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Summary: Sphingolipid metabolism is increasingly recognised as a therapeutic target in cancer due to its regulation of cell proliferation and apoptosis. The sphingolipid rheostat is proposed to control cell fate through maintaining balance between pro‐apoptotic and pro‐survival sphingolipids. This balance is regulated by metabolising enzymes involved in sphingolipid production. One such enzyme, sphingosine kinase‐2 (SPHK2), produces pro‐survival sphingosine 1‐phosphate (S1P) by phosphorylation of pro‐apoptotic sphingosine. Elevated SPHK2 has been found in multiple cancer types and contributes to cell survival, chemotherapeutic resistance and apoptosis resistance. We have previously shown elevation of S1P in large granular lymphocyte (LGL) leukaemia serum and cells isolated from patients. Here, we examined SPHK2 expression in LGL leukaemia and found SPHK2 mRNA and protein upregulation in a majority of LGL leukaemia patient samples. Knockdown of SPHK2 with siRNA in LGL leukaemia cell lines decreased proliferation. Additionally, the use of ABC294640 or K145, both SPHK2‐specific inhibitors, decreased viability of LGL leukaemia cell lines. ABC294640 selectively induced apoptosis in LGL cell lines and freshly isolated LGL leukaemia patient cells compared to normal controls. Mechanistically, SPHK2 inhibition downregulated pro‐survival myeloid cell leukaemia‐1 (Mcl‐1) protein through proteasomal degradation. Targeting of SPHK2 therefore provides a novel therapeutic approach for the treatment of LGL leukaemia. [ABSTRACT FROM AUTHOR]

Natural killer ( NK) large granular lymphocyte ( LGL) leukaemia features a clonal proliferation of CD3− NK cells that can be classified into either aggressive or chronic categories. The NKL cell line, derived from an aggressive Asian NK cell leukaemia, and patient samples from chronic NK- LGL leukaemia were used in our study to probe for synergistic efficacy of the epigenetic drugs vorinostat ( SAHA) and cladribine in this disease. We demonstrate that histone deacetylases ( HDACs) are over-expressed in both aggressive and chronic NK leukaemia. Administration of the HDAC inhibitor SAHA reduces class I and II HDAC expression and enhances histone acetylation in leukaemic NK cells. In vitro combination treatment with SAHA and cladribine dose-dependently exerts synergistic cytotoxic and apoptotic effects on leukaemic NK cells. Expression profiling of apoptotic regulatory genes suggests that both compounds led to caspase-dependent apoptosis through activation of intrinsic mitochondrial and extrinsic death receptor pathways. Collectively, these data show that combined epigenetic therapy, using HDAC and DNA methyltransferase inhibitors, may be a promising therapeutic approach for NK- LGL leukaemia. [ABSTRACT FROM AUTHOR]

We have demonstrated that pancreatitis-associated ascitic fluid contributes to hepatocyte injury during acute pancreatitis; a phenomenon independent of ascites'' enzymatic content and Kupffer cell-derived cytokines. Our aim is to characterize the mechanisms of pancreatitis-associated ascitic fluid induced hepatocyte death. NIH mice were injected intraperitoneally with pathogen-free pancreatitis-associated ascitic fluid. Twenty-four hours later, serum AST, ALT, LDH, and hepatocyte apoptosis (TUNEL) were measured. Human hepatocytes (CCL-13) were treated with pancreatitis-associated ascitic fluid ±SB203580 or caspase-3 inhibitor-II. Mitochondrial membrane integrity was determined by DiOC6 staining. Apoptosis was measured by TUNEL staining and flow cytometry after dual labeling with Annexin-V/7-AAD. Data are mean ± SEM of triplicates. Pancreatitis-associated ascitic fluid increased serum AST, ALT, LDH, and apoptotic cells in the mouse liver (all P < 0.03 vs. sham). In CCL-13 cells, pancreatitis-associated ascitic fluid induced a time and dose-dependent increase in apoptosis, in addition to p38-MAPK phosphorylation (P = 0.02 vs. control), caspase-3 cleavage (P < 0.03 vs. control) and decreased DiOC6 mitochondrial staining (P < 0.01 vs. control). Both caspase-3 inhibitor-II and SB203580 decreased apoptosis, but the former had no effect on DiOC6 staining. Pancreatitis-associated ascitic fluid induces liver injury and hepatocyte apoptosis by activating p38-MAPK and caspase-3 dependent pro-apoptotic pathways. ( J Gastrointest Surg 2003;7:200–208.) [Copyright &y& Elsevier]