| Background:Multiple myeloma (MM), a B-cell malignancy characterized by proliferation of monoclonal plasma cell in bone marrow, leading to osteolytic bone destruction and impaired hematopoiesis, is an incurable cancer. Despite newer effective agents such as bortezomib, lenalidomide, and thalidomide are changing the therapy of multiple myeloma patients, there is still no cure at present, and multiple myeloma patients ultimately progresses and the treatment of patients with relapsed and refractory remains challenging. Therefore, new treatment approaches and novel drugs are needed to improve patient outcome. Honokiol (HNK) is a natural product derived from Magnolia, which is used as medicinal herb in traditional Chinese medicine, and it has a good effect on the treatment of fever, neurological disorders and gastrointestinal disorders. In recent years, its effects of anti-tumor were concerned by domestic and international. And it is demonstrated to affect tumor cell growth and induce apoptosis in different cancer cell lines. Furthermore it has synergistic anti-tumor activity with the traditional chemotherapy drugs.Objective:To investigate the anti-proliferation and apoptosis effect induced by Honokiol and Bortezomib on KM3 human multiple myeloma cell line in vitro, and explore the precise mechanisms of the effects by measuring the expression of apoptosis related proteins and genes.Methods:Samples were divided into control group and experimental group, and the experimental group was further divided into HNK treating group (at concentration of 4,6,8,10,12 and 15μg/mL was B, C, D, E, F and G groups, respectively), bortezomib treating group (at concentration of 4,6,8,10,12,15μg/mL, was H, I, J groups, respectively), and combined treating groups (HNK 4μg/mL+bortezomib 5 ng/mL, HNK4μg/mL+bortezomib 10 ng/mL, HNK4μg/mL+bortezomib 20 ng/mL, was K,L, M group, respectively). MTT colorimetric assay were performed to assess the cell proliferation. The cell apoptosis and the cell cycle were determined by flow cytometry. The caspase-3 and caspase-9 colorimetric assay kits were used to measure the activity of caspase. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of apoptosis-related genes in cells. And apoptosis-related proteins were detected with Western blot.Results:Honokiol at concentrations between 4-15ug/mL showed significant inhibiting effect on KM3 cell proliferation. At 24h,48h, and 72h, the inhibition rate rising from 20.38%,27.45% and 44.94% to 80.54%,89.99% and 90.91%, respectively. There were significant differences among different groups, or at different time. The inhibition rates of honokiol on KM3 cells at 48h in H, I, J, K, L, M were 3.13%,36.22%,53.99%,52.68%,69.99%,78.53%, respectively. There were considerable differences between K and H, L and I, or M and J (p<0.05). Apoptosis rate of KM3 cells in B, C, E at 24h and 48h were 6.92%,16.15%,60.70% and 18.84%,37.21%,86.07%, respectively. There were significant differences in apoptosis among different groups at the same time (p<0.05). Apoptosis rate of KM3 cells in H and I group at 24h were 9.27% and 17.87%, at 48h the rate were 11.92% and 53.51%. Statistical differences were found in apoptosis between H and I group (p<0.05). Apoptosis rates of KM3 cells in K and L group at 24h were 15.75% and 22.18%, at 48h the rate were 35.96% and 74.70%. The apoptosis rates in K and L group were significantly higher than H and I group (p<0.05). It was observed that 27.25% of cells were in the G0/G1 -phase and more than 70% honokiol of cells were in the S-phase in the control group, after treated with HNK, G0/G1 -phase of KM3 cells were significantly augmented with the increase of HNK concentration(p<0. 05). And caspase-3 and caspase-9 activity were increased with the increase of HNK concentration (p<0.05). There were no statistical differences between control group and experimental groups in the expression of apoptosis-related genes. Furthermore, the cleavage of caspase-3 and caspase-9 were observed after treatmented with honokiol. And the protein level of Mcl-1 was decreased when cells were exposed to honokiol for 48 hours compared to control group, the protein level of Bax was not changed when treated with honokiol for 48 hours in this study.Conclusion:Honokiol and bortezomib could inhibit the proliferation of KM3 cells and induce cell apoptosis respectively, thus the combination of honokiol and bortezomib could increase the apoptosis rates and enhance cytotoxic activity of KM3 cells. The mechanism of Honokiol inducing apoptosis may be achieved through the mitochondrial pathway. |
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