| Background&ObjectivePlenty of cancer cells develop resistance against their chemotherapeutic agents which are structurally and mechanistically various. For example, paclitaxel and adriamycin, widely used in ovarian cancer chemotherapy treatment, come out unsatisfactory only because the tumor lost the sensibility to the chemotherapeutic agents, which is currently defined as multi-drug resistance (MDR). Intrinsic and acquired MDR mainly results from the overexpression of cell membrane-bound ATP-binding cassette (ABC) transporters, which actively extrude a variety of chemotherapeutic drugs out of the cancer cells. Importantly, P-glycoprotein(p-gp), encoded by MDR1gene, is able to transport a wide range of anticancer agents such as the anthracyclines, vinca alkaloids, taxanes, and epipodophyllotoxins, thereby may be responsible for intrinsic and acquired drug resistance in numerous human cancers. Recently, P-gp associated MDR is proved to be effectively overcome by either blocking its drug-pump function or inhibiting its expression. Thus, suppression of P-gp function and expression may certainly reverse the P-gp associated MDR in cancer cells that comes to increase the efficacy of chemotherapy.Since P-gp associated MDR was first identified exceed semi-century ago, researches on new effective P-gp inhibitors have attracted extensive attention worldwide. The discovery of verapamil reversal MDR by blocking P-gp in1980s led to a wave of P-gp inhibitor development, and various agents including designed compounds have been reported to suppress P-gp. However, most of these agents necessitated high doses which caused serious side-effects and the intrinsic cytotoxicity, for the designed compounds, due to dose-limiting toxicity, consequently, relevant clinical trial showed failed results. Although new generation of P-gp inhibitors have been developing, novel approaches in overcoming P-gp/MDR1mediated MDR by blocking its function and expression are still needed. In this case, natural supplement agents are gaining increasingly interest in cancer supplementary therapy.MDR1expression has been verified in a certain cancer cells, including human ovarian cancer cells A2780and its multidrug resistant subline A2780/T. Molecularly, the P-gp/MDR1expression is mediated by nuclear factor κ-light-chain-enhancer of activated B cells(NF-κB), cylooxygenases-2, CYP3A4, the mitogen-activated protein kinase(MAPK) pathway, and phosphoinositide3-kinase(PI3K). Among these, NF-κB and MAPK/ERK pathway are the most important factors in terms of their molecular mechanisms related to induction of MDR. The NF-κB pathway responds actively to MDR1induction due to its activation by the generation of reactive oxygen species, the activation of IκB kinase, and the degradation of IκB. Besides, NF-κB is bound at nucleotide position-159of the MDR1promoter mediating the transcription of MDR1. Similarly, the MAPK pathway, comprising extracellular signal-regulated kinase(ERK), c-Jun NH2-terminal kinase(JNK)/stress-activate protein kinase(SAPK), and p38MAPK subfamilies, also plays critical roles in the transmission of signals provided by various kinds of stimulus to regulate the expression of MDR1. Recently, a number of studies showed that the over-expression of P-gp appears to be closely associated with the nuclear localization of Y-box binding protein1(YB-1) in various solid tumors such as breast cancer, ovarian cancer, prostate cancer, and osteosarcoma. Meanwhile, it’s reported that MAPK/ERK pathway regulated the phosphorylation of YB-1by ERK phosphorylation. However, the interaction of natural flavonoids agents and MAPK/ERK mediated YB-1activity has not been reported yet.Grape seed procyanidin(GSP), a class of polyphenol flavonoids compounds, naturally contained in fruits, vegetables, nuts, seeds, flowers and barks, is reported to exhibit a wide range of biological effects including antibacterial, antiviral, anti-inflammatory, antiallergic and vasodilatory actions. GSP as a main ingredient of Pyconogenol(PYC, Horphag Research Ltd, UK) is used as a popular dietary supplement to overcome inflammatory and degenerative diseases and wound healing. Recently, GSP is most widely studied in terms of their bioactivities. It’s found that GSP could inhibit the function of P-gp on blood-brain barrier. But the role of GSP in reversing cancer cells MDR by inhibiting P-gp expression as well as the mechanism remains unreported. Therefore, we investigated whether GSP can enhance the cytotoxicity of paclitaxel along with adriamycin and down-regulate possibly the expression with the transcription of P-gp/MDR1in A2780/T cells. Furthermore, we demonstrated whether the MDR1expression inhibited by GSP has involved in the inhibitory effect of NF-ｋB activity and MAPK/ERK pathway mediated YB-1in A2780/T cells.Content&ResultsTo confirm the reported overexpression of MDR1in A2780/T cells, the MDR1mRNA and protein expression levels were compared between A2780/T and A2780cells by RT-PCR and Western blot analysis respectively, which showed that MDR1mRNA and protein levels were both overexpressed in A2780/T cells. A2780or A2780/T cells were exposed to paclitaxel(0～100μM) and adriamycin(0～80μM) for48h. The IC50value in A2780/T or A2780cells was significantly (P<0.001) difference of paclitaxel (100.36±1.68μM vs.0.23±0.02μM) and adriamycin (15.08±0.39μM vs.0.65±0.02μM), indicating that A2780/T cells was the paclitaxel resistant subline of A2780, and MDR1was overexpressed in A2780/T cells.The viability of A2780and A2780/T cells against GSP(3.125～200μM) treated for48h was determined using the MTT assay, which suggested that the concentration groups of GSP used in the study(0～40μM) had insignificant effect to the cells viability. Then the effect of GSP on paclitaxel and adriamycin cytotoxicity was examined in A2780and A2780/T cells. The cells were treated in GSP(0～40μM) combining with various concentrations of paclitaxel(0～20μM for A2780, and0～100μM for A2780/T) and adriamycin(0～8μM for A2780, and0～80μM for A2780/T) for48h. The viability of the cells was analyzed by the MTT assay. GSP significantly enhanced the cytotoxicity of paclitaxel and adriamycin against A2780/T cells, but had almost no effect on the cytotoxicity of paclitaxel and adriamycin against A2780cells. These results indicate that GSP selectively increased the cytotoxic effect of paclitaxel and adriamycin in A2780/T cells in a dose-dependent manner.Overexpression of MDR1mRNA and protein levels is associated with the MDR phenotype. RT-PCR and Western blot analysis were performed to detect the change in MDR1mRNA and protein levels upon treatment with GSP, respectively. Results showed, the MDR1mRNA level markedly was decreased in both dose-and time-dependent manner by GSP treatment. Similarly, GSP decreased the MDR1protein level in both dose-and time-dependent manner. Thus, GSP suppressed P-gp/MDR1expression in A2780/T cells.The P-gp-dependent efflux of fluorescent Rho-123was extensively used to determine the efflux from drug-resistant cell lines expressing P-gp. The accumulation of Rho-123in A2780/T cells was measured to determine whether the changes observed in MDR1expression were correlated with the changes in P-gp function. Intracellular Rho-123in A2780/T cells after treatment with GSP significantly accumulated in a dose-dependent manner, but the intracellular Rho-123in A2780cells insignificantly accumulated, reflecting the decreased P-gp efflux function in MDR1overexpression cells rather than sensitive cells.To elucidate the effects of GSP on MDR1promoter activity, A2780/T cells were transfected with MDR1promoter reporter plasmids containing NF-κB responsive region and were then treated with various concentrations of GSP and paclitaxel. As a result, GSP was observed not only to have dramatically inhibited MDR1promoter activity in a dose-dependent manner, but also to have dose-dependently suppressed paxlitaxel-activated MDR1promoter activity.To evaluate whether NF-κB is involved in the suppression of MDR1expression by GSP, the effect of GSP on NF-κB activity and LPS or RANKL induced NF-κB activation. Lysates of the GSP-treated cells were analysed by Western blotting with anti-phospho-ser32of IκBα, anti-phospho-ser473of AKT and anti-p65antibodies. GSP partially inhibited phospho-AKT and phospho-IκBα in a time-and dose-dependent manner. Consistent with this, the amount of the nuclear p65subunit of NF-κB was significantly decreased with its increasing in nucleus. Furthermore, nuclear translocation of p65induced by LPS and RANKL was evaluated by immunofluorescence. The LPS and RANKL treatment induced p65nuclear translocation in A2780/T cells. However, the pre-treatment with GSP before LPS or RANKL stimulation prevented the nulear translocation of p65, indicating that GSP suppressed LPS and RANKL induced NF-κB activation. To further examine the GSP inhibitory of P-gp mediated by NF-κB, A2780/T cells were exposed to40μM GSP0to6h or0～40μM GSP24h prior to incubation with LPS for30min. P-gp expression and activity were induced by LPS, and this effect was reduced in the presence of40μM GSP6h or different concentrations of GSP24h. Also, GSP could inhibit LPS-induced phosphorylation of AKT and IκB-α as well as p65nuclear translocation, further confirming GSP could inhibit the LPS-induced NF-κB activation in A2780/T cells. PDTC as a NF-κB activation inhibitor, showed the same tendency of GSP. These data indicate that GSP suppressed MDR1by inhibiting NF-κB activation.The A2780/T cells treated with GSP demonstrated the time-dependent inhibition of ERK1/2phosphorylation and YB-1nuclear translocation, as well as, dose-dependent effect, indicating that GSP may suppress MAPK/ERK mediated YB-1activation in A2780/T cells. Furthermore, to confirm the role of MAPK pathway in MDR1suppression by GSP, the MAPK/ERK pathway was activated by LPS in A2780/T cells. Incubated with either different concentrations of GSP for6h or40μM GSP0to6h, A2780/T cells were treated with1μg/ml LPS for30min, the LPS treated A2780/T cells were significantly induced the MAPK/ERK pathway. Treatment with40μM GSP0～6h and0～40μM GSP for6h markedly inhibited the LPS-induced ERK1/2phosphorylation and YB-1nuclear translocation, compared with phosphor-ERK1/2level and YB-1nucleus expression levelin GSP-free group, respectively. ERK1/2inhibitor U1026was used as a positive control, showed the same tendency of GSP. These results indicate that GSP suppression of MDR1by restraining the YB-1activation via MAPK/ERK pathway inhibition.Discussion&ConclusionP-glycoprotein, encoded by MDR1and located on7, q21.1chromosome, has attracted great interest because of its crucial role in MDR in a variety of cancers. P-glycoprotein overexpresses in many cancer cells including ovarian cancer cells is known to actively reduce the efficacy of chemotherapeutic agents and even result in treatment failure ultimately in ovarian cancer patients. Therefore, blocking MDR1is believed to enhance the efficacy of chemotherapy in these patients. Different kinds of P-gp inhibitors were developed from generation to generation. Although these agents worked successfully in vitro, most results of clinical trials were disappointing, because some of these synthetic compounds did not work in vivo or had unexpected side-effects. Due to the low cytotoxicity and food-bored, investigators turned their focus to natural plants sourced compounds.Recently, a series of studies have been reported that the extensive bioactivities of GSP which used as a natural supplement for regulating oxidative balance. In these studies, GSP is found to reverse the MDR effect in blood-brain barrier by blocking the function of P-gp but the MDR reversal effect of GSP in chemotherapeutic agent resistant cancer cells and even the mechanism of MDR reversal is not clear. Additionally, there’s no report of GSO on the influence of P-gp expression, and also the mechanism is unknown. Because of the structural similarities of GSP to the other dimmer flavonoids, which have been reported to reverse MDR by inhibiting both function and expression of P-gp, hence, it is reasonable to speculate that GSP may reverse MDR by blocking the expression of P-gp as well. Furthermore, researchers have found that GSP inhibited epithelial-mesenchymal transition by NF-κB and MAPK pathway. However, there’s no report on whether NF-κB and MAPK/ERK pathway is involved in the inhibition of P-gp expression by GSP. Besides, co-expression of YB-1and P-gp may be a promising relevant bio marker for unfavorable prognosis in ovarian cancer, the interventional effect of YB-1on GSP regulating P-gp expression in ovarian cancer cells is also unknown yet.Therefore, this study provides the first trial on GSP to explore its role in reversing MDR in paclitaxel resistant ovarian cancer cells by attenuating the expression and function of P-gp/MDR1. Our results showed that GSP markedly increased the cytotoxicity of paclitaxel in paclitaxel resistant P-gp overexpression A2780/T cells but faintly in paclitaxel sensitive A2780cells, which indicates that GSP may reverses MDR by mediating overexpressed P-gp. Similarly, GSP also markedly increased the cytotoxicity of adriamycin in A2780/T but A2780, illustrating that GSP enhanced the cytotoxicity of paclitaxel is a non-specifical drug interaction but MDR reversal. Meanwhile, GSP significantly increases intracellular Rho-123accumulation in A2780/T cells indicating that GSP inhibits the function of P-gp. Furthermore, we found that GSP down-regulated the expression of P-gp in both mRNA and protein level in a dose-and time-dependent manner, as we speculated. Additionally, GSP inhibited the MDR1promoter activity and paclitaxel-induced MDR1promoter activation. These results fully evidence that GSP has capable to suppress the activity of MDR1transcription. The reduction of MDR1expression probably at both translational and transcriptional levels has been proposed as one mechanism by which some agents reverse the MDR phenotype. Thus, GSP reverses MDR phenotype by partly inhibiting both translation and transcription of MDR1, as a result of decreasing the function and expression of P-gp.Inactive NF-κB dimmers, composed of p65and p50subunits, are sequestered in the cytoplasma in association with the inhibitory molecules of the IκB family. Stimulation of cells with LPS and RANKL causes phosphorylation of the inhibitor IκBα, leading to its polyubiquitination and proteasome-mediated degradation and the release of active NF-κB. Furthermore, AKT phosphorylation mediates the phosphorylation of IκBα to activate NF-κB pathway. NF-κB induces the transcription of MDR1by translocating into nucleus binding the respondent region of MDR1promoter and then influences the expression of P-gp. The current study was designed to assess the possible role of NF-κB activation in GSP reducing the expression of P-gp. First, GSP was shown to inhibit NF-κB pathway by restraining the AKT and IκBα phosphorylation so as to control decomposition of IκBα, the inhibitor of NF-κB. Then, GSP was found to increase the expression of p65(a NF-κB subunit) in cytosol while decrease nuclear p65expression to block the nuclear NF-κB mediated MDR1transcription. Moreover, GSP obstructed LPS and RANKL induced NF-κB nuclear translocation, manifesting its subunits reversely translocated from nucleus to cytoplasma. These findings indicate that the inhibition of NF-κB activation may be involved in GSP suppressing the expression of P-gp.On the other hand, YB-1is an oncogenic transcription/translation factor that is over-expressed in a number of cancer types. Previous studies have shown that YB-1activated by Akt and the activation of MAPK/ERK appeared YB-1of normally localizing in the cytoplasm translocated into nucleus, leading to activation of the MDR1expression. Additionally, many reports supported that MDR1gene could be activated by external signals via MAPK/ERK. In this study, GSP reduced the phosphorylation of ERK1/2and inhibited the YB-1nuclear translocation while GSP suppressed P-gp expression. Furthermore, other studies showed that the specific inhibitors of ERK significantly reversed P-gp-mediated MDR. Thus, GSP is prospected to down-regulate the expression of P-gp probably associated with the inhibition of MAPK/ERK pathway. To test this prospection, LPS was also used in our study to induce P-gp, MAPK/ERK and YB-1activation so as to identify whether GSP can attenuate the induced phosphorylation of ERK as an ERK inhibitor U1026. Our findings showed that GSP attenuated LPS-induced ERK1/2phosphorylation and YB-1nuclear translocation respectively, which further testified our hypothesis, suggestting that GSP suppressed P-gp expression was regulated by YB-1, in part, through regulating the action of MAPK/ERK.Taking together, our current studies show that GSP significantly increases the efficacy of paclitaxel and adriamycin in A2780/T cells by reversing the phenotype of MDR via blocking the function of P-gp and inhibiting the expression and transcription of MDR1. Furthermore, GSP can suppress the NF-κB activity and MAPK/ERK pathway mediated YB-1nuclear translocation, which may have a correlation with the expression-down-regulated P-gp. Although we have preliminarily verified the possible mechanism of GSP inhibiting P-gp, the specific target of GSP interacting P-gp via NF-κB and MAPK/ERK pathway is unknown. Moreover, our results are only limited to human ovarian cancer cells of A2780/T and A2780in vitro. It’s worth our further investigating whether GSP could reverse MDR in other ovarian cancer cells or another cancer cell model. Besides, the in vivo assay should also be performed in the future.In conclusion, our data demonstrates that GSP reverses MDR in A2780/T cells by inhibiting both function and expression of P-gp and inhibits P-gp expression in A2780/T cells possibly via the suppression of NF-κB activity and MAPK/ERK pathway medicated YB-1activation. This study first verifies the effect and mechanism of a natural supplement GSP reversing MDR by interacting P-gp, suggesting that GSP may be used in combination with conventional P-gp substrate chemotherapeutic drugs to overcome MDR in ovarian cancer patients, providing a new use of such natural supplements as a chemo-sensitizer. |
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