PHâ…¡-7, One Of Indirubin Derivatives, Induces Apoptosis And Reverses P-glycoprotein-mediated Multidrug Resistance Via AP-1Transcription Factors In A Human Breast Cancer Cell Line MCF-7/ADR

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...