Clinicopathological characteristics associated with the engraftment of patient lymphoma cells in NOG mice.

Patient-derived xenograft (PDX) mouse models are useful for deepening our understanding of the biology of malignant lymphoma; however, factors associated with the success of the PDX lymphoma model are largely unknown. We retrospectively analyzed the characteristics of 66 xenotransplantations from 65 patients. In all, 43 (65%) specimens were obtained from patients aged > 60 years, and 42 (64%) specimens were obtained at diagnosis. Specimens were obtained from patients with the following diseases: diffuse large B-cell lymphoma (n = 30), intravascular large B-cell lymphoma (n = 12), follicular lymphoma (n = 8), peripheral T-cell lymphoma (n = 7), mantle cell lymphoma (n = 2), and other (n = 7). The specimens were sourced mainly from bone marrow (n = 31, 47%) and extranodal tumors (n = 13, 20%). Engraftment was successful in 33/66 (50%) xenotransplantations. The median age of patients who provided successful specimens was significantly higher than that for unsuccessful specimens (p = 0.013). Specimens with a high proportion of tumor cells in the graft and those obtained from patients with relapsed/refractory disease showed higher tendencies toward successful engraftment. Taken together, these data suggest that tumor cells with a highly malignant potential might have a high likelihood of engraftment.

Exosomes secreted from cancer-associated fibroblasts elicit anti-pyrimidine drug resistance through modulation of its transporter in malignant lymphoma.

The tumor microenvironment is deeply involved in the process of tumor growth and development. In this study, we focused on cancer-associated fibroblasts (CAFs) and their derived exosomes on the lymphoma microenvironment to uncover their clinical significance. CAFs were established from primary lymphoma samples, and exosomes secreted from CAFs were obtained by standard procedures. We then investigated the roles of CAFs and their derived exosomes in the survival and drug resistance of lymphoma cells. CAFs supported the survival of lymphoma cells through increased glycolysis, and the extent differed among CAFs. Exosomes were identified as a major component of the extracellular vesicles from CAFs, and they also supported the survival of lymphoma cells. The suppression of RAB27B, which is involved in the secretion of exosomes, using a specific siRNA resulted in reduced exosome secretion and decreased survival of lymphoma cells. Moreover, anti-pyrimidine drug resistance was induced in the presence of exosomes through the suppression of the pyrimidine transporter, equilibrative nucleoside transporter 2 (ENT2), and the suppression of ENT2 was significant in in vivo experiments and clinical samples. RNA sequencing analysis of miRNAs in exosomes identified miR-4717-5p as one of the most abundant miRNAs in the exosome, which suppressed the expression of ENT2 and induced anti-pyrimidine drug resistance in vitro. Our results suggest that exosomes including miR-4717-5p secreted from CAFs play a pivotal role in the lymphoma microenvironment, indicating that they are a promising therapeutic target.

PDE3 inhibitor and EGCG combination treatment suppress cancer stem cell properties in pancreatic ductal adenocarcinoma.

Recurrence following chemotherapy is observed in the majority of patients with pancreatic ductal adenocarcinoma (PDAC). Recent studies suggest that cancer stem cells (CSCs) may be involved in PDAC recurrence and metastasis. However, an efficient approach to targeting pancreatic CSCs remains to be established. Here we show that in cancer cells overexpressing the 67-kDa laminin receptor (67LR)-dependent cyclic GMP (cGMP) inducer, epigallocatechin-3-O-gallate (EGCG) and a phosphodiesterase 3 (PDE3) inhibitor in combination significantly suppressed the Forkhead box O3 and CD44 axis, which is indispensable for the CSC properties of PDAC. We confirmed that the EGCG and PDE3 inhibitor in combination strongly suppressed tumour formation and liver metastasis in vivo. We also found that a synthesized EGCG analog capable of inducing strong cGMP production drastically suppressed the CSC properties of PDAC and extended the survival period in vivo. In conclusion, the combination treatment of EGCG and a PDE3 inhibitor as a strong cGMP inducer could be a potential treatment candidate for the eradication of CSCs of PDAC.

The FOXO3/PGC-1ß signaling axis is essential for cancer stem cell properties of pancreatic ductal adenocarcinoma.

In 95% of patients with pancreatic ductal adenocarcinoma, recurrence is observed following chemotherapy. Findings from several studies have indicated that cancer stem cells (CSCs) are resistant to anticancer agents and may be involved in cancer recurrence and metastasis. The CD44 protein is a major CSC marker, and CD44 also plays an indispensable role in the CSC properties in several cancers, including pancreatic cancer; however, no clinical approach exists to inhibit CD44 activity. Here, we have performed knock-in/knockdown experiments, and we demonstrate that the forkhead box O3 (FOXO3)/liver kinase B1 (LKB1)/AMP-activated protein kinase/peroxisome proliferator-activated receptor-γ co-activator-1ß (PGC-1ß)/pyruvate dehydrogenase-A1 pathway is essential for CD44 expression and CSC properties. We observed that patients exhibiting high pyruvate dehydrogenase-A1 expression have a poor prognosis. Systemic PGC-1ß knock-out mice are fertile and viable and do not exhibit an overt phenotype under normal conditions. This suggests that cGMP induction and PGC-1ß inhibition represent potential strategies for treating patients with pancreatic ductal adenocarcinoma.

Green tea polyphenol epigallocatechin-O-gallate induces cell death by acid sphingomyelinase activation in chronic myeloid leukemia cells.

An epidemiological study showed that green tea consumption is associated with a reduced risk of hematopoietic malignancy. The major green tea polyphenol epigallocatechin­3-O-gallate (EGCG) is reported to have anticancer effects. Chronic myeloid leukemia (CML) is a major hematopoietic malignancy characterized by expansion of myeloid cells. In the present study, we showed EGCG-induced acid sphingomyelinase (ASM) activation and lipid raft clustering in CML cells. The ASM inhibitor desipramine significantly reduced EGCG-induced cell death. Protein kinase Cδ is a well­known kinase that plays an important role in ASM activation. We observed EGCG-induced phosphorylation of protein kinase Cδ at Ser664. Importantly, EGCG-induced ASM activation was significantly reduced by pretreatment of CML cells with the soluble guanylate cyclase inhibitor NS2028, suggesting that EGCG induced ASM activation through the cyclic guanosine monophosphate (cGMP)-dependent pathway. Indeed, pharmacological inhibition of a cGMP-negative regulator enhanced the anti-CML effect of EGCG. These results indicate that EGCG-induced cell death via the cGMP/ASM pathway in CML cells.

Metabolic profiling-based data-mining for an effective chemical combination to induce apoptosis of cancer cells.

Green tea extract (GTE) induces apoptosis of cancer cells without adversely affecting normal cells. Several clinical trials reported that GTE was well tolerated and had potential anti-cancer efficacy. Epigallocatechin-3-O-gallate (EGCG) is the primary compound responsible for the anti-cancer effect of GTE; however, the effect of EGCG alone is limited. To identify GTE compounds capable of potentiating EGCG bioactivity, we performed metabolic profiling of 43 green tea cultivar panels by liquid chromatography-mass spectrometry (LC-MS). Here, we revealed the polyphenol eriodictyol significantly potentiated apoptosis induction by EGCG in vitro and in a mouse tumour model by amplifying EGCG-induced activation of the 67-kDa laminin receptor (67LR)/protein kinase B/endothelial nitric oxide synthase/protein kinase C delta/acid sphingomyelinase signalling pathway. Our results show that metabolic profiling is an effective chemical-mining approach for identifying botanical drugs with therapeutic potential against multiple myeloma. Metabolic profiling-based data mining could be an efficient strategy for screening additional bioactive compounds and identifying effective chemical combinations.

The enhancing effect of fucoidan derived from Undaria pinnatifida on immunoglobulin production by mouse spleen lymphocytes.

In this study, we revealed that a Mekabu (Udaria pinnantifida) extract enhanced immunoglobulin (Ig) production of mouse spleen lymphocytes. Furthermore, it was suggested that water-soluble and high molecular weight ingredients in the Mekabu extract have significant enhancing effect on Ig production. Therefore, fucoidan was estimated as the active component.

Phosphodiesterase 5 inhibitor acts as a potent agent sensitizing acute myeloid leukemia cells to 67-kDa laminin receptor-dependent apoptosis.

(-)-Epigallocatechin-3-O-gallate (EGCG), a polyphenol in green tea, induces apoptosis in acute myeloid leukemia (AML) cells without affecting normal cells. In this study, we observed that cGMP acts as a cell death mediator of the EGCG-induced anti-AML effect through acid sphingomyelinase activation. EGCG activated the Akt/eNOS axis, a well-known mechanism in vascular cGMP upregulation. We also observed that a major cGMP negative regulator, phosphodiesterase 5, was overexpressed in AML cells, and PDE5 inhibitor, an anti-erectile dysfunction drug, synergistically enhanced the anti-AML effect of EGCG. This combination regimen killed AML cells via overexpressed 67-kDa laminin receptors.

Cardiac and skeletal muscle abnormality in taurine transporter-knockout mice.

Taurine, a sulfur-containing beta-amino acid, is highly contained in heart and skeletal muscle. Taurine has a variety of biological actions, such as ion movement, calcium handling and cytoprotection in the cardiac and skeletal muscles. Meanwhile, taurine deficiency leads various pathologies, including dilated cardiomyopathy, in cat and fox. However, the essential role of taurine depletion on pathogenesis has not been fully clarified. To address the physiological role of taurine in mammalian tissues, taurine transporter-(TauT-) knockout models were recently generated. TauTKO mice exhibited loss of body weight, abnormal cardiac function and the reduced exercise capacity with tissue taurine depletion. In this chapter, we summarize pathological profile and histological feature of heart and skeletal muscle in TauTKO mice.

Connective tissue growth factor induces cardiac hypertrophy through Akt signaling.

In the process of cardiac remodeling, connective tissue growth factor (CTGF/CCN2) is secreted from cardiac myocytes. Though CTGF is well known to promote fibroblast proliferation, its pathophysiological effects in cardiac myocytes remain to be elucidated. In this study, we examined the biological effects of CTGF in rat neonatal cardiomyocytes. Cardiac myocytes stimulated with full length CTGF and its C-terminal region peptide showed the increase in cell surface area. Similar to hypertrophic ligands for G-protein coupled receptors, such as endothelin-1, CTGF activated amino acid uptake; however, CTGF-induced hypertrophy is not associated with the increased expression of skeletal actin or BNP, analyzed by Northern-blotting. CTGF treatment activated ERK1/2, p38 MAPK, JNK and Akt. The inhibition of Akt by transducing dominant-negative Akt abrogated CTGF-mediated increase in cell size, while the inhibition of MAP kinases did not affect the cardiac hypertrophy. These findings indicate that CTGF is a novel hypertrophic factor in cardiac myocytes.

Taurine depletion caused by knocking out the taurine transporter gene leads to cardiomyopathy with cardiac atrophy.

The sulfur-containing beta-amino acid, taurine, is the most abundant free amino acid in cardiac and skeletal muscle. Although its physiological function has not been established, it is thought to play an important role in ion movement, calcium handling, osmoregulation and cytoprotection. To begin examining the physiological function of taurine, we generated taurine transporter- (TauT-) knockout mice (TauTKO), which exhibited a deficiency in myocardial and skeletal muscle taurine content compared with their wild-type littermates. The TauTKO heart underwent ventricular remodeling, characterized by reductions in ventricular wall thickness and cardiac atrophy accompanied with the smaller cardiomyocytes. Associated with the structural changes in the heart was a reduction in cardiac output and increased expression of heart cardiac failure (fetal) marker genes, such as ANP, BNP and beta-MHC. Moreover, ultrastructural damage to the myofilaments and mitochondria was observed. Further, the skeletal muscle of the TauTKO mice also exhibited decreased cell volume, structural defects and a reduction of exercise endurance capacity. Importantly, the expression of Hsp70, ATA2 and S100A4, which are upregulated by osmotic stress, was elevated in both heart and skeletal muscle of the TauTKO mice. Taurine depletion causes cardiomyocyte atrophy, mitochondrial and myofiber damage and cardiac dysfunction, effects likely related to the actions of taurine. Our data suggest that multiple actions of taurine, including osmoregulation, regulation of mitochondrial protein expression and inhibition of apoptosis, collectively ensure proper maintenance of cardiac and skeletal muscular structure and function.