当浓度增加到400μg/L时,对GST-π mRNA的抑制幅度与200μg/L时对比,无显著性差异(p>0.05)。
2. ELISA结果：
P-gp, LRP, GST-π蛋白的表达在对照组中分别是49.04±2.07ug/L、119.35±1.04ug/L和58.62±1.38ug/L；与对照组相比浓度为50ug/L的TRAIL组P-gp, LRP和GST-π的含量分别为40.42±1.89ug/L、112.51±1.19ug/L和57.56±1.19ug/L(与对照组相比均p<0.05)。浓度为100ug/L的TRAIL组P-gp,LRP和GST-π的含量分别为36.49±0.94ug/L、106.69±1.51ug/L和52.30±0.80ug/L,也均较对照组显著降低(p<0.05)。同时浓度为100ug/L的TRAIL组与50ug/L TRAIL组相比也显著降低(均p<0.05)。浓度为200ug/LTRAIL组P-gp, LRP, GST-π的对应数值为25.15±0.69ug/L、83.54±2.15ug/L和31.41±1.65ug/L,比对照组显著降低(p<0.05)。与浓度为100ug/L TRAIL组相比也显著降低(均p<0.05)。浓度400ug/L TRAIL组P-gp, LRP, GST-π的水平相应是24.45±1.41ug/L、82.63±1.35ug/L、30.80±1.34ug/L,与对照组、50ug/L TRAIL组、100ug/L TRAIL组比较,均显著降低(p<0.05；)。然而浓度400ug/L TRAIL组与200ug/L TRAIL组比较,P-gp, LRP和GST-π的降低并无显著性差异(p>0.05)。
结论：
1. MDR1, LRP, GST-π在胃癌耐药细胞株SGC7901/VCR均有表达。
2.在四种浓度的TRAIL的干预下,SGC7901/VCR中耐药基因MDR1、LRP和GST-π的表达均较对照组显著下降且下降幅度与浓度有关。
3. TRAIL除了促进肿瘤细胞的凋亡作用,它还可能通过降低肿瘤耐药细胞株中耐药基因MDR1, LRP, GST-π在的表达而降低胃癌的耐药,增强化疗药物的疗效。
论文第二部分：TRAIL联合顺铂杀伤胃癌细胞株SGC-7901/VCR和对耐药基因MDR1表达的干预研究
目的：
探讨肿瘤坏死因子相关凋亡配体(TRAIL)和小剂量化疗药物顺铂(DDP)对胃癌细胞株SGC-7901/VCR的活性和联合应用对多药耐药基因1(MDR1)的影响。
方法：
运用MTT法检测不同浓度的TRAIL和顺铂对胃癌耐药细胞株SGC7901/VCR的活性或增殖的影响。筛选出TRAIL及顺铂的亚毒性剂量。采用流式细胞术检测TRAIL(?)(?)顺铂的亚毒性剂量下单用及联合应用后的细胞凋亡率。TRAIL和顺铂联合应用后,用RT-PCR技术检测胃癌耐药细胞株耐药基因MDR1mRNA(?)的表达。同时用ELISA法检测耐药蛋白P-gp(?)勺表达含量,研究TRAIL对胃癌耐药细胞株多药耐药基因表达的影响。
结果：
1.MTT法检测药物对SGC-7901/VCR细胞活力的影响的结果显示：
顺铂和TRAIL都可一定程度上杀伤胃癌耐药细胞SGC-7901/VCR,随着药物浓度的增加,细胞活力下降。经计算48h顺铂的IC10(活性抑制10%)为0.535g/L,以MTT结果选取200μg/L作为TRAIL浓度较合适。流式细胞术结果显示对照组、顺铂组、TRAIL组、TRAIL顺铂联合用药组中,作用48h后胃癌细胞凋亡率以联合用药组细胞凋亡显著升高,差异有统计学意义(P<0.05)。
2.RT-PCR结果：
对照组中胃癌耐药细胞株SGC7901/VCR的耐药基因MDR1呈阳性。胃癌耐药细胞株被TRAIL和顺铂联合作用48小时后,胃癌耐药细胞株中的MDR1mRNA的表达被抑制,较对照组显著降低(p<0.05)。联合用药组细胞凋亡明显升高,与对照及单药组比较,差异有统计学意义(P<0.05)。
3. ELISA法结果：
与单药组比较,联合用药组协同作用明显,P-gp蛋白表达下降,差异有统计学意义(P<0.05)
结论：
1.胃癌耐药细胞株SGC7901/VCR中耐药基因MDR1高表达,MDR1是胃癌多药耐药的耐药基因。
2. TRAIL与顺铂联合具有协同抑制胃癌耐药细胞株SGC7901/VCR活性、显著增加凋亡率,下调MDR1基因和P-gp蛋白表达的作用。
3.推测TRAIL与顺铂协同杀伤胃癌耐药细胞株SGC7901/VCR的机制可能是通过抑制MDR1基因的表达从而逆转胃癌细胞的多药耐药实现的。
Background
Remains high incidence of gastric cancer, the diagnosis and treatment of gastric cancer is still one of the challenges faced in the cancer diagnosis and treatment. Diagnosis and treatment of early gastric cancer,10-year survival rate of nearly90%, but the domestic early detection of gastric cancer is still low. Already in the middle and late diagnosis of the majority of patients with gastric cancer and had to undergo chemotherapy which for many years a important method of treatment of gastric cancer. But a frustrating problem in cancer chemotherapy patients is on chemotherapy drug resistance problem, also known as multi-drug resistant (multidrug resistance, MDR). Therefore, the phenomenon of drug resistant gastric cancer as the research direction, exploring its internal mechanism and looking for intervention against drug-resistant tumor cells was of great value, and lay the foundation for the screening of drugs against resistant or reversal of drug resistance.
The previous series of studies have shown that resistance genes such as LRP. GST-π high expression was positively correlated with COX-2in human gastric cancer cells. The equally resistant gastric cancer cell lines in vitro SGC7901/VCR also was observed a similar phenomenon. With COX-2inhibitors celecoxib down this expression, confirmed that COX-2activity relationship of high expression of the resistance genes, at the same time show that the intervention of foreign material from the resistant gene is effective. Tumor necrosis factor-related apoptosis-inducing ligand on normal cells almost no effect, but can induce apoptosis in some types of tumor cells, while also direct killing effect on tumor cells. Recently found that some of the phenomena that TRAIL has increased the effectiveness of chemotherapy drugs kill tumor cells,that the tumor cells become more sensitive to chemotherapeutic drugs. TRAIL mechanism to increase the effectiveness of chemotherapy drugs killing tumor cells lie? Is the classic role of the pro-apoptotic effect on tumor cell, and the synergistic effect of the antineoplastic agents or by inhibiting the expression of the resistance genes of the tumor cells thereby reversing the drug resistance of the tumor cells? By the results of our previous studies, we assume that the tumor cells by inhibiting tumor cell resistance gene leave to become sensitive to chemotherapeutic drugs. If this assumption is true, it will provide good prospects solve troubled by stomach cancer chemotherapy drug problem.
This study is divided into two parts:Part Ⅰ:TRAIL resistant gastric cancer cell lines SGC7901/VCR common culture, to explore the effect of TRAIL on the expression of MDR1, LRP and GST-π in drug-resistant gastric cancer Cell SGC7901/VCR; Part Ⅱ:Explore sub-toxic doses of tumor necrosis factor-related apoptosis ligand (TRAIL) in combination with low-dose chemotherapy drug cisplatin (DDP) on gastric cancer cell lines SGC-7901/VCR activity, apoptosis rate and multidrug resistance gene1(MDR1) expression.
Part Ⅰ:The effect of TRAIL on the expression of multidrug resistant genes MDR1, LRP and GST-π in drug-resistant gastric cancer Cell SGC7901/VCR
Objective:
Research TRAIL on gastric cancer cell line SGC7901/VCR multidrug resistance gene MDR1, LRP and GST-π expression of, and to explore the possible mechanism of action. The research applications of TRAIL to provide experimental evidence to resolve the multidrug resistance of gastric cancer cells.
Method:
Resistant gastric cancer cell line SGC7901/VCR with different concentrations of TRAIL cultured48h, no dosing cell line as a control, RT-PCR was used to detect the groups resistant gastric cancer cell line resistance gene MDR1, LRP, GST-πimRNA expression.
ELISA was used to detect the three types of resistance protein P-gp, LRP, GST-π content of resistant gastric cancer cell lines, t-test and one-way ANOVA statistical methods for analysis. The purpose is to understand the TRAIL on resistant gastric cancer cell line expression of multidrug resistance gene.
Results:
1. RT-PCR results:
The control group three resistant gastric cancer cell line resistant gene MDR1, LRP, GST-π were positive and mRNA values were0.88±0.01,0.91±0.04and1.01±0.13. With TRAIL action, gastric resistant cell line of MDR1, LRP, GST-π mRNA expression was inhibited, and with the increase of concentration, inhibited stronger, but did not show a linear relationship. The TRAIL group MDR1mRNA value in four concentrations of the experimental group than in the control group was significantly lower (0.78±0.02;0.69±0.05;0.55±0.01and0.53±0.01,respectively, all four groups are p<0.01). With increasing concentrations of TRAIL, MDR1mRNA be suppressed amplitude increasing trend, showed a significant difference (P<0.01) among the respective groups. But when the concentration was increased to400μg/L and200μg/L, inhibition of MDR1mRNA amplitude contrast, there was no significant difference (p>0.05). Similarly, the TRAIL-group LRPmRNA value in the experimental group of four concentrations significantly lower than those of the control group, the values were0.85±0.05;0.76±0.05;0.59±0.04and0.58±0.02, all four groups p<0.01. With TRAIL concentration of50μg/L,100μg/L.200μg/L increments. LRPmRNA decreases in each concentration groups showed a significant differences(P<0.01). But when the concentration was increased to400μg/L, the inhibition of LRPmRNA compared with200μg/L, no significant difference (p>0.05).
Performance similar to the first two: The TRAIL-group GST-πmRNA value in the experimental group of four concentrations significantly lower than those of the control group (respectively0.89±0.04,0.77±0.08,0.65±0.06and0.61±0.03, p <0.01). With the TRAIL concentration of50ug/L,100μg/L,200ug/L increments, GST-π mRNA was inhibited more significantl and showed a significant difference among various concentrations (P <0.01). But when the concentration was increased to400μg/L and200μg/L, contrast, there was no significant difference inhibition of GST-πmRNA amplitude (p>0.05).
2. ELISA results:
P-gp, LRP, GST-π protein expression in the control group were49.04±2.07ug/L,119.35±1.04ug/L and58.62±1.38ug/L. Compared with the control group, the concentration of50ug/L of TRAIL group of P-gp, LRP and GST-π content were40.42±1.89ug/L,112.51±19ug/L and57.56±1.19ug/L (all p <0.01). Concentration of100ug/L of TRAIL group of P-gp, LRP and GST-71content were36.49±0.94ug/L,106.69±1.51ug/L,52.30±0.80ug/L and also significantly lower than the control group (p <0.01). At the same time the concentration of100ug/L TRAIL group compared with50ug/L TRAIL group was also significantly decreased (p <0.01). For200ug/L TRAIL group, P-gp, LRP and GST-π values were25.15±0.69ug/L,83.54±2.15ug/L and31.41±1.65ug/L. They were significantly lowered than the control group (p <0.01). P-gp, LRP and GST-π values in200ug/L TRAIL group was significantly lower than in100ug/L TRAIL group also (p <0.01). P-gp, LRP and GST-π levels in400ug/L TRAIL group were24.45±1.41ug/L.82.63±1.35ug/L.30.80±1.34ug/L respectively and they were significantly lowered than in the control group,50ug/L TRAIL group and100ug/L TRAIL (p <0.01;). However, there was no significant difference of P-gp, LRP and GST-π's values between400ug/L TRAIL group and200ug/L TRAIL group(p>0.05).
Conclusion:
1. The gastric cancer resistance gene MDR1, LRP, GST-π in gastric cancer resistant cell line SGC7901/VCR expression.
2. Four concentrations of TRAIL intervention, SGC7901/VCR resistance gene MDR1,LRP and GST-π expression decreased and their decrease amplitudes were related to the TRAIL concentrations.
3. In addition to promoting apoptosis of tumor cells, TRAIL may also reduce MDR1, LRP, GST-πexpressions of tumors resistant cell line SGC7901/VCR and enhanced the efficacy of chemotherapy drugs.
Part II:Effect of TRAIL in combination with DDP on the expression of MDR1gene in gastric cancer cells
Objective:
To study the effect of tumor necrosis factor related apoptosis inducing ligand (TRAIL) combined with chemotherapeutic drug cisplatin(DDP) on activity, apoptosis rate, and expression of multidrug resistance gene MDR1in the gastric cancer cell line SGC-7901/VCR.
Methods:
SGC-7901/VCR cells were cultured with DDP and TRAIL in different concentrations. The apoptosis rate was separately measured by a flow cytometer in DDP (sub-toxic dose) alone, TRAIL ((200μg/L) alone and in the combination of the two. Expression level of MDR1mRNA and P-gp protein were detected by RT-PCR and ELISA analysis, respectively.
Results:
The apoptosis rate in combination group was significantly higher than that in the other groups (P<0.05). According to the results of RT-PCR and ELISA, the expression of MDR1mRNA and P-gp protein of combination group were statistically significant different compared with other groups (P<0.05).
Conclusion:
1. MDR1high expression in resistant gastric cancer cell line SGC7901/VCR MDR1resistance gene indicated that MDR1was resistance gene in gastric cancer With relevant secondary multidrug resistance.
2. TRAIL can enhance the effect of DDP in anticancer therapy. Cisplatin and TRAIL synergistic down MDRlgene expression.
3. The down-regulate of multidrug resistance gene MDR1may play a potential role in overcoming the chemotherapeutic resistance of gastric cancer cells.
胃癌的发病率仍较高,胃癌的诊治仍是肿瘤诊治中面临的挑战之一。早期胃癌诊治效果良好,十年生存率近90%,但国内早期胃癌的检出率仍较低。大部分患者诊断时已经处于中晚期而不得不接受化疗。化疗多年来仍是胃癌治疗的一个比较重要的方法。但胃癌化疗中一个令人沮丧的问题是胃癌细胞对化疗药物的耐药问题,又称为多药耐药(multidrug resistance, MDR)。因此,将胃癌耐药现象作为研究方向,探索其可能的内在机制和寻找对抗肿瘤细胞耐药的干预物将具有很大的价值,为筛选出抗耐药或逆转耐药的药物打下基础。
我们在以前的应用免疫组化的方法的研究中观察到在人的胃癌细胞中耐药基因如LRP、GST-π高表达且与COX-2呈正相关。同样ELISA法在体外培养的胃癌耐药细胞株SGC7901/VCR中,也观察到上述基因对应的蛋白表达被抑制的现象。用COX-2抑制剂塞来昔布能下调这种表达,证实COX-2高表达与上述耐药基因的活性的关系,显示用外来物对耐药基因的干预可能是有效的。肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor related apoptosis-inducing ligand, TRAIL)能诱导某些种类的肿瘤细胞凋亡,还对一些肿瘤细胞有细胞毒作用。近来还发现TRAIL和一些抗肿瘤药物联合应用时表现出协调效应,使耐药或部分耐药的肿瘤细胞变得对药物有敏感性了。TRAIL增加化疗药物杀伤肿瘤细胞的效力的机制何在?是经典的对肿瘤细胞的促凋亡作用、与抗肿瘤药物的协同杀伤肿瘤细胞作用还是通过抑制了肿瘤细胞耐药基因进而逆转肿瘤细胞的耐药性而增强了对肿瘤细胞的杀伤力。通过我们既往的研究结果,我们假设可能是后者即通过抑制肿瘤细胞耐药基因而使肿瘤细胞的耐药性发生逆转。若果真如此,将对解决困扰胃恶性肿瘤化疗的耐药问题提供较好的切入点。因此,本课题分为两个研究部分：第一部分我们采用不同浓度的TRAIL与胃癌耐药细胞株SGC7901/VCR共同培养,观察TRAIL对耐药基因MDR1,LRP和GST-π有无作用效果,上调还是下调?目的是对TRAIL在耐药基因的调节中扮演的角色进行探索；第二部分我们将TRAIL联合化疗药物顺铂加入胃癌细胞株SGC-7901/VCRTRAIL中,探讨亚毒性剂量的肿瘤坏死因子相关凋亡配体(TRAIL)联合小剂量化疗药物顺铂(DDP)能否协同抑制胃癌细胞株SGC-7901/VCR的活性、提高SGC-7901/VCR的凋亡率和干预其多药耐药基因1(MDR1)勺表达,为进一步走向临床研究打下基础。
第一部分：TRAILI对胃癌耐药细胞株SGC7901/VCR中MDR1, LRP和GST-π耐药基因表达的影响
目的：
研究TRAIL对胃癌细胞株SGC7901/VCR多药耐药基因MDR1, LRP和GST-π的表达的影响,为最终在临床上使用TRAIL逆转胃癌耐药,解决胃癌细胞多药耐药问题提供初步实验依据。
方法：
胃癌耐药细胞株SGC7901/VCR与不同浓度的TRAIL(50,100,200,400ug/L)培养48h,没有任何干预措施的SGC7901/VCR细胞株作为对照,RT-PCR检测各组胃癌耐药细胞株耐药基因MDRl,LRP,GST-π mRNA的表达。同时用ELISA法检测各组胃癌耐药细胞株中三种耐药蛋白P-gp、LRP和GST-π的表达含量：用t检验及单因素方差分析的统计学方法进行分析,研究TRAIL对胃癌耐药细胞株多药耐药基因表达的影响。
结果：
1. RT-PCR结果：
对照组中胃癌耐药细胞株SGC7901/VCR中的三个耐药基因MDR1,LRP,GST-π均呈阳性,mRNA值分别为0.88±0.01,0.91±0.04和1.01±0.13。胃癌耐药细胞株被四种浓度的TRAIL作用,48h后,胃癌耐药细胞株中的MDR1,LRP,GST-πmRNA的表达被抑制,并随着浓度的增高,抑制效益愈强,但未显示出线性关系。TRAIL组的MDR1mRNA值在四个浓度的实验组均较对照组显著降低(分别为0.78±0.02；0.69±0.05；0.55±0.01和0.53±0.01,与对照组相比,四组均p<0.05)。随着TRAIL的浓度50μ g/L、100μ g/L、200μ g/L递增,MDR1mRNA被抑制幅度有逐渐递增的趋势,各个浓度组之间均显示出显著性差别(P<0.05)。但当浓度增加到400μg/L时,对MDR1mRNA的抑制程度与200μ g/L时对比,无显著性差异(p>0.05)。同样, TRAIL组的LRPmRNA值在50,100,200,400μ g/L四个浓度的实验组均较对照组显著降低,其数值分别是0.85±0.05；0.76±0.05；0.59±0.04和0.58±0.02,全部四种浓度均较对照组显著降低p<0.05)。随着TRAIL的浓度50μg/L、100μ g/L、200μ g/L递增,LRPmRNA被抑制程度有逐渐递增的趋势,各个浓度组之间均显示出显著性差别(P<0.05)。但当浓度增加到400μ g/L时,对LRP mRNA的抑制程度与200μ g/L时对比,亦无显著性差异(p>0.05)。与前两者表现相似的是：TRAIL组的GST-π mRNA值在四个浓度的实验组均较对照组显著降低(分别为0.89±0.04；0.77±0.08；0.65±0.06；0.61±0.03。与对照组相比,四种浓度均显著降低,p<0.05)。随着TRAIL的浓度50μg/L、100μg/L、200μg/L递增,GST-π mRNA被抑制程度有逐渐递增的趋势,各个浓度组之间均显示出显著性差别(P<0.05)。但当浓度增加到400μg/L时,对GST-π mRNA的抑制幅度与200μg/L时对比,无显著性差异(p>0.05)。
2. ELISA结果：
P-gp, LRP, GST-π蛋白的表达在对照组中分别是49.04±2.07ug/L、119.35±1.04ug/L和58.62±1.38ug/L；与对照组相比浓度为50ug/L的TRAIL组P-gp, LRP和GST-π的含量分别为40.42±1.89ug/L、112.51±1.19ug/L和57.56±1.19ug/L(与对照组相比均p<0.05)。浓度为100ug/L的TRAIL组P-gp,LRP和GST-π的含量分别为36.49±0.94ug/L、106.69±1.51ug/L和52.30±0.80ug/L,也均较对照组显著降低(p<0.05)。同时浓度为100ug/L的TRAIL组与50ug/L TRAIL组相比也显著降低(均p<0.05)。浓度为200ug/LTRAIL组P-gp, LRP, GST-π的对应数值为25.15±0.69ug/L、83.54±2.15ug/L和31.41±1.65ug/L,比对照组显著降低(p<0.05)。与浓度为100ug/L TRAIL组相比也显著降低(均p<0.05)。浓度400ug/L TRAIL组P-gp, LRP, GST-π的水平相应是24.45±1.41ug/L、82.63±1.35ug/L、30.80±1.34ug/L,与对照组、50ug/L TRAIL组、100ug/L TRAIL组比较,均显著降低(p<0.05；)。然而浓度400ug/L TRAIL组与200ug/L TRAIL组比较,P-gp, LRP和GST-π的降低并无显著性差异(p>0.05)。
结论：
1. MDR1, LRP, GST-π在胃癌耐药细胞株SGC7901/VCR均有表达。
2.在四种浓度的TRAIL的干预下,SGC7901/VCR中耐药基因MDR1、LRP和GST-π的表达均较对照组显著下降且下降幅度与浓度有关。
3. TRAIL除了促进肿瘤细胞的凋亡作用,它还可能通过降低肿瘤耐药细胞株中耐药基因MDR1, LRP, GST-π在的表达而降低胃癌的耐药,增强化疗药物的疗效。
论文第二部分：TRAIL联合顺铂杀伤胃癌细胞株SGC-7901/VCR和对耐药基因MDR1表达的干预研究
目的：
探讨肿瘤坏死因子相关凋亡配体(TRAIL)和小剂量化疗药物顺铂(DDP)对胃癌细胞株SGC-7901/VCR的活性和联合应用对多药耐药基因1(MDR1)的影响。
方法：
运用MTT法检测不同浓度的TRAIL和顺铂对胃癌耐药细胞株SGC7901/VCR的活性或增殖的影响。筛选出TRAIL及顺铂的亚毒性剂量。采用流式细胞术检测TRAIL(?)(?)顺铂的亚毒性剂量下单用及联合应用后的细胞凋亡率。TRAIL和顺铂联合应用后,用RT-PCR技术检测胃癌耐药细胞株耐药基因MDR1mRNA(?)的表达。同时用ELISA法检测耐药蛋白P-gp(?)勺表达含量,研究TRAIL对胃癌耐药细胞株多药耐药基因表达的影响。
结果：
1.MTT法检测药物对SGC-7901/VCR细胞活力的影响的结果显示：
顺铂和TRAIL都可一定程度上杀伤胃癌耐药细胞SGC-7901/VCR,随着药物浓度的增加,细胞活力下降。经计算48h顺铂的IC10(活性抑制10%)为0.535g/L,以MTT结果选取200μg/L作为TRAIL浓度较合适。流式细胞术结果显示对照组、顺铂组、TRAIL组、TRAIL顺铂联合用药组中,作用48h后胃癌细胞凋亡率以联合用药组细胞凋亡显著升高,差异有统计学意义(P<0.05)。
2.RT-PCR结果：
对照组中胃癌耐药细胞株SGC7901/VCR的耐药基因MDR1呈阳性。胃癌耐药细胞株被TRAIL和顺铂联合作用48小时后,胃癌耐药细胞株中的MDR1mRNA的表达被抑制,较对照组显著降低(p<0.05)。联合用药组细胞凋亡明显升高,与对照及单药组比较,差异有统计学意义(P<0.05)。
3. ELISA法结果：
与单药组比较,联合用药组协同作用明显,P-gp蛋白表达下降,差异有统计学意义(P<0.05)
结论：
1.胃癌耐药细胞株SGC7901/VCR中耐药基因MDR1高表达,MDR1是胃癌多药耐药的耐药基因。
2. TRAIL与顺铂联合具有协同抑制胃癌耐药细胞株SGC7901/VCR活性、显著增加凋亡率,下调MDR1基因和P-gp蛋白表达的作用。
3.推测TRAIL与顺铂协同杀伤胃癌耐药细胞株SGC7901/VCR的机制可能是通过抑制MDR1基因的表达从而逆转胃癌细胞的多药耐药实现的。
Background
Remains high incidence of gastric cancer, the diagnosis and treatment of gastric cancer is still one of the challenges faced in the cancer diagnosis and treatment. Diagnosis and treatment of early gastric cancer,10-year survival rate of nearly90%, but the domestic early detection of gastric cancer is still low. Already in the middle and late diagnosis of the majority of patients with gastric cancer and had to undergo chemotherapy which for many years a important method of treatment of gastric cancer. But a frustrating problem in cancer chemotherapy patients is on chemotherapy drug resistance problem, also known as multi-drug resistant (multidrug resistance, MDR). Therefore, the phenomenon of drug resistant gastric cancer as the research direction, exploring its internal mechanism and looking for intervention against drug-resistant tumor cells was of great value, and lay the foundation for the screening of drugs against resistant or reversal of drug resistance.
The previous series of studies have shown that resistance genes such as LRP. GST-π high expression was positively correlated with COX-2in human gastric cancer cells. The equally resistant gastric cancer cell lines in vitro SGC7901/VCR also was observed a similar phenomenon. With COX-2inhibitors celecoxib down this expression, confirmed that COX-2activity relationship of high expression of the resistance genes, at the same time show that the intervention of foreign material from the resistant gene is effective. Tumor necrosis factor-related apoptosis-inducing ligand on normal cells almost no effect, but can induce apoptosis in some types of tumor cells, while also direct killing effect on tumor cells. Recently found that some of the phenomena that TRAIL has increased the effectiveness of chemotherapy drugs kill tumor cells,that the tumor cells become more sensitive to chemotherapeutic drugs. TRAIL mechanism to increase the effectiveness of chemotherapy drugs killing tumor cells lie? Is the classic role of the pro-apoptotic effect on tumor cell, and the synergistic effect of the antineoplastic agents or by inhibiting the expression of the resistance genes of the tumor cells thereby reversing the drug resistance of the tumor cells? By the results of our previous studies, we assume that the tumor cells by inhibiting tumor cell resistance gene leave to become sensitive to chemotherapeutic drugs. If this assumption is true, it will provide good prospects solve troubled by stomach cancer chemotherapy drug problem.
This study is divided into two parts:Part Ⅰ:TRAIL resistant gastric cancer cell lines SGC7901/VCR common culture, to explore the effect of TRAIL on the expression of MDR1, LRP and GST-π in drug-resistant gastric cancer Cell SGC7901/VCR; Part Ⅱ:Explore sub-toxic doses of tumor necrosis factor-related apoptosis ligand (TRAIL) in combination with low-dose chemotherapy drug cisplatin (DDP) on gastric cancer cell lines SGC-7901/VCR activity, apoptosis rate and multidrug resistance gene1(MDR1) expression.
Part Ⅰ:The effect of TRAIL on the expression of multidrug resistant genes MDR1, LRP and GST-π in drug-resistant gastric cancer Cell SGC7901/VCR
Objective:
Research TRAIL on gastric cancer cell line SGC7901/VCR multidrug resistance gene MDR1, LRP and GST-π expression of, and to explore the possible mechanism of action. The research applications of TRAIL to provide experimental evidence to resolve the multidrug resistance of gastric cancer cells.
Method:
Resistant gastric cancer cell line SGC7901/VCR with different concentrations of TRAIL cultured48h, no dosing cell line as a control, RT-PCR was used to detect the groups resistant gastric cancer cell line resistance gene MDR1, LRP, GST-πimRNA expression.
ELISA was used to detect the three types of resistance protein P-gp, LRP, GST-π content of resistant gastric cancer cell lines, t-test and one-way ANOVA statistical methods for analysis. The purpose is to understand the TRAIL on resistant gastric cancer cell line expression of multidrug resistance gene.
Results:
1. RT-PCR results:
The control group three resistant gastric cancer cell line resistant gene MDR1, LRP, GST-π were positive and mRNA values were0.88±0.01,0.91±0.04and1.01±0.13. With TRAIL action, gastric resistant cell line of MDR1, LRP, GST-π mRNA expression was inhibited, and with the increase of concentration, inhibited stronger, but did not show a linear relationship. The TRAIL group MDR1mRNA value in four concentrations of the experimental group than in the control group was significantly lower (0.78±0.02;0.69±0.05;0.55±0.01and0.53±0.01,respectively, all four groups are p<0.01). With increasing concentrations of TRAIL, MDR1mRNA be suppressed amplitude increasing trend, showed a significant difference (P<0.01) among the respective groups. But when the concentration was increased to400μg/L and200μg/L, inhibition of MDR1mRNA amplitude contrast, there was no significant difference (p>0.05). Similarly, the TRAIL-group LRPmRNA value in the experimental group of four concentrations significantly lower than those of the control group, the values were0.85±0.05;0.76±0.05;0.59±0.04and0.58±0.02, all four groups p<0.01. With TRAIL concentration of50μg/L,100μg/L.200μg/L increments. LRPmRNA decreases in each concentration groups showed a significant differences(P<0.01). But when the concentration was increased to400μg/L, the inhibition of LRPmRNA compared with200μg/L, no significant difference (p>0.05).
Performance similar to the first two: The TRAIL-group GST-πmRNA value in the experimental group of four concentrations significantly lower than those of the control group (respectively0.89±0.04,0.77±0.08,0.65±0.06and0.61±0.03, p <0.01). With the TRAIL concentration of50ug/L,100μg/L,200ug/L increments, GST-π mRNA was inhibited more significantl and showed a significant difference among various concentrations (P <0.01). But when the concentration was increased to400μg/L and200μg/L, contrast, there was no significant difference inhibition of GST-πmRNA amplitude (p>0.05).
2. ELISA results:
P-gp, LRP, GST-π protein expression in the control group were49.04±2.07ug/L,119.35±1.04ug/L and58.62±1.38ug/L. Compared with the control group, the concentration of50ug/L of TRAIL group of P-gp, LRP and GST-π content were40.42±1.89ug/L,112.51±19ug/L and57.56±1.19ug/L (all p <0.01). Concentration of100ug/L of TRAIL group of P-gp, LRP and GST-71content were36.49±0.94ug/L,106.69±1.51ug/L,52.30±0.80ug/L and also significantly lower than the control group (p <0.01). At the same time the concentration of100ug/L TRAIL group compared with50ug/L TRAIL group was also significantly decreased (p <0.01). For200ug/L TRAIL group, P-gp, LRP and GST-π values were25.15±0.69ug/L,83.54±2.15ug/L and31.41±1.65ug/L. They were significantly lowered than the control group (p <0.01). P-gp, LRP and GST-π values in200ug/L TRAIL group was significantly lower than in100ug/L TRAIL group also (p <0.01). P-gp, LRP and GST-π levels in400ug/L TRAIL group were24.45±1.41ug/L.82.63±1.35ug/L.30.80±1.34ug/L respectively and they were significantly lowered than in the control group,50ug/L TRAIL group and100ug/L TRAIL (p <0.01;). However, there was no significant difference of P-gp, LRP and GST-π's values between400ug/L TRAIL group and200ug/L TRAIL group(p>0.05).
Conclusion:
1. The gastric cancer resistance gene MDR1, LRP, GST-π in gastric cancer resistant cell line SGC7901/VCR expression.
2. Four concentrations of TRAIL intervention, SGC7901/VCR resistance gene MDR1,LRP and GST-π expression decreased and their decrease amplitudes were related to the TRAIL concentrations.
3. In addition to promoting apoptosis of tumor cells, TRAIL may also reduce MDR1, LRP, GST-πexpressions of tumors resistant cell line SGC7901/VCR and enhanced the efficacy of chemotherapy drugs.
Part II:Effect of TRAIL in combination with DDP on the expression of MDR1gene in gastric cancer cells
Objective:
To study the effect of tumor necrosis factor related apoptosis inducing ligand (TRAIL) combined with chemotherapeutic drug cisplatin(DDP) on activity, apoptosis rate, and expression of multidrug resistance gene MDR1in the gastric cancer cell line SGC-7901/VCR.
Methods:
SGC-7901/VCR cells were cultured with DDP and TRAIL in different concentrations. The apoptosis rate was separately measured by a flow cytometer in DDP (sub-toxic dose) alone, TRAIL ((200μg/L) alone and in the combination of the two. Expression level of MDR1mRNA and P-gp protein were detected by RT-PCR and ELISA analysis, respectively.
Results:
The apoptosis rate in combination group was significantly higher than that in the other groups (P<0.05). According to the results of RT-PCR and ELISA, the expression of MDR1mRNA and P-gp protein of combination group were statistically significant different compared with other groups (P<0.05).
Conclusion:
1. MDR1high expression in resistant gastric cancer cell line SGC7901/VCR MDR1resistance gene indicated that MDR1was resistance gene in gastric cancer With relevant secondary multidrug resistance.
2. TRAIL can enhance the effect of DDP in anticancer therapy. Cisplatin and TRAIL synergistic down MDRlgene expression.
3. The down-regulate of multidrug resistance gene MDR1may play a potential role in overcoming the chemotherapeutic resistance of gastric cancer cells.
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17. Zhu W, Shan X, Wang TS,et al. miR-181b modulates multidrug resistance by targeting BCL2 in human cancer cell lines. Int J Cancer 2010,127:2520-2529.
18. Follet J, Corcos L, Baffet G, et al. The association of statins and taxanes:an efficient combination trigger of cancer cell apoptosis. Br J Cancer 2012,106(4): 685-92.
19. Gianni L. Anthracycline resistance:the problem and its current definition. Semin Oncol 1997,24(4):10-17.
20. Borska S. Chmielewska M, Wysocka T.et al. In vitro effect of quercetin on human gastric carcinoma:Targeting cancer cells death and MDR. Food and Chemical Toxicology 2012.(50): 3375-3383.
21. Duarte N, Lage H, Abrantes M.et al. Phenolic compounds as selective antineoplasic agents against multidrug-resistant human cancer cells. Planta Med 2010,76(10):975-80.
22. Tozawa K, Oshima T, Kobayashi T, et al. Oxaliplatin in treatment of the cisplatin-resistant MKN45 cell line of gastric cancer. Anticancer Res 2008,28(4B):2087-2092.
23. Shi LX, Ma R, Lu R, et al. Reversal effect of tyroservatide (YSV) tripeptide on multidrug resistance in resistant human hepatocellular carcinoma cell line BEL-7402/5-FU. Cancer Lett 2008,269(1):101-10.
24. Song X, Wang JB, Yin DL, et al. Down-regulation of lung resistance related protein by RNA interference targeting survivin induces the reversal of chemoresistances in hepatocellular carcinoma. Chin Med J.2009, 122(21):2636-42.
25. HAO Yi-xin, HE Zheng-wenl, DU Nan, et al. Suppression of MDR1 gene expression and reversal of cisplatin resistance in renal carcinoma cells by RNA inter-ference. ZHONGLIU 2010,30(2):115-118.
26. Komdeur R, Plaat BE, Hoekstra HJ, et al. Expression of P-glycoprotein, multidrug resistance-associated protein 1, and lung resistance-related protein in human soft tissue sarcomas before and after hyperthermic isolated limb perfusion with tumor necrosis factor related apoptosis inducing ligand-alpha and melphalan. Cancer 2001,91(10):1940-8.
27. Wang Q, Chen XP, Hai S, et al. TNF-alpha induced reversal of multidrug resistance in human hepatocellular carcinoma cells. Zhonghua Wai Ke Za Zhi 2007,45(9):602-4.
28. Wu Y, Fan Y, Xue B, et al. Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals. Oncogene 2006.25(42):5787-800.
29. Cretney E, Takeda K, Smyth MJ. Cancer:novel therapeutic strategies that exploit the TNF-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor pathway. Int J Biochem Cell Biol 2007.39(2):280-286.
30. Wu GS. TRAIL as a target in anti-cancer therapy. Cancer Lett 2009,285(1):1-5.
31.苏艳新,王立.多西他赛联合TRAIL诱导胃癌细胞SGC7901凋亡的作用.中国癌症杂志2008,18(4)：262-266.
32. Sadarangani A, Kato S, Espinoza N, et al. TRAII mediates apoptosis in cancerous but not norm-al prim ary cultured cells of the human reproductive tract. Apoptosis 2007,12(1):73-85.
33. Cuello M, Ettenberg SA, Nau MM, et al. Synergistic induction of apoptosis by the combination of TRAIL and chemotherapy in chemoresistant ovarian cancer cells. Gynecol Oncol.2001,81(3):380-90.
34. Abdulghani J, El-Deiry WS. TRAIL receptor signaling and therapeutics. Expert Opin Ther Targets 2010,14(10):1091-108.
35. Liu FT, Agrawal SG, Gribben JG, et al. Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL. Blood 2008,111(5):2797-805.
36. Shenoy K, Wu Y, Pervaiz S. LY303511 enhances TRAIL sensitivity of SHEP-1 neuroblastoma cells via hydrogen peroxide-mediated mitogen-activated protein kinase activation and up-regulation of death receptors. Cancer Res 2009:69(5):1941-50.
37. Seo SB, Hur JG, Kim MJ, et al. TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3beta pathway and activation of caspases. Mol Cancer 2010, 9:199-212.
38. Zhu GX, Qu Q, Chen QJ, et al. Expression and significance of DR5, DcRl protein for TNF-related apoptosis-inducing ligand and P-glycoprotein in endometrial carcinoma. Journal of Shanxi Medical University.2006:37(8):800-803.
39. Shen X, Zhen JY, Shi H, et al. Survivin Knockdown Enhances Gastric Cancer Cell Sensitivity to Radiation and Chemotherapy In Vitro and in Nude Mice. Am J Med Sci 2012,344(1):52-8.
40.刘玲,费素娟TRAIL联合PTDC对SGC-7901细胞生长抑制和凋亡诱导的实验研究.徐州医学院学报2009,29(11)：71821.
41. Huang C. Xu D. Xia Q, et al. Reversal of P-glycoprotein-mediated multidrug resistance of human hepatic cancer cells by Astragaloside Ⅱ. J Pharm Pharmacol 2012,64(12):1741-50.
42. Li X, Li JP, Yuan HY, et al. Recent advances in P-glycoprotein-mediated multidrug resistance reversal mechanisms. Methods Find Exp Clin Pharmacol 2007,29:607-617.
43. Lu D, Shi HC, Wang ZX, et al. Multidrug resistance-associated biomarkers PGP, GST-pi, Topo-Ⅱ and LRP as prognostic factors in primary ovarian carcinoma. Br J Biomed Sci 2011,68(2):69-74.
44. Hu WQ, Peng CW, Li Y. The expression and significance of P-glycoprotein, lung resistance protein and multidrug resistance-associated protein in gastric cancer. Exp Clin Cancer Res.2009,28:144.
45. Ding L; Chen XP; Zhang ZW, et al. Synergistic effect of bromocriptine and tumor necrosis factor-a onreversing hepatocellular carcinoma multidrug resistance in nude mouseMDRl model of liver neoplasm. WJG 2005,11(36):5621-5626.
46. Xu Z, Zhu H, Luk JM, et al. Clinical significance of SOD2 and GST-π gene polymorphisms in Chinese patients with gastric cancer. Cancer 2012, 118(22):5489-5498.
47.魏素菊,刘海英,史健,韩文峰.重组改构人TNF逆转卵巢癌耐药细胞株SKOV3/DDP的耐药性及其机制.中国肿瘤生物治疗杂志2008,15(2)：150-154.
1. Yang L. Incidence and mortality of gastric cancer in China [J]. World J Gastroenterol 2006; 12(1):17-20.
2. Guo P, Huang ZL, Yu P, Li K. Trends in cancer mortality in China:an update. Ann Oncol 2012; 23(10):2755-62.
3. Sheng X, Zhang L, Tong N, Luo D, Wang M, Zhang Z. MDR1 C3435T polymorphism and cancer risk:a meta-analysis based on 39 case-control studies. Mol Biol Rep 2012; 39(7):7237.
4. Lee SW, Lee YL, Lee YJ, Park SY, Kim IS, Choi TH, Ha JH, Ahn BC, Lee J. Enhanced antitumor effects by combination gene therapy using MDR1 gene shRNA and HSV1-tk in a xenograft mouse model. Cancer Lett 2010; 291(1): 83-9.
5. Lin X, Zhang X, Wang Q, Li J, Zhang P, Zhao M, Li X. Perifosine downregulates MDR1 gene expression and reverses multidrug-resistant phenotype by inhibiting PI3K/Akt/NF-kB signaling pathway in a human breast cancer cell line. Neoplasma 2012; 59(3):248-56.
6. CretneyE,Takeda K, SmythMJ. Cancer:noveltherapeu-tic strategiesthat exploit the TNF related apoptosis-indudng ligand(TRAIL)/TRAI1 receptor pathway[J]. Int J Biochem CellPio 2007,39(2):280-286.
7.喻龙姗,张开光,崔喻芳.TRAIL联合顺铂对胃癌多药耐药基因GST-π的影响[J].安徽医科大学学报,2013,48(2)：111-5.
8. Zhang K G, Qin C Y, Wang H Q, et al. The effect of TRAIL on the expression of multidrug resistant genes MDR1, LRP and GST-p in drug-resistant gastric cancer cell SGC7901/VCR [J]. Hepatogastroenterology 2012,59(120):2672-6.
9. Tahara T, Shibata T, Yamashita H, et al. Promoter methylation status of multidrug resistance 1 (MDR1) gene in noncancerous gastric mucosa correlates with Helicobacter Pylori infection and gastric cancer occurrence [J]. Cancer Invest 2010,28(7):711-716.
10. Lacueva J,Perez-Ramos M.et al.Multidrug resistance-associated protein (MRP1) gene is strongly expression in gastric carcinomas.Analysis by immunohistochemisty and real-time quantitative RT-PCR. Histopathology 2005,46(4):389-95.
11. Takakuwa O, Oguri T, Ozasa H, Uemura T, Kasai D, Miyazaki M, Maeno K, Sato S. Over-expression of MDR1 in amrubicinol-resistant lung cancer cells. Cancer Chemother Pharmacol 2011; 68(3):669-76.
12. Testa U. TRAIL/TRAIL-R in hematologic malignancies. J Cell Biochem 2010; 110(1):21-34.
13. Younes A, Kadin ME. Emerging applications of the tumor necrosis factor family of ligands and receptors in cancer therapy. J Clin Oncol 2003; 21(18):3526-34.
14.王慧群,张开光等.TRAIL抑制胃癌多药耐药基因MDR/P-gp表达的研究.安徽医科大学学报,2012,47(1)：31-34.
15.苏艳新,王立.多西他赛联合TRAIL诱导胃癌细胞SGC 7901凋亡的作用[J].中国癌症杂志,2008,18(4)：262-6.
16. Dolloff N G, Mayes P A, Hart L S, et al. Off-target lapatinib activity sensitizes colon cancer cells through TRAIL death receptor up-regulation[J]. Sci Transl Med 2011,3(86):86ra50.
17. Moon D O, Kang C H, Kang S H, et al. Capsaicin sensitizes TRAIL-induced apoptosis through Spl-mediated DR5 up-regulation:involvement of Ca2+ influx [J]. Toxicol Appl Pharmacol 2012.259(1):87-95.
18. Lacour S. Hammann A. Wotawa A, et al. Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand mediated caspase-8 activation and apoptosis [J]. Cancer Rees 2001,61(4):1645-51.
19. Wu Y. Fan Y. Xue B, et al. Human glutathione S-transferase PI-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals [J]. Oncogene 2006,25(42): 5787-800.
20. Li X, Li JP, Yuan HY, Gao X, Qu XJ, Xu WF, Tang W. Recent advances in P-glycoprotein-mediated multidrug resistance reversal mechanisms. Methods Find Exp Clin Pharmacol 2007,29(9):607-17.
21. Li X, Fan R, Zou X et al. Reversal of multidrug resistance of gastric cancer cells by down-regulation of CIAPIN1 with CIAPIN1 siRNA. Mol Biol (Mosk) 2008,42(1):102-9.
22. Li X, Zhang Y, Xiong C,et al. Overexpression of a new gene P28GANK confers multidrug resistance of gastric cancer cells. Cancer Invest 2009,27(2):129-39.
23. Song W,Jiang R, Zhao CM. Role of Integrin-Linked Kinase in Multi-drug Resistance of Human Gastric Carcinoma SGC7901/DDP Cells. Asian Pacific Journal of Cancer Prevention 2012,13(11):5619-25.
24. Huang SL, Chen M, Shen YH et al. Inhibition of activated Stat3 reverses drug resistance to chemotherapeutic agents in gastric cancer cells. Cancer Letters 2012,315(2):198-205
2. Hohenberger P,Gretschel S.Gastric cancer. Lancet 2003,362(9380):305-15.
3.孙秀娣,牧人,周友尚,等.中国胃癌死亡率20年变化情况分析及其发展趋势预测.中华肿瘤杂志.2004,26(1)：4-9.
4. Zhang D, Fan D. New insights into the mechanisms of gastric cancer multidrug resistance and future perspectives. Future Oncol 2010,(6):527-537.
5. Tahara T, Shibata T, Yamashita H, et al. Promoter methylation status of multidrug resistance 1 (MDR1) gene in noncancerous gastric mucosa correlates with Helicobacter Pylori infection and gastric cancer occurrence. Cancer Invest 2010,28(7):711-716.
6. Yu PF, Guo JM, Xu Q, et al. Significance of multidrug resistance gene-associated proteins in the postoperative adjuvant chemotherapy for gastric carcinoma and the prognosis. Zhonghua Wei Chang Wai Ke Za Zhi 2010,13(4):289-293.
7. Scheffer GL,Wijingard PLG, Flens MJ, et al. The drug resistace-related protein LRP is the vault protein. Nature Med 1995,1:578-582.
8. Hu WQ, Peng CW, Li Y. The expression and significance of P-glycoprotein, lung resistance protein and multidrug resistance-associated protein in gastric cancer. Exp Clin Cancer Res 2009,28:144.
9. Tanner M, Isola J, Wiklund T, et al. Topoisomerase IIalpha gene amplification predicts favorable treatment response to tailored and dose-escalated anthracycline-based adjuvant chemotherapy in HER-2/neu-amplified breast cancer. J Clin Oncol 2006,24(16):2428-36.
10. Walther W, Stein U, Fichtner I, et al. Mdrl promoter driven tumor necrosis factor alpha expression for a chemotherapy controllable combined in vivo gene therapy and chemotherapy of tumors. Cancer Gene Ther 2000,7(6):893-900.
11. SchaeferU, Voloshanenko O, W ilienD, et al. TRAIL:amultifunctional cytokihe[J]. Front Piosc 2007,12:3813-3824.
12. CretneyE, Takeda K, SmythMJ. Cancer:noveltherapeu-tic strategiesthat exploit the TNF related apoptosis-indudng ligand(TRAIL)/TRAI1 receptor pathway. Int J Biochem CellPio 2007,39(2):280-286.
13. Wu GS. TRAIL as a target in anti-cancer therapy. CancerLett 2009,285(1):1-5.
14. Takamizawa S, Okamoto S, Bishop W, et al. Differential apoptosis gene expression in pediatric tumors of the kidney. Pediatr Surg2000,35(2):390-395.
15. Zhu W, Zhu DX, Lu SQ,et al. miR-497 modulates multidrug resistance of human cancer cell lines by targeting BCL2. Med Oncol 2012,29:384-391.
16. Song X, Wang JB, Yin DL, et al. Jiang HC.Down-regulation of lung resistance related protein by RNA interference targeting survivin induces the reversal of chemoresistances in hepatocellular carcinoma. Chin Med J 2009, 122(21):2636-42.
17. Zhu W, Shan X, Wang TS,et al. miR-181b modulates multidrug resistance by targeting BCL2 in human cancer cell lines. Int J Cancer 2010,127:2520-2529.
18. Follet J, Corcos L, Baffet G, et al. The association of statins and taxanes:an efficient combination trigger of cancer cell apoptosis. Br J Cancer 2012,106(4): 685-92.
19. Gianni L. Anthracycline resistance:the problem and its current definition. Semin Oncol 1997,24(4):10-17.
20. Borska S. Chmielewska M, Wysocka T.et al. In vitro effect of quercetin on human gastric carcinoma:Targeting cancer cells death and MDR. Food and Chemical Toxicology 2012.(50): 3375-3383.
21. Duarte N, Lage H, Abrantes M.et al. Phenolic compounds as selective antineoplasic agents against multidrug-resistant human cancer cells. Planta Med 2010,76(10):975-80.
22. Tozawa K, Oshima T, Kobayashi T, et al. Oxaliplatin in treatment of the cisplatin-resistant MKN45 cell line of gastric cancer. Anticancer Res 2008,28(4B):2087-2092.
23. Shi LX, Ma R, Lu R, et al. Reversal effect of tyroservatide (YSV) tripeptide on multidrug resistance in resistant human hepatocellular carcinoma cell line BEL-7402/5-FU. Cancer Lett 2008,269(1):101-10.
24. Song X, Wang JB, Yin DL, et al. Down-regulation of lung resistance related protein by RNA interference targeting survivin induces the reversal of chemoresistances in hepatocellular carcinoma. Chin Med J.2009, 122(21):2636-42.
25. HAO Yi-xin, HE Zheng-wenl, DU Nan, et al. Suppression of MDR1 gene expression and reversal of cisplatin resistance in renal carcinoma cells by RNA inter-ference. ZHONGLIU 2010,30(2):115-118.
26. Komdeur R, Plaat BE, Hoekstra HJ, et al. Expression of P-glycoprotein, multidrug resistance-associated protein 1, and lung resistance-related protein in human soft tissue sarcomas before and after hyperthermic isolated limb perfusion with tumor necrosis factor related apoptosis inducing ligand-alpha and melphalan. Cancer 2001,91(10):1940-8.
27. Wang Q, Chen XP, Hai S, et al. TNF-alpha induced reversal of multidrug resistance in human hepatocellular carcinoma cells. Zhonghua Wai Ke Za Zhi 2007,45(9):602-4.
28. Wu Y, Fan Y, Xue B, et al. Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals. Oncogene 2006.25(42):5787-800.
29. Cretney E, Takeda K, Smyth MJ. Cancer:novel therapeutic strategies that exploit the TNF-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor pathway. Int J Biochem Cell Biol 2007.39(2):280-286.
30. Wu GS. TRAIL as a target in anti-cancer therapy. Cancer Lett 2009,285(1):1-5.
31.苏艳新,王立.多西他赛联合TRAIL诱导胃癌细胞SGC7901凋亡的作用.中国癌症杂志2008,18(4)：262-266.
32. Sadarangani A, Kato S, Espinoza N, et al. TRAII mediates apoptosis in cancerous but not norm-al prim ary cultured cells of the human reproductive tract. Apoptosis 2007,12(1):73-85.
33. Cuello M, Ettenberg SA, Nau MM, et al. Synergistic induction of apoptosis by the combination of TRAIL and chemotherapy in chemoresistant ovarian cancer cells. Gynecol Oncol.2001,81(3):380-90.
34. Abdulghani J, El-Deiry WS. TRAIL receptor signaling and therapeutics. Expert Opin Ther Targets 2010,14(10):1091-108.
35. Liu FT, Agrawal SG, Gribben JG, et al. Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL. Blood 2008,111(5):2797-805.
36. Shenoy K, Wu Y, Pervaiz S. LY303511 enhances TRAIL sensitivity of SHEP-1 neuroblastoma cells via hydrogen peroxide-mediated mitogen-activated protein kinase activation and up-regulation of death receptors. Cancer Res 2009:69(5):1941-50.
37. Seo SB, Hur JG, Kim MJ, et al. TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3beta pathway and activation of caspases. Mol Cancer 2010, 9:199-212.
38. Zhu GX, Qu Q, Chen QJ, et al. Expression and significance of DR5, DcRl protein for TNF-related apoptosis-inducing ligand and P-glycoprotein in endometrial carcinoma. Journal of Shanxi Medical University.2006:37(8):800-803.
39. Shen X, Zhen JY, Shi H, et al. Survivin Knockdown Enhances Gastric Cancer Cell Sensitivity to Radiation and Chemotherapy In Vitro and in Nude Mice. Am J Med Sci 2012,344(1):52-8.
40.刘玲,费素娟TRAIL联合PTDC对SGC-7901细胞生长抑制和凋亡诱导的实验研究.徐州医学院学报2009,29(11)：71821.
41. Huang C. Xu D. Xia Q, et al. Reversal of P-glycoprotein-mediated multidrug resistance of human hepatic cancer cells by Astragaloside Ⅱ. J Pharm Pharmacol 2012,64(12):1741-50.
42. Li X, Li JP, Yuan HY, et al. Recent advances in P-glycoprotein-mediated multidrug resistance reversal mechanisms. Methods Find Exp Clin Pharmacol 2007,29:607-617.
43. Lu D, Shi HC, Wang ZX, et al. Multidrug resistance-associated biomarkers PGP, GST-pi, Topo-Ⅱ and LRP as prognostic factors in primary ovarian carcinoma. Br J Biomed Sci 2011,68(2):69-74.
44. Hu WQ, Peng CW, Li Y. The expression and significance of P-glycoprotein, lung resistance protein and multidrug resistance-associated protein in gastric cancer. Exp Clin Cancer Res.2009,28:144.
45. Ding L; Chen XP; Zhang ZW, et al. Synergistic effect of bromocriptine and tumor necrosis factor-a onreversing hepatocellular carcinoma multidrug resistance in nude mouseMDRl model of liver neoplasm. WJG 2005,11(36):5621-5626.
46. Xu Z, Zhu H, Luk JM, et al. Clinical significance of SOD2 and GST-π gene polymorphisms in Chinese patients with gastric cancer. Cancer 2012, 118(22):5489-5498.
47.魏素菊,刘海英,史健,韩文峰.重组改构人TNF逆转卵巢癌耐药细胞株SKOV3/DDP的耐药性及其机制.中国肿瘤生物治疗杂志2008,15(2)：150-154.
1. Yang L. Incidence and mortality of gastric cancer in China [J]. World J Gastroenterol 2006; 12(1):17-20.
2. Guo P, Huang ZL, Yu P, Li K. Trends in cancer mortality in China:an update. Ann Oncol 2012; 23(10):2755-62.
3. Sheng X, Zhang L, Tong N, Luo D, Wang M, Zhang Z. MDR1 C3435T polymorphism and cancer risk:a meta-analysis based on 39 case-control studies. Mol Biol Rep 2012; 39(7):7237.
4. Lee SW, Lee YL, Lee YJ, Park SY, Kim IS, Choi TH, Ha JH, Ahn BC, Lee J. Enhanced antitumor effects by combination gene therapy using MDR1 gene shRNA and HSV1-tk in a xenograft mouse model. Cancer Lett 2010; 291(1): 83-9.
5. Lin X, Zhang X, Wang Q, Li J, Zhang P, Zhao M, Li X. Perifosine downregulates MDR1 gene expression and reverses multidrug-resistant phenotype by inhibiting PI3K/Akt/NF-kB signaling pathway in a human breast cancer cell line. Neoplasma 2012; 59(3):248-56.
6. CretneyE,Takeda K, SmythMJ. Cancer:noveltherapeu-tic strategiesthat exploit the TNF related apoptosis-indudng ligand(TRAIL)/TRAI1 receptor pathway[J]. Int J Biochem CellPio 2007,39(2):280-286.
7.喻龙姗,张开光,崔喻芳.TRAIL联合顺铂对胃癌多药耐药基因GST-π的影响[J].安徽医科大学学报,2013,48(2)：111-5.
8. Zhang K G, Qin C Y, Wang H Q, et al. The effect of TRAIL on the expression of multidrug resistant genes MDR1, LRP and GST-p in drug-resistant gastric cancer cell SGC7901/VCR [J]. Hepatogastroenterology 2012,59(120):2672-6.
9. Tahara T, Shibata T, Yamashita H, et al. Promoter methylation status of multidrug resistance 1 (MDR1) gene in noncancerous gastric mucosa correlates with Helicobacter Pylori infection and gastric cancer occurrence [J]. Cancer Invest 2010,28(7):711-716.
10. Lacueva J,Perez-Ramos M.et al.Multidrug resistance-associated protein (MRP1) gene is strongly expression in gastric carcinomas.Analysis by immunohistochemisty and real-time quantitative RT-PCR. Histopathology 2005,46(4):389-95.
11. Takakuwa O, Oguri T, Ozasa H, Uemura T, Kasai D, Miyazaki M, Maeno K, Sato S. Over-expression of MDR1 in amrubicinol-resistant lung cancer cells. Cancer Chemother Pharmacol 2011; 68(3):669-76.
12. Testa U. TRAIL/TRAIL-R in hematologic malignancies. J Cell Biochem 2010; 110(1):21-34.
13. Younes A, Kadin ME. Emerging applications of the tumor necrosis factor family of ligands and receptors in cancer therapy. J Clin Oncol 2003; 21(18):3526-34.
14.王慧群,张开光等.TRAIL抑制胃癌多药耐药基因MDR/P-gp表达的研究.安徽医科大学学报,2012,47(1)：31-34.
15.苏艳新,王立.多西他赛联合TRAIL诱导胃癌细胞SGC 7901凋亡的作用[J].中国癌症杂志,2008,18(4)：262-6.
16. Dolloff N G, Mayes P A, Hart L S, et al. Off-target lapatinib activity sensitizes colon cancer cells through TRAIL death receptor up-regulation[J]. Sci Transl Med 2011,3(86):86ra50.
17. Moon D O, Kang C H, Kang S H, et al. Capsaicin sensitizes TRAIL-induced apoptosis through Spl-mediated DR5 up-regulation:involvement of Ca2+ influx [J]. Toxicol Appl Pharmacol 2012.259(1):87-95.
18. Lacour S. Hammann A. Wotawa A, et al. Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand mediated caspase-8 activation and apoptosis [J]. Cancer Rees 2001,61(4):1645-51.
19. Wu Y. Fan Y. Xue B, et al. Human glutathione S-transferase PI-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals [J]. Oncogene 2006,25(42): 5787-800.
20. Li X, Li JP, Yuan HY, Gao X, Qu XJ, Xu WF, Tang W. Recent advances in P-glycoprotein-mediated multidrug resistance reversal mechanisms. Methods Find Exp Clin Pharmacol 2007,29(9):607-17.
21. Li X, Fan R, Zou X et al. Reversal of multidrug resistance of gastric cancer cells by down-regulation of CIAPIN1 with CIAPIN1 siRNA. Mol Biol (Mosk) 2008,42(1):102-9.
22. Li X, Zhang Y, Xiong C,et al. Overexpression of a new gene P28GANK confers multidrug resistance of gastric cancer cells. Cancer Invest 2009,27(2):129-39.
23. Song W,Jiang R, Zhao CM. Role of Integrin-Linked Kinase in Multi-drug Resistance of Human Gastric Carcinoma SGC7901/DDP Cells. Asian Pacific Journal of Cancer Prevention 2012,13(11):5619-25.
24. Huang SL, Chen M, Shen YH et al. Inhibition of activated Stat3 reverses drug resistance to chemotherapeutic agents in gastric cancer cells. Cancer Letters 2012,315(2):198-205