Anti-tumour Effect And Molecular Mechanisms Of Icaritin On Multiple Myeloma Cells

Multiple myeloma (MM) is a hematologic neoplastic plasma cell disorder that is characterized by clonal proliferation, blocked apoptosis and accumulation of malignant plasma cells in bone marrow, resulted in the destroy of bone, secretion of monoclonal proteins into the blood or urine, and associated organ dysfunction. In spite of the extended overall survival by the application of novel chemotherapy regimens, the availability of novel agents such as bortezomib and the introduction of autologous stem-cell transplantation, multiple myeloma it is still an incurable malignancy in present.Icaritin is a hydrolytic product of icariin, which is extracted from the Chinese herbal Epimedium brevicornum. As one flavanoid, icaritin can activate estrogen receptor; reduce bone destruction caused by various factors, promote differentiation of cardiac muscle cell and neurocyte, enhance cell-mediated immunity, inhibit cell growth and induce apoptosis in several cancers including prostate cancer, breast cancer, hepatocellular carcinoma and endometrial cancer cells.In this study, to provide experimental basis for further clinical research of using icaritin as a novel agent for multiple myeloma treatment, we mainly examined the anti-malignancy effect of icaritin and investigated the potential molecular mechanism of it in multiple myeloma in vitro.Firstly, we obtained freshly heparinized bone marrow samples from patients with multiple myeloma and with non-bone marrow disease. The mononuclear cells were isolated by Ficoll-Hypaque separation. The samples from patients with non-bone marrow disease were used as normal control in following experiments. The purified CD-138positive cells were acquired from the bone marrow mononuclear cells (BMMNCs) of multiple myeloma patients using the CD-138positive selection immunomagnetic beads. Flow cytometric detection proved that the purity of all selected samples were over80%. MTT assay showed that icaritin significantly inhibited cell proliferation of BMMNCs and purified CD138positive cells from MM patients in a dose-dependent manner. Compared to the BMMNCs from MM patients and control patients, inhibition of purified CD138positive cells caused by icaritin is the most marked. This phenomenon indicated the specific effect of icaritin on the clonal malignant plasma cells. Moreover, FCM detection displayed that icaritin induced apoptosis remarkably in purified CD138positive cells in dose dependent manner. These findings demonstrated the specific cytotoxicity of icaritin to malignant plasma cells.We treated the MM cell lines, KM3cell line and dexamethasone-resistant U266cell line, with different concentrations of icaritin for various times. MTT assay revealed that icaritin inhibited proliferation of both cell lines markedly in dose and time dependent manners. FCW assay showed increased apoptosis in both KM3and U266cells lines with increased concentrations of icaritin. The Wright-Giemsa staining displayed that icaritin caused the apoptotic morphological changes in U266cell line, including the chromatin condensation, increase of cytoplasmic vacuoles, fuzzy and ruptures of cell membrane. Poly (ADP-ribose) polymerase (PARP) is an important protein involved in DNA repair, which is inactivated by Caspase cleavage and considered as a molecular mark of programmed cell death. Western blot detection showed the accumulation of cleaved PARP with icaritin increasing in U266cells. The aforementioned findings verified the anti-MM effect of icaritin in various aspects.To investigate the potential molecular mechanism by which icaritin impair MM cells, we observed the influence of icaritin on cell cycle distribution and apoptotic signal pathway in U266cell line. Here, we found that icaritin caused S-phase arrest in U266cells accompanied with downregulation of cyclinD1. To apoptotic signal proteins, icaritin can up-regulated the pro-apoptotic Bax and Bak proteins of Bcl-2family and down-regulated the anti-apoptotic Bcl-xl protein of Bcl-2family, induced activation of Caspase9and Caspase3, and evoked release of cytochrome C from mitochondrion to cytoplasm.Dietary flavonoids show proteasome-inhibitory activity. Here, we demonstrated that icaritin is also has proteasome-inhibitory activity. It indicated that proteasome-inhibitory activity of icaritin may be involved in its anti-MM effect.IL-6is an important cytokine for the resistance of MM cells to dexamethasone. The autocrine-loop of IL-6in U266cells is related to its resistance to dexamethasone induced apoptosis. We found that icaritin reduced the level of IL-6in culture supernatant of U266persistently. On the contrary, treatment with dexamethasone accompanied with ascension of IL-6in culture supernatant. In the presence of dexamethasone, icaritin can still reduce the IL-6in culture supernatant, suggesting the potential reversal effect on resistance to dexamethasone in U266cell line.MAPK cell signal pathway is related to survival and proliferation of MM cells. Erk is an important downstream protein of IL-6and is associated with proliferation of MM cells. Previous studies proved that anti-tumor capability of icaritin is related to sustained phosphorylation of Erk or JNK pathway. Activation of c-jun can induce apoptosis in MM cells, too. Here, we demonstrated that icaritin caused sustained activation of Erk and JNK in U266cells, upregulated and activated c-jun, and indicated that icaritin induced apoptosis might be related to Erk/JNK signal pathway.Finally, we examined the cooperativity of icaritin and dexamethasone or bortezomib; found that bortezomib and icaritin showed the strongest inhibition of cell viability in U266cells. These findings indicated that the combination of icaritin and bortezomib might be a potential regimen for MM therapy.