Yoko Nakagawa

β-Catenin Small Interfering RNA Successfully Suppressed Progression of Multiple Myeloma in a Mouse Model

Purpose: β-catenin is the downstream effector of the Wnt signaling pathway, and it regulates cell proliferation. β-catenin overexpression correlates positively with prognosis in several types of malignancies. We herein assessed its effects on growth of multiple myeloma cells using a xenograft model. Experimental Design: We first investigated the expression of β-catenin in multiple myeloma cell lines and multiple myeloma cells obtained from patients. Next, we investigated the growth inhibitory effects of β-catenin small interfering RNA on the growth of multiple myeloma cells in vivo. Six-week-old male BALB/c nu/nu mice were inoculated s.c. in the right flank with 5 × 106 RPMI8226 cells, followed by s.c. injections of β-catenin small interfering RNA, scramble small interfering RNA, or PBS/atelocollagen complex twice a week for a total of eight injections. Results: Significantly higher levels of β-catenin expression were observed in multiple myeloma cell lines and in samples from patients with multiple myeloma than those found in mononuclear cells obtained from healthy volunteers. In in vivo experiments, no inhibitory effects were observed following treatment with scramble small interfering RNA or PBS/atelocollagen complexes, whereas treatment with β-catenin small interfering RNA/atelocollagen complex significantly inhibited growth of multiple myeloma tumors (P < 0.05). Conclusions: β-catenin small interfering RNA treatment inhibited the growth of multiple myeloma tumors in a xenograft model. To our knowledge, this is the first report showing that the treatment with β-catenin small interfering RNA produces an inhibitory effects on growth of hematologic malignancies in vivo. Because treatment with β-catenin small interfering RNA inhibited growth of multiple myeloma cells, β-catenin is the attractive novel target for treating multiple myeloma.

AV-65, a novel Wnt/β-catenin signal inhibitor, successfully suppresses progression of multiple myeloma in a mouse model.

Multiple myeloma (MM) is a malignant neoplasm of plasma cells. Although new molecular targeting agents against MM have been developed based on the better understanding of the underlying pathogenesis, MM still remains an incurable disease. We previously demonstrated that β-catenin, a downstream effector in the Wnt pathway, is a potential target in MM using RNA interference in an in vivo experimental mouse model. In this study, we have screened a library of more than 100 000 small-molecule chemical compounds for novel Wnt/β-catenin signaling inhibitors using a high-throughput transcriptional screening technology. We identified AV-65, which diminished β-catenin protein levels and T-cell factor transcriptional activity. AV-65 then decreased c-myc, cyclin D1 and survivin expression, resulting in the inhibition of MM cell proliferation through the apoptotic pathway. AV-65 treatment prolonged the survival of MM-bearing mice. These findings indicate that this compound represents a novel and attractive therapeutic agent against MM. This study also illustrates the potential of high-throughput transcriptional screening to identify candidates for anticancer drug discovery.

Anti-myeloma effect of homoharringtonine with concomitant targeting of the myeloma-promoting molecules, Mcl-1, XIAP, and β-catenin

Since a variety of cell intrinsic and extrinsic molecular abnormalities cooperatively promote tumor formation in multiple myeloma (MM), therapeutic approaches that concomitantly target more than one molecule are increasingly attractive. We herein demonstrate the anti-myeloma effect of a cephalotaxus alkaloid, homoharringtonine (HHT), an inhibitor of protein synthesis, through the induction of apoptosis. HHT significantly reduced Mcl-1, a crucial protein involved in myeloma cell survival, in all three myeloma cell lines examined, whereas certain BH3-only proteins, such as Bim, Bik, and Puma, remained unchanged following HHT treatment, and their expression levels depended on the cell type. HHT also reduced the levels of c-FLIPL/S, activated caspase-8, and induced active truncated-Bid. Thus, HHT-induced apoptosis appears to be mediated via both intrinsic and extrinsic apoptosis pathways, and the resultant imbalance between BH3-only proteins and Mcl-1 may be pivotal for apoptosis by HHT. In addition, HHT treatment resulted in reduced levels of β-catenin and XIAP proteins, which also contribute to disease progression and resistance to chemotherapy in MM. In combination, HHT enhanced the effects of melphalan, bortezomib, and ABT-737. These results suggest that HHT could constitute an attractive option for MM treatment though its ability to simultaneously target multiple tumor-promoting molecules.

Galectin-9 ameliorates acute GVH disease through the induction of T-cell apoptosis

Galectins comprise a family of animal lectins that differ in their affinity for β‐galactosides. Galectin‐9 (Gal‐9) is a tandem‐repeat‐type galectin that was recently shown to function as a ligand for T‐cell immunoglobin domain and mucin domain‐3 (Tim‐3) expressed on terminally differentiated CD4+ Th1 cells. Gal‐9 modulates immune reactions, including the induction of apoptosis in Th1 cells. In this study, we investigated the effects of Gal‐9 in murine models of acute GVH disease (aGVHD). First, we demonstrated that recombinant human Gal‐9 inhibit MLR in a dose‐dependent manner, involving both Ca2+ influx and apoptosis in T cells. Next, we revealed that recombinant human Gal‐9 significantly inhibit the progression of aGVHD in murine BM transplantation models. In conclusion, Gal‐9 ameliorates aGVHD, possibly by inducing T‐cell apoptosis, suggesting that gal‐9 may be an attractive candidate for the treatment of aGVHD.

Rakicidin A effectively induces apoptosis in hypoxia adapted Bcr-Abl positive leukemic cells

Treatment with Abl tyrosine kinase inhibitors (TKI) drastically improves the prognosis of chronic myelogenous leukemia (CML) patients. However, quiescent CML cells are insensitive to TKI and can lead to relapse of the disease. Thus, research is needed to elucidate the properties of these quiescent CML cells, including their microenvironment, in order to effectively target them. Hypoxia is known to be a common feature of solid tumors that contributes to therapeutic resistance. Leukemic cells are also able to survive and proliferate in severely hypoxic environments. The hypoxic conditions in the bone marrow (BM) allow leukemic cells that reside there to become insensitive to cell death stimuli. To target leukemic cells in hypoxic conditions, we focused on the hypoxia‐selective cytotoxin, Rakicidin A. A previous report showed that Rakicidin A, a natural product produced by the Micromonospora strain, induced hypoxia‐selective cytotoxicity in solid tumors. Here, we describe Rakicidin A‐induced cell death in hypoxia‐adapted (HA)‐CML cells with stem cell‐like characteristics. Interestingly, apoptosis was induced via caspase‐dependent and ‐independent pathways. In addition, treatment with Rakicidin A in combination with the TKI, imatinib, resulted in synergistic cytotoxicity against HA‐CML cells. In conclusion, Rakicidin A is a promising compound for targeting TKI‐resistant quiescent CML stem cells in the hypoxic BM environment. (Cancer Sci 2011; 102: 591–596)

Allograft inflammatory factor-1 is overexpressed and induces fibroblast chemotaxis in the skin of sclerodermatous GVHD in a murine model

Allograft inflammatory factor (AIF)-1 has been identified in chronic rejection of rat cardiac allografts and is thought to be involved in the immune response. We previously showed that AIF-1 was strongly expressed in synovial tissues in rheumatoid arthritis and that rAIF-1 increased the IL-6 production of synoviocytes and peripheral blood mononuclear cells. Recently, the expression of AIF-1 has been reported in systemic sclerosis (SSc) tissues, whose clinical features and histopathology are similar to those of chronic graft-vs-host disease (GVHD). To clarify the pathogenic mechanism of fibrosis, we examined the expression and function of AIF in sclerodermatous (Scl) GVHD mice. We demonstrated that immunoreactive AIF-1 and IL-6 were significantly expressed in infiltrating mononuclear cells and fibroblasts in thickened skin of Scl GVHD mice compared with control. The immunohistochemical findings were confirmed by Western blot analysis. Wound healing assay also revealed that rAIF-1 increased the migration of normal human dermal fibroblasts (NHDF) directly, but cell growth assay did not show that rAIF-1 increased the proliferation of them. These findings suggest that AIF-1, which can induce the migration of fibroblasts and the production of IL-6 in affected skin tissues, is an important molecule promoting fibrosis in GVHD. Although the biological function of AIF-1 has not been completely elucidated, AIF-1 can induce IL-6 secretion on mononuclear cells and fibroblast chemotaxis. AIF-1 may accordingly provide an attractive new target for antifibrotic therapy in SSc as well as Scl GVHD.

A combination of a DNA-chimera siRNA against PLK-1 and zoledronic acid suppresses the growth of malignant mesothelioma cells in vitro

Although novel agents effective against malignant mesothelioma (MM) have been developed, the prognosis of patients with MM is still poor. We generated a DNA-chimeric siRNA against polo-like kinase-1 (PLK-1), which was more stable in human serum than the non-chimeric siRNA. The chimeric PLK-1 siRNA inhibited MM cell proliferation through the induction of apoptosis. Next, we investigated the effects of zoledronic acid (ZOL) on MM cells, and found that ZOL also induced apoptosis in MM cells. Furthermore, ZOL augmented the inhibitory effects of the PLK-1 siRNA. In conclusion, combining a PLK-1 siRNA with ZOL treatment is an attractive strategy against MM.

Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells

Umbilical cord blood (UCB) has advantages over other tissues because it can be obtained without an invasive procedure and complex processing. We explored the availability of cryopreserved UCB cells as a source of mesenchymal stromal/stem cells (MSCs). MSCs were successfully isolated from six of 30 UCB units (median volume, 34.0 mL; median nucleated cell number, 4.4×108) that were processed and cryopreserved using CP-1/human serum albumin. This isolation rate was lower than that (57%) from non-cryopreserved UCB cells. The number of nucleated cells before and after hydroxyethyl starch separation, UCB unit volume, and cell viability after thawing did not significantly differ between UCB units from which MSCs were successfully isolated and those from which they were not. When CryoSure-DEX40 was used as a cryoprotectant, MSCs were isolated from two of ten UCB units. Logistic regression analysis demonstrated that the cryopreservation method was not significantly associated with the success of MSC isolation. The isolated MSCs had a similar morphology and surface marker expression profile as bone marrow-derived MSCs and were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. In summary, MSCs can be isolated from cryopreserved UCB cells. However, the cryopreservation process reduces the isolation rate; therefore, freshly donated UCB cells are preferable for the isolation of MSCs.

Multiple myeloma cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-β/Smad pathway activation

The emergence of new molecular targeting agents has improved the prognosis of patients with multiple myeloma (MM). However, MM remains incurable because MM stem cells are likely resistant to these agents. Thus, it is important to further investigate the biology of MM stem cells, which reside in the hypoxic bone marrow niche. In this study, we established and investigated the characteristics of hypoxia-adapted MM (HA-MM) cells, which could proliferate for more than six months under hypoxic conditions (1% O2). The G0 fraction of HA-MM cells was larger than that of parental MM cells under normoxic conditions (20% O2). HA-MM cells possess enhanced tumorigenicity in primary and secondary transplantation studies. HA-MM cells also exhibited increased mRNA levels of stem cell markers and an enhanced self-renewal ability, and thus demonstrated characteristics of MM stem cells. These cells overexpressed phosphorylated Smad2, and treatment with a transforming growth factor (TGF)-β/Smad signaling inhibitor decreased their clonogenicity in a replating assay. In conclusion, MM cells adapted to long-exposure of hypoxia exhibit stem cell characters with TGF-β/Smad pathway activation.