靛玉红衍生物PHII-7诱导乳腺癌耐药株MCF-7/ADR凋亡并通过转录因子AP-1逆转其多药耐药
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摘要
乳腺癌是女性易患的恶性肿瘤之一,发病率居女性肿瘤第二位,且呈逐年上升趋势,严重危害广大妇女的健康。化疗是临床上常用的治疗手段,但多药耐药(Multidrug resistance, MDR)仍是导致乳腺癌化疗失败的重要原因之一。
MDR形成机制复杂,其中P一糖蛋白(P-glycoprotein, P-gp)的过表达介导的药物外排作用是主要的耐药机制。P-gp属于ATP结合盒(ATP-binding cassette, ABC)转运蛋白超家族成员之一,由mdrl基因编码,是一种相对分子质量为170KDa的跨膜糖蛋白。P-gp的过度表达形成药物的转运泵,可与细胞内化疗药物结合并将药物泵到细胞外,以维持细胞内药物浓度在亚致死量的范围,从而产生耐药性。在大约30-40%的原发性乳腺癌和多于50%的转移性乳腺癌患者P-gp都高表达,因此研制能抑制P-gp表达及其功能的新型抗癌药是目前研究的热点。目前被证实具有逆转P-gp介导的MDR作用的药物和制剂很多,如维拉帕米(verapamil)、环胞多肽(cyclosporine)、三氟拉嗪(trifluroperazine)等,由于其有效逆转浓度往往超出临床应用的安全范围,在临床推广中存在不足。因此,寻找高效、低毒、作用靶点广、逆转作用显著的肿瘤MDR逆转剂刻不容缓。
近期,人们发现一些传统中药在作为抗癌药同时具有逆转耐药效应,相对于西药,因其有效性和安全性而受到青睐。靛玉红是传统中药(tranditional Chinese medicine, TCM)复方“当归龙荟丸”(danggui longhui wan)的活性成分。在中国,当归龙荟丸被用于治疗慢性粒细胞性白血病(chronic myelogenous leukemia,CML)。靛玉红的确切作用机制虽尚不清楚,但其对CML的治疗取得了令人欣慰的结果,同时抗实体瘤的效应也被研究。研究表明,靛玉红衍生物对多种人类肿瘤细胞也具有抗癌作用。一些靛玉红衍生物,如indirubin-5-sulfonate和indirubin-3-monoxime I临床试验已取得可喜成绩。基于靛玉红属于氧化吲哚类这一事实,我们设计和合成了一系列的氧化吲哚类衍生物：Ⅰ,Ⅱ,Ⅲ类,并经过构效分析,最终得到了抗癌效应最强的化合物：PH Ⅱ-7。
前期研究发现,PH Ⅱ-7可抑制多种人肿瘤细胞生长,对多药耐药肿瘤细胞同样有效,体内实验证实对多种移植瘤有效。PH Ⅱ-7的一个主要的特征就是对多药耐药的肿瘤细胞同样有效。本实验选用乳腺癌敏感株MCF-7和高表达P-gp的多药耐药细胞株MCF-7/ADR,观察PHⅡ-7的抗癌和耐药逆转作用。结果表明,耐药株MCF-7/ADR相对于敏感株MCF-7对ADR的耐药倍数将近40倍,而PH Ⅱ-7对MCF-7/ADR和MCF-7均有生长抑制作用,IC50值接近,分别为6.07μM和5.51μM。用凋亡试剂盒Annexin-V FITC-PI则试结果表明,4μM的PHⅡ-7可诱导MCF-7/ADR将近20%的凋亡率。MTT和凋亡结果表明,PHⅡ-7可以显著抑制MCF-7/ADR细胞的增殖活性,并诱导其凋亡。
用verapamil作为阳性对照,PH Ⅱ-7的逆转活性被进一步检测。在非毒性的浓度0.5μM.1μM和2μM, PHⅡ-7明显提高MCF-7/ADR对ADR的敏感性,表现为IC50值的浓度依赖性降低。相对于verapamil的54.68的耐药逆转倍数,0.5μM、1μM和2μM PHⅡ-7分别将MCF-7/ADR的IC50值137.80μgM降至108.20μM,18.64μM和0.55μM,逆转倍数分别为1.27,7.39,250.55。PH Ⅱ-7对MCF-7/ADR的逆转机制尚不清楚,因此,接下来的实验主要检测了PH Ⅱ-7对mdrl/P-gp的表达和外排功能的影响。
Rhodamine123作为P-gp的荧光性底物,被用于检测P-gp的外排泵功能。结果表明,同verapamil相同,(0.5μM,2μM) PHⅡ-7可明显增加MCF-7/ADR细胞内Rhodamine123的蓄积量,但对敏感株MCF-7无明显影响,提示PH Ⅱ-7可抑制P-gp的外排泵功能。进一步的研究表明,经PH Ⅱ-7作用后,MCF-7ADR细胞mdrl/P-gp表达明显降低。流式细胞仪检测对P-gp蛋白的检测结果表明,10μM verapamil导致了25.83%的P-gp表达下调,而0.5μM、IμM、2μM和PHⅡ-7对P-pg抑制率分别为1.35%、5.39%、14.72%和25.87%。4μM PHⅡ-7对P-gp的抑制率同10μM verapamil接近,但2μM PHⅡ-7的逆转效应是10μM verapamil的4.58倍。对P-gp表达和功能的双重抑制、诱导凋亡共同参与了PHⅡ-7的耐药逆转作用,强大的逆转效应保证了其应用的安全性。
mdrl在转录水平可被多个转录因子调节。活化蛋白-1(activating protein-1, AP-1)是调节应激反应的重要的转录因子之一,由Fos和Jun蛋白家族的二聚体组成。AP-1是多个信号转导途径中的关键因子,在细胞分化、增生、凋亡过程中都起非常重要的作用。在乳腺癌细胞,AP-1已被证实为乳腺癌浸润和转移的重要的调节因子。此外,其他几条证据也使我们去调查AP-1在mdrl转录调节中的可能作用：(1) mdrl启动子区包含AP-1的重复位点,且位于启动子起始转录所必须的区域。(2)在几株人多药耐药细胞上已证实了c-fos、c-jun的过表达和AP-1活性的增强。AP-1活性的降低可增加对抗癌药的敏感性,提示了AP-1在MDR信号转导途径中的重要作用。
作为AP-1的组成成分之一,c-fos在肿瘤发生、肿瘤细胞增生、转化、血管生成、肿瘤浸和转移中起非常重要的作用。在乳腺癌,c-fos的过度表达和预后成反比。c-fos基因的敲除能够延长乳腺癌移植瘤的存活时间,抑制乳腺癌细胞的增生和浸润。而且,研究表明,一些用于诱导多药耐药的药物如阿霉素、长春新碱(vincristine)、依托泊甙(VP-16)和秋水仙碱(colchicine),在应用过程中也引起c-fos表达水平的升高。这提示在多药耐药形成早期,转录机制的加强促进了P-gp表达增高。
相对于敏感株MCF-7,耐药株MCF-7/ADR高表达c-fos mRNA和蛋白。而PHⅡ-7以时间和浓度依赖性的方式抑制MCF-7/ADR细胞的c-fos表达。4μM PHⅡ-7处理MCF-7/ADR24h后,c-fos蛋白水平明显下降,这和4μM PHⅡ-7对P-gp25.87%的抑制率相一致。为进一步确证c-fos在P-gp介导的多药耐药中的作用,用重组的干扰载体pSilencer3.1/sic-fos转染MCF-7/ADR细胞,干扰c-fos基因。并筛选出两株稳定单克隆细胞,MCF-7/ADR/sic-fos-8B和-3D,观察c-fos基因的抑制引起的耐药性的变化。转染结果表明,c-fos基因的抑制可下调mdrl和P-gp的表达,增加细胞内的Rhodamine123蓄积量,对ADR的敏感性增加。和c-fos的干扰程度相一致,MCF-7/ADR/sic-fos-3D因为c-fos干扰得更明显,因此表现出对ADR更强的敏感性、更多的细胞内Rhodamine123的蓄积、较少的mdrl/P-gp表达,这些结果更进一步确定了c-fos对P-gp介导的多药耐药的调节作用。
综上所述,PH Ⅱ-7作为靛玉红衍生物之一,一方面可诱导耐药的乳腺癌细胞凋亡,另一方面可通过抑制转录因子AP-1,下调P-gp表达,逆转MCF-7/ADR的多药耐药。因此,PHⅡ-7具有抗癌和逆转耐药的双重作用。虽然靛玉红衍生物的抗癌作用已被广泛研究,但本研究首次报道了靛玉红衍生物具有逆转耐药作用。且强大的逆转耐药作用保证了其应用的安全性,优于传统的逆转剂verapamil。同时,我们的研究结果提示了c-fos可以作为耐药逆转剂的潜在的靶点之一。另外,本研究结果也为PHⅡ-7的临床应用(单独或与抗癌药联合应用)治疗多药耐药肿瘤提供了实验基础。
Breast cancer is one of the most common female malignancies in the world. Its incidence ranks second as a cause of cancer death in women (after lung cancer). Although a definite therapeutic effect has been achieved by existing treatment modalities of surgery, hormonal therapy, chemotherapy and radiation, multidrug resistance (MDR) is one of the most significant impediments to successful chemotherapy of progressive breast cancer.
There are a number of mechanisms by which a cell can acquire MDR. It is generally thought that the overexpression of P-gp, a product of the mdrl gene family, is a predominant cause of the acquisition of an MDR phenotype. Human P-gp is a170kDa plasma membrane glycoprotein, and functions as an ATP-dependent transmembrane drug transporter that reduces intracellular drug accumulation by pumping drugs out of the cells. In about30-40%of primary and more than50%of metastatic breast cancer patient samples, P-gp has been found to be overexpressed. Therefore, the identification of novel agents which can inhibit pump function or/and expression of P-gp is of utmost interest in cancer research. In the past years, a number of experimental agents, including verapamil, cyclosporine and trifluroperazine, have been studied with various efficacies in reversing MDR phenotype in cancer cells. However, the application of these compounds is very limited due to their unfavorable toxicities and/or their significant effects on the metabolism and pharmacokinetics of the concurrently administered chemotherapeutic agents. Therefore, development or discovery of safe and effective MDR reversal agents is urgently required.
Recently, the use of traditional Chinese medicine (TCM) was found to be a fertile area in which to look for antitumor agents with activity against MDR because of their potency and safety. Indirubin has been identified as an active component of a CTM, Danggui Longhui Wan, which is used for the treatment of chronic myelogenous leukemia (CML). Indirubin has undergone clinical trials in China to treat CML with promising results, although its mechanism of action remains unclear. In addition, anti-solid tumor activity of indirubin has also been investigated. Several studies have recently demonstrated that indirubin derivatives had antitumor activity on a variety of human cancer cells. Some indirubin derivatives, such as, indirubin-5-sulfonate and indirubin-3-monoxime, have undergone clinical trials with promising results. Based on the fact that indirubins belong to the chemical class of oxindole, we designed and synthesized a series of derivatives of oxindole:Ⅰ, Ⅱ, Ⅲ. Through further SAR (structure-activity relationship) analysis on the-X substitute, and finally got the most potent antitumor compound PH Ⅱ-7.
The previous data in our lab indicated that this compound had significant antitumor activity against malignant tumor cells and xenografts (not yet printed). One of the major features of PHⅡ-7was its activity against MDR tumor cells. In this study, we chose breast cancer cells MCF-7and its resistant MCF-7/ADR cells, a classic multidrug-resistant cell subline expressing high level of P-glycoprotein (P-gp), as a model, and found that the IC50of ADR for MCF-7cells and MCF-7/ADR cells were3.45μM and137.80μM, respectively, So MCF-7/ADR cells in these experiments were about40-fold resistant to ADR in comparison with the parental MCF-7cells, In contrast, both the parent and the MDR lines were almost equally sensitive to PH Ⅱ-7with IC50values6.07μM and5.51μM, respectively. The cytotoxicity of PHⅡ-7toward MCF-7/ADR cells was also evaluated by quantification of apoptotic cells.4μM PH Ⅱ-7treatment significantly induced apoptosis in MCF-7/ADR cells, and the percentage of apoptosis was about20%. The results indicated that PH Ⅱ-7significantly inhibited the growth and induced its apoptosis in MCF-7/ADR cells.
PH Ⅱ-7was examined for its reversal activity, in comparison to verapamil, in MCF-7/ADR cells. At nontoxic concentrations of0.5,1, and2μM, PHⅡ-7induced a significant decrease of the IC50of ADR against MCF-7/ADR cells in a concentration dependent manner. In detail,10μM verapamil produced a54.68-fold ADR sensitization in MCF-7/ADR cells, while0.5,1, and2μM PHⅡ-7lowered the IC50of ADR from 137.80μM to108.20,18.64, and0.55μM, and this gave a1.27-,7.39-, and250.55-fold reversal of MDR, respectively. The mechanism by which this occurs was unknown, the effects of PHⅡ-7on expression and function of P-gp were examined.
Rhodamine123, a fluorescent substrate of P-gp, was used to monitor the efflux activity of P-gp. Incubation with (0.5,2μM) PH Ⅱ-7or with (10μM) verapamil effectively enhanced the intracellular accumulation of Rhodamine123in MDR cells, but showed no significant effects in the parental sensitive cells. These results demonstrated that like verapamil, PH Ⅱ-7was able to interfere with P-gp function. The variation of P-gp protein expression level was analyzed by flow cytometry. In comparison to25.83%P-gp downregulation by10μM verapamil,0.5μM,1μM,2μM and4μM PH Ⅱ-7gave a1.35%,5.39%,14.72%and25.87%P-gp decrease, respectively.4μM PH Ⅱ-7had the same inhibiton rate of P-gp expression as10μM verapamil. Interestingly,2μM PH Ⅱ-7completely reversed MDR in MCF-7/ADR cells with a much better potency (about4.58fold) than10μM verapamil. Inhibition of P-gp expression and function, induction of apoptosis were involved in the potent MDR reversal of PH Ⅱ-7, and the pentency suggested a greater degree of safety.
mdrl is regulated at the transcriptional level by multiple factors. A well known mediator of stress response pathways is the transcription factor activating protein-1(AP-1), which is composed of dimers from the Jun and Fos protein families. AP-1is a key component of many signal transduction pathways, which has been implicated in many different biological processes including cell differentiation, proliferation, and apoptosis. In breast cancer cells, the AP-1proteins have been identified as important regulators of growth and invasion. In addition, several lines of evidence led us to investigate a possible role for AP-1in the transcriptional regulation of mdrl. First, the promoter region of mdrl contains a putative site for AP-1located within a region of the promoter that has been demonstrated to be necessary for gene induction. Second, constitutive overexpression of both c-fosand c-Jun and increased AP-1binding activity have been observed in several human multidrug resistant cell lines, while reduced AP-1binding activity has been associated with increased drug sensitivity, suggesting a possible role for AP-1in the signal pathway leading to MDR.
As a member of the AP-1transcription factor complex, c-fos is considered to play a critical role in tumorigenesis, proliferation and transformation, angiogenesis, tumor invasion, and metastasis, and its expression is associated with poor clinical outcomes. In breast cancer, increased c-fos protein expression was associated with poor prognosis. Knockdown of c-fos expression was able to prolong survival and inhibit the proliferation and invasiveness of breast cancer xenografts. Furthermore, several studies indicated that ADR resistance was associated with overexpression of the protooncogene c-fos in tumors. The additional finding that drugs like ADR, vincristine, VP-16, and colchicine, which are used to select cells for MDR, also cause a rise in the level of c-fos message suggests a model wherein an early event in the acquisition of MDR is the boosting of the transcriptional machinery, which allows for increased expression of P-gp.
MCF-7/ADR cells displayed increased c-fos mRNA and protein levels compared to MCF-7cells, whereas PHⅡ-7inhibited c-fos expression in a time-and dose-dependent manner in MCF-7/ADR cells. After treatment of MDR cells with4μM PH Ⅱ-7for24h, c-fos protein levels decreased significantly, which was consistent with25.87%inhibition rate of P-gp by4μM PHⅡ-7.To comfirm furtherly the role of c-fos on P-gp mediated MDR, we performed the stable transfection of MCF-7/ADR cells with the pSilencer3.1-sic-fos construct, generating MCF-7/ADR/sic-fos cells. Two clones, MCF-7/ADR/sic-fos-8B and-3D, were selected for further expansion and characterization. The transfected results demonstrated that c-fos shRNA could downregulate expression of mdrl and P-gp, increase intracellular ADR concentration and sensitivity, Morever, consistent with c-fos interference level, MCF-7/ADR/sic-fos-3D displayed more sensitive to ADR, more Rhodamine123accumulation and mdrl/P-gp expression than MCF-7/ADR/sic-fos-8B cells, which confirming furtherly that c-fos may modulate P-gp expression in MCF-7/ADR cells.
In conclusion, PH Ⅱ-7, one of indirubin derivatives, could induce apoptosis in MDR breast cancer cells; On the other hand, PH Ⅱ-7reversed MDR by downregulating P-gp through AP-1transcription factors in MCF-7/ADR cells. Therefore, PHⅡ-7is of dual functions of antitumor and reversing MDR. Meanwhile, the findings suggested c-fos might be a potential drug target for circumventing tumor MDR. In addition, the present results provide a biochemical basis for possible clinical application of PH Ⅱ-7alone, or in combination with conventional antineoplastic agents in treating MDR tumors.
In conclusion, PH Ⅱ-7, one of indirubin derivatives, could induce apoptosis in MDR breast cancer cells; On the other hand, PH Ⅱ-7reversed MDR by downregulating P-gp through AP-1transcription factors in MCF-7/ADR cells. Therefore, PHⅡ-7is of dual functions of antitumor and reversing MDR. The anti-tumor activity of indirubin derivatives has also been investigated in many studies, however, the activity against MDR was firstly demonstrated. Meanwhile, the findings suggested c-fos might be a potential drug target for circumventing tumor MDR. In addition, the present results provide a biochemical basis for possible clinical application of PH Ⅱ-7alone, or in combination with conventional antineoplastic agents in treating MDR tumors
MDR形成机制复杂,其中P一糖蛋白(P-glycoprotein, P-gp)的过表达介导的药物外排作用是主要的耐药机制。P-gp属于ATP结合盒(ATP-binding cassette, ABC)转运蛋白超家族成员之一,由mdrl基因编码,是一种相对分子质量为170KDa的跨膜糖蛋白。P-gp的过度表达形成药物的转运泵,可与细胞内化疗药物结合并将药物泵到细胞外,以维持细胞内药物浓度在亚致死量的范围,从而产生耐药性。在大约30-40%的原发性乳腺癌和多于50%的转移性乳腺癌患者P-gp都高表达,因此研制能抑制P-gp表达及其功能的新型抗癌药是目前研究的热点。目前被证实具有逆转P-gp介导的MDR作用的药物和制剂很多,如维拉帕米(verapamil)、环胞多肽(cyclosporine)、三氟拉嗪(trifluroperazine)等,由于其有效逆转浓度往往超出临床应用的安全范围,在临床推广中存在不足。因此,寻找高效、低毒、作用靶点广、逆转作用显著的肿瘤MDR逆转剂刻不容缓。
近期,人们发现一些传统中药在作为抗癌药同时具有逆转耐药效应,相对于西药,因其有效性和安全性而受到青睐。靛玉红是传统中药(tranditional Chinese medicine, TCM)复方“当归龙荟丸”(danggui longhui wan)的活性成分。在中国,当归龙荟丸被用于治疗慢性粒细胞性白血病(chronic myelogenous leukemia,CML)。靛玉红的确切作用机制虽尚不清楚,但其对CML的治疗取得了令人欣慰的结果,同时抗实体瘤的效应也被研究。研究表明,靛玉红衍生物对多种人类肿瘤细胞也具有抗癌作用。一些靛玉红衍生物,如indirubin-5-sulfonate和indirubin-3-monoxime I临床试验已取得可喜成绩。基于靛玉红属于氧化吲哚类这一事实,我们设计和合成了一系列的氧化吲哚类衍生物：Ⅰ,Ⅱ,Ⅲ类,并经过构效分析,最终得到了抗癌效应最强的化合物：PH Ⅱ-7。
前期研究发现,PH Ⅱ-7可抑制多种人肿瘤细胞生长,对多药耐药肿瘤细胞同样有效,体内实验证实对多种移植瘤有效。PH Ⅱ-7的一个主要的特征就是对多药耐药的肿瘤细胞同样有效。本实验选用乳腺癌敏感株MCF-7和高表达P-gp的多药耐药细胞株MCF-7/ADR,观察PHⅡ-7的抗癌和耐药逆转作用。结果表明,耐药株MCF-7/ADR相对于敏感株MCF-7对ADR的耐药倍数将近40倍,而PH Ⅱ-7对MCF-7/ADR和MCF-7均有生长抑制作用,IC50值接近,分别为6.07μM和5.51μM。用凋亡试剂盒Annexin-V FITC-PI则试结果表明,4μM的PHⅡ-7可诱导MCF-7/ADR将近20%的凋亡率。MTT和凋亡结果表明,PHⅡ-7可以显著抑制MCF-7/ADR细胞的增殖活性,并诱导其凋亡。
用verapamil作为阳性对照,PH Ⅱ-7的逆转活性被进一步检测。在非毒性的浓度0.5μM.1μM和2μM, PHⅡ-7明显提高MCF-7/ADR对ADR的敏感性,表现为IC50值的浓度依赖性降低。相对于verapamil的54.68的耐药逆转倍数,0.5μM、1μM和2μM PHⅡ-7分别将MCF-7/ADR的IC50值137.80μgM降至108.20μM,18.64μM和0.55μM,逆转倍数分别为1.27,7.39,250.55。PH Ⅱ-7对MCF-7/ADR的逆转机制尚不清楚,因此,接下来的实验主要检测了PH Ⅱ-7对mdrl/P-gp的表达和外排功能的影响。
Rhodamine123作为P-gp的荧光性底物,被用于检测P-gp的外排泵功能。结果表明,同verapamil相同,(0.5μM,2μM) PHⅡ-7可明显增加MCF-7/ADR细胞内Rhodamine123的蓄积量,但对敏感株MCF-7无明显影响,提示PH Ⅱ-7可抑制P-gp的外排泵功能。进一步的研究表明,经PH Ⅱ-7作用后,MCF-7ADR细胞mdrl/P-gp表达明显降低。流式细胞仪检测对P-gp蛋白的检测结果表明,10μM verapamil导致了25.83%的P-gp表达下调,而0.5μM、IμM、2μM和PHⅡ-7对P-pg抑制率分别为1.35%、5.39%、14.72%和25.87%。4μM PHⅡ-7对P-gp的抑制率同10μM verapamil接近,但2μM PHⅡ-7的逆转效应是10μM verapamil的4.58倍。对P-gp表达和功能的双重抑制、诱导凋亡共同参与了PHⅡ-7的耐药逆转作用,强大的逆转效应保证了其应用的安全性。
mdrl在转录水平可被多个转录因子调节。活化蛋白-1(activating protein-1, AP-1)是调节应激反应的重要的转录因子之一,由Fos和Jun蛋白家族的二聚体组成。AP-1是多个信号转导途径中的关键因子,在细胞分化、增生、凋亡过程中都起非常重要的作用。在乳腺癌细胞,AP-1已被证实为乳腺癌浸润和转移的重要的调节因子。此外,其他几条证据也使我们去调查AP-1在mdrl转录调节中的可能作用：(1) mdrl启动子区包含AP-1的重复位点,且位于启动子起始转录所必须的区域。(2)在几株人多药耐药细胞上已证实了c-fos、c-jun的过表达和AP-1活性的增强。AP-1活性的降低可增加对抗癌药的敏感性,提示了AP-1在MDR信号转导途径中的重要作用。
作为AP-1的组成成分之一,c-fos在肿瘤发生、肿瘤细胞增生、转化、血管生成、肿瘤浸和转移中起非常重要的作用。在乳腺癌,c-fos的过度表达和预后成反比。c-fos基因的敲除能够延长乳腺癌移植瘤的存活时间,抑制乳腺癌细胞的增生和浸润。而且,研究表明,一些用于诱导多药耐药的药物如阿霉素、长春新碱(vincristine)、依托泊甙(VP-16)和秋水仙碱(colchicine),在应用过程中也引起c-fos表达水平的升高。这提示在多药耐药形成早期,转录机制的加强促进了P-gp表达增高。
相对于敏感株MCF-7,耐药株MCF-7/ADR高表达c-fos mRNA和蛋白。而PHⅡ-7以时间和浓度依赖性的方式抑制MCF-7/ADR细胞的c-fos表达。4μM PHⅡ-7处理MCF-7/ADR24h后,c-fos蛋白水平明显下降,这和4μM PHⅡ-7对P-gp25.87%的抑制率相一致。为进一步确证c-fos在P-gp介导的多药耐药中的作用,用重组的干扰载体pSilencer3.1/sic-fos转染MCF-7/ADR细胞,干扰c-fos基因。并筛选出两株稳定单克隆细胞,MCF-7/ADR/sic-fos-8B和-3D,观察c-fos基因的抑制引起的耐药性的变化。转染结果表明,c-fos基因的抑制可下调mdrl和P-gp的表达,增加细胞内的Rhodamine123蓄积量,对ADR的敏感性增加。和c-fos的干扰程度相一致,MCF-7/ADR/sic-fos-3D因为c-fos干扰得更明显,因此表现出对ADR更强的敏感性、更多的细胞内Rhodamine123的蓄积、较少的mdrl/P-gp表达,这些结果更进一步确定了c-fos对P-gp介导的多药耐药的调节作用。
综上所述,PH Ⅱ-7作为靛玉红衍生物之一,一方面可诱导耐药的乳腺癌细胞凋亡,另一方面可通过抑制转录因子AP-1,下调P-gp表达,逆转MCF-7/ADR的多药耐药。因此,PHⅡ-7具有抗癌和逆转耐药的双重作用。虽然靛玉红衍生物的抗癌作用已被广泛研究,但本研究首次报道了靛玉红衍生物具有逆转耐药作用。且强大的逆转耐药作用保证了其应用的安全性,优于传统的逆转剂verapamil。同时,我们的研究结果提示了c-fos可以作为耐药逆转剂的潜在的靶点之一。另外,本研究结果也为PHⅡ-7的临床应用(单独或与抗癌药联合应用)治疗多药耐药肿瘤提供了实验基础。
Breast cancer is one of the most common female malignancies in the world. Its incidence ranks second as a cause of cancer death in women (after lung cancer). Although a definite therapeutic effect has been achieved by existing treatment modalities of surgery, hormonal therapy, chemotherapy and radiation, multidrug resistance (MDR) is one of the most significant impediments to successful chemotherapy of progressive breast cancer.
There are a number of mechanisms by which a cell can acquire MDR. It is generally thought that the overexpression of P-gp, a product of the mdrl gene family, is a predominant cause of the acquisition of an MDR phenotype. Human P-gp is a170kDa plasma membrane glycoprotein, and functions as an ATP-dependent transmembrane drug transporter that reduces intracellular drug accumulation by pumping drugs out of the cells. In about30-40%of primary and more than50%of metastatic breast cancer patient samples, P-gp has been found to be overexpressed. Therefore, the identification of novel agents which can inhibit pump function or/and expression of P-gp is of utmost interest in cancer research. In the past years, a number of experimental agents, including verapamil, cyclosporine and trifluroperazine, have been studied with various efficacies in reversing MDR phenotype in cancer cells. However, the application of these compounds is very limited due to their unfavorable toxicities and/or their significant effects on the metabolism and pharmacokinetics of the concurrently administered chemotherapeutic agents. Therefore, development or discovery of safe and effective MDR reversal agents is urgently required.
Recently, the use of traditional Chinese medicine (TCM) was found to be a fertile area in which to look for antitumor agents with activity against MDR because of their potency and safety. Indirubin has been identified as an active component of a CTM, Danggui Longhui Wan, which is used for the treatment of chronic myelogenous leukemia (CML). Indirubin has undergone clinical trials in China to treat CML with promising results, although its mechanism of action remains unclear. In addition, anti-solid tumor activity of indirubin has also been investigated. Several studies have recently demonstrated that indirubin derivatives had antitumor activity on a variety of human cancer cells. Some indirubin derivatives, such as, indirubin-5-sulfonate and indirubin-3-monoxime, have undergone clinical trials with promising results. Based on the fact that indirubins belong to the chemical class of oxindole, we designed and synthesized a series of derivatives of oxindole:Ⅰ, Ⅱ, Ⅲ. Through further SAR (structure-activity relationship) analysis on the-X substitute, and finally got the most potent antitumor compound PH Ⅱ-7.
The previous data in our lab indicated that this compound had significant antitumor activity against malignant tumor cells and xenografts (not yet printed). One of the major features of PHⅡ-7was its activity against MDR tumor cells. In this study, we chose breast cancer cells MCF-7and its resistant MCF-7/ADR cells, a classic multidrug-resistant cell subline expressing high level of P-glycoprotein (P-gp), as a model, and found that the IC50of ADR for MCF-7cells and MCF-7/ADR cells were3.45μM and137.80μM, respectively, So MCF-7/ADR cells in these experiments were about40-fold resistant to ADR in comparison with the parental MCF-7cells, In contrast, both the parent and the MDR lines were almost equally sensitive to PH Ⅱ-7with IC50values6.07μM and5.51μM, respectively. The cytotoxicity of PHⅡ-7toward MCF-7/ADR cells was also evaluated by quantification of apoptotic cells.4μM PH Ⅱ-7treatment significantly induced apoptosis in MCF-7/ADR cells, and the percentage of apoptosis was about20%. The results indicated that PH Ⅱ-7significantly inhibited the growth and induced its apoptosis in MCF-7/ADR cells.
PH Ⅱ-7was examined for its reversal activity, in comparison to verapamil, in MCF-7/ADR cells. At nontoxic concentrations of0.5,1, and2μM, PHⅡ-7induced a significant decrease of the IC50of ADR against MCF-7/ADR cells in a concentration dependent manner. In detail,10μM verapamil produced a54.68-fold ADR sensitization in MCF-7/ADR cells, while0.5,1, and2μM PHⅡ-7lowered the IC50of ADR from 137.80μM to108.20,18.64, and0.55μM, and this gave a1.27-,7.39-, and250.55-fold reversal of MDR, respectively. The mechanism by which this occurs was unknown, the effects of PHⅡ-7on expression and function of P-gp were examined.
Rhodamine123, a fluorescent substrate of P-gp, was used to monitor the efflux activity of P-gp. Incubation with (0.5,2μM) PH Ⅱ-7or with (10μM) verapamil effectively enhanced the intracellular accumulation of Rhodamine123in MDR cells, but showed no significant effects in the parental sensitive cells. These results demonstrated that like verapamil, PH Ⅱ-7was able to interfere with P-gp function. The variation of P-gp protein expression level was analyzed by flow cytometry. In comparison to25.83%P-gp downregulation by10μM verapamil,0.5μM,1μM,2μM and4μM PH Ⅱ-7gave a1.35%,5.39%,14.72%and25.87%P-gp decrease, respectively.4μM PH Ⅱ-7had the same inhibiton rate of P-gp expression as10μM verapamil. Interestingly,2μM PH Ⅱ-7completely reversed MDR in MCF-7/ADR cells with a much better potency (about4.58fold) than10μM verapamil. Inhibition of P-gp expression and function, induction of apoptosis were involved in the potent MDR reversal of PH Ⅱ-7, and the pentency suggested a greater degree of safety.
mdrl is regulated at the transcriptional level by multiple factors. A well known mediator of stress response pathways is the transcription factor activating protein-1(AP-1), which is composed of dimers from the Jun and Fos protein families. AP-1is a key component of many signal transduction pathways, which has been implicated in many different biological processes including cell differentiation, proliferation, and apoptosis. In breast cancer cells, the AP-1proteins have been identified as important regulators of growth and invasion. In addition, several lines of evidence led us to investigate a possible role for AP-1in the transcriptional regulation of mdrl. First, the promoter region of mdrl contains a putative site for AP-1located within a region of the promoter that has been demonstrated to be necessary for gene induction. Second, constitutive overexpression of both c-fosand c-Jun and increased AP-1binding activity have been observed in several human multidrug resistant cell lines, while reduced AP-1binding activity has been associated with increased drug sensitivity, suggesting a possible role for AP-1in the signal pathway leading to MDR.
As a member of the AP-1transcription factor complex, c-fos is considered to play a critical role in tumorigenesis, proliferation and transformation, angiogenesis, tumor invasion, and metastasis, and its expression is associated with poor clinical outcomes. In breast cancer, increased c-fos protein expression was associated with poor prognosis. Knockdown of c-fos expression was able to prolong survival and inhibit the proliferation and invasiveness of breast cancer xenografts. Furthermore, several studies indicated that ADR resistance was associated with overexpression of the protooncogene c-fos in tumors. The additional finding that drugs like ADR, vincristine, VP-16, and colchicine, which are used to select cells for MDR, also cause a rise in the level of c-fos message suggests a model wherein an early event in the acquisition of MDR is the boosting of the transcriptional machinery, which allows for increased expression of P-gp.
MCF-7/ADR cells displayed increased c-fos mRNA and protein levels compared to MCF-7cells, whereas PHⅡ-7inhibited c-fos expression in a time-and dose-dependent manner in MCF-7/ADR cells. After treatment of MDR cells with4μM PH Ⅱ-7for24h, c-fos protein levels decreased significantly, which was consistent with25.87%inhibition rate of P-gp by4μM PHⅡ-7.To comfirm furtherly the role of c-fos on P-gp mediated MDR, we performed the stable transfection of MCF-7/ADR cells with the pSilencer3.1-sic-fos construct, generating MCF-7/ADR/sic-fos cells. Two clones, MCF-7/ADR/sic-fos-8B and-3D, were selected for further expansion and characterization. The transfected results demonstrated that c-fos shRNA could downregulate expression of mdrl and P-gp, increase intracellular ADR concentration and sensitivity, Morever, consistent with c-fos interference level, MCF-7/ADR/sic-fos-3D displayed more sensitive to ADR, more Rhodamine123accumulation and mdrl/P-gp expression than MCF-7/ADR/sic-fos-8B cells, which confirming furtherly that c-fos may modulate P-gp expression in MCF-7/ADR cells.
In conclusion, PH Ⅱ-7, one of indirubin derivatives, could induce apoptosis in MDR breast cancer cells; On the other hand, PH Ⅱ-7reversed MDR by downregulating P-gp through AP-1transcription factors in MCF-7/ADR cells. Therefore, PHⅡ-7is of dual functions of antitumor and reversing MDR. Meanwhile, the findings suggested c-fos might be a potential drug target for circumventing tumor MDR. In addition, the present results provide a biochemical basis for possible clinical application of PH Ⅱ-7alone, or in combination with conventional antineoplastic agents in treating MDR tumors.
In conclusion, PH Ⅱ-7, one of indirubin derivatives, could induce apoptosis in MDR breast cancer cells; On the other hand, PH Ⅱ-7reversed MDR by downregulating P-gp through AP-1transcription factors in MCF-7/ADR cells. Therefore, PHⅡ-7is of dual functions of antitumor and reversing MDR. The anti-tumor activity of indirubin derivatives has also been investigated in many studies, however, the activity against MDR was firstly demonstrated. Meanwhile, the findings suggested c-fos might be a potential drug target for circumventing tumor MDR. In addition, the present results provide a biochemical basis for possible clinical application of PH Ⅱ-7alone, or in combination with conventional antineoplastic agents in treating MDR tumors
引文
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