Tomonori Nakazato

Induction of Apoptosis in Leukemic Cells by Homovanillic Acid Derivative, Capsaicin, through Oxidative Stress Implication of Phosphorylation of p53 at Ser-15 Residue by Reactive Oxygen Species

Capsaicin (N-vanillyl-8-methyl-1-nonenamide) is a homovanillic acid derivative found in pungent fruits. Several investigators have reported the ability of capsaicin to inhibit events associated with the promotion of cancer. However, the effects of capsaicin on human leukemic cells have never been investigated. We investigated the effects of capsaicin on leukemic cells in vitro and in vivo and further examined the molecular mechanisms of capsaicin-induced apoptosis in myeloid leukemic cells. Capsaicin suppressed the growth of leukemic cells, but not normal bone marrow mononuclear cells, via induction of G0-G1 phase cell cycle arrest and apoptosis. Capsaicin-induced apoptosis was in association with the elevation of intracellular reactive oxygen species production. Interestingly, capsaicin-sensitive leukemic cells were possessed of wild-type p53, resulting in the phosphorylation of p53 at the Ser-15 residue by the treatment of capsaicin. Abrogation of p53 expression by the antisense oligonucleotides significantly attenuated capsaicin-induced cell cycle arrest and apoptosis. Pretreatment with the antioxidant N-acetyl-l-cystein and catalase, but not superoxide dismutase, completely inhibited capsaicin-induced apoptosis by inhibiting phosphorylation of Ser-15 residue of p53. Moreover, capsaicin effectively inhibited tumor growth and induced apoptosis in vivo using NOD/SCID mice with no toxic effects. We conclude that capsaicin has potential as a novel therapeutic agent for the treatment of leukemia.

Green Tea Component, Catechin, Induces Apoptosis of Human Malignant B Cells via Production of Reactive Oxygen Species

Purpose: Green tea polyphenol, (−)-epigallocatechin-3-gallate, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of (−)-epigallocatechin-3-gallate as a novel therapeutic agent for the patients with B-cell malignancies including multiple myeloma. Experimental Design: We investigated the effects of (−)-epigallocatechin-3-gallate on the induction of apoptosis in HS-sultan as well as myeloma cells in vitro and further examined the molecular mechanisms of (−)-epigallocatechin-3-gallate-induced apoptosis. Results: (−)-Epigallocatechin-3-gallate rapidly induced apoptotic cell death in various malignant B-cell lines in a dose- and time-dependent manner. (−)-Epigallocatechin-3-gallate-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials (Δψm); the release of cytochrome c, Smac/DIABLO, and AIF from mitochondria into the cytosol; and the activation of caspase-3 and caspase-9. Elevation of intracellular reactive oxygen species (ROS) production was also shown during (−)-epigallocatechin-3-gallate-induced apoptosis of HS-sultan and RPMI8226 cells as well as fresh myeloma cells. Antioxidant, catalase, and Mn superoxide dismutase significantly reduced ROS production and (−)-epigallocatechin-3-gallate-induced apoptosis, suggesting that ROS plays a key role in (−)-epigallocatechin-3-gallate-induced apoptosis in B cells. Furthermore, a combination with arsenic trioxide (As2O3) and (−)-epigallocatechin-3-gallate significantly enhanced induction of apoptosis compared with As2O3 alone via decreased intracellular reduced glutathione levels and increased production of ROS. Conclusions: (−)-Epigallocatechin-3-gallate has potential as a novel therapeutic agent for patients with B-cell malignancies including multiple myeloma via induction of apoptosis mediated by modification of the redox system. In addition, (−)-epigallocatechin-3-gallate enhanced As2O3-induced apoptosis in human multiple myeloma cells.

Emodin has a cytotoxic activity against human multiple myeloma as a Janus-activated kinase 2 inhibitor

Emodin is an active component of a traditional Chinese and Japanese medicine isolated from the root and rhizomes of Rheum palmatum L. Here, we show that emodin significantly induces cytotoxicity in the human myeloma cells through the elimination of myeloid cell leukemia 1 (Mcl-1). Emodin inhibited interleukin-6–induced activation of Janus-activated kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription 3 (STAT3), followed by the decreased expression of Mcl-1. Activation of caspase-3 and caspase-9 was triggered by emodin, but the expression of other antiapoptotic Bcl-2 family members, except Mcl-1, did not change in the presence of emodin. To clarify the importance of Mcl-1 in emodin-induced apoptosis, the Mcl-1 expression vector was introduced into the human myeloma cells by electroporation. Induction of apoptosis by emodin was almost abrogated in Mcl-1–overexpressing myeloma cells as the same level as in parental cells, which were not treated with emodin. In conclusion, emodin inhibits interleukin-6–induced JAK2/STAT3 pathway selectively and induces apoptosis in myeloma cells via down-regulation of Mcl-1, which is a good target for treating myeloma. Taken together, our results show emodin as a new potent anticancer agent for the treatment of multiple myeloma patients. [Mol Cancer Ther 2007;6(3):987–94]

Zerumbone, a bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas- and mitochondria-mediated pathway

We demonstrated here for the first time that zerumbone (ZER), a natural cyclic sesquiterpene, significantly suppressed the proliferation of promyelocytic leukemia NB4 cells among several leukemia cell lines, but not human umbilical vein endothelial cells (HUVECs), by inducing G2/M cell cycle arrest followed by apoptosis with 10 µM of IC50. Treatment of NB4 cells with growth‐suppressive concentrations of ZER resulted in G2/M cell cycle arrest that was associated with a decline of Cyclin B1 protein, but with the phosphorylation of ATM/Chk1/Chk2. In addition, ZER induced the phosphorylation of Cdc25C at the Thr48 residue and Cdc2 at the Thr14/Tyr15 residues. Furthermore, ZER‐induced apoptosis in NB4 cells was initiated by the expression of Fas (CD95)/Fas Ligand (CD95L), concomitant with the activation of caspase‐8. ZER was also found to induce the cleavage of Bid, a mediator that is known to connect the Fas/CD95 cell death receptor to the mitochondrial apoptosis pathway. ZER also induced the cleavage of Bax and Mcl‐1 proteins, but not Bcl‐2 or Bcl‐XL. ZER‐induced apoptosis took place in association with a loss of the mitochondrial transmembrane potential as well as the activation of caspase‐3 and ‐9, resulting in the degradation of the proteolytic poly (ADP‐ribose) polymerase (PARP). ZER also triggered a release of cytochrome c into the cytoplasm. Both antagonistic anti‐Fas antibody ZB4 and pan‐caspase inhibitor Z‐VAD inhibited ZER‐induced apoptosis in NB4 cells. Taken together, ZER is an inducer of apoptosis in leukemic cells that specifically triggers the Fas/CD95‐ and mitochondria‐mediated apoptotic signaling pathway. (Cancer Sci 2007; 98: 118–126)

Cantharidin induces apoptosis of human multiple myeloma cells via inhibition of the JAK/STAT pathway

Multiple myeloma is an incurable B‐cell malignancy requiring new therapeutic strategies in clinical settings. Interleukin (IL)–6 signaling pathways play a critical role in the pathogenesis of multiple myeloma. The traditional Chinese medicine cantharidin (CTD) has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of CTD as a novel therapeutic agent for the patients with multiple myeloma. We investigated the in vitro effects of CTD for its antimyeloma activity, and further examined the molecular mechanisms of CTD‐induced apoptosis. CTD inhibited the cellular growth of human myeloma cell lines as well as freshly isolated myeloma cells in patients. Cultivation with CTD induced apoptosis of myeloma cells in a cell‐cycle‐independent manner. Treatment with CTD induced caspase‐3, –8, and –9 activities, and it was completely blocked by each caspase inhibitor. We further examined the effect of CTD on the IL‐6 signaling pathway in myeloma cells, and found that CTD inhibited phosphorylation of STAT3 at tyrosine 705 residue as early as 1 h after treatment and down‐regulated the expression of the antiapoptotic bcl‐xL protein. STAT3 directly bound and activated the transcription of bcl‐xL gene promoter, resulting in the induction of the expression of bcl‐xL in myeloma cells. The essential role of STAT3 in CTD effects was confirmed by transfection with the constitutively active and dominant negative form of STAT3 in U266 cells. In conclusion, we have demonstrated that CTD is a promising candidate to be a new therapeutic agent in signal transduction therapy. (Cancer Sci 2008; 99: 1820–1826)

Caffeine induces G2/M arrest and apoptosis via a novel p53-dependent pathway in NB4 promyelocytic leukemia cells.

Methylxantine derivative, caffeine, is known to prevent the p53‐dependent apoptosis pathway via inhibition of ATM (ataxia telangiectasia mutated) kinase, which activates p53 by phosphorylation of the Ser‐15 residue. In contrast, it has been reported that caffeine induces p53‐mediated apoptosis through Bax protein in non‐small‐cell lung cancer cells. Therefore, the effects of caffeine on cellular growth in malignant cells are controversial. We investigated the effects of caffeine on cell proliferation, cell cycle progression, and induction of apoptosis in NB4 promyelocytic leukemia cells containing wild‐type p53. Caffeine suppressed the cellular growth of NB4 cells in a dose‐ and time‐dependent manner. Caffeine induced G2/M phase cell cycle arrest in NB4 cells in association with the induction of phosphorylation at the Ser‐15 residue of p53 and induction of tyrosine phosphorylation of cdc2. Expression of Bax protein was increased in NB4 cells after treatment with caffeine. Interestingly, the antisense oligonucleotides for p53 significantly reduced p53 expression and caffeine‐induced G2/M phase cell cycle arrest in NB4 cells. These results suggest that caffeine induces cell cycle arrest and apoptosis in association with activation of p53 by a novel pathway to phosphorylate the Ser‐15 residue and induction of phosphorylation of cdc 2 in leukemic cells with normal p53.

Dose-adjusted preemptive therapy for cytomegalovirus disease based on real-time polymerase chain reaction after allogeneic hematopoietic stem cell transplantation

We have prospectively evaluated the efficacy of real-time PCR-guided preemptive therapy for CMV diseases in allogeneic hematopoietic stem cell transplant recipients with grades II–IV acute GVHD. The dose of ganciclovir was adjusted according to the viral load determined by real-time polymerase chain reaction (PCR). On detecting CMV reactivation in the plasma, ganciclovir was initiated at a dose of 5 mg/kg body weight once daily, and the dose was increased to twice daily if viral load continued to increase after initiating ganciclovir. In 39 evaluable patients, CMV reactivation assessed by real-time PCR became positive in 30 (77%). One developed CMV gastroenteritis before PCR became positive. Thus the remaining 29 patients were treated preemptively with ganciclovir. The dose of ganciclovir was increased in 12 patients (41%) of preemptively treated patients for increasing viral load. CMV diseases were diagnosed in two patients (one gastroenteritis and one retinitis), and late CMV disease was diagnosed in one patient (gastritis). The treatment was generally well-tolerated, but three patients (10%) developed neutropenia (neutrophil count less than 1.0 × 109/l). In conclusion, real-time PCR-guided preemptive therapy with decreased dose of ganciclovir is feasible and does not increase the frequency of CMV diseases if the dose is adjusted according to the viral load.

Myeloperoxidase Is a Key Regulator of Oxidative Stress–Mediated Apoptosis in Myeloid Leukemic Cells

Purpose: We reported previously that reactive oxygen species (ROS) are key mediators of apoptosis induced by a polyphenol, (−)-epigallocatechin-3-gallate (EGCG), in myeloid leukemic cells. This study aimed to further examine the mechanism of ROS-mediated apoptosis induced by EGCG and its relationship to the heme enzyme myeloperoxidase (MPO). Experimental Design: We established stably transfected K562 cells expressing wild-type and mutant MPO. Then, sensitivity against EGCG and other ROS-inducing agent was examined and further investigated the detailed molecular mechanism of ROS-inducing apoptosis in MPO-positive leukemic cells. Results: EGCG rapidly induced apoptosis in MPO-positive leukemia cells. Preincubation of myeloid leukemic cells with the MPO-specific inhibitor, 4-aminobenzoic acid hydrazide, and the heme biosynthesis inhibitor, succinylacetone, resulted in inhibition of the intracellular MPO activity, ROS production, and induction of apoptosis following addition of EGCG. Overexpression of MPO sensitized EGCG-resistant K562 cells to apoptosis induced by EGCG. In contrast, an enzymatically inactive MPO mutant–expressing K562 cell could not respond to EGCG, suggesting that MPO is important for determining the sensitivity to EGCG-induced oxidative stress. Hypochlorous acid scavengers and the hydroxyl radical (·OH) scavenger inhibited EGCG-induced apoptosis in myeloid leukemic cells. The fluorescence intensity of both aminophenyl fluorescein– and hydroxyphenyl fluorescein–loaded myeloid leukemic cells significantly increased on stimulation with EGCG, indicating that EGCG generated highly toxic ROS in myeloid leukemic cells. Conclusions: These results indicated that highly toxic ROS such as ·OH generated via the hydrogen peroxide/MPO/halide system induce apoptosis and that ROS may be the direct mediators of EGCG-induced apoptosis in MPO-positive leukemic cells.

Induction of apoptosis in human myeloid leukemic cells by 1'-acetoxychavicol acetate through a mitochondrial- and Fas-mediated dual mechanism.

Purpose: The purpose of this investigation was to determine the antileukemic effects of 1′-acetoxychavicol acetate (ACA) obtained from rhizomes of the commonly used ethno-medicinal plant Languas galanga (Zingiberaceae). Experimental Design: We evaluated the effects of ACA on various myeloid leukemic cells in vitro and in vivo. We further examined the molecular mechanisms of ACA-induced apoptosis in myeloid leukemic cells. Results: Low-dose ACA dramatically inhibited cellular growth of leukemic cells by inducing apoptosis. Because NB4 promyelocytic leukemic cells were most sensitive to ACA, we used NB4 cells for further analyses. Production of reactive oxygen species triggered ACA-induced apoptosis. ACA-induced apoptosis in NB4 cells was in association with the loss of mitochondrial transmembrane potential (ΔΨm) and activation of caspase-9, suggesting that ACA-induced death signaling is mediated through a mitochondrial oxygen stress pathway. In addition, ACA activated Fas-mediated apoptosis by inducing of casapse-8 activity. Pretreatment with the thiol antioxidant N-acetyl-l-cysteine (NAC) did not inhibit caspase-8 activation, and the antagonistic anti-Fas antibody ZB4 did not block generation of reactive oxygen species, indicating that both pathways were involved independently in ACA-induced apoptosis. Furthermore, ACA had a survival advantage in vivo in a nonobese diabetic/severe combined immunodeficient mice leukemia model without any toxic effects. Conclusions: We conclude that ACA induces apoptosis in myeloid leukemic cells via independent dual pathways. In addition, ACA has potential as a novel therapeutic agent for the treatment of myeloid leukemia.

Effects of immunosuppressive agents on magnesium metabolism early after allogeneic hematopoietic stem cell transplantation

Background. The calcineurin inhibitors, cyclosporine A (CSA) and tacrolimus, cause hypomagnesemia by suppressing reabsorption of magnesium (Mg) from renal tubules. To assess whether the effect on Mg metabolism after allogeneic hematopoietic stem cell transplantation (HSCT) differs among calcineurin inhibitors, we prospectively evaluated the Mg metabolism in recipients of allogeneic HSCT who received CSA or tacrolimus Methods. Patients who underwent allogeneic HSCT were enrolled. CSA and tacrolimus were given by continuous infusion starting from day –1. Serum Mg and the total amount of urinary Mg excretion were measured once before starting of CSA or tacrolimus, and once weekly after HSCT for 4 weeks. Mg was supplemented with magnesium l-aspartate by continuous infusion to maintain the serum Mg level >1.4 mEq/L. Results. Thirty-six patients were evaluated (12 in the CSA group, 24 in the tacrolimus group). The serum Mg level began to decrease in both groups at the first week after HSCT, and the mean serum Mg levels were significantly lower in the tacrolimus group than in the CSA group from the first to the third week. The total amount of urinary Mg excretion and Mg supplementation began to increase in both groups at the second week after HSCT, and the amounts in the tacrolimus group were significantly higher than those in the CSA group. Conclusions. Although both calcineurin inhibitors increased urinary Mg excretion and caused hypomagnesemia shortly after HSCT, the effect was more significant with tacrolimus than with CSA. This observation may explain the higher incidence of renal impairment and encephalopathy in patients receiving tacrolimus.