Lipid from Infective <i>L</i>. <i>donovani</i> Regulates Acute Myeloid Cell Growth via Mitochondria Dependent MAPK Pathway

The microbial source, which includes live, attenuated, or genetically modified microbes or their cellular component(s) or metabolites, has gained increasing significance for therapeutic intervention against several pathophysiological conditions of disease including leukemia, which remains an incurable disease till now despite recent advances in the medical sciences. We therefore took up the present study to explore if the leishmanial lipid (pLLD) isolated from L. donovani can play an anti-neoplastic role in acute myeloid leukemia cells by regulating cellular growth. Indeed pLLD significantly inhibited cell proliferation of four AML cell lines (HL-60, MOLT-4, U937, and K562). Scanning electron microscopy and DNA fragmentation analysis revealed that it significantly induced apoptosis of U937 cells through morphological alteration. Occurrence of apoptosis was checked by using Annexin exposure and this established that the cell cycle was arrested at G0/G1 phase in time-dependent manner. pLLD increased the intracellular ROS with alteration of mitochondrial membrane potential, as detected using DCFDA. It also regulated the expression of apoptosis-related proteins like Bax, Bcl2, Bad and t-Bid besides causing cleavage of PARP as determined by western blot analysis. Treatment of U937 cells with pLLD induced the activation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK)1/2, p38, and caspases 9/3. The results suggest that pLLD induces apoptosis in acute myeloid leukemia cells possibly via increasing intracellular ROS and regulating the MAPK pathway.包含活的、减毒或转基因微生物及其细胞组分或代谢产物的微生物来源物，在针对包括白血病在内的多种疾病病理生理状态的治疗干预领域日益凸显其重要价值——即便近年医学科学已取得诸多进展，白血病至今仍属不治之症。为此本研究旨在探究从杜氏利什曼原虫（L. donovani）中分离得到的利什曼脂质（pLLD）能否通过调控细胞增殖，对急性髓系白血病细胞发挥抗肿瘤作用。实验结果证实，pLLD可显著抑制四种急性髓系白血病细胞系（HL-60、MOLT-4、U937及K562）的增殖活性。扫描电子显微镜观察与DNA片段化分析结果显示，pLLD可通过诱导细胞形态学改变，显著诱导U937细胞发生凋亡。通过膜联蛋白染色验证凋亡发生后，进一步发现细胞周期呈时间依赖性阻滞于G0/G1期。采用二氯荧光素二乙酸酯（DCFDA）检测发现，pLLD可升高细胞内活性氧（ROS）水平并改变线粒体膜电位。蛋白质免疫印迹分析结果显示，pLLD除可引发多聚ADP核糖聚合酶（PARP）的剪切外，还可调控Bax、Bcl2、Bad及t-Bid等凋亡相关蛋白的表达。对U937细胞施加pLLD处理后，可激活细胞外信号调节激酶（ERK）1/2、c-Jun氨基末端激酶（JNK）1/2、p38丝裂原活化蛋白激酶以及半胱天冬酶9/3。综上，本研究结果表明，pLLD或通过升高细胞内活性氧水平并调控丝裂原活化蛋白激酶（MAPK）通路，诱导急性髓系白血病细胞发生凋亡。