Disulfide-Mediated Apoptosis of HTLV-I-Infected Cells in Patients with HTLV-I-Associated Myelopathy/Tropical Spastic Paraparesis

Background: This study was conducted to construct a basis for the therapeutic strategy against human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) using a compound containing the disulfide moiety prosultiamine, which is a homologue of allithiamine originally synthesized by allicin and thiamine-thiol, for the targeting of HTLV-I-infected cells. Methods: 1) Analyses of the apoptotic pathway in allicin or prosultiamine treatment against an HTLV-I-infected T cell line derived from an HAM/TSP patient (HCT-1) by flow cytometry and western blot. 2) Evaluation of the effect of the targeting of HTLV-I-infected cells in a prosultiamine in vitro treatment and clinical trial in HAM/TSP patients by quantitative PCR analysis of HTLV-I proviral load. Results: Prosultiamine, like allicin, induced caspase-dependent apoptosis against HCT-1. The fact that the loss of mitochondrial membrane potential was recovered in z-VAD-fmk-pretreated HCT-1 with prosultiamine treatment suggested that prosultiamine can induce caspase-dependent apoptosis through the mitochondrial pathway. Based on the data showing that prosultiamine in vitro treatment against peripheral blood CD4 T cells of HAM/TSP patients induced a significant decrease of HTLV-I proviral copy numbers by apoptosis of HTLV-I-infected cells, we treated 6 HAM/TSP patients with intravenous administration of prosultiamine for 14 days. As a result of this treatment, the copy numbers of HTLV-I provirus in peripheral blood decreased to about 30-50% of their pretreatment levels with some clinical benefits in all patients. Conclusions: Our results suggest that prosultiamine has the potential to be a new therapeutic tool focusing on the targeting of HTLV-I-infected cells in HAM/TSP. Nishiura et al., (re-revised) page 3 Introduction Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic progressive myelopathy characterized by the involvement of bilateral pyramidal tracts with sphincteric disturbances [1]. The primary pathological feature of HAM/TSP is chronic inflammation of the spinal cord characterized by perivascular cuffing and parenchymal infiltration of lymphocytes [2]. Although the exact mechanism of the pathogenesis of HAM/TSP is still obscure, immunological abnormalities, such as the increased number of activated T cells and Th1 activation arising from a high HTLV-I proviral load, etc., in the peripheral blood play an important role in the pathological process of spinal cord lesions in HAM/TSP patients [3, 4]. Based on this evidence, therefore, immunomodulatory therapy, such as prednisolone [1, 5] and interferon- [6, 7] treatment, has been the main treatment administered to HAM/TSP patients. Indeed, since it is conceivable that the immune-activated status of the peripheral blood involved in the process of chronic inflammation of the spinal cord is one of the targets of the treatments, these treatments produced good results in their own ways. However, their efficacy is still controversial. In addition, whether or not these treatments are tolerable as a long-term or lifelong treatment is uncertain. In the therapeutic strategy against HAM/TSP, most importantly, the ideal treatment is the elimination of HTLV-I-infected cells themselves from the peripheral blood because HTLV-I-infected CD4 T cells are the first responders in the immunopathogenesis of HAM/TSP[4, 8]. Allicin (diallyl thiosulfinate) (Figure 1), a natural organosulfur compound derived from garlic (Allium sativum), has diverse biological activities, including anticarcinogenic, antibacterial, and antifungal activity [9, 10]. Although the mechanisms Nishiura et al., (re-revised) page 4 by which cytotoxic effects are induced by organosulfur compounds are poorly understood, a disulfide moiety in their structures seems to play an important role in triggering cell death [11]. The disruption of the intracellular redox system induced by the chemical reaction of a disulfide moiety with thiol-containing intracellular molecules, such as thioredoxin (Trx), Trx reductase, and glutathione (GSH), might be involved in the cytotoxicity [9, 11]. However, allicin is a very unstable compound and disappears rapidly after intravenous administration [12, 13]. Therefore, its use in therapeutic trials for HAM/TSP is limited. Prosultiamine (N-[(4-amino-2-methyl-5-pyrimidinyl) methyl]N-[4-hydroxy-1-methyl-2-(propyldithio)-1-butenyl]-formamide) (Alinamin), a product of Takeda Pharma Co. Inc. (Osaka, Japan), is a homologue of allithiamine, originally synthesized by thiol-type vitamin B1 and allicin [14]. To enhance stability in the blood and the efficient access of vitamin B1 to the tissues, prosultiamine was developed after allyl disulfide derived from allicin was substituted with propyl disulfide in the structure of allithiamine [15]. Thus, prosultiamine has a disulfide moiety in its structure, like allicin (Figure 1). The fact that prosultiamine is reduced to a part of thiamine and propyl disulfide by the intracellular reducing system after the penetration to the cells might indicate that this compound is expected to have a pharmacological action similar to that of allicin in the cells. Importantly, prosultiamine is pharmacologically stable and is very frequently prescribed for vitamin B1 deficiency in Japan. As such, this drug is potentially available for immediate clinical trials in HAM/TSP patients. Considering the above, we first investigated the cytotoxic effect of prosultiamine against various HTLV-I-infected T cell lines derived from HAM/TSP patients compared to that of allicin through analyses of apoptotic signaling. Secondly, based on the data Nishiura et al., (re-revised) page 5 showing that prosultiamine in vitro treatment against peripheral blood CD4 T cells of HAM/TSP patients induced a significant decrease in HTLV-I proviral copy numbers via apoptosis of HTLV-I-infected cells, we treated 6 HAM/TSP patients with intravenous administration of prosultiamine. As a result of this treatment, the copy numbers of HTLV-I provirus in peripheral blood decreased to about 30-50% of their levels at pretreatment with some clinical benefits in all patients. We herein propose prosultiamine treatment as a new therapeutic strategy against HAM/TSP. Subjects and Methods 1) Cell lines and peripheral blood CD4 T cells HTLV-I-infected T cell lines derived from the cerebrospinal fluid of three HAM/TSP patients (HCT-1, HCT-4, and HCT-5) were used [16]. These cell lines were HTLV-I-producing cell lines. Jurkat cells were also used as an HTLV-I-non-infected T cell line. HCT-1, HCT-4, and HCT-5 were interleukin (IL)-2-dependent and were maintained in RPMI 1640 containing 20% fetal bovine serum (FBS) supplemented with 100 units/ml of recombinant human IL-2 (kindly provided by Shionogi, Japan). Jurkat cells were maintained in RPMI 1640 containing 10% FBS. The peripheral blood CD4-enriched T cells of HAM/TSP patients were separated in the negative selection by the depletion of CD8 T cells from macrophage/B cell-depleted mononuclear cells using magnetic beads coated with anti-CD8 monoclonal antibody (Dynabeads CD8) (Dynal Biotech, Oslo, Norway) as previously described [17]. CD4 -enriched T cells were used as CD4 T cells. 2) Cytotoxicity assay Nishiura et al., (re-revised) page 6 Each cell line was cultured at the concentration of 2 x 10 /ml for 24 hr in the presence of allicin (LKT Labs Inc., MN) or prosultiamine (kindly provided by Takeda Pharma Co. Inc., Osaka, Japan) at various concentrations in 96-well culture plates. As a control, each cell line was cultured in the presence of vehicle alone. Cultures were studied in quadriplicate. Cell viability was determined by MTS nonradioactive cell proliferation assay (Promega Madison, WI) [17]. 3) Apoptosis assay a) Flow cytometry HCT-1 was cultured at the concentration of 2 x 10/ml in the presence of 40 M prosultiamine or vehicle for 1, 3, 5, 8, and 24 hr. After HCT-1 was collected at each time point and stained either by the potential sensitive fluorescent dye DiOC6 (3) (3,3’-dihexyloxacarbocyanine iodide) (Nacalai Tesque, Kyoto, Japan) as previously described [18] or by FITC-conjugated annexin V (BD Biosciences, San Diego, CA) according to the instructions provided by the manufacturer, the loss of the mitochondrial membrane potential (m) or the percentages of apoptotic cells, respectively, was analyzed by flow cytometry (Epics XL System II, Beckman Coulter, Fullerton, CA). b) Effect of pretreatment with z-VAD-fmk against apoptosis To examine whether or not apoptosis is dependent on caspase activation, after HCT-1 pretreated with 200 M z-VAD-fmk (the pan-caspase inhibitor) (Calbiochem, La Jolla, CA) for 1 hr was treated with 40 M of either allicin, prosultiamine or vehicle for 24 hr, the cell viability and the loss of m were determined by MTS assay and flow cytometry, respectively. Nishiura et al., (re-revised) page 7 4) Western blot analysis HCT-1 was collected at various time points after treatment with 40 M allicin, prosultiamine or vehicle, and lysed by M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) supplemented with protease inhibitor cocktail (Sigma, Saint Louis, MO). Insoluble material was removed by centrifugation at 13,000 rpm for 30 min at 4C, and the supernatant was used for western blotting. An identical amount of protein for each lysate (10 g) was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (ATTO, Tokyo). Proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. After overnight blocking with TBS with 0.1% Tween-20 (TBST) containing 5% skim milk (Difco) at 4°C, the PVDF membrane was incubated in the presence of mouse anti-caspase-3 antibody (1:1000 dilution, Cell Signaling, Danvers, MA) at 4°C overnight. After overnight incubation with donkey anti-mouse IgG, horseradish peroxidase-linked species-specific whol