氯化两面针碱促进骨髓瘤细胞凋亡的机制研究

Mechanism of nitidine chloride on promoting apoptosis of myeloma cells

摘要:

目的 探究氯化两面针碱促进骨髓瘤细胞凋亡的机制。方法 将人多发性骨髓瘤（MM）细胞株RPMI 8226培养至对数生长期，采用CCK-8法检测细胞活性并测定半数抑制浓度（IC₅₀）。将MM细胞株RPMI 8226随机分为空白组、硼替佐米（BTZ）组、氯化两面针碱（NCe）组和BTZ+NCe组。BTZ组加入500 μL 10 nmol/L BTZ溶液，参照IC₅₀在NCe组加入500 μL 4.5 μmol/L NCe，BTZ+NCe组加入250 μL 10 nmol/L BTZ和250 μL 4.5 μmol/L NCe，继续培养24 h。采用流式细胞术检测各组细胞凋亡情况，实时荧光定量聚合酶链反应、Western blotting检测各组细胞STK4、YAP1、Cl-caspase-3 mRNA和蛋白的表达。结果 与空白孔相比，其余各孔的细胞存活率均降低（P <0.05）；与0 μmol/L NCe孔比较，1 μmol/L、2 μmol/L、4 μmol/L、8 μmol/L NCe孔的细胞存活率均降低（P <0.05）。孵育24 h的IC₅₀为（4.56±0.64）μmol/L。与空白组比较，BTZ组、NCe组及BTZ+NCe组细胞凋亡率升高（P <0.05）。与空白组比较，BTZ组、NCe组及BTZ+NCe组STK4 mRNA和蛋白相对表达量降低（P <0.05），YAP1和Cl-caspase-3 mRNA和蛋白相对表达量升高（P <0.05）。结论 氯化两面针碱可以抑制骨髓瘤细胞增殖，促进其凋亡，该作用可能通过抑制STK4/YAP1通路来实现。

Abstract:

Objective To explore the mechanism of nitidine chloride on promoting apoptosis of myeloma cells.Methods Multiple myeloma (MM) cell lines RPMI 8226 were cultured to the logarithmic phase, and the cell viability and the half-maximal inhibitory concentration (IC₅₀) were detected via the CCK8 assay. RPMI 8226 cells were randomly divided into blank group, bortezomib (BTZ) group, nitidine chloride (NCe) group and BTZ + NCe group. The cells in the BTZ group, Nce group and the BTZ + NCe group were then cultured for another 24 hours in the presence of 500 μL 10 nmol/L of BTZ, 500 μL 4.5 μmol/L of NCe according to the IC₅₀, and 250 μL 10 nmol/L of BTZ plus 250 μL 4.5 μmol/L of NCe, respectively. Annexin V-FITC/PI double labeling was performed to measure the cell apoptosis, while quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were applied to detect the relative mRNA and protein expressions of STK4, YAP1, and Cl-caspase-3 in each group, respectively.Results Compared with the blank wells, the cell survival rates of other wells decreased (P < 0.05). Compared with the wells without NCe, the cell survival rates of wells with 1 μmol/L, 2 μmol/L, 4 μmol/L and 8 μmol/L of NCe also decreased (P < 0.05), and the IC₅₀ at 24 h of culture was (4.56 ± 0.64) μmol/L. Compared with the blank group, the apoptosis rates were higher, the relative mRNA and protein expressions of STK4 were lower (P < 0.05), and the relative mRNA and protein expressions of YAP1 and Cl-caspase-3 were higher in the BTZ group, NCe group and BTZ + NCe group (P < 0.05).Conclusions Nitidine chloride can inhibit the proliferation of myeloma cells and promote their apoptosis, which may be achieved by regulating the STK4/YAP1 pathway.