HI-TOPK-032 (cat. no. 614849) was purchased from Calbiochem (San Diego, CA, USA), dissolved in dimethyl sulfoxide (cat. no. 13407-45, Nacalai Tesque, Inc., Kyoto, Japan). Fucoidan was prepared from the brown algae Cladosiphon okamuranus Tokida cultivated in Okinawa as previously described in detail (12). Antibodies against PBK/TOPK (cat. no. 4942), phospho-PBK/TOPK (Thr9) (cat. no. 4941), Bcl-xL (cat. no. 2762), Bax (cat. no. 2772), Bak (cat. no. 3814), survivin (cat. no. 2808), phosphatase and tensin homologue (PTEN) (cat. no. 9556), phospho-PTEN (Ser380) (cat. no. 9551), Akt (cat. no. 9272), phospho-Akt (Thr308) (cat. no. 13038), phospho-Akt (Ser473) (cat. no. 4060), phospho-IκBα (Ser32 and 36) (cat. no. 9246), IκB kinase (IKK)α (cat. no. 2682), IKKβ (cat. no. 2684), phospho-IKKα/β (Ser176/180 and Ser177/181) (cat. no. 2694), cleaved caspase-3 (cat. no. 9664), cleaved caspase-8 (cat. no. 9496), cleaved caspase-9 (cat. no. 9501) and poly(ADP-ribose) polymerase (PARP) (cat. no. 9541) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against cyclin B1 (cat. no. MS-868), CDK1 (cat. no. MS-275), CDK6 (cat. no. MS-398), retinoblastoma protein (pRb) (cat. no. MS-107) and actin (cat. no. MS-1295) were obtained from Neomarkers, Inc. (Fremont, CA, USA). Antibodies against XIAP (cat. no. M044-3) and phospho-pRb (Ser780) (cat. no. M054-3S) were purchased from Medical & Biological Laboratories, Co. (Aichi, Japan). Antibodies against c-IAP2 (cat. no. sc-7944), Mcl-1 (cat. no. sc-819), IκBα (cat. no. sc-371), JunB (cat. no. sc-46), JunD (cat. no. sc-74), IFN-induced protein with tetratricopeptide repeats (IFIT)1 (cat. no. sc-134948), IFIT2 (cat. no. sc-390724), IFIT3 (cat. no. sc-393396) and NF-κB subunits p50 (cat. no. sc-114X), p52 (cat. no. sc-298X), RelA (cat. no. sc-109X), c-Rel (cat. no. sc-70X) and RelB (cat. no. sc-226X) and AP-1 subunits c-Fos (cat. no. sc-52X), FosB (cat. no. sc-48X), Fra-1 (cat. no. sc-605X), Fra-2 (cat. no. sc-604X), c-Jun (cat. no. sc-45X), JunB (cat. no. sc-46X) and JunD (cat. no. sc-74X) for supershift assay were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Lt-4, an antibody against Tax, was previously described (13).

The HTLV-1-transformed MT-2, MT-4, C5/MJ, SLB-1 and HUT-102, and ATLL-derived MT-1, TL-OmI and ED-40515(−) T-cell lines, were maintained in RPMI-1640 medium (cat. no. 30264-56; Nacalai Tesque, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% penicillin/streptomycin (cat. no. 09367-34, Nacalai Tesque, Inc.) at 37°C in a humidified incubator under 5% CO₂. The C5/MJ, HUT-102 and MT-1 cells were obtained from Fujisaki Cell Center, Hayashibara Laboratories, Inc. (Okayama, Japan). The MT-2 and MT-4 cells were kindly provided by Dr Naoki Yamamoto (Tokyo Medical and Dental University, Tokyo, Japan). The SLB-1 and ED-40515(−) cells were provided by Dr Diane Prager (UCLA School of Medicine, Los Angeles, CA, USA) and Dr Michiyuki Maeda (Kyoto University, Kyoto, Japan), respectively. The TL-OmI cell line was provided by Dr Masahiro Fujii (Niigata University, Niigata, Japan). The study also included peripheral blood mononuclear cells (PBMCs) obtained from healthy donors (cat. no. HC-0001; Lifeline Cell Technology, Frederick, MD, USA). The PBMCs were stimulated with 20 µg/ml of phytohemagglutinin (PHA) (cat. no. L8754; Sigma-Aldrich Co., St. Louis, MO, USA) for 72 h.

The MT-2 cells that produce viral particles were pre-treated with 200 µg/ml of mitomycin C (MMC) (cat. no. M0503; Sigma-Aldrich Co.) for 1 h, pipetted vigorously, and washed 3 times with phosphate-buffered saline. PBMCs from healthy donors (Lifeline Cell Technology) and MMC-treated MT-2 cells were co-cultured in the presence of 10 ng/ml of interleukin (IL)-2 (kindly provided by Takeda Pharmaceutical Company Ltd., Osaka, Japan). The culture medium was half-changed with fresh medium supplemented with IL-2 every 3 days. As the MT-2 cells were pre-treated extensively with MMC, no discernible MT-2 cells were found.

Whole cell extracts were prepared by subjecting the treated cells to lysis with lysis buffer [62.5 mM Tris-HCl (pH 6.8) (cat. no. 35434-21), 2% sodium dodecyl sulfate (cat. no. 31607-65), 10% glycerol (cat. no. 17045-65), 6% 2-mercaptoethanol (cat. no. 21438-82; all from Nacalai Tesque, Inc.) and 0.01% bromophenol blue (cat. no. 021-02911; Wako Pure Chemical Industries, Osaka, Japan)]. Protein concentrations were determined using a commercial kit (DC Protein Assay, cat. no. 5000116JA; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Lysate protein (20 µg) was subjected to 8–15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), electrotransferred onto polyvinylidene difluoride membranes (cat. no. IPVH00010EMD; Millipore, Darmstadt, Germany). The membranes were blocked with 4% non-fat dry milk (cat no. 9999; Cell Signaling Technology, Inc.) for 1 h at room temperature, and blotted overnight with the respective antibody (1:1,000). Finally, the blots were hybridized with horseradish peroxidase-conjugated secondary anti-mouse (1:1,000) (cat. no. 7076; Cell Signaling Technology, Inc.) or anti-rabbit IgG antibody (1:1,000) (cat. no. 7074; Cell Signaling Technology, Inc.), and visualized using an enhanced chemiluminescence reagent (cat. no. RPN2232; Amersham Biosciences Corp., Piscataway, NJ, USA).

The HUT-102 and MT-2 cells were treated with 5 µM of HI-TOPK-032. After 24 h, total RNA was extracted using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany). Microarray analysis using a SurePrint G3 Human GE 8×60 K Microarray kit version 3.0 (Agilent Technologies, Inc., Waldbronn, Germany) was performed as previously described (14).

TRIzol reagent (cat. no. 15596026; Invitrogen Life Technologies, Carlsbad, CA, USA) was used to extract total RNA from the cells. A total of 1 µg RNA was reverse transcribed into cDNA using a PrimeScript RT-PCR kit (cat. no. RR014A; Takara Bio, Inc., Otsu, Japan). The sequence-specific primers used for RT-PCR are summarized in Table I. The PCR cycling conditions were as follows: 94°C for 2 min, followed by 25–35 cycles of 94°C for 1 min, 54–60°C for 1 min and 72°C for 1 min, with a final extension step of 72°C for 5 min.

The water-soluble tetrazolium (WST)-8 uptake method was used to assess the cell proliferative and toxic effects of HI-TOPK-032 (0.31–20 µM) and fucoidan (0.13–6 mg/ml) for 48 h. The cells were plated in 96-well flat microtiter plates in triplicate and treated as indicated for 48 h prior to the WST-8 assay. A total of 10 µl of the WST-8 reagent (cat. no. 07553-44; Nacalai Tesque, Inc.) were added to each well. After a 4-h reaction, WST-8 reduction was measured at 450 nm using a Wallac 1420 Multilabel Counter (PerkinElmer, Inc., Waltham, MA, USA). The values were normalized to the untreated control samples.

The cells were treated with HI-TOPK-032 (0.63–20 µM) for 24 h and then permeabilized by incubation on ice for 20 min with 100 µg/ml of digitonin, and treated with the phycoerythrin-conjugated APO2.7 antibody (cat. no. IM2088; Beckman Coulter, Inc., Marseille, France) for 15 min at room temperature. After staining with APO2.7 antibody, the induction of apoptosis was determined by Epics XL flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). In addition, for the analysis of morphological changes in the nuclei, the cells were stained by 10 µg/ml of Hoechst 33342 (cat. no. 346-07951; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and observed under a Leica DMI6000 microscope (Leica Microsystems, Wetzlar, Germany).

Caspase activity was assessed using Colorimetric Caspase Assay kits (cat. nos. 4800, 4805 and 4810; Medical & Biological Laboratories, Co.). Briefly, the cell extracts were recovered using the cell lysis buffer supplied with the kit, and assessed for caspase−3, −8 and −9 activities using respective colorimetric probe. The kits are based on the detection of chromophore ϱ-nitroanilide after cleavage from caspase-specific labeled substrates. Colorimetric readings were determined with a microplate reader (Wallac 1420 Multilabel Counter; PerkinElmer, Inc.).

The cells were stained with the CycleTEST Plus DNA Reagent kit (cat. no. 340242; Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA). The cell cycle distribution was analyzed by an Epics XL flow cytometry that uses the MultiCycle software (version 3.0; Phoenix Flow Systems, San Diego, CA, USA). The population of nuclei at each phase of the cell cycle was determined, and apoptotic cells with hypodiploid DNA content were detected in the sub-G₁ region.

To determine NF-κB and AP-1 activation, we prepared nuclear extracts from the HI-TOPK-032-treated cells and performed EMSA, as previously described in detail (15). Nuclear extracts were incubated with ³²P-labeled probes. The top strand sequences of the oligonucleotide probes or competitors were as follows: For a typical NF-κB element of the IL-2 receptor α chain (IL-2Rα) gene, 5′-GATCCGGCAGGGGAATCTCCCTCTC-3′ and for the consensus AP-1 element of the IL- 8 gene, 5′-GATCGTGATGACTCAGGTT-3′. The above underlined sequences are the NF-κB and AP-1 binding sites, respectively. In the competition experiments, nuclear extracts were pre-incubated for 15 min with 100-fold excess of unlabeled oligonucleotides. For supershift assays, nuclear extracts were incubated with antibodies against NF-κB or AP-1 subunits for 45 min at room temperature before the complex was analyzed by EMSA. The dried gels were visualized using a Pharos FX Plus System (Bio-Rad Laboratories Inc.).

Eighteen 5-week-old female C.B-17/Icr-severe combined immunodeficient (SCID) mice were obtained from Kyudo, Co. (Tosu, Japan). The HUT-102 cells (1×10⁷/0.2 ml RPMI-1640 medium) were inoculated subcutaneously into the post-auricular region of the SCID mice. The mice were divided into 2 groups (n=9, each). HI-TOPK-032 was solubilized in 5.2% polyethylene glycol 400 (cat. no. 161-09065; Wako Pure Chemical Industries) and 5.2% Tween-80 (cat. no. 231181; Becton-Dickinson, Franklin Lakes, NJ, USA), and administrated at a dose of 12.5 mg/kg intraperitoneally 5 times a week. The treatment was continued for 4 weeks, beginning on the day after cell inoculation. The control group received the vehicle (5.2% polyethylene glycol 400 and 5.2% Tween-80) only. The tumor diameter was measured weekly with a shifting caliper and tumor volume was calculated. The body weights were also measured weekly. All mice were sacrificed on day 28. The tumors were excised and their weights measured. Blood samples were collected from mice under deep terminal anesthesia by cardiac puncture, and the sera were stored at −80°C until assayed for soluble IL-2Rα [soluble cluster of differentiation 25 (sCD25)] and sCD30. This experiment was performed according to the Guidelines for Animal Experimentation of the University of the Ryukyus (Nishihara, Japan), and was approved by the Animal Care and Use Committee of the University (reference no. A2016073).

The tumor specimens were collected from the treated or untreated mice, fixed in formalin solution (Wako Pure Chemical Industries), dehydrated through graded ethanol series (Japan Alcohol Selling Co., Tokyo, Japan), and embedded in paraffin (cat. no. 09620; Sakura Finetek Japan Co., Tokyo, Japan). The paraffin-embedded specimens of ATLL tumors were stained with hematoxylin and eosin (H&E, cat. nos. 234-12 and 1159350025; Merck, Darmstadt, Germany), and examined histologically. The analysis of DNA fragmentation by TUNEL assay was performed using a commercial kit (cat. no. 11684817910; Roche Applied Science, Penzberg, Germany) according to the instructions supplied by the manufacturer. The cells were examined under a light microscope (Axioskop 2 Plus) with an Achroplan 40x/0.65 lens (both from Zeiss, Hallbergmoos, Germany). Images were acquired with an AxioCam 503 color and AxioVision LE64 software (Zeiss).

Serum concentrations of human sCD25 (cat. no. 950.500.048; Diaclone SAS, Besançon, France) and human sCD30 (cat. no. BMS240; Affymetrix eBioscience, San Diego, CA, USA) were assessed in the treated and untreated mice by enzyme-linked immunosorbent assay (ELISA), according to the protocol supplied by the manufacturer.

The results are expressed as the means ± standard deviation (SD). The Student's t-test or ANOVA with Tukey-Kramer statistical tests were used to evaluate the data of 2 groups or more than 2 groups, respectively. Differences were considered statistically significant at P<0.05.

To assess PBK/TOPK expression in HTLV-1-infected T-cell lines, we examined the protein levels of PBK/TOPK. The protein levels were determined in 5 HTLV-1-transformed T-cell lines (Fig. 1A, lanes 1–5) and in 3 ATLL-derived T-cell lines (Fig. 1A, lanes 6–8) and compared with those in PBMCs from two healthy donors (Fig. 1A, lanes 9 and 10), using western blot analysis. HTLV-1-transformed T-cell lines constitutively expressed Tax at protein and mRNA levels (Fig. 1A and B). Western blot analysis revealed that PBK/TOPK protein expression was markedly higher in the HTLV-1-infected T-cell lines than in the PBMCs from healthy donors (Fig. 1A). RT-PCR analysis confirmed the elevated mRNA expression of PBK/TOPK in the HTLV-1-infected T-cell lines compared to PBMCs from healthy donors (Fig. 1B). The phosphorylation of PBK/TOPK by CDK1/cyclin B1 is required for its mitotic activity (9,10). We found that PBK/TOPK was constitutively phosphorylated and CDK1/cyclin B1 was strongly expressed in all HTLV-1-infected T-cell lines. By contrast, no phosphorylation of PBK/TOPK and no expression of CDK1/cyclin B1 were detected by western blot analysis in the PBMCs of healthy donors (Fig. 1A, lanes 9 and 10). Notably, PHA stimulation induced the expression of PBK/TOPK at the protein and mRNA levels, the phosphorylation of PBK/TOPK, and the protein expression of CDK1/cyclin B1 in PBMCs (Fig. 1A and B).

To examine whether HTLV-1 infection can induce PBK/TOPK expression, we co-cultured PBMCs and MMC-treated MT-2 cells. At 7 days after co-cultivation, the PBMCs were harvested for the assessment of viral gene expression by RT-PCR. The PBMCs co-cultured with MMC-treated MT-2 cells expressed Tax mRNA (Fig. 1C). Furthermore, the expression levels of PBK/TOPK and IL-2Rα, one of the known target genes of Tax (16), increased in these cells following induction of Tax expression. These results suggest that HTLV-1 infection induces the expression of PBK/TOPK in PBMCs.

The PBK/TOPK inhibitor, HI-TOPK-032, is known to bind the active site of PBK/TOPK (17). In this study, HI-TOPK-032 suppressed the phosphorylation of PBK/TOPK (Fig. 2A). To examine the effects of HI-TOPK-032, HTLV-1-infected T-cell lines were treated with various concentrations of HI-TOPK-032 for 48 h, and cell viability was assessed by WST-8 assay. Cell viability was inhibited in a dose-dependent manner (Fig. 2B). The effect of HI-TOPK-032 on the viability of PBMCs was less pronounced at the 5 µM concentration (Fig. 2C).

First, the morphological changes induced by HI-TOPK-032 were examined using microscopy. The cells treated with HI-TOPK-032 for 48 h demonstrated morphological characteristics of apoptosis, such as chromatin condensation and nuclear fragmentation (Fig. 3A). Next, the induction of apoptosis by HI-TOPK-032 was measured using three commonly used assays: APO2.7 staining (Fig. 3B and C), the cleavage of caspases (Fig. 3D) and the activation of caspases (Fig. 3E). The mitochondrial membrane antigen, APO2.7, is known to be expressed during apoptosis (18). Treatment with HI-TOPK-032 resulted in an increase in the proportion of APO2.7-positive apoptotic cells in a dose-dependent manner (Fig. 3B and C). Western blots of the cells treated with HI-TOPK-032 revealed that apoptosis was accompanied by an increase in the cleavage of caspase−3, −8, −9 and PARP, a substrate for caspase-3 (Fig. 3D). The activation of caspase−3, −8 and −9 was also detected in the cells treated with 2.5 µM of HI-TOPK-032 (Fig. 3E).

To examine the effects of HI-TOPK-032 on cell cycle progression, cell cycle analysis was performed on the cells. The cells were treated with 1.25 or 2.5 µM of HI-TOPK-032 for 24–48 h. The percentages of cells in the sub-G₁ phase of the high-dose HI-TOPK-032 (2.5 µM) treatment group were higher than those of the control group (Fig. 4A and B). In comparison, the low-dose HI-TOPK-032 (1.25 µM) treatment group exhibited a reduced proportion of cells in the S phase, but an increased proportion of cells in the G₁ phase population (Fig. 4A and C). These results indicate that high-dose and low-dose HI-TOPK-032 induces the apoptosis of and G₁ cell cycle arrest in HTLV-1-infected T-cell lines.

We assessed the effects of HI-TOPK-032 on the molecular cascades of apoptosis in HUT-102 cells. As shown in Fig. 5, HI-TOPK-032 decreased the expression of the anti-apoptotic proteins, Mcl-1, c-IAP2, XIAP and survivin in the HUT-102 cells. By contrast, HI-TOPK-032 enhanced the expression of pro-apoptotic protein, Bak. It had no significant effects on the Bcl-xL and Bax protein levels. To examine the mechanisms through which HI-TOPK-032 induces G₁ cell cycle arrest in the HUT-102 cells, the phosphorylation state of pRb was evaluated by western blot analysis. HI-TOPK-032 decreased the levels of the phosphorylated forms of pRb, and altered the hyperphosphorylated form to a hypophosphorylated form of pRb (Fig. 5). Next, we examined the expression of CDK6 to clarify pRb phosphorylation. Treatment with HI-TOPK-032 decreased the expression of CDK6 (Fig. 5).

Transcription factors are proteins that bind at a specific promoter region of the DNA and regulate the expression of various genes. NF-κB and AP-1 are closely linked with cell survival, proliferation and transformation. Therefore, they are important targets for therapeutic intervention in ATLL (3). Notably, Mcl-1, c-IAP2, XIAP, survivin and CDK6 are NF-κB-regulated gene products (19–23). As shown in Fig. 6A, the DNA-binding of NF-κB and AP-1 was observed in the HUT-102 and MT-2 cells. These binding reactions were specific since cold competitors, but not unrelated oligonucleotides, competed with each DNA-binding activity (Fig. 6A, lanes 2 and 3). The components of NF-κB and AP-1 DNA-binding complexes were analyzed with specific antibodies against 5 NF-κB family proteins and 7 AP-1 family proteins. The activated complexes of NF-κB and AP-1 consisted of p50/p65/c-Rel/RelB (Fig. 6A, lanes 4–6 and 8) and JunB/JunD (Fig. 6A, lanes 9 and 10), respectively. We investigated whether HI-TOPK-032 inhibits the constitutive NF-κB and AP-1 activation in HTLV-1-infected T-cell lines. HI-TOPK-032 reduced the DNA-binding of NF-κB and AP-1 in a dose-dependent manner (Fig. 6B). Next, we examined the effect of HI-TOPK-032 on JunB/JunD expression, as JunB/JunD are functional components of AP-1 in HTLV-1-infected T-cell lines. Western blot analysis revealed that HI-TOPK-032 inhibited the AP-1 signaling pathway through the suppression of JunB/JunD protein levels (Fig. 5, right panel).

In the unstimulated state, NF-κB is sequestered in the cytoplasm by binding to IκBα. In response to a variety of stimuli including viruses, the IKK complex comprised of IKKα/IKKβ/IKKγ is activated, and phosphorylates IκBα, which is sequentially ubiquitinated and subjected to proteasomal degradation (24). As PBK/TOPK was identified as a novel upstream kinase of IκBα regulating NF-κB (25), we examined whether HI-TOPK-032 affects IκBα phosphorylation. HI-TOPK-032 inhibited the phosphorylation and degradation of IκBα (Fig. 5). In addition, the inhibition of PBK/TOPK by HI-TOPK-032 led to a slight decrement in the total IKKβ levels and a significant reduction in phosphorylated IKKα/β levels. The phosphoinositide 3-kinase (PI3K)-Akt signal is a component of a pathway important for cell survival and growth during carcinogenesis, and Akt utilizes IKKs for the activation of NF-κB (26). The tumor suppressor, PTEN, dephosphorylates phosphatidylinositol 3,4,5-triphosphate (PIP3) to PI(4,5)P2, and opposes the PI3K-Akt signaling (27). The loss of the expression/activity of PTEN is among the most frequently occurring in cancer (27). ATLL cells do not harbor genetic changes in PTEN, but express high levels of PTEN that is highly phosphorylated (28). Its phosphorylation maintains PTEN in an inactive form (29). It has been suggested that PBK/TOPK promotes Akt activation by inducing PTEN phosphorylation (30). Thus, we hypothesized that PBK/TOPK may regulate the PTEN-Akt pathway in ATLL. The decreased PTEN phosphorylation levels upon HI-TOPK-032 treatment were associated with a decreased Akt phosphorylation (Fig. 5), suggesting that the PBK/TOPK-mediated phosphorylation of PTEN may lead to PTEN inactivation and Akt activation in ATLL. Tax activates NF-κB by interacting with IKKγ (5). In addition, Tax activates Akt by inhibition of PTEN (31). We then examined the effect of HI-TOPK-032 on Tax expression. As shown in Fig. 5, the amount of Tax in HUT-102 cells decreased progressively with the increasing concentrations of HI-TOPK-032. Therefore, Tax may also be a target of HI-TOPK-032.

To address the vital mechanisms of HI-TOPK-032-induced cell death, we also used DNA microarray analysis and compared the gene expression profiles of treated and untreated cells. We selected the evaluation of only genes that were upregulated by >20-fold (see the list of these genes in Table II). One notable conclusion that can be drawn from Table II is that genes associated with IFN regulation (IFIT1-3, OASL and IFI44) are the most highly upregulated by HI-TOPK-032 in two cell lines. IFIT1 plays a key role in suppression of growth and promotion of apoptosis of cancer cells (32). IFIT2 promotes apoptosis through a mitochondrial pathway dependent on the action of Bcl-2 proteins (33). Bax and Bak are required for apoptosis in response to IFIT2 (33). IFIT3 is also an anti-proliferative IFN-induced protein (34). It was important to confirm the microarray results with RT-PCR. Similar to the results of the microarray analysis, the IFIT1 and 2 genes were overexpressed in the HUT-102 and MT-2 cells (Fig. 7A). The IFIT1-3 protein levels were also increased in the HUT-102 cells treated with HI-TOPK-032 (Fig. 7B). These results suggest the involvement of IFITs in HI-TOPK-032-induced apoptosis. In addition, similar to the results of western blot analysis, the downregulation of c-IAP2, survivin and Tax, and the upregulation of Bak were confirmed by RT-PCR (Fig. 7A). The HTLV-1 basic leucine zipper factor (HBZ), which is encoded by the minus-strand of the provirus, is linked to oncogenic transformation in addition to Tax (35). Thus, we examined the level of HBZ expression. However, HI-TOPK-032 did not affect the expression of HBZ (Fig. 7A).

We previously reported that fucoidan, a sulfated polysaccharide isolated from brown algae Cladosiphon okamuranus Tokida, induced apoptosis through the suppression of NF-κB and AP-1 in HTLV-1-infected T-cell lines (36). A recent study indicated that fucoidan directly interacts with PBK/TOPK and inhibits its kinase activity (37). As shown in Fig. 8A, fucoidan decreased the viability of HTLV-1-infected T-cell lines in a dose-dependent manner. Furthermore, fucoidan increased the cleavage of caspase−3, −8, −9 and PARP in the MT-2 cells (Fig. 8B). Based on these results, we investigated whether fucoidan influences the PBK/TOPK-Akt signaling axis. As expected, the decreased phosphorylation of PBK/TOPK and downregulation of kinase Akt was observed in the cells treated with fucoidan (Fig. 8C). On the other hand, the expression of Tax was not affected by fucoidan.

In addition to the above-mentioned experiments in the cultured cells, we examined the antitumor activity of HI-TOPK-032 in mice. HUT-102 cells were inoculated subcutaneously into the post-auricular region of SCID mice. The mice were injected intraperitoneally with either the vehicle or HI-TOPK-032 at 12.5 mg/kg 5 times a week over a period of 4 weeks. Multiple tumors were not observed in any of the mice. The maximum diameter of a single tumor and the maximum tumor volume observed were 18×18×5 mm and 1,620 mm³, respectively. Treatment of mice with HI-TOPK-032 significantly inhibited HUT-102 tumor volume and weight compared with the vehicle-treated group (Fig. 9A and B). In addition, mice appeared to tolerate the treatment with HI-TOPK-032 without any overt signs of toxicity or significant loss of body weight (16.4–18.5 and 19.3–22.5 g upon purchase and upon sacrifice, respectively), similar to the vehicle-treated group (16.4–18.2 and 19.5–21.6 g upon purchase and upon sacrifice) (Fig. 9C). These results confirmed that HI-TOPK-032 inhibited tumor growth through the inhibition of PBK/TOPK. Next, we used ELISA to determine the circulating levels of surrogate tumor markers sCD25 (38) and sCD30 (39) secreted by HUT-102 tumor xenografts. The HI-TOPK-032-treated mice exhibited a 29 and 11% decrease in sCD25 and sCD30 levels, respectively, compared with the control group, although the observed changes in these levels were not statistically signifi-cant (Fig. 9D).

H&E staining revealed a significant increase in apoptosis in mice treated with HI-TOPK-032 compared with the control group. Apoptosis was characterized by cytoplasmic condensation, chromatin hyperchromatism and condensation, and nuclear fragmentation. TUNEL assay confirmed these findings and demonstrated increased apoptosis signals in HI-TOPK-032-treated tumors (Fig. 9E). These findings support the interpretation that the observed decline in tumor growth in the HI-TOPK-032-treated tumors in vivo was caused by an increase in apoptosis.