| Objective： Multiple myeloma(MM) is a late-stage clonal B-cellmalignancy characterized by the infiltration of aberrant plasma cells in bonemarrow and is associated with high monoclonal protein in the blood. Itaccounts for about10%of the hematological malignancy. The standardtreatments for MM are high-dose chemotherapy and stem cell transplantation.Although many new drugs,including bortezomib,thalidomide, andlenalidomide (Revlimid) have been recently approved, multiple myelomaremains an incurable disease. The development of drug resistance andintolerable toxicity are major problems. Therefore, there is a clear need fornovel agents of high performance and low toxicity, or the identification ofnovel compounds which can enhance the effects of current chemotherapeuticagents.Norcantharidin(NCTD), the demethylated analog of cantharidin isolatedfrom the traditional Chinese medicine cantharidin, has significant anti-canceractivity and less nephrotoxicity, especially the digestive system. Our previousstudies have confirmed that NCTD can inhibit the growth of MM cellsthrough apoptosis. Bortezomib(BTZ) is the first proteasome inhibitorapproved to treat relapsed multiple myeloma(MM). To reduce its side effectsand enhance antitumor activity,our present study investigate the combinationof BTZ with NCTD in myeloma cells in vitro and in nude mice. The purposeis to provide a new clinical method in patients with MM.Methods:1MTT assay was used to detect the anti-preliferation effect of BTZ andNCTD,either alone or their combination in U266cells.U266cells were treated with different concentrations of NCTD (0,1.25,2.5,5,10,20μmol/L), BTZ(0,1.25,2.5,5,10,20nmol/L)or their combinationat fixed molar concentration ratio of1000:1for72h. Cell viability wasdetected by MTT assay. Isobologram analyses was used to determine thelevel of interaction (synergistic,additive, or antagonistic)between thesimultaneous combination of NCTD and BTZ.2Groups: The following exprements were divided into four groups: controlgroup, NCTD group(5μmol/L), BTZ group(5nmol/L) and combinationgroup(5μmol/L NCTD+5nmol/L BTZ).3Flow cytometry were used to determine cell apoptosis and cell cycle：U266cells were treated with NCTD5μmol/L and BTZ5nmol/L alone or incombination for24h. Annexin-V and PI were used in combination todetermine apoptosis. The change of cell cycle before and after treatmentwere analysis by flow cytometry also.4RT–q PCR was used to examine the expression of NF-κB P65,Survivin,Bcl-2and Bax mRNA in control, NCTD group, BTZ group or theircombination for24h.5The protein expression of nuclear factor-κappa B P65(NF-κB P65),phosphorylated NF-κB p65(p-NF-κB p65), NF-κB inhibitor IkBα,phosphorylated IκBα (p-IκBα),Survivin,Bcl-2and Bax were determinedby Western blot.6Nude mouse xenograft assay: Animal model simulating human multiplemyeloma was established by subcutaneous implantation U266cells intonude mice. The mice were then randomized into4groups: The controlgroup was treated intraperitoneally (IP) with normal saline3×/week for4weeks. The NCTD group was administered20mg/kg,3×/week for4weeks.The BTZ group received BTZ (0.25mg/kg) once per week for4weeks. Thecombination group was treated with a combination of NCTD (20mg/kg,3×/week) and bortezomib (0.25mg/kg, once a week). Tumor growth andanimal body weights were monitored every three days. The tumor growthinhibition rate was calculated by using the formula IR (%)=(1-TWt/TWc) ×100, where the TWt and TWc were the mean tumor weight of treatmentand control groups.Results：1Both NCTD and BTZ showed dose-dependent inhibition to the growth ofU266cells. Simultaneous exposure of U266cells to differentconcentrations of NCTD and BTZ for72h showed a strong synergisticeffect (CI <1). We found that the inhibition rates of2.5μmol/L NCTD,2.5nmol/L BTZ and2.5μmol/L NCTD plus2.5nmol/L BTZ were(10.50±2.40)%,(17.50±1.40)%and (68.10±2.00)%, respectively.2Flow cytometry showed that the apoptosis rates in the control,NCTD(5μmol//L),BTZ (5nmol/L) and combination group were (5.28±1.19)%,(13.37±2.17)%,(19.42±3.35)%and (53.26±4.86)%, respectively. Cellcycle analysis showed that both NCTD and BTZ increased the percentageof cells in G2/M phase and the combination of the two drugs reinforced theindividual effects.3Combination of BTZ and NCTD decreased the mRNA expression ofNF-κB P65,Survivin and Bcl-2, while increased the expression of Bax.The mRNA expression of NF-κB P65,Survivin and Bcl-2decreasedfrom(1.03±0.10),(1.06±0.04) and (0.88±0.17) to (0.29±0.04),(0.35±0.01) and (0.28±0.06), while the expression of Bax increased from(2.53±0.19) to (5.66±0.44), respectively in BTZ group and combinationgroup.4Combination of BTZ and NCTD further decreased the protein expressionof nuclear NF-κB P65and cytoplasm p-NF-κB P65induced by BTZ. Theexpression of nuclear NF-κB P65and cytoplasm p-NF-κB P65decreasedfrom (1.08±0.11) and (0.20±0.08) to (0.61±0.08) and (0.01±0.005),respectively, while the expression of the cytoplasm NF-κB P65wasunchanged in the BTZ group and the combined group. NCTD and BTZ stillcan inhibit phosphorylation IkBα, and promote the cytoplasm IκBαexpression.5BTZ inhibit the protein expression of Survivin and Bcl-2while improving the expression of Bax, NCTD potentiated its effects. The expression ofSurvivin and Bcl-2decreased from (0.91±0.18) and (0.56±0.10) to(0.31±0.07) and (0.20±0.05), while the expression of Bax increased from(0.78±0.10) to (2.43±0.34), respectively in BTZ group and combinationgroup.6Both NCTD and BTZ could inhibit the growth of nude mice xenograft.Tumor volume in combination group(476.30±340.92)mm3was smallerthan ones in BTZ group(1006.67±240.58)mm3. The tumor inhibition ratein combination group was84%, while67%in BTZ group, showing thatNCTD potentiate bortezomib-antitumor effects in nude mice xenografts.Conclusions：1NCTD showed a growth inhibition effect on U266cells with adose-dependent manner and potentiated the anti-myeloma effects induced byBTZ.The inhibition effect could be related to the apoptosis effect.2Both NCTD and BTZ could inhibit the growth of xenografts in nude mice.The inhibitory effect of BTZ could be potentiated by the combination ofNCTD.3NCTD could potentialize the chemosensitivity of multiple myeloma cellsto BTZ through regulating NF-κB/IκBα signaling pathway andNF-κB-regulated gene products including Survivin, Bcl-2,and Bax. |
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