The Effect And Mechanism Study Of Asclepiasterol, A Novel C₂₁ Steroidal Glycoside Derived From Asclepias Curassavica Reversing P-gp-mediated Multidrug Resistance

Background and ObjectiveMultidrug resistance（MDR） mediated by P-glycoprotein（P-gp） is confirmed to be one of the major cause of failure for cancer therapy. The ATP-binding cassette（ABC） transporters, represent by P-gp, pump a wide range of structurally and functionally diverse amphipathic anticancer drugs out of tumor cells and finally result in failure of chemotherapy due to the low intracellular drug concentration. Historically, researches on ABC-transporter-mediated multidrug resistance has been focused on the P-glycoprotein/P-gp. P-gp is the first and classical ABC transporter that is overexpressing in many human solid and hematologic cancers, and is confirmed to be a marker of chemo-resistance or decreased survival in leukemias, lymphomas, osteosarcomas, small-cell lung cancers and breast cancers, etc. Therefore developing ABCB1/Pgp inhibitors appears to be very important for chemotherapy. Many P-gp inhibitors have been discovered during these years, and were found to show significant effect antagonizing P-gp function both in vitro and in vivo. However, phase III trials of these agents have been disappointing and no significant survival benefit of these P-gp inhibitors has yet been achieved. Thus, searching for new MDR modulators with higher efficacy and low toxicity is warrant.Throughout history, human kind has used natural products from plants, animal and microorganisms to treat diseases. Even now, 80% of the world’s populations still use herbal medicines. During recent years, the industrial reservoirs of chemical lead structures have begun to dwindle and increasing attention has been paid to natural products. Keeping in mind that most of the established resistance-modifying agents（RMA） are synthetic compounds and are too toxic at the required dose, the search for P-glycoprotein inhibitors from natural products may be an alternative approach. Many natural products have been confirmed to show P-glycoprotein inhibitory activities, indicating that searching for new natural products capable of potent modulating P-glycoprotein might be promising. The natural products reported with Pglycoprotein inhibitory activities belong to baicalin and its derivatives, tryptamine ketone, alkaloids, terpene and so on. In 2015, many research groups reported that chemical synthesis of cardiac glycosides and cardiotonic steroids are capable of reversing P-gp-mediated MDR in vitro. In our previous study, we carried out a systematic study on the chemical constituents of Asclepias curassavia L, a milkweed plant as a source of food for butterflies, and performed an extensive screening for P-gp-mediated MDR reversal activities of these compounds. We identified a novel steroidal compound asclepiasterol capable of reversing P-gp-mediated MDR. Based on the reports and our previous study, the main purpose of this study is to investigate the effect of asclepiasterol on reversing P-gp-mediated MDR in P-gp-overexpressing cancer cells and elucidated the underlying mechanisms.Methods:This study try to investigate the effect of asclepiasterol on reversing P-gp-mediated MDR in P-gp-overexpressing cancer cells and elucidated the underlying mechanisms by the following three aspects: asclepiasterol and the function of P-gp, asclepiasterol and the protein expression level of P-gp, asclepiasterol and the prsttranslational regulation of P-gp in the MDR cells.1. MTT assay was used to test the cytotoxic effect of asclepiasterol on the MDR cells（MCF-7/ADR, Hep G-2/ADM, K562/ADR and K562/ADM） and their corresponding parental cell lines（MCF-7, Hep G-2 and K562）, and the non-tumor cell lines HEK293, Chang, and LO₂.2. Investigation of asclepiasterol’s modulating effect on MDR cells was conducted in both resistance and the corresponding parental cell lines by MTT assay and CCK-8 assay. The IC50 values of the chemotherapeutic agents including Dox, EPI, DNR, paclitaxel and cisplatin for sensitive and resistant cells in the presence or absence of asclepiasterol were calculated.3. Annexin V/PI assay was used to detect the apoptosis induced by paclitaxel in the MDR cells.4. Since the MDR cells with higher expression levels of P-gp were reported to be more malignant and resistant to traditional chemotherapeutic agents, we investigated the colony formation capacity of the MDR cells treated with 3.0 μM Dox and its combination with asclepiasterol by soft agar assay.5. Western Blotting and Real-Time PCR assay were used to detect the relative protein and gene expression level in the MDR after asclepiasterol treatmet.6. P-gp ATPase systems kit was used to test the effect of asclepiasterol on P-gp function.7. The effect of asclepiasterol on Dox and Rh123 accumulation in P-gp overexpressing MCF-7/ADR, Hep G-2/ADM and their corresponding parental cells MCF-7 and Hep G-2 weredetected by flow cytometry.8. Si RNA assay and relative protein inhibitors were used to analyze the relationship between Pgp and the other protein markers.9. Co-Immunoprecipitation assay was used to analyze the interaction of P-gp and the other protein marker.Results:1. Asclepiasterol was low cytotoxic on both MDR tumor and non-tumor cells. Treatment with increasing concentrations of asclepiasterol between 0 and 10.0 μM for 48 h didn’t inhibit the proliferation of the MDR cells（MCF-7/ADR、Hep G-2/ADM、K562/ADR and K562/ADM） and their corresponding parental cell lines（MCF-7, Hep G-2 and K562）, and the non-tumor cell lines HEK293, Chang, and LO₂. Specifically, more than 95% of cells were viable under 5.0 μM of asclepiasterol.2. Asclepiasterol at 2.5 and 5.0 μM significantly enhanced the cytotoxicity of the chemotherapeutic agents（Dox, EPI, DNR and paclitaxel） to the MDR cells.3. Asclepiasterol enhanced the anticancer efficacy of chemotherapeutic agents in MDR cells. the MDR cells were found to be more responsive to paclitaxel in the presence of asclepiasterol, and the anchorage independent soft agar colony formation capacity of the MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin.4. Asclepiasterol treatment remarkably increased the intracellular accumulation of doxorubicin and rhodamine 123（Rh123） in MDR cells.5. Asclepiasterol decreased the expression of P-gp protein level without stimulating or suppressing the MDR1 m RNA level.6. Asclepiasterol affected the accumulation of Rh123 in MDR cells during the short time（4h）, and the P-gp ATPase assay indicated that asclepiasterol inhibited hydrolysis of ATP by influenting the P-gp ATPase acitivity.7. Asclepiasterol inhibited the serine/threonine protein kinase Pim-1 expression levels in MDR cells, which was also comfired by the si RNA and protein inhibitor assay that P-gp expression level could be affected by inhibiting the Pim-1 expression level.8. Asclepiasterol could inhibit the effect of Pim-1 protecting the P-gp stability in cell surface.9. MAPK/ERK signal pathway was activated in the P-gp overexpressing cells MCF-7/ADR and Hep G-2/ADM as compared with the corresponding parental cells（MCF-7 and Hep G-2）. Asclepiasterol treatment led to a significant reduction of phosphorylation ERK1/2 especially at 5.0 μM without stimulating or suppressing the total ERK1/2 level in the MDR cells. The MAPK/ERK pathway inhibitor U0126-Et OH was found to significantly suppress the phosphorylation of ERK1/2 and the MDR cells treated with U0126-Et OH showed lower expression levels of P-gp. EGF, an activator of the MAPK/ERK pathway activated MAPK/ERK pathway and reversed asclepiasterol-mediated P-gp down-regulation in MDR cells.10. Ubiqutin-proteasome pathway was involoved in the asclepiasterol-mediated P-gp downregulation, and the ubiquitin-proteasome pathway inhibitor MG-132 could reverse the asclepiasterol-mediated P-gp down-regulation in MDR cells.Conclusions:1. Asclepiasterol, a novel natural C₂₁ steroidal glycoside derived from Asclepias curassavica resversed the P-gp-mediated multidrug resistance, which was the first C₂₁ steroid glycoside that was found to reverse MDR in our our best acknowledgement.2. Asclepiasterol reversed P-gp-mediated MDR by both inhibiting the P-gp function and expression levels in MDR cells.3. The serine/threonine protein kinase Pim-1, MAPK/ERK signal pathway and ubiqutinproteasome pathway were involoved in the asclepiasterol-mediated P-gp down-regulation.