Hideo Uchida

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.

Thalidomide for the treatment of refractory multiple myeloma: association of plasma concentrations of thalidomide and angiogenic growth factors with clinical outcome.

Recent reports showed that thalidomide has anti‐angiogenic activity and is effective for the treatment of refractory multiple myeloma (MM). We examined the relationship between the clinical efficacy and adverse effects of thalidomide and the plasma concentrations of this drug as well as angiogenic growth factors in refractory MM. Ten out of twenty‐four evaluable patients (42%) showed more than 25% reduction of M‐protein, and eight (33%) achieved more than 50% reduction. These changes were associated with restoration of anemia and recovery of normal immunoglobulin level. Somnolence and headache, constipation, peripheral neuropathy and skin rash were frequently observed, but were well tolerated. However, grade 2–4 severe neutropenia was also observed in nine cases. These adverse effects other than neutropenia occurred more frequently in the patients with higher plasma concentrations of thalidomide (≥2.0 μg/ml at 12 h after the last administration) and were readily alleviated by dose reduction. In contrast, neutropenia developed regardless of the plasma concentration. Plasma concentrations of angiogenic growth factors were frequently elevated before treatment. After thalidomide treatment, these growth factor levels tend to decrease to near‐normal ranges in responders but were still high in most non‐responders. After thalidomide treatment, plasma vascular endothelial growth factor (VEGF) level was significantly reduced in responders (P=0.025), but not in non‐responders (P=0.37). Reduction of plasma VEGF level might be an important indicator for anti‐myeloma effect of thalidomide.

Activation of the p21(CIP1/WAF1) promoter by bone morphogenetic protein-2 in mouse B lineage cells.

BMPs exert a negative growth effect on various types of cells. We have previously reported that BMP-2 inhibited the growth of HS-72 mouse hybridoma cells by inducing p21CIP1/WAF1 expression. In the present study, we demonstrated that BMP-2 activated the mouse p21CIP1/WAF1 promoter in HS-72 cells, and that a 29-base pair (b) region of the promoter (−1928/−1900 relative to the TATA box), conserved between mice and humans, was responsive to BMP-2 as well as expression of Smad1, Smad4, and constitutively active mutants of BMP type I receptors. Furthermore, an oligonucleotide containing the 29-b region was found to be associated with Smad4 and phosphorylated Smad1 in the nuclear extract of BMP-2-stimulated HS-72 cells. These results suggested that BMP-2 might activate p21CIP1/WAF1 transcription by inducing a binding of Smad4 and Smad1 to the 29-b region in HS-72 cells.

Novel variant isoform of G-CSF receptor involved in induction of proliferation of FDCP-2 cells: relevance to the pathogenesis of myelodysplastic syndrome.

Recent studies have shown that point mutations in granulocyte colony‐stimulating factor receptor (G‐CSFR) are involved in the pathogenesis of severe congenital neutropenia (SCN) and in the transformation of SCN to acute myelogenous leukemia (AML). It is reasonably speculated that the abnormalities in the signal transduction pathways for G‐CSF could be partly responsible for the pathogenesis and the development to AML in patients with myelodysplastic syndromes (MDS). Therefore, we investigated the structural and functional abnormalities of the G‐CSFR in 14 patients with MDS and 10 normal subjects. In in vitro colony forming assay, MDS samples showed reduced response to growth factors. However, G‐CSF, but not GM‐CSF and IL‐3, enhanced clonal growth in three cases of high risk patients with MDS (RAEB, RAEB‐t, and MDS having progressed to acute myeloid leukemia (AML)) and one low risk patient (RA). Eight out of 14 patients including above 4 patients demonstrated a common deletion of the G‐CSFR cDNA; a deletion of three nucleotides (2128–2130) in the juxtamembrane domain of the G‐CSFR, which resulted in a conversion of Asn630Arg631 to Lys630. To assess the functional activities of this deletion in the G‐CSFR isoform, a mutant with the same three‐nucleotide deletion was constructed by site‐directed mutagenesis. FDCP‐2 cells expressing the G‐CSFR isoform responded to G‐CSF, and exhibited proliferative responses than did those cells having wild‐type G‐CSFR. Moreover, these isoforms showed prolonged activation of STAT3 in response to G‐CSF than did the wild‐type. These results suggest that the deletion in the juxtamembrane domain of the G‐CSFR gives a growth advantage to abnormal MDS clones and may contribute to the pathogenesis of MDS.

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.

The role of TC-PTP (PTPN2) in modulating sensitivity to imatinib and interferon-α in CML cell line, KT-1 cells

T-cell protein tyrosine phosphatase (TC-PTP, also known as PTPN2) is a negative regulator of the JAK/STAT pathway. STAT5 is activated by BCR-ABL kinase and STAT1 is an important transcription factor for interferon (IFN)-α-induced signaling in chronic myeloid leukemia (CML). We used siRNA to delete TC-PTP in the CML cell line, KT-1, and examined changes in the sensitivity to imatinib and IFN-α. Suppression of TC-PTP induced activation of STAT5, leading to imatinib resistance, while prolonged phosphorylation of STAT1 was induced by IFN-α, triggering cell death in KT-1 cells. These findings suggest that TC-PTP modulates sensitivity to imatinib and IFN-α in CML.

New therapeutic approaches to acute myeloid leukemia.

Background: The heterogeneity of acute myeloid leukemia (AML) has been established by many new insights into the pathogenesis and treatment of patients with AML. Understanding the basic cellular and molecular pathogenesis of leukemic cells is vital to the development of new treatment approaches. Objective/methods: To review progress until now with agents that are showing promise in the treatment of AML, we summarize the published preclinical and clinical trials that have been completed. Results: Based on recent progress of investigations, more specifically targeted agents have been developed for the treatment of AML such as tyrosine kinase inhibitors, monoclonal antibodies, epigenetic agents, antiangiogenic agents, and farnesyl transferase inhibitors. Conclusion: In the future, in addition to performing therapeutic trials of these agents, it will be important to identify other highly specific therapeutic agents based on our evolving understanding of the biology of AML.