Abstract 2563: Combination of triptolide and prednisolone to induce apoptosis in acute lymphoblastic leukemia cells

Abstract

Abstract Background: Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children. Although considered highly curable, about 20% of children with ALL relapse and majority of them will die of the resistant disease. Sensitivity to the glucocorticoid, prednisolone (PRED), is critical for cure and the development of combination therapy with PRED may improve treatment outcome. Triptolide, a diterpene triepoxide extracted from the Chinese plant Tripterygium wilfordii has been reported to be immunosuppressive, anti-inflammatory, and anti-proliferative in a broad spectrum of diseases. In this study, we investigate the effect of triptolide to induce apoptosis in ALL cells in combination with prednisolone. Methods: Three clinical important leukemia cells lines 697, Sup-B15 and RS4;11 that represent TCF3-PBX1, BCR-ABL1 and MLL-AF4 respectively were used. They were exposed to triptolide as well as in combination with PRED. Cell viability was determined by MTT assay (Promega). Cell Death Detection ELISA (Roche) was used for measuring apoptosis levels. Western blot was used to detect protein expression levels. Caspase-3 and -9 activities were measured using Caspase-Glo Assay kits (Promega). Cell cycle analysis was performed by Propidium Iodide (PI) staining using flow cytometry. Results: Triptolide can induce cell death at ED50 of 15nM for 697, 13nM for Sup-B15, and 13nM for RS4;11 at 24h time point. The ED50 further decreased to 3nM for 697, 4nM for Sup-B15 and 5nM for RS4;11 at 48h time point. Apoptosis induction and cell cycle arrest was observed upon triptolide treatment in these three cell lines. The levels of caspase-3 and caspase-9 were significantly increased with triptolide treatment, suggesting that triptolide induced apoptosis was caspase-dependent. Although PRED treatment as a single agent can also induce apoptosis, cell cycle arrest and caspase activation in these three cell lines, the co-treatment of PRED and triptolide significantly enhanced these effects compared to single treatment (p<0.05). To understand the combination mechanisms, we evaluated the Bcl-2 family member expression level changes by triptolide using Western blot and results showed that triptolide could reduce the expression level of anti-apoptotic members Mcl-1 and Bcl-xl. On the other hand, PRED could only induce pro-apoptotic protein BIM upregulation. Interestingly, single treatment of triptolide could induce glucocorticoid receptor activation by phosphorylation at Ser211 site which is also a well-known mechanism through which PRED induces apoptosis. Therefore, these mechanisms may contribute to the enhancement of PRED-induced apoptosis by triptolide in these cells. Conclusion: Triptolide can induce apoptosis in ALL cell lines. Combined therapy with PRED and triptolide is a novel promising therapy for this hematological malignancy that warrants further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2563. doi:10.1158/1538-7445.AM2011-2563