Work

SphK1 inhibitor potentiates the anti‐cancer effect of EGCG on leukaemia cells

Year: 2016

Type: article

Abstract: Chronic lymphocytic leukaemia (CLL), the most prevalent adult leukaemia in Western countries, is characterized by the co-expression of the B-cell antigens CD19, CD20 and CD23, and the T-cell antigen CD5 (Oscier et al, 2012). Considering the risk/benefit ratio in patients with early-stage CLL who have a risk for disease progression, no applicable method meets the accepted indications for treatment according to the National Cancer Institute International Workshop on Chronic Lymphocytic Leukemia criteria (Hallek et al, 2008); moreover, the current standard treatment of patients with early disease is a watch-and-wait strategy (Eichhorst et al, 2015). Therefore, a novel therapeutic strategy is required. Polyphenon E™ is a botanical drug containing 50% (−)-epigallocatechin-O-3-gallate (EGCG) that has been approved by the US Food and Drug Administration (FDA) for the treatment of external genital warts. In a phase II clinical study, Polyphenon E showed positive biological responses in 69% of patients with early-stage CLL (29 of 42 patients) without causing severe side effects (Shanafelt et al, 2013). Previous reports have shown that EGCG induces its anti-cancer effects through the 67-kD laminin receptor (67LR), which is overexpressed in several types of cancer (Britschgi et al, 2010; Tsukamoto et al, 2012). Because we previously reported on the overexpression of 67LR in CLL cells compared with that in normal cells (Kumazoe et al, 2015) and that acid sphingomyelinase (ASM) plays a central role in EGCG-induced 67LR-dependent apoptosis (Kumazoe et al, 2013; Huang et al, 2015), we hypothesized that negative regulators of ceramide protects cancer cells from EGCG-induced apoptosis. Relative to normal B cells, our Western blot analysis suggested that sphingosine kinase 1 (SPHK1), one of the negative regulators of ceramide that catalyses the phosphorylation of sphingosine and subsequently promotes ceramide metabolism, was overexpressed in primary CLL B cells that were derived from 22 of 24 different patients (Fig 1A). We subsequently examined whether safingol (L-threo-dihydrosphingosine), which first entered clinical trials as an anti-cancer agent and is a potent competitive inhibitor of SPHK1, potentiated EGCG-induced cell death in CLL cells. Surprisingly, safingol significantly potentiated the anti-cancer effects of EGCG in all 10 patient-derived CLL B cells (Fig 1B, C). The EGCG/safingol combination resulted in a greater increase in the death of CLL cells, which was attributed to apoptosis (Fig 1C–E). Taken together, the EGCG/SPHK1 inhibitor combination may be a novel therapeutic strategy for CLL patients with the overexpression of 67LR and SPHK1. Based on previous evidence showing the crucial role played by ASM in EGCG-induced cell death in chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) cells (Kumazoe et al, 2013; Huang et al, 2015), we hypothesized that safingol potentiates the effects of EGCG in these cells. Relative to normal peripheral blood mononuclear cells (PBMCs), our Western blot analysis demonstrated significantly increased levels of 67LR, ASM and SPHK1 in the CML cell line [K562] and AML cell lines [HL-60, Kasumi-1 (subtype M2) and Kasumi-3 (subtype M0)] (Fig 1F). Co-treatment with safingol significantly increased EGCG-induced cell death in K562, HL-60 and Kasumi-3 cells (Fig 1G). Although Kasumi-3 cells (AML M0) are known to acquire resistance to cytarabine, a drug that is used to treat AML, the EGCG/safingol combination significantly suppressed their viable cell number (Fig 1H). To confirm the role of SPHK1 in EGCG-resistance, we determined that the effect of SPHK1 silencing on EGCG-induced cell death in Kasumi-3 cells. Our data suggested that SPHK1 suppression was sufficient to potentiate EGCG-induced cell death (Fig 1I). Safingol has limited single-agent activity in vivo, and strategies to increase its effectiveness in cancer treatment without increasing toxicity are urgently required. Cisplatin has been reported to potentiate the anti-cancer effect of safingol (Dickson et al, 2011). However, considering the major adverse effects of cisplatin on the risk/benefit balance and patient quality of life, we believe that it is not an appropriate candidate for combination therapy with safingol in patients with early-stage CLL. Given that Polyphenon E™ has shown generally positive response and is well tolerated by patients with early-stage CLL, the combination of Polyphenon E™ and safingol therapy is a potentially useful therapy for patients who are being managed using the watch-and-wait strategy. These data suggest that, by targeting 67LR and SPHK1 overexpression in patients with CLL, EGCG and SPHK1 inhibitor combination therapy may be a novel therapeutic strategy, and clinical testing of this approach should be considered. The authors declare no conflict of interest. This work was kindly supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science to H. Tachibana. (Grant Number 22228002 and 15H02448); International Myeloma Foundation (IMF) Japan's grants to H. Tachibana; This work was also supported in part by Grant-in-Aid for JSPS Fellows to M.K. (PD) and JSPS KAKENHI grant 15K18821. S.T., C.L. and N.E performed the experiments; M.K., S.T., Y.H., T.D. and H.T. designed the study; M.K. and S.T. analysed the data; M.K. and S.T. created the figure and M.K. and H.T. wrote the manuscript.

Source: British Journal of Haematology

DOI: 10.1111/bjh.14119