【摘要】 目的 探讨石蒜碱对伊马替尼耐药的慢性粒细胞白血病（chronic myeloid leukemia, CML）细胞促凋亡的作用机制，通过旧药新用策略为临床克服耐药提供新的治疗思路。方法 采用细胞增殖和毒性实验（cell counting kit-8, CCK-8）检测不同浓度梯度伊马替尼作用下K562和K562/G01细胞的增殖活性。采用石蒜碱与CML细胞共培养，运用CCK-8法检测CML细胞的增殖活性，以光学显微镜观察CML细胞的形态学变化，采用流式细胞术检测CML细胞的凋亡率，利用液相色谱-串联质谱检测CML细胞中腺苷（adenosine,A）和N6-甲基腺苷（N6-methyladenosine, m6A）含量，通过Western blot法检测METTL14、Bcl-xL/Bcl-xS蛋白比值的表达变化。结果 耐伊马替尼K562/G01细胞的耐药性得到证实，以伊马替尼作用CML细胞48 h,K562/G01细胞的IC₅₀值约为K562细胞的24倍（P<0.05）。石蒜碱呈剂量依赖性抑制K562和K562/G01细胞的活性，但石蒜碱对K562/G01细胞活性的抑制作用更强，使细胞呈碎片化，数量明显减少，体积大小不一，而K562细胞形态和数量无明显变化。流式细胞术结果显示，与未处理组比，石蒜碱可提高K562/G01细胞的凋亡率（P=0.01），降低其整体m6A甲基化水平（P=0.03），而石蒜碱对K562细胞的凋亡和m6A甲基化修饰均无明显抑制效果（P>0.05）。同时Western blot实验结果表明，石蒜碱呈剂量依赖性下调METTL14蛋白的表达，在降低抗凋亡蛋白Bcl-xL表达的同时升高促凋亡蛋白Bcl-xS的表达，Bcl-xL/Bcl-xS蛋白比值显著降低。石蒜碱联合伊马替尼作用于耐药CML细胞，能显著降低K562/G01细胞的增殖活性（P<0.01）。结论 石蒜碱联合伊马替尼协同下调METTL14表达，从而影响Bcl-x的剪接模式，促进Bcl-xL向Bcl-xS的转换，诱导细胞凋亡，从而逆转K562/G01细胞的耐药性。更多还原

【Abstract】 Objective To investigate the pro-apoptotic mechanism of lycorine in imatinib-resistant chronic myeloid leukemia（CML） cells and to provide new therapeutic strategies for overcoming clinical drug resistance through drug repurposing. Methods The proliferative activity of K562 and K562/G01 cells treated with graded concentrations of imatinib was assayed using the Cell Counting Kit-8（CCK-8）. After co-culture with lycorine, CML cell proliferation was measured by CCK-8. Morphological changes were observed under a light microscope, and apoptosis rates were analyzed by flow cytometry. The levels of adenosine（A） and N6-methyladenosine（m6A）were quantified using liquid chromatography-tandem mass spectrometry. Protein expression of METTL14 and the Bcl-xL/Bcl-xS ratio was assessed by Western blot. Results The drug resistance of K562/G01 cells was confirmed, with an IC₅₀value approximately 24-fold higher than that of K562 cells after 48-hour imatinib treatment（P<0.05）. Lycorine dose-dependently inhibited the viability of both cell lines, with a more pronounced effect on K562/G01 cells, inducing cell fragmentation, reduced cell numbers, and irregular cell sizes, whereas K562 cells showed no significant morphological changes. Flow cytometry revealed that lycorine significantly increased apoptosis in K562/G01 cells（P=0.01）and decreased the overall m6A methylation level（P=0.03）, whereas it had no significant effect on apoptosis or m6A methylation modification in K562 cells（P>0.05）. Western blot analysis revealed that lycorine dose-dependently downregulated METTL14 protein expression, decreased the anti-apoptotic protein Bcl-xL, increased the pro-apoptotic protein Bcl-xS, and thus significantly lowered the Bcl-xL/Bcl-xS ratio.Combination treatment with lycorine and imatinib synergistically suppressed K562/G01 cell proliferation（P<0.01）. Conclusion The combination of lycorine and imatinib synergistically downregulates METTL14 expression, thereby modulating Bcl-x splicing patterns to promote the shift from antiapoptotic Bcl-xL to pro-apoptotic Bcl-xS, which induces apoptosis and reverses drug resistance in K562/G01 cells.更多还原