Xi Rong-gang, Huang Jian, Li Dan, Wang Xiao-bo, Wu Li-jun
School of Traditional Chinese Medicines, Shenyang Pharmaceutical University, Shenyang 110016, China.
Acta Pharmacol Sin. 2008 Mar;29(3):355-63. doi: 10.1111/j.1745-7254.2008.00759.x.
To study the mechanism by which nanoparticle realgar powders (NRP) induce human histocytic lymphoma U937 cell apoptosis.
After the U937 cells were treated with various doses of NRP, the viability of the NRP-induced U937 cells was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Granular apoptotic bodies with membrane blebbing and condensed nuclei were observed by fluorescence microscopy. The apoptotic ratio induced by NRP was measured by lactate dehydrogenase (LDH) activity-based assay. Caspase-3 and the expressions of Akt, p-Akt, a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase (SIRT1), p53, and p-p53 were detected by Western blot analysis.
The growth-inhibitory activity of NRP for U937 cells was in a time- and dose-dependent manner. After treatment with various concentrations of NRP for 24 h, the majority of U937 cells underwent apoptosis as measured by LDH assay. In the presence of NRP, wortmannin, the inhibitor of phosphoinositide 3-kinase (PI3-K), and Akt inhibitor KP372-1 augmented the NRP-induced cell apoptosis. When the U937 cells were treated with NRP for the indicated time periods, procaspase-3 was gradually degraded and the activated caspase-3 was significantly increased. The expressions of anti-apoptotic proteins Akt and p-Akt were downregulated. Importantly, the inhibition of SIRT1 contributed to the activation of p53 and the inactivation of the PI3-K/Akt signaling pathway increased the expression of the p53 protein and downregulated the SIRT1 protein expression.
The PI3-K/Akt signaling pathway plays an important role in NRP-induced U937 cell apoptosis. The reduced SIRT1 expression and activated p53 might be partially due to the inhibition of the PI3-K/Akt pathway triggered by the NRP-induced initiation of U937 cell apoptosis.
研究纳米雄黄粉（NRP）诱导人组织细胞淋巴瘤U937细胞凋亡的机制。
用不同剂量的NRP处理U937细胞后，采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法检测NRP诱导的U937细胞活力。通过荧光显微镜观察到有膜泡形成和核浓缩的颗粒状凋亡小体。采用基于乳酸脱氢酶（LDH）活性的方法测定NRP诱导的凋亡率。通过蛋白质免疫印迹分析检测半胱天冬酶-3以及Akt、p-Akt、烟酰胺腺嘌呤二核苷酸（NAD+）依赖性组蛋白脱乙酰酶（SIRT1）、p53和p-p53的表达。
NRP对U937细胞的生长抑制活性呈时间和剂量依赖性。用不同浓度的NRP处理24小时后，通过LDH检测发现大多数U937细胞发生凋亡。在存在NRP的情况下，磷酸肌醇3-激酶（PI3-K）抑制剂渥曼青霉素和Akt抑制剂KP372-1增强了NRP诱导的细胞凋亡。当用NRP处理U937细胞指定时间段后，procaspase-3逐渐降解，活化的半胱天冬酶-3显著增加。抗凋亡蛋白Akt和p-Akt的表达下调。重要的是，SIRT1的抑制导致p53激活以及PI3-K/Akt信号通路失活增加了p53蛋白的表达并下调了SIRT1蛋白表达。
PI3-K/Akt信号通路在NRP诱导的U /937细胞凋亡中起重要作用。SIRT1表达降低和p53激活可能部分归因于NRP诱导U937细胞凋亡引发的PI3-K/Akt通路抑制。
