| Background:Multiple myeloma (MM) is a malignant disease characterized by aberrant plasma cell infiltration in the bone marrow.It accounts for approximately10%of all hematologic malignancies. The disease has been able to achieve good quality responses with the introduction of proteasome inhibitor, strengthened support therapy and hematopoietic stem cell transplantation. However, most patients eventually relapse or become refractory to treatment, and MM remains incurable due to drug resistance. Honokiol (HNK) is an active compound isolated from the Magnolia officinalis. It has been shown antiproliferative effects in numerous hematological (e.g multiple myeloma and acute leukemia) and solid tumor malignancies (e.g melanoma and breast cancer). Recent studies show honokiol combined with chemotherapy drugs also can exert significant anti-neoplastic activity in drug-resistant tumor cells. Hopefully, the coming few years will see potential use of honokiol for reversing drug-resistance.Objective:In this study, we will establish the KM3/BTZ cell line resistant to bortezomib according to the KM3cell line. We can investigate the primary drug-resistant mechanism of the KM3/BTZ cell line. Meanwhile, we will explore the reversing effect of Honokiol on the KM3/BTZ cell line.Methods:1. The bortezomib-resistant KM3/BTZ cell line was established from the parental KM3cell line in vitro, by stepwise increasing concentrations of bortezomib.2. We observed the morphological changes of KM3and KM3/BTZ cells through an inverted microscope, drew the cell growth curve and calculated the population doubling time of KM3and KM3/BTZ cells.3. KM3and KM3/BTZ cells were treated with bortezomib at different concentration and detected the proliferation inhibitory rate by MTT assays. We investigated the stability of the resistant character and calculated the resistance index of KM3/BTZ cells.4. The expression of MDR-1mRNA was determined by RT-PCR in parental and drug-resistant cell lines.5. The cytotoxic effect of HNK at different concentrations was detected by MTT assays on KM3and KM3/BTZ cells. We identified the less-cytoxic dose of honokiol (cell’s proliferation inhibitory rate<20%).6. KM3/BTZ cells were treated with honokiol of less-cytoxic dose and bortezomib at different concentrations and investigated the proliferation inhibitory effect by MTT assays. Then we calculated the reversal fold.7. Apoptosis was observed after KM3/BTZ cells were treated with bortezomib with or without honokiol of less-cytoxic dose by flow cytometry.Results:1. We established the KM3/BTZ cell line resistant to bortezomib successfully.2.The doubling time of KM3and KM3/BTZ cell lines was (27.33±1.61) h and (38.26±1.22) h respectively. As compared with the parental cells, the KM3/BTZ cells exhibited a significant longer doubling time and grew more slowly (P<0.05)3. MTT assays showed that the IC50of bortezomib to KM3and KM3/BTZ cells were (17.82±1.03) ng/mLand (351.26±3.51) ng/mL respectively. The KM3/BTZ exhibited a19.7-fold level of resistance to bortezomib as compared to the KM3(P<0.05)4. RT-PCR showed that the expression of MDR-1mRNA was not observed in either parental or resistant cells.5. MTT assays showed that the IC50of honokiol to KM3and KM3/BTZ cells were (5.97±1.56) ng/mL and (7.82±1.05) ng/mL respectively. The honokiol with the dose of4μg/mL could cause an inhibitory rate less than20%, and it was chosen for the reversal experiment.6. MTT assays showed honokiol (4μg/mL) could exhibit a2.08-fold reversal to KM3/BTZ cells. The IC50of bortezomib were (351.26±3.51) ng/mL and (168.53±3.77) ng/ml in single and combined groups (P<0.05)7. Flow cytometry assays showed that the apotosis rates in combined groups were significantly higher than the single-bortezomib groups (P<0.05)Conclusion:We have successfully established bortezomib-resistant cell line KM3/BTZ and its drug-resistant character is stable. The KM3/BTZ cell line might serve as an ideal model to investigate the mechanisms of drug-resistant and explore reversing effect. The expression of MDR-1mRNA is not observed in either resistant or parent cells. Honokiol could restore the sensitivity of bortezomib in KM3/BTZ cell line, but it still needs further study to elaborate the reversal mechanism. |
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