Raf-1/Mdr-1 siRNAs联合转染对口腔上皮癌多药耐药性逆转的研究
摘要
化疗是目前治疗肿瘤的有效方法之一,但化疗后肿瘤细胞产生多药耐药性阻碍了化疗药物高效杀灭癌细胞,成为口腔上皮鳞癌化疗失败的主要原因。多药耐药性(MDR)是指肿瘤细胞能对多种结构、功能及杀伤机制各异的化疗药物均产生耐药。目前大量的研究表明,肿瘤多药耐药性的发生机制复杂多样,是多基因、多步骤综合作用的结果。
在众多因素中,耐药相关蛋白P-糖蛋白(P-glycoprotein,Pgp)的表达增强在耐药性的发生、发展过程中起到了非常重要的作用,也是逆转肿瘤多药耐药性方面研究的重点。另外多药耐药相关蛋白(MRP),谷胱甘肽转移酶(GST),转录因子NF-κB等的异常表达都与肿瘤细胞接受化疗后多药耐药性的产生有关。因为涉及的环节很多,实践证明仅针对一个环节进行阻断,如单一阻断多药耐药基因Mdr-1的表达尚不能取得令人满意的治疗效果。因此国内外学者通过多种方法试图阻断多药耐药蛋白或耐药相关蛋白的异常表达,有效逆转肿瘤细胞的多药耐药性,例如通过使用多种中药的多阻断方法、反义RNA技术以及基因水平阻断耐药相关蛋白的有效表达等等手段。RNA干扰(RNA interference,RNAi)是新近发展起来的一种有效阻断基因表达的技术。它是将双链小分子RNA片段转染入靶细胞,使靶mRNA降解,从而抑制基因的表达。在肿瘤多药耐药方面的研究中,将RNAi技术用于封闭肿瘤多药耐药基因,干扰其在多药耐药性肿瘤细胞内的表达,从而实现肿瘤细胞多药耐药性的逆转,使其重新对化疗药物敏感成为目前的研究热点。本试验期望通过采用RNAi技术并结合双阻断的方法对多药耐药口腔上皮癌细胞实现更为有效的逆转,从而使多药口腔上皮癌细胞重新对较低浓度化疗药物敏感。
Raf-1基因的异常表达与多种肿瘤的发生密切相关,被激活的Raf蛋白参与细胞生长、细胞周期停滞甚至与凋亡有关。Raf突变在多种恶性肿瘤中被探测到,因此是肿瘤介入治疗的重要靶点。近年来有研究表明,Raf的异常表达也影响着肿瘤细胞耐药性的产生。例如研究发现Raf-1通过调整肿瘤细胞中药泵Mdr-1的表达来参与乳腺癌、前列腺癌细胞多药耐药性的发生。但Raf-1蛋白激酶在口腔上皮癌多药耐药性发生中所起的作用的研究尚未见报道。本研究通过检测Raf-1基因在口腔上皮癌多药耐药株(KBV)及敏感株(KB)中表达的差异,探讨Raf-1基因表达在口腔上皮癌多药耐药性发生机制中所起的作用;运用RNAi技术分别单阻断KBV细胞中Raf-1、Mdr-1基因的转录和蛋白表达,检测各自逆转细胞多药耐药性的效果;采用联合转染即同时将Raf-1/Mdr-1 siRNAs转染进入KBV细胞中的双阻断方法降低KBV细胞的多药耐药性,检测联合转染的逆转效果并与Raf-1 siRNA、Mdr-1 siRNA单一转染的逆转效果相比较,期望找到能更有效逆转口腔上皮癌多药耐药性、进一步提高口腔上皮癌化疗成功率的方法。
本研究采用体外培养的口腔上皮癌药物敏感株KB及多药耐药株KBV作为一对亲本细胞及多药耐药细胞。实验通过RNAi技术将Mdr-1 siRNA,Raf-1 siRNA ,Raf-1/ Mdr-1 siRNAs及control siRNA转染进入KBV细胞内,获得Mdr-1 siRNA单一转染细胞、Raf-1 siRNA单一转染细胞、Raf-1/ Mdr-1 siRNAs联合转染细胞,control siRNA转染细胞作为空白对照;通过RT-PCR技术及免疫荧光技术、Western blot技术检测Raf-1及Mdr-1基因在各组细胞中转录及翻译水平的差异,通过四唑盐(MTT)比色法及流式细胞检测技术(FCM)测定各组细胞的半数致死率IC_(50)及凋亡率,以检测各组细胞对长春新碱(VCR)敏感度的差异。
本研究分为四部分进行,第一部分是Raf-1基因在KBV细胞多药耐药性发生中作用的研究;第二部分是Mdr-1 siRNA转染对KBV细胞多药耐药性的逆转作用的研究;第三部分是Raf-1 siRNA转染对KBV细胞多药耐药性的逆转作用的研究;第四部分是Raf-1/ Mdr-1 siRNAs联合转染对KBV细胞多药耐药性的逆转作用的研究。
第一部分实验RT-PCR检测、Western blot检测结果显示KB细胞在Raf-1、Mdr-1基因转录和翻译水平均非常显著地低于KBV细胞,统计学差异非常显著(P<0.01);免疫荧光图像显示KBV细胞中Raf-1和P-gp蛋白表达显著强于KB细胞;MTT法结果显示KB与KBV细胞的IC_(50)分别是21.33±2.25μg/L、352.68±12.36μg/L,耐药指数RI(KBV/KB)=16.5,KBV细胞的IC_(50)显著高于KB细胞,统计学差异非常显著(P<0.01);流式细胞仪检测结果显示KB细胞凋亡率达98.8±1.2%;KBV细胞凋亡率达23.5±2.1%,KB细胞凋亡率显著高于KBV细胞,统计学差异非常显著(P<0.01)。实验结果提示在口腔上皮癌多药耐药性的发生、发展过程,Mdr-1无疑起着非常重要的作用,而Raf-1基因可能也参与了口腔上皮癌多药耐药性的发生和发展。这一结果为我们以后寻找阻断口腔上皮癌多药耐药发生的新靶点提供了新途径。
第二部分实验RT-PCR检测、Western blot检测结果显示Mdr-1 RNAi非常显著地下调KBV细胞Mdr-1基因在转录和翻译水平的表达,显著低于空白转染KBV组,差异具有统计学意义(P<0.01);免疫荧光图像显示Mdr-1 siRNA转染细胞中P-gp蛋白表达显著弱于空白转染KBV细胞;MTT法测定结果显示Mdr-1 siRNA、KBV、KB及空白转染细胞的IC_(50)分别是130.68±6.35μg/L、352.68±12.36μg/L、21.33±2.25μg/L、356.47±14.23μg/L,耐药指数RI(Mdr-1 siRNA转染/KB)=6.1,RI(KBV/KB) =16.5,Mdr-1 siRNA转染细胞的IC_(50)及RI显著低于空白转染KBV细胞,差异具有统计学意义(P<0.01);流式细胞仪检测结果显示Mdr-1 siRNA转染细胞凋亡率达67.2±3.7%,KB细胞凋亡率达98.8±1.2%;KBV细胞凋亡率达23.5±2.1%,空白转染细胞达24.3±1.6%,经Mdr-1 siRNA转染的KBV细胞凋亡率非常显著地高于空白转染KBV细胞,差异具有统计学意义(P<0.01)。实验结果提示Mdr-1 siRNA转染是能对口腔上皮癌多药耐药性实现部分逆转,并提高耐药细胞对化疗药物敏感性的有效方法,为临床治疗提供了理论基础。
第三部分实验RT-PCR检测、Western blot检测结果及免疫荧光图像显示Raf-1 RNAi非常显著地下调KBV细胞中Raf-1、Mdr-1基因在转录和翻译水平的表达,且均显著地低于空白转染KBV细胞,差异具有统计学意义(P<0.01);免疫荧光图像显示Raf-1 siRNA转染细胞中Raf-1、P-gp蛋白表达显著弱于空白转染KBV细胞;MTT实验结果显示Raf-1 siRNA转染细胞、KB、KBV细胞及空白转染细胞的IC_(50)分别是201.38±7.13μg/L、21.33±2.25μg/L、352.68±12.36μg/L、356.47±14.23μg/L,RI(KBV/KB) =16.5,RI(Raf-1 siRNA转染/KB)=9.5,Raf-1 siRNA转染KBV细胞的IC_(50)及RI显著低于空白转染KBV细胞,差异具有统计学意义(P<0.01);流式细胞仪检测结果显示Raf-1 siRNA转染KBV细胞凋亡率达49.2±3.2%,空白转染KBV细胞达24.3±1.6%,KB细胞凋亡率达98.8±1.2%;KBV细胞凋亡率达23.5±2.1%,Raf-1 siRNA转染细胞凋亡率非常显著地高于空白转染细胞,差异具有统计学意义(P<0.01)。实验结果进一步明确了Raf-1基因与口腔上皮癌多药耐药性发生之间的关系密切,Raf-1基因积极参与口腔上皮癌多药耐药的发生过程,干扰Raf-1基因的有效表达可以引起口腔上皮癌多药耐药性显著下降。本实验同时提示Raf-1基因参与口腔上皮癌多药耐药性的发生可能是通过增强细胞中Mdr-1基因表达来实现的。因此采用Raf-1 siRNA转染技术是阻断Raf-1及Mdr-1基因表达,部分逆转口腔上皮癌细胞的多药耐药性,使其部分恢复对化疗药物敏感性的有效方法之一。
第四部分实验RT-PCR检测、Western blot检测结果显示采用联合转染Mdr-1/Raf-1 siRNAs进入KBV细胞中,即双阻断的方法逆转KBV细胞的多药耐药性,与单阻断Mdr-1或Raf-1基因有效表达的方法相比,无论在转录还是在蛋白表达水平,均更大程度地下调了耐药基因Mdr-1的表达,Raf-1 / Mdr-1 siRNAs联合转染细胞中Mdr-1基因的表达显著低于Raf-1 siRNA单一转染、Mdr-1 siRNA单一转染细胞,差异具有统计学意义(P<0.01);免疫荧光图像显示Raf-1 / Mdr-1 siRNAs联合转染细胞中P-gp蛋白表达显著弱于Raf-1 siRNA单一转染及Mdr-1 siRNA单一转染KBV细胞;MTT法测定结果显示Raf-1 / Mdr-1 siRNAs联合转染KBV细胞、Raf-1 siRNA转染、Mdr-1 siRNA转染细胞、KB细胞、KBV细胞及空白转染细胞的IC_(50)分别是58.76±3.27μg/L、201.38±7.13μg/L、130±6μg/L、21.33±2.25μg/L、352.68±12.36μg/L、356.47±14.23μg/L ;耐药指数RI(KBV/KB) =16.5 , RI(Raf-1 siRNA转染/KB)=9.5,RI(Mdr-1 siRNA转染/KB)=6.1,RI[(Raf-1 / Mdr-1 siRNA转染)/KB]=2.8,统计学分析显示Raf-1 / Mdr-1 siRNA联合转染的IC_(50)及RI显著低于Raf-1 siRNA单一转染、Mdr-1 siRNA单一转染KBV细胞,差异具有统计学意义( P< 0.01);流式细胞仪检测结果显示Raf-1/ Mdr-1siRNA联合转染细胞凋亡率达88.2%±4.3%,Raf-1 siRNA转染组细胞凋亡率达49.2±3.2%, Mdr-1 siRNA转染组细胞凋亡率达67.2%±3.7%,KB细胞凋亡率达98.8±1.2%,KBV细胞凋亡率达23.5±2.1%,空白转染组达24.3±1.6%,统计分析Raf-1/ Mdr-1siRNA联合转染细胞凋亡率非常显著地高于Raf-1 siRNA转染细胞及Mdr-1 siRNA转染细胞,差异具有统计学意义(P<0.01)。实验结果提示Mdr-1 /Raf-1 siRNAs联合转染与Raf-1 RNAi、Mdr-1 RNAi的单阻断方法相比较,更大程度地降低了KBV细胞中Mdr-1基因的表达,更有效地逆转了KBV细胞的多药耐药性,使KBV细胞重新对低浓度化疗药物敏感。如果应用于临床会大大降低化疗药物的用药浓度,减少化疗对患者机体的损伤,有利于提高肿瘤化疗的成功率。
综上所述,本研究结果提示Mdr-1的过表达作为多种肿瘤细胞产生多药耐药性的主要原因,同样成为口腔上皮癌多药耐药性发生的主要原因;Raf-1基因也积极参与了口腔上皮癌多药耐药性的发生过程,而且可能是通过增强Mdr-1的表达来实现的,Raf-1基因成为引起口腔上皮癌细胞多药耐药的重要原因之一。Raf-1 RNAi或Mdr-1 RNAi的单一阻断方法都是逆转口腔上皮癌多药耐药性的有效方法。由于肿瘤多药耐药性的产生是一个多步骤、多因素共同作用的结果,是一个非常复杂的过程,所以仅通过单一阻断Raf-1或Mdr-1多药耐药相关基因的表达均未能取得理想效果。本研究结果显示Raf-1/ Mdr-1 siRNAs联合转染的双阻断方法与Raf-1 RNAi或Mdr-1 RNAi的单一阻断方法相比,更显著地下调KBV细胞中多药耐药基因Mdr-1在转录和蛋白水平的表达,更有效地逆转KBV细胞的多药耐药性,使其重新对低浓度化疗药物敏感,为临床治疗提供了理论基础。
Multidrug resistance(MDR) is a major barrier for chemotherapy of most patients with oral squamous cell carcinoma. MDR means that tumor cells develop the resistance to many kinds of chemotherapy agents with various structures and functions.
The multidrug resistance is of a multisteps process and a very complex mechanism. The overexpression of P-glycoprotein(P-gp) encoded by mdr-1 gene is one of the most important reasons for MDR development..The human Pgp(Mdr-1) on cell membrane as efflux pump belongs to the ATP-binding cassette transporter expelling out antitumor agents from the cytoplasm , reducing intracellular drug concentrations in the cells to sublethal ,so that P-pg is a important target for reversing MDR. Besides,there are many other factors impacting the genesis of the MDR,for example, glutathlone S-transferase(GST) which acts by detoxification of some chemotherapeutic drugs,multidrug resistance-associated protein(MRP), transcription factor nuclear factor-kappa B(NF-κB) and so on. So many kinds of the agents such as antisense RNA and traditional medicines were released to have partly effect on reversing MDR of the tumor cells because MDR mechanism is too complex. we cannot attain ideal effect if we merely block one target or signaling pathway related to MDR. Researchers has adoptted many methods to reverse MDR,such as traditional Chinese drugs,anti-RNA, blockade of the gene expressions or signal transductions related to MDR.
In recent years,RNA interference(RNAi) has widely used as an experimental tool to analyse the function of mammalian genes. Double- stranded RNA(dsRNA) reagents are used to bind to and promote the degradation of target RNAs,resulting in knockdown of the expression of specific genes.In the field of reversal of multidrug resistance,small interfering double-stranded RNAs (siRNA) were designed to target the factors such as P-gp or GST mRNA as a strategy to inhibit resistant gene expression at the mRNA level in order to restore sensitivity to chemotherapeutic drugs.
Raf-1 is a cytoplasmic serine/threonine protein kinase that plays an important role in the tumorigenesis and inhibiton of apoptosis by modulating transcriptional activation and mitogenesis.Some experiments has indicated that Raf-1 kinase is associated with drug resistance in human breast cancer and prostate cancer and ectopic expression of Raf1 will increase the levels of the Mdr-1 drug pump.This increased expression of Mdr-1 most likely occurs by a transcriptional mechanism.The present research was to investigate the effect of Raf-1 on the multidrug resistance in multidrug resistant human oral squamous cell carcinoma (KBV) cells and detect the effect of Raf-1 siRNA, Mdr-1 siRNA and Raf-1/Mdr-1 siRNAs (Mdr-1 siRNA and Raf-1siRNA transfected into drug resistant KBV cells at the same time) on the reversal of multidrug resistance in KBV cells. We hoped that we might put forward a more effective method to reverse the multidrug resistance in KBV cells by the present study.
In this study, we investigated Mdr-1 and Raf-1 genes expression and abilities to resist the chemotherapeutic drug vincristine (VCR) in six groups: Mdr-1 siRNA transfected KBV cells,Raf-1 siRNA transfected KBV cells,Mdr1 /Raf-1 two siRNAs transfected KBV cells, KBV, KB and Control siRNA as control. Using RT-PCR , western blotting assay and immuno- fluorescent method the changes of the RNA and protein levels of both Mdr-1 and Raf-1 genes were studied.Using tetrazolium-based chemosensitivity assay(MTT) and flow cytometry (FCM) the changes of chemosensitivity to VCR in six groups of cells were detected.
This study includes four sections: (1) detection of relationships between Raf-1 and MDR in KBV cells; (2)the effect of Mdr-1 siRNA transfection on MDR in KBV cells; (3) the effect of Raf-1 siRNA transfection on MDR in KBV cells; (4) the effect of Mdr1 /Raf-1 siRNAs transfection on MDR in KBV cells.
The first section was to detect the relationgships between Raf-1 and multidrug resistance in KBV cells.The changes of the mRNA and protein levels of both Mdr-1 and Raf-1 genes in KBV and KB cells were determined by RT-PCR ,western blotting assay and immunofluorescent method.By MTT and FCM, IC_(50) ratios and apoptosis rates in both KBV and KB cells were detected.The findings indicated that expressions of both Raf-1 and Mdr1 mRNA and protein levels in KBV cells were lower than those in KB cells statistically (P<0.01). Immunofluorescent image showed that both Mdr1 and Raf-1 protein expressions in KBV cells were much stronger than those in KB cells.The IC_(50) ratio in KB cells was 21.33±2.25μg/L and that in KBV cells was 352.68±12.36μg/L.The resistance Index (RI) was 16.5.The apoptosis rate in KB cells was 98.8±1.2% and that in KBV cells was 23.5±2.1%.These results showed that differences in the chemosensitivity to VCR between KBV cells and KB cells were significant statistically (P<0.01). This study suggested that Raf-1 might play an important role in the process of developing the multidrug resistance in the human oral squamous carcinoma.Mdr-1 gene overexpression was still closely related with MDR in KBV cells.
The second section was to investigate the effect of Mdr-1 siRNA on MDR in KBV cells. Mdr-1 siRNA was transfected into KBV cells. The changes of the Mdr-1 and raf-1 gene expression at the mRNA and protein levels in Mdr-1 siRNA transfected KBV cells were determined by RT-PCR ,western blotting assay and immunofluorescent method. Using MTT and FCM IC_(50) and apoptosis rates in Mdr-1 siRNA transfected KBV cells were determined. The findings suggested that the mRNA and protein levels of Mdr-1 genes in Mdr1 siRNA transfected KBV cells were much lower than those in KBV cells statistically (P<0.01). Immunofluorescent image showed that P-gp expression in Mdr-1 siRNA transfected KBV cells were much weaker than that in KBV cells.The IC_(50) in Mdr-1 siRNA transfected KBV cells,KBV,KB and Control siRNA transfected KBV cells were respectively 130.68±6.35μg/L, 352.68±12.36μg/L, 21.33±2.25μg/L, 356.47±14.23μg/L. RI(Mdr-1 siRNA/KB) was 6.1 and RI(KBV/KB) was 16.5.The apoptosis rates in Mdr-1 siRNA transfected KBV cells was 67.2±3.7% ,24.3±1.6% in Control siRNA transfected KBV cells,98.8± 1.2% in KB cells and 23.5±2.1% in KBV cells.These results showed that the chemosensitivity to VCR in Mdr-1 siRNA transfected KBV cells was higher than that in KBV cells statistically (P<0.01). This study suggested that Mdr-1 siRNA was effective inhibitor of Mdr-1 gene expression to reverse the resistance of human oral squamous cell carcinoma.
The third section was to explore the effect of Raf-1 siRNA on MDR in KBV cells. Raf-1 siRNA was transfected into KBV cells.The Mdr-1 and Raf-1 genes expression at the mRNA and protein levels in Raf-1 siRNA transfected KBV cells were determined using RT-PCR ,western blotting assay and immunofluorescent method. Using MTT and FCM IC_(50) ratios and apoptosis rates in Raf-1 siRNA transfected KBV cells were determined. The findings suggested that the mRNA and protein levels of both Raf-1 and Mdr-1 genes in Raf-1 siRNA transfected KBV cells were lower than those in KBV cells statistically (P<0.01). Immunofluorescent image showed that P-gp and Raf-1 proteins expression in Raf-1 siRNA transfected KBV cells were much weaker than those in Control siRNA transfected KBV cells.The IC_(50) in Raf-1 siRNA transfected KBV cells ,KBV,KB and Control siRNA transfected KBV cells were respectively 201.38±7.13μg/L, 352.68±12.36μg/L, 21.33±2.25μg/L, 356.47±14.23μg/L.RI(Raf-1 siRNA/KB) was 9.5 and RI(KBV/KB) was 16.5.The apoptosis rate in Raf-1 siRNA transfected KBV cells was 49.2±3.2% , 24.3±1.6% in Control siRNA transfected KBV cells,98.8±1.2% in KB cells and 23.5±2.1% in KBV cells..These results showed that differences in the chemosensitivity to VCR between Raf-1 siRNA transfected KBV cells and KBV cells were significant statistically (P<0.01). This study suggested that Raf-1 siRNA was also effective inhibitor of Raf-1 and Mdr-1 gene expression to reverse the resistance of KBV cells.In multidrug resistant human oral squamous cell carcinoma, overexpression of Raf-1 could increase the levels of the Mdr-1 drug pump, which might be the reversal mechanism of Raf-1.
The fourth section was to investigate the the effect of Mdr-1 /Raf-1 siRNAs transfection on MDR in KBV cells. Mdr-1 /Raf-1 siRNAs were transfected into KBV cells at the same time. The Mdr-1 and Raf-1 genes expression at the mRNA and protein levels were assayed by RT-PCR, western blotting assay and immunofluorescent method. By MTT and FCM IC_(50) ratios and apoptosis rates in groups of cells were determined. The findings suggested that the mRNA and protein levels of Mdr-1 genes in Mdr-1 /Raf-1 siRNAs transfected KBV cells were much lower than those in Mdr-1 siRNA transfected KBV cells or Raf-1 siRNA transfected KBV cells statistically (P < 0.01). Immunofluorescent image showed that P-gp expression in Mdr-1 /Raf-1 siRNAs transfected KBV cells was much weaker than those in Raf-1 siRNA or Mdr-1 siRNA transfected KBV cells.The IC_(50) in Mdr-1 /Raf-1 siRNAs transfected KBV cells,Raf-1 siRNA transfected KBV cells,Mdr-1 siRNA transfected KBV cells,KB,KBV and Control siRNA transfected KBV cells were respectively 58.76±3.27μg/L, 201.38±7.13μg/L, 130±6μg/L, 21.33±2.25μg/L, 352.68±12.36μg/L, 356.47±14.23μg/L.RI (KBV/KB) was 16.5, RI(Raf-1 siRNA/KB) was 9.5,RI(Mdr-1 siRNA/KB) was 6.1 and RI[(Raf-1 / Mdr-1 siRNAs)/KB] was 2.8.The apoptosis rate in Raf-1 / Mdr-1 siRNAs transfected KBV cells was 88.2%±4.3%, 49.2±3.2% in Raf-1 siRNA transfected KBV cells, 67.2%±3.7% in Mdr-1 siRNA transfected KBV cells,98.8±1.2% in KB cells;23.5±2.1% in KBV cells and 24.3±1.6% in Control siRNA transfected KBV cells respectively.These results showed that the chemosensitivity to VCR in Raf-1 / Mdr-1 siRNAs KBV cells was much higher than Raf-1 siRNA transfected KBV cells or Mdr-1 siRNA transfected KBV cells statistically (P< 0.01). This study suggested that Raf-1 / Mdr-1 siRNAs transfection that block Mdr-1 genes expression more effectively has proved to be highly beneficial in reversing the resistance of KBV cells,compared with Raf-1 siRNA or Mdr-1 siRNA single transfection.
In sum, Raf-1 might play an important role in the process of developing the multidrug resistance in the human oral squamous carcinoma.Mdr-1 gene expression was still closely related to MDR in KBV cells. Mdr1 siRNA was effective inhibitor of Mdr-1 gene expression to reverse the resistance of human oral squamous cell carcinoma. Raf-1 siRNA could down regulated both Raf-1 and Mdr-1 genes expression and reverse the resistance of KBV cells. Raf1 could modulate the levels of the Mdr-1 drug pump in KBV cells, which might be the reversal mechanism of Raf1. Mdr1 is still a main target for therapeutic intervention. Raf-1 / Mdr-1 siRNAs transfection could block Mdr-1 gene expression and reverse the resistance of KBV cells more effectively compared with Raf-1 siRNA or Mdr-1 siRNA single transfection.
在众多因素中,耐药相关蛋白P-糖蛋白(P-glycoprotein,Pgp)的表达增强在耐药性的发生、发展过程中起到了非常重要的作用,也是逆转肿瘤多药耐药性方面研究的重点。另外多药耐药相关蛋白(MRP),谷胱甘肽转移酶(GST),转录因子NF-κB等的异常表达都与肿瘤细胞接受化疗后多药耐药性的产生有关。因为涉及的环节很多,实践证明仅针对一个环节进行阻断,如单一阻断多药耐药基因Mdr-1的表达尚不能取得令人满意的治疗效果。因此国内外学者通过多种方法试图阻断多药耐药蛋白或耐药相关蛋白的异常表达,有效逆转肿瘤细胞的多药耐药性,例如通过使用多种中药的多阻断方法、反义RNA技术以及基因水平阻断耐药相关蛋白的有效表达等等手段。RNA干扰(RNA interference,RNAi)是新近发展起来的一种有效阻断基因表达的技术。它是将双链小分子RNA片段转染入靶细胞,使靶mRNA降解,从而抑制基因的表达。在肿瘤多药耐药方面的研究中,将RNAi技术用于封闭肿瘤多药耐药基因,干扰其在多药耐药性肿瘤细胞内的表达,从而实现肿瘤细胞多药耐药性的逆转,使其重新对化疗药物敏感成为目前的研究热点。本试验期望通过采用RNAi技术并结合双阻断的方法对多药耐药口腔上皮癌细胞实现更为有效的逆转,从而使多药口腔上皮癌细胞重新对较低浓度化疗药物敏感。
Raf-1基因的异常表达与多种肿瘤的发生密切相关,被激活的Raf蛋白参与细胞生长、细胞周期停滞甚至与凋亡有关。Raf突变在多种恶性肿瘤中被探测到,因此是肿瘤介入治疗的重要靶点。近年来有研究表明,Raf的异常表达也影响着肿瘤细胞耐药性的产生。例如研究发现Raf-1通过调整肿瘤细胞中药泵Mdr-1的表达来参与乳腺癌、前列腺癌细胞多药耐药性的发生。但Raf-1蛋白激酶在口腔上皮癌多药耐药性发生中所起的作用的研究尚未见报道。本研究通过检测Raf-1基因在口腔上皮癌多药耐药株(KBV)及敏感株(KB)中表达的差异,探讨Raf-1基因表达在口腔上皮癌多药耐药性发生机制中所起的作用;运用RNAi技术分别单阻断KBV细胞中Raf-1、Mdr-1基因的转录和蛋白表达,检测各自逆转细胞多药耐药性的效果;采用联合转染即同时将Raf-1/Mdr-1 siRNAs转染进入KBV细胞中的双阻断方法降低KBV细胞的多药耐药性,检测联合转染的逆转效果并与Raf-1 siRNA、Mdr-1 siRNA单一转染的逆转效果相比较,期望找到能更有效逆转口腔上皮癌多药耐药性、进一步提高口腔上皮癌化疗成功率的方法。
本研究采用体外培养的口腔上皮癌药物敏感株KB及多药耐药株KBV作为一对亲本细胞及多药耐药细胞。实验通过RNAi技术将Mdr-1 siRNA,Raf-1 siRNA ,Raf-1/ Mdr-1 siRNAs及control siRNA转染进入KBV细胞内,获得Mdr-1 siRNA单一转染细胞、Raf-1 siRNA单一转染细胞、Raf-1/ Mdr-1 siRNAs联合转染细胞,control siRNA转染细胞作为空白对照;通过RT-PCR技术及免疫荧光技术、Western blot技术检测Raf-1及Mdr-1基因在各组细胞中转录及翻译水平的差异,通过四唑盐(MTT)比色法及流式细胞检测技术(FCM)测定各组细胞的半数致死率IC_(50)及凋亡率,以检测各组细胞对长春新碱(VCR)敏感度的差异。
本研究分为四部分进行,第一部分是Raf-1基因在KBV细胞多药耐药性发生中作用的研究;第二部分是Mdr-1 siRNA转染对KBV细胞多药耐药性的逆转作用的研究;第三部分是Raf-1 siRNA转染对KBV细胞多药耐药性的逆转作用的研究;第四部分是Raf-1/ Mdr-1 siRNAs联合转染对KBV细胞多药耐药性的逆转作用的研究。
第一部分实验RT-PCR检测、Western blot检测结果显示KB细胞在Raf-1、Mdr-1基因转录和翻译水平均非常显著地低于KBV细胞,统计学差异非常显著(P<0.01);免疫荧光图像显示KBV细胞中Raf-1和P-gp蛋白表达显著强于KB细胞;MTT法结果显示KB与KBV细胞的IC_(50)分别是21.33±2.25μg/L、352.68±12.36μg/L,耐药指数RI(KBV/KB)=16.5,KBV细胞的IC_(50)显著高于KB细胞,统计学差异非常显著(P<0.01);流式细胞仪检测结果显示KB细胞凋亡率达98.8±1.2%;KBV细胞凋亡率达23.5±2.1%,KB细胞凋亡率显著高于KBV细胞,统计学差异非常显著(P<0.01)。实验结果提示在口腔上皮癌多药耐药性的发生、发展过程,Mdr-1无疑起着非常重要的作用,而Raf-1基因可能也参与了口腔上皮癌多药耐药性的发生和发展。这一结果为我们以后寻找阻断口腔上皮癌多药耐药发生的新靶点提供了新途径。
第二部分实验RT-PCR检测、Western blot检测结果显示Mdr-1 RNAi非常显著地下调KBV细胞Mdr-1基因在转录和翻译水平的表达,显著低于空白转染KBV组,差异具有统计学意义(P<0.01);免疫荧光图像显示Mdr-1 siRNA转染细胞中P-gp蛋白表达显著弱于空白转染KBV细胞;MTT法测定结果显示Mdr-1 siRNA、KBV、KB及空白转染细胞的IC_(50)分别是130.68±6.35μg/L、352.68±12.36μg/L、21.33±2.25μg/L、356.47±14.23μg/L,耐药指数RI(Mdr-1 siRNA转染/KB)=6.1,RI(KBV/KB) =16.5,Mdr-1 siRNA转染细胞的IC_(50)及RI显著低于空白转染KBV细胞,差异具有统计学意义(P<0.01);流式细胞仪检测结果显示Mdr-1 siRNA转染细胞凋亡率达67.2±3.7%,KB细胞凋亡率达98.8±1.2%;KBV细胞凋亡率达23.5±2.1%,空白转染细胞达24.3±1.6%,经Mdr-1 siRNA转染的KBV细胞凋亡率非常显著地高于空白转染KBV细胞,差异具有统计学意义(P<0.01)。实验结果提示Mdr-1 siRNA转染是能对口腔上皮癌多药耐药性实现部分逆转,并提高耐药细胞对化疗药物敏感性的有效方法,为临床治疗提供了理论基础。
第三部分实验RT-PCR检测、Western blot检测结果及免疫荧光图像显示Raf-1 RNAi非常显著地下调KBV细胞中Raf-1、Mdr-1基因在转录和翻译水平的表达,且均显著地低于空白转染KBV细胞,差异具有统计学意义(P<0.01);免疫荧光图像显示Raf-1 siRNA转染细胞中Raf-1、P-gp蛋白表达显著弱于空白转染KBV细胞;MTT实验结果显示Raf-1 siRNA转染细胞、KB、KBV细胞及空白转染细胞的IC_(50)分别是201.38±7.13μg/L、21.33±2.25μg/L、352.68±12.36μg/L、356.47±14.23μg/L,RI(KBV/KB) =16.5,RI(Raf-1 siRNA转染/KB)=9.5,Raf-1 siRNA转染KBV细胞的IC_(50)及RI显著低于空白转染KBV细胞,差异具有统计学意义(P<0.01);流式细胞仪检测结果显示Raf-1 siRNA转染KBV细胞凋亡率达49.2±3.2%,空白转染KBV细胞达24.3±1.6%,KB细胞凋亡率达98.8±1.2%;KBV细胞凋亡率达23.5±2.1%,Raf-1 siRNA转染细胞凋亡率非常显著地高于空白转染细胞,差异具有统计学意义(P<0.01)。实验结果进一步明确了Raf-1基因与口腔上皮癌多药耐药性发生之间的关系密切,Raf-1基因积极参与口腔上皮癌多药耐药的发生过程,干扰Raf-1基因的有效表达可以引起口腔上皮癌多药耐药性显著下降。本实验同时提示Raf-1基因参与口腔上皮癌多药耐药性的发生可能是通过增强细胞中Mdr-1基因表达来实现的。因此采用Raf-1 siRNA转染技术是阻断Raf-1及Mdr-1基因表达,部分逆转口腔上皮癌细胞的多药耐药性,使其部分恢复对化疗药物敏感性的有效方法之一。
第四部分实验RT-PCR检测、Western blot检测结果显示采用联合转染Mdr-1/Raf-1 siRNAs进入KBV细胞中,即双阻断的方法逆转KBV细胞的多药耐药性,与单阻断Mdr-1或Raf-1基因有效表达的方法相比,无论在转录还是在蛋白表达水平,均更大程度地下调了耐药基因Mdr-1的表达,Raf-1 / Mdr-1 siRNAs联合转染细胞中Mdr-1基因的表达显著低于Raf-1 siRNA单一转染、Mdr-1 siRNA单一转染细胞,差异具有统计学意义(P<0.01);免疫荧光图像显示Raf-1 / Mdr-1 siRNAs联合转染细胞中P-gp蛋白表达显著弱于Raf-1 siRNA单一转染及Mdr-1 siRNA单一转染KBV细胞;MTT法测定结果显示Raf-1 / Mdr-1 siRNAs联合转染KBV细胞、Raf-1 siRNA转染、Mdr-1 siRNA转染细胞、KB细胞、KBV细胞及空白转染细胞的IC_(50)分别是58.76±3.27μg/L、201.38±7.13μg/L、130±6μg/L、21.33±2.25μg/L、352.68±12.36μg/L、356.47±14.23μg/L ;耐药指数RI(KBV/KB) =16.5 , RI(Raf-1 siRNA转染/KB)=9.5,RI(Mdr-1 siRNA转染/KB)=6.1,RI[(Raf-1 / Mdr-1 siRNA转染)/KB]=2.8,统计学分析显示Raf-1 / Mdr-1 siRNA联合转染的IC_(50)及RI显著低于Raf-1 siRNA单一转染、Mdr-1 siRNA单一转染KBV细胞,差异具有统计学意义( P< 0.01);流式细胞仪检测结果显示Raf-1/ Mdr-1siRNA联合转染细胞凋亡率达88.2%±4.3%,Raf-1 siRNA转染组细胞凋亡率达49.2±3.2%, Mdr-1 siRNA转染组细胞凋亡率达67.2%±3.7%,KB细胞凋亡率达98.8±1.2%,KBV细胞凋亡率达23.5±2.1%,空白转染组达24.3±1.6%,统计分析Raf-1/ Mdr-1siRNA联合转染细胞凋亡率非常显著地高于Raf-1 siRNA转染细胞及Mdr-1 siRNA转染细胞,差异具有统计学意义(P<0.01)。实验结果提示Mdr-1 /Raf-1 siRNAs联合转染与Raf-1 RNAi、Mdr-1 RNAi的单阻断方法相比较,更大程度地降低了KBV细胞中Mdr-1基因的表达,更有效地逆转了KBV细胞的多药耐药性,使KBV细胞重新对低浓度化疗药物敏感。如果应用于临床会大大降低化疗药物的用药浓度,减少化疗对患者机体的损伤,有利于提高肿瘤化疗的成功率。
综上所述,本研究结果提示Mdr-1的过表达作为多种肿瘤细胞产生多药耐药性的主要原因,同样成为口腔上皮癌多药耐药性发生的主要原因;Raf-1基因也积极参与了口腔上皮癌多药耐药性的发生过程,而且可能是通过增强Mdr-1的表达来实现的,Raf-1基因成为引起口腔上皮癌细胞多药耐药的重要原因之一。Raf-1 RNAi或Mdr-1 RNAi的单一阻断方法都是逆转口腔上皮癌多药耐药性的有效方法。由于肿瘤多药耐药性的产生是一个多步骤、多因素共同作用的结果,是一个非常复杂的过程,所以仅通过单一阻断Raf-1或Mdr-1多药耐药相关基因的表达均未能取得理想效果。本研究结果显示Raf-1/ Mdr-1 siRNAs联合转染的双阻断方法与Raf-1 RNAi或Mdr-1 RNAi的单一阻断方法相比,更显著地下调KBV细胞中多药耐药基因Mdr-1在转录和蛋白水平的表达,更有效地逆转KBV细胞的多药耐药性,使其重新对低浓度化疗药物敏感,为临床治疗提供了理论基础。
Multidrug resistance(MDR) is a major barrier for chemotherapy of most patients with oral squamous cell carcinoma. MDR means that tumor cells develop the resistance to many kinds of chemotherapy agents with various structures and functions.
The multidrug resistance is of a multisteps process and a very complex mechanism. The overexpression of P-glycoprotein(P-gp) encoded by mdr-1 gene is one of the most important reasons for MDR development..The human Pgp(Mdr-1) on cell membrane as efflux pump belongs to the ATP-binding cassette transporter expelling out antitumor agents from the cytoplasm , reducing intracellular drug concentrations in the cells to sublethal ,so that P-pg is a important target for reversing MDR. Besides,there are many other factors impacting the genesis of the MDR,for example, glutathlone S-transferase(GST) which acts by detoxification of some chemotherapeutic drugs,multidrug resistance-associated protein(MRP), transcription factor nuclear factor-kappa B(NF-κB) and so on. So many kinds of the agents such as antisense RNA and traditional medicines were released to have partly effect on reversing MDR of the tumor cells because MDR mechanism is too complex. we cannot attain ideal effect if we merely block one target or signaling pathway related to MDR. Researchers has adoptted many methods to reverse MDR,such as traditional Chinese drugs,anti-RNA, blockade of the gene expressions or signal transductions related to MDR.
In recent years,RNA interference(RNAi) has widely used as an experimental tool to analyse the function of mammalian genes. Double- stranded RNA(dsRNA) reagents are used to bind to and promote the degradation of target RNAs,resulting in knockdown of the expression of specific genes.In the field of reversal of multidrug resistance,small interfering double-stranded RNAs (siRNA) were designed to target the factors such as P-gp or GST mRNA as a strategy to inhibit resistant gene expression at the mRNA level in order to restore sensitivity to chemotherapeutic drugs.
Raf-1 is a cytoplasmic serine/threonine protein kinase that plays an important role in the tumorigenesis and inhibiton of apoptosis by modulating transcriptional activation and mitogenesis.Some experiments has indicated that Raf-1 kinase is associated with drug resistance in human breast cancer and prostate cancer and ectopic expression of Raf1 will increase the levels of the Mdr-1 drug pump.This increased expression of Mdr-1 most likely occurs by a transcriptional mechanism.The present research was to investigate the effect of Raf-1 on the multidrug resistance in multidrug resistant human oral squamous cell carcinoma (KBV) cells and detect the effect of Raf-1 siRNA, Mdr-1 siRNA and Raf-1/Mdr-1 siRNAs (Mdr-1 siRNA and Raf-1siRNA transfected into drug resistant KBV cells at the same time) on the reversal of multidrug resistance in KBV cells. We hoped that we might put forward a more effective method to reverse the multidrug resistance in KBV cells by the present study.
In this study, we investigated Mdr-1 and Raf-1 genes expression and abilities to resist the chemotherapeutic drug vincristine (VCR) in six groups: Mdr-1 siRNA transfected KBV cells,Raf-1 siRNA transfected KBV cells,Mdr1 /Raf-1 two siRNAs transfected KBV cells, KBV, KB and Control siRNA as control. Using RT-PCR , western blotting assay and immuno- fluorescent method the changes of the RNA and protein levels of both Mdr-1 and Raf-1 genes were studied.Using tetrazolium-based chemosensitivity assay(MTT) and flow cytometry (FCM) the changes of chemosensitivity to VCR in six groups of cells were detected.
This study includes four sections: (1) detection of relationships between Raf-1 and MDR in KBV cells; (2)the effect of Mdr-1 siRNA transfection on MDR in KBV cells; (3) the effect of Raf-1 siRNA transfection on MDR in KBV cells; (4) the effect of Mdr1 /Raf-1 siRNAs transfection on MDR in KBV cells.
The first section was to detect the relationgships between Raf-1 and multidrug resistance in KBV cells.The changes of the mRNA and protein levels of both Mdr-1 and Raf-1 genes in KBV and KB cells were determined by RT-PCR ,western blotting assay and immunofluorescent method.By MTT and FCM, IC_(50) ratios and apoptosis rates in both KBV and KB cells were detected.The findings indicated that expressions of both Raf-1 and Mdr1 mRNA and protein levels in KBV cells were lower than those in KB cells statistically (P<0.01). Immunofluorescent image showed that both Mdr1 and Raf-1 protein expressions in KBV cells were much stronger than those in KB cells.The IC_(50) ratio in KB cells was 21.33±2.25μg/L and that in KBV cells was 352.68±12.36μg/L.The resistance Index (RI) was 16.5.The apoptosis rate in KB cells was 98.8±1.2% and that in KBV cells was 23.5±2.1%.These results showed that differences in the chemosensitivity to VCR between KBV cells and KB cells were significant statistically (P<0.01). This study suggested that Raf-1 might play an important role in the process of developing the multidrug resistance in the human oral squamous carcinoma.Mdr-1 gene overexpression was still closely related with MDR in KBV cells.
The second section was to investigate the effect of Mdr-1 siRNA on MDR in KBV cells. Mdr-1 siRNA was transfected into KBV cells. The changes of the Mdr-1 and raf-1 gene expression at the mRNA and protein levels in Mdr-1 siRNA transfected KBV cells were determined by RT-PCR ,western blotting assay and immunofluorescent method. Using MTT and FCM IC_(50) and apoptosis rates in Mdr-1 siRNA transfected KBV cells were determined. The findings suggested that the mRNA and protein levels of Mdr-1 genes in Mdr1 siRNA transfected KBV cells were much lower than those in KBV cells statistically (P<0.01). Immunofluorescent image showed that P-gp expression in Mdr-1 siRNA transfected KBV cells were much weaker than that in KBV cells.The IC_(50) in Mdr-1 siRNA transfected KBV cells,KBV,KB and Control siRNA transfected KBV cells were respectively 130.68±6.35μg/L, 352.68±12.36μg/L, 21.33±2.25μg/L, 356.47±14.23μg/L. RI(Mdr-1 siRNA/KB) was 6.1 and RI(KBV/KB) was 16.5.The apoptosis rates in Mdr-1 siRNA transfected KBV cells was 67.2±3.7% ,24.3±1.6% in Control siRNA transfected KBV cells,98.8± 1.2% in KB cells and 23.5±2.1% in KBV cells.These results showed that the chemosensitivity to VCR in Mdr-1 siRNA transfected KBV cells was higher than that in KBV cells statistically (P<0.01). This study suggested that Mdr-1 siRNA was effective inhibitor of Mdr-1 gene expression to reverse the resistance of human oral squamous cell carcinoma.
The third section was to explore the effect of Raf-1 siRNA on MDR in KBV cells. Raf-1 siRNA was transfected into KBV cells.The Mdr-1 and Raf-1 genes expression at the mRNA and protein levels in Raf-1 siRNA transfected KBV cells were determined using RT-PCR ,western blotting assay and immunofluorescent method. Using MTT and FCM IC_(50) ratios and apoptosis rates in Raf-1 siRNA transfected KBV cells were determined. The findings suggested that the mRNA and protein levels of both Raf-1 and Mdr-1 genes in Raf-1 siRNA transfected KBV cells were lower than those in KBV cells statistically (P<0.01). Immunofluorescent image showed that P-gp and Raf-1 proteins expression in Raf-1 siRNA transfected KBV cells were much weaker than those in Control siRNA transfected KBV cells.The IC_(50) in Raf-1 siRNA transfected KBV cells ,KBV,KB and Control siRNA transfected KBV cells were respectively 201.38±7.13μg/L, 352.68±12.36μg/L, 21.33±2.25μg/L, 356.47±14.23μg/L.RI(Raf-1 siRNA/KB) was 9.5 and RI(KBV/KB) was 16.5.The apoptosis rate in Raf-1 siRNA transfected KBV cells was 49.2±3.2% , 24.3±1.6% in Control siRNA transfected KBV cells,98.8±1.2% in KB cells and 23.5±2.1% in KBV cells..These results showed that differences in the chemosensitivity to VCR between Raf-1 siRNA transfected KBV cells and KBV cells were significant statistically (P<0.01). This study suggested that Raf-1 siRNA was also effective inhibitor of Raf-1 and Mdr-1 gene expression to reverse the resistance of KBV cells.In multidrug resistant human oral squamous cell carcinoma, overexpression of Raf-1 could increase the levels of the Mdr-1 drug pump, which might be the reversal mechanism of Raf-1.
The fourth section was to investigate the the effect of Mdr-1 /Raf-1 siRNAs transfection on MDR in KBV cells. Mdr-1 /Raf-1 siRNAs were transfected into KBV cells at the same time. The Mdr-1 and Raf-1 genes expression at the mRNA and protein levels were assayed by RT-PCR, western blotting assay and immunofluorescent method. By MTT and FCM IC_(50) ratios and apoptosis rates in groups of cells were determined. The findings suggested that the mRNA and protein levels of Mdr-1 genes in Mdr-1 /Raf-1 siRNAs transfected KBV cells were much lower than those in Mdr-1 siRNA transfected KBV cells or Raf-1 siRNA transfected KBV cells statistically (P < 0.01). Immunofluorescent image showed that P-gp expression in Mdr-1 /Raf-1 siRNAs transfected KBV cells was much weaker than those in Raf-1 siRNA or Mdr-1 siRNA transfected KBV cells.The IC_(50) in Mdr-1 /Raf-1 siRNAs transfected KBV cells,Raf-1 siRNA transfected KBV cells,Mdr-1 siRNA transfected KBV cells,KB,KBV and Control siRNA transfected KBV cells were respectively 58.76±3.27μg/L, 201.38±7.13μg/L, 130±6μg/L, 21.33±2.25μg/L, 352.68±12.36μg/L, 356.47±14.23μg/L.RI (KBV/KB) was 16.5, RI(Raf-1 siRNA/KB) was 9.5,RI(Mdr-1 siRNA/KB) was 6.1 and RI[(Raf-1 / Mdr-1 siRNAs)/KB] was 2.8.The apoptosis rate in Raf-1 / Mdr-1 siRNAs transfected KBV cells was 88.2%±4.3%, 49.2±3.2% in Raf-1 siRNA transfected KBV cells, 67.2%±3.7% in Mdr-1 siRNA transfected KBV cells,98.8±1.2% in KB cells;23.5±2.1% in KBV cells and 24.3±1.6% in Control siRNA transfected KBV cells respectively.These results showed that the chemosensitivity to VCR in Raf-1 / Mdr-1 siRNAs KBV cells was much higher than Raf-1 siRNA transfected KBV cells or Mdr-1 siRNA transfected KBV cells statistically (P< 0.01). This study suggested that Raf-1 / Mdr-1 siRNAs transfection that block Mdr-1 genes expression more effectively has proved to be highly beneficial in reversing the resistance of KBV cells,compared with Raf-1 siRNA or Mdr-1 siRNA single transfection.
In sum, Raf-1 might play an important role in the process of developing the multidrug resistance in the human oral squamous carcinoma.Mdr-1 gene expression was still closely related to MDR in KBV cells. Mdr1 siRNA was effective inhibitor of Mdr-1 gene expression to reverse the resistance of human oral squamous cell carcinoma. Raf-1 siRNA could down regulated both Raf-1 and Mdr-1 genes expression and reverse the resistance of KBV cells. Raf1 could modulate the levels of the Mdr-1 drug pump in KBV cells, which might be the reversal mechanism of Raf1. Mdr1 is still a main target for therapeutic intervention. Raf-1 / Mdr-1 siRNAs transfection could block Mdr-1 gene expression and reverse the resistance of KBV cells more effectively compared with Raf-1 siRNA or Mdr-1 siRNA single transfection.
引文
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9. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature, 2001, 411 (6836):494-498
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12. McCaffrey AP, Meuse L, Pham TTT, et al. RNA interference in adultmice. Nature,2002,418(6869):38-39
13. Wu H, Hait WN, Yang HM. Small interfering RNA-induced suppression of MDR1 restores sensitivity to multidrug-resistant cancer cells. Cancer Research, 2003 ,63(7):1515-1519
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1. Lin Li, Jianfeng Xu, Taishan Min, et al.Reversal of MDR1 gene-dependent multidrug resistance using low concentration of endonuclease-prepared small interference RNA. Eur. J. Pharmacol., 2006; 536(1-2) 93-97
2. Vérène Stierlé, Alain Laigle, Béatrice Jollès.Modulation of MDR1 gene expression in multidrug resistant MCF7 cells by low concentrations of small interfering RNAs. Biochem. Pharmacol., 2005;70(10) 1424-1430
3. Michael Lee, Woo Suk Koh, Sang Seop Han.Down-regulation of Raf-1 kinase is associated with paclitaxel resistance in human breast cancer MCF-7/Adr cells.Cancer Lett., 2003; 193(1) 57-64
4. Neil Thompson, John Lyons .Recent progress in targeting the Raf/MEK/ERK pathway with inhibitors in cancer drug discovery. Curr. Opin. Pharmacol., 2005;5(4) 350-356
5. Sung-Tsai Yu, Tzer-Ming Chen, Shih-Yun Tseng, et al.Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells. Biochem. Biophys. Res. Commun., 2007;358(1) 79-84
6. Attila Kónya, Attila Andor, Péter Sátorhelyi, et al.Inhibition of the MDR1 transporter by new phenothiazine derivatives. Biochem. Biophys. Res. Commun.,2006; 346(1) 45-50
7. Ray K.M. Leung, Paul A. Whittaker .RNA interference: From gene silencing to gene-specific therapeutics. Pharmacol. Ther.,2005;107(2) 222-239
8. Christoph K. Thoeringer, Thomas Wultsch, Anaid Shahbazian, et al.Multidrug-resistance gene 1-type p-glycoprotein (MDR1 p-gp) inhibition by tariquidar impacts on neuroendocrine and behavioral processing of stress. Psychoneuroendocrinology,2007; 32 (8-10) 1028-1040
9. Karnati R. Roy, Kalle M. Arunasree, Amit Dhoot, et al. C-Phycocyanin inhibits 2-acetylaminofluorene-induced expression of MDR1 in mouse macrophage cells: ROS mediated pathway determined via combination of experimental and In silico analysis. Arch. Biochem. Biophys.,2007; 459(2)169-177
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