Institute of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
Acta Pharmacol Sin. 2011 Apr;32(4):503-11. doi: 10.1038/aps.2010.237. Epub 2011 Mar 28.
To examine the effects of triptolide (TPL) on T-cell leukemia cells and identify their underlying mechanisms.
The cytotoxicity of TPL was assessed by MTT assay. Cell apoptosis was determined using annexin V and DAPI staining and analyzed by flow cytometry or fluorescence microscopy. The activation of caspase pathways and the expression of nuclear factor κB (NF-κB) p65 were examined by Western blotting. Differences in microRNA (miRNA) expression in Molt-4 and Jurkat cells before and after TPL treatment were identified using microarrays and real-time RT-PCR, respectively.
TPL 20-160 nmol/L treatment potently inhibited cell growth and induced apoptosis in T-cell lymphocytic leukemia cell lines. Molt-4 and Jurkat cells, however, were more sensitive to TPL than L428 and Raji cells. After 24 h of treatment, bortezomib abrogated the growth of Molt-4 and Jurkat cells with an IC(50) of 15.25 and 24.68 nmol/L, respectively. Using Molt-4 cells, we demonstrated that treatment 20-80 nmol/L inhibited the translocation of NF-κB p65 from the cytoplasm to the nucleus and that phosphorylated NF-κB p65 in nuclear extracts was down-regulated in a dose-dependent manner. Similar results were also seen in Jurkat cells but not in L428 cells, as these cells are resistant to TPL and bortezomib (a NF-κB inhibitor). Twenty-three miRNAs were differentially expressed after TPL treatment. Functional analysis revealed that TPL treatment could inhibit expression of miR-16-1* and that transfection of miR-16-1* led to significantly decreased apoptosis induced by TPL.
Our in vitro studies suggest that TPL might be an effective therapeutic agent for treatment of T-cell lymphocytic leukemia and that its cytotoxic effects could be associated with inhibition of NF-κB and down-regulation of miR-16-1*.
研究雷公藤红素（TPL）对 T 细胞白血病细胞的作用，并探讨其作用机制。
采用 MTT 法检测 TPL 的细胞毒性，用 Annexin V 和 DAPI 染色，流式细胞术或荧光显微镜检测细胞凋亡，Western blot 检测半胱氨酸天冬氨酸蛋白酶（caspase）途径的激活和核因子κB（NF-κB）p65 的表达。采用微阵列和实时 RT-PCR 分别检测 Molt-4 和 Jurkat 细胞在 TPL 处理前后微小 RNA（miRNA）的表达差异。
TPL（20-160 nmol/L）显著抑制 T 细胞淋巴瘤细胞系的细胞生长并诱导细胞凋亡。与 L428 和 Raji 细胞相比，Molt-4 和 Jurkat 细胞对 TPL 更为敏感。24 h 后，硼替佐米对 Molt-4 和 Jurkat 细胞的生长抑制作用的 IC50 分别为 15.25 和 24.68 nmol/L。用 Molt-4 细胞实验表明，TPL（20-80 nmol/L）处理可抑制 NF-κB p65 从细胞质向核内转移，且核提取物中磷酸化的 NF-κB p65 呈剂量依赖性下调。在 Jurkat 细胞中也观察到类似的结果，但在 L428 细胞中未见此结果，因为这些细胞对 TPL 和硼替佐米（NF-κB 抑制剂）具有抗性。TPL 处理后有 23 个 miRNA 表达差异。功能分析表明，TPL 处理可抑制 miR-16-1的表达，转染 miR-16-1可显著减少 TPL 诱导的细胞凋亡。
本体外研究提示 TPL 可能是治疗 T 细胞淋巴瘤的有效治疗药物，其细胞毒性作用可能与抑制 NF-κB 和下调 miR-16-1*有关。
