Triptolide inhibits the proliferation of cells from lymphocytic leukemic cell lines in association with downregulation of NF-κB activity and miR-16-1

Aim： To examine the effects of triptolide （TPL） on T-cell leukemia cells and identify their underlying mechanisms. Methods： 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 KB （NF-KB） 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. Results： 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 IC5o of 15.25 and 24.68 nmol/L, respectively. Using Molt-4 cells, we demonstrated that TPL 20-80 nmol/L inhibited the translocation of NF-κB p65 from the cytoplasm to the nucleus and that phosphorylated NF-KB 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. Conclusion： 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-KB and down-regulation of miR-16-1＊.