Morihiko Sagawa

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)

The gold compound auranofin induces apoptosis of human multiple myeloma cells through both down-regulation of STAT3 and inhibition of NF-κB activity

Constitutive activation of NF-κB and STAT3 plays an important role in the cellular proliferation and survival of multiple myeloma cells. We first found that auranofin (AF), a coordinated gold compound, induced a significant level of cell cycle arrest at G1 phase and subsequent apoptosis of myeloma cells. Further, AF inhibited constitutive and IL-6-induced activation of JAK2 and phosphorylation of STAT3 followed by the decreased expression of Mcl-1. AF down-regulated the activation of NF-κB, and the combination of AF and a specific NF-κB inhibitor resulted in a marked decrease of Mcl-1 expression. These results suggest that AF inhibits both IL-6 induced-JAK/STAT pathway and NF-κB activation in myeloma cells.

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.

Clinicopathological analyses in patients with other iatrogenic immunodeficiency-associated lymphoproliferative diseases and rheumatoid arthritis

Abstract Despite numerous attempts to uncover the mechanism of other iatrogenic immunodeficiency-associated lymphoproliferative diseases (OIIA-LPDs), this mechanism remains poorly understood, especially in rheumatoid arthritis (RA) patients. We analyzed the data on 23 patients with LPDs and RA. Patients were categorized into three groups according to whether they had methotrexate (MTX); MTX-regressive LPDs, MTX-persistent LPDs or other drugs-mediated LPDs. The LPDs seen in OIIA-LPDs-RA might have a unique behavior to think about several rare phenotypes. The overall survival of all patients was 74% at 5 years, and those of the three groups were 100%, 64% and 60%, respectively. Among the 6 patients who died, 4 had LPDs that were detected late, and thus adequate treatment was not given. In addition, several patients with diffuse large B cell lymphoma with a complex karyotype achieved complete remission (CR). Only one among the 17 patients who achieved CR relapsed. OIIA-LPDs-RA appeared to have a better prognosis than other more common types of lymphomas. Regarding RA treatment, various anti-RA drugs were given to the patients after developing LPDs, including MTX, but no recurrent patients were documented.

A new disulfide-linked dimer of a single-chain antibody fragment against human CD47 induces apoptosis in lymphoid malignant cells via the hypoxia inducible factor-1α pathway

CD47 belongs to the immunoglobulin superfamily and is associated with β‐integrins. Recently it was reported that CD47 ligation rapidly induces apoptosis in B‐chronic lymphocytic leukemia (CLL) cells. Chronic lymphocytic leukemia is still an incurable hematological malignancy even with the novel therapeutic agents; therefore, new and effective agents for the treatment of CLL in clinical settings are urgently needed. We generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Subsequently, we created a disulfide‐stabilized dimer of a single‐chain antibody fragment of MABL (S‐S diabody) to get rid of the adverse effect of MABL such as hemagglutination. In this study, we analyzed the effects of this new antibody on cellular proliferation, and the molecular mechanism of CD47‐mediated apoptosis in human lymphoid malignant cells. Treatment with S‐S diabody alone induced apoptosis of CD47‐positive primary B‐CLL and leukemic cells (MOLT‐4 and JOK‐1). In addition, administration of S‐S diabody significantly prolonged the survival of severe combined immunodeficiency (SCID) mice inoculated with JOK‐1 cells. In gene expression profiling of the S‐S diabody‐treated MOLT‐4 cells, hypoxia inducible factor (HIF)‐1α downstream genes (RTP801 and BNIP3) were upregulated, and the mRNA expression levels of HIF‐1α, RTP801 and BNIP3 were increased. Knockdown of HIF‐1α by siRNA repressed S‐S diabody‐induced apoptosis in MOLT4 cells. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies, and S‐S diabody might have potential as a novel therapeutic agent for B‐CLL. (Cancer Sci 2011; 102: 1208–1215)

Quinone methide tripterine, celastrol, induces apoptosis in human myeloma cells via NF-κB pathway

Multiple myeloma is still an incurable hematological malignancy despite the development of high-dose chemotherapy with stem cell transplantation. However, the therapeutic approach for multiple myeloma has progressed significantly in the last decade. Novel agents such as bortezomib, thalidomide and lenalidomide have been introduced in clinics as expanded treatment options and have improved the outcomes of patients with multiple myeloma. More recently, the development of novel agents with better effects and lower side-effects for the treatment of multiple myeloma has became necessary in the clinical setting. Celastrol is a quinone methide triterpene derived from the medicinal plant Tripterygium wilfordii, which has been used to treat chronic inflammatory and autoimmune diseases. It also has been reported that celastrol has potential as an anticancer agent; however, the effects of celastrol against myeloma have never been reported. It has been reported that the mechanisms of action occur via the NF-κB pathway. However, the effects of celastrol against multiple myeloma have never been reported. The recent clinical success of proteasome inhibitor bortezomib, which acts by inhibiting the NF-κB activity in patients with multiple myeloma led us to investigate the effects of celastrol on myeloma cells. Here we found for the first time that celastrol induces cell cycle arrest at the G1 phase followed by apoptosis in human myeloma cell line U266 cells. In addition, we showed that celastrol induces apoptosis of myeloma cells via activation of the caspase-3 and NF-κB pathways. These results suggest that celastrol would be an effective therapeutic agent in signal transduction therapy for the treatment of patients with multiple myeloma.

Induction of apoptosis via the modulation of reactive oxygen species (ROS) production in the treatment of myeloid leukemia.

Recent advances in genetic and molecular biology have provided greater insight into the biology of acute myeloid leukemia (AML). These investigations have shown that AML is a heterogeneous disease of biologically different entities. Current therapeutic approaches to AML are based on chemotherapy, but the side effects of the drugs used and various complications, including infections and bleeding, are sometimes fatal. In addition, responses to therapy and long-term outcome differ depending on the subentity in question. Therefore, it is essential to develop new therapeutic strategies such as biology adapted treatment based on the individual molecular pathogenesis of AML. Natural compounds appear to be safer than the current chemotherapeutic drugs, and we have therefore sought new potential agents among various natural compounds with the ability to induce the apoptosis of myeloid leukemic cells. Recently, we found that a highly toxic reactive oxygen species (ROS) generated via the hydrogen peroxide/myeloperoxidase [H(2)O(2)/MPO/halide] system by natural compounds induces apoptosis in MPO-positive leukemic cells. This result is of great interest in establishing novel therapeutic approaches to AML mediated through ROS-induced apoptosis of leukemic cells.

Gossypol induces apoptosis in multiple myeloma cells by inhibition of interleukin-6 signaling and Bcl-2/Mcl-1 pathway

Multiple myeloma (MM) is a clonal plasma cell disorder affecting the immune system with various systemic symptoms. MM remains incurable even with high dose chemotherapy using conventional drugs, thus necessitating development of novel therapeutic strategies. Gossypol (Gos) is a natural polyphenolic compound extracted from cotton plants, and has been shown to possess anti-neoplastic activity against various tumors. Recent studies have shown that Gos is an inhibitor for Bcl-2 or Bcl-XL acting as BH3 mimetics that interfere interaction between pro-apoptotic BH3-only proteins and Bcl-2/Bcl-XL. Since most of the patients with MM overexpress Bcl-2 protein, we considered Gos might be a promising therapeutic agent for MM. We herein show that Gos efficiently induced apoptosis and inhibited proliferation of the OPM2 MM cell line, in a dose- and time-dependent manner. Gos induced activation of caspase-3 and cytochrome c release from mitochondria, showing mitochondrial dysfunction pathway is operational during apoptosis. Further investigation revealed that phosphorylation of Bcl-2 at serine-70 was attenuated by Gos treatment, while protein levels were not affected. In addition, Mcl-1 was downregulated by Gos. Interestingly, phosphorylation of JAK2, STAT3, ERK1/2 and p38MAPK was inhibited by Gos-treatment, indicating that Gos globally suppressed interleukin-6 (IL-6) signals. Moreover, JAK2 inhibition mimicked the effect of Gos in OPM2 cells including Bcl-2 dephosphorylation and Mcl-1 downregulation. These results demonstrated that Gos induces apoptosis in MM cells not only through displacing BH3-only proteins from Bcl-2, but also through inhibiting IL-6 signaling, which leads to Bcl-2 dephosphorylation and Mcl-1 downregulation.

Triptolide induces apoptotic cell death of multiple myeloma cells via transcriptional repression of Mcl-1

Triptolide, a diterpenoid trioxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been used as a natural medicine in China for hundreds of years. Several reports have demonstrated that triptolide inhibits the proliferation of cancer cells in vitro and reduces the growth of several types of tumors in vivo. To address the potential of triptolide as a novel therapeutic agent for patients with multiple myeloma, we investigated the effects of triptolide on the induction of apoptosis in human multiple myeloma cells in vitro. Triptolide rapidly induces apoptotic cell death in various myeloma cell lines. Triptolide-induced apoptosis in myeloma cells is associated with the loss of mitochondrial transmembrane potential (∆ψm), the release of cytochrome c and Smac/DIABLO from mitochondria into the cytosol, and the activation of caspase-3 and caspase-9. Furthermore, triptolide induces a rapid decline in the levels of Mcl-1 protein that correlates with caspase activation and induction of apoptosis. Inhibition of Mcl-1 synthesis by triptolide occurs at the level of mRNA transcription and is associated with an inhibition of phosphorylation of RNA polymerase II CTD. These results indicate that Mcl-1 is an important target for triptolide-induced apoptosis in myeloma cells that occurs via inhibition of Mcl-1 mRNA transcription coupled with rapid protein degradation through the ubiquitin-proteasome pathway.

Successful treatment with a modified bortezomib schedule of weekly and longer intervals for patients with refractory/resistance multiple myeloma

Bortezomib is a potent agent for multiple myeloma (MM); however, severe treatment-related toxicities such as peripheral neuropathy have been observed in conjunction with its use. In this study, we present the cases of 9 patients with refractory MM whose administration schedule was modified from twice weekly to an interval of once weekly or longer mainly due to adverse events. The average duration from diagnosis to the time of bortezomib induction was 56 months. The schedule was changed to the modified administration according to the physicians discretion. The average duration of modified treatment was 16 months. Six patients with IgG or IgA subtype showed more than a minor response. One patient with BJP had stable disease for 3 years, and the other BJP-type patient with extramedullary plasmacytomas showed remarkable tumor regression. The treatment-related toxicities of this strategy were mild and tolerable. To our knowledge, this is the first report of the administration of bortezomib at intervals longer than once weekly.