Chu-Tse Wu

Treatment of myocardial ischemia with bone marrow-derived mesenchymal stem cells overexpressing hepatocyte growth factor.

Mesenchymal stem cells could differentiate into cardiomyocytes in vitro and have been shown to reconstitute the impaired myocardium in vivo. Hepatocyte growth factor, a recognized angiogenic factor and endothelial cell chemoattractant, has been applied in the treatment of myocardial ischemia. In this study, we used a ligation model of proximal left anterior descending coronary artery of rats to evaluate the effect of mesenchymal stem cells overexpressing hepatocyte growth factor in the treatment of myocardial ischemia. Bone marrow-derived mesenchymal stem cells were isolated, expanded, characterized, and infected with adenovirus carrying human hepatocyte growth factor cDNA (Ad-HGF). Mesenchymal stem cells infected by Ad-HGF released soluble HGF protein at a high level, which was maintained at least for 2 weeks. Implantation of mesenchymal stem cells overexpressing hepatocyte growth factor into left anterior descending risk areas improved the functions of impaired myocardium, including diminishing the area of ischemia, increasing the number of capillaries, and reducing collagen content. By using the sry gene as a marker, we also demonstrated that the engrafted cells or their progeny incorporated into ischemic cardiac muscle. These results showed that treatment of myocardial ischemia with bone marrow-derived mesenchymal stem cells overexpressing hepatocyte growth factor could be a novel strategy that can both restore local blood flow and regenerate lost cardiomyocytes.

Hepatocyte Growth Factor Gene-Modified Mesenchymal Stem Cells Reduce Radiation-Induced Lung Injury

Abstract Effective therapeutic strategies for radiation-induced lung injury (RILI) are lacking. Mesenchymal stem cells (MSCs), as gene therapy delivery vehicles, possess the ability to repair injured lung. In this study, we conducted MSC-based hepatocyte growth factor (HGF) gene therapy for RILI. Mice received single-dose radiation with 20 Gy of γ rays locally to the lung, and then were administered normal sodium, Ad-HGF-modified MSCs, or Ad-Null-modified MSCs. Ad-HGF-modified MSCs (MSCs-HGF) improved histopathological and biochemical markers of lung injury. MSCs-HGF could reduce secretion and expression of proinflammatory cytokines, including tumor necrosis factor-α, interferon-γ, interleukin (IL)-6, and intercellular adhesion molecule-1, and increase the expression of antiinflammatory cytokine IL-10. It could also decrease expression levels of profibrosis factors transforming growth factor-β, Col1a1 (collagen type 1, α1), and Col3a1, and inhibit fibrosis progress. MSCs-HGF could promote proliferation of lung epithelial cells and protect them from apoptosis, and improve the expression of endogenous HGF and its receptor c-Met significantly. We also found that sphingosine-1-phosphate receptor-1 expression was increased in injured lung. These results suggest MSC-based HGF gene therapy not only reduces inflammation but also inhibits lung fibrosis.

Sphingosine 1-phosphate induces Mcl-1 upregulation and protects multiple myeloma cells against apoptosis.

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. However, neither the distribution of S1P receptors nor the effects of S1P on multiple myeloma (MM) cells are fully understood. Here, we show that MM cells express the S1P receptors, S1P1, S1P2, and S1P3. Furthermore, S1P protects MM cells against Dex-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time- and concentration-dependent manner in human MM cell lines. Treatment of MM cells with pertussis toxin (PTX), a pan-S1P receptor inhibitor, results in blockage of S1P-induced upregulation of Mcl-1. These data demonstrate that S1P upregulates the expression of Mcl-1 and protects MM cells from Dex-induced apoptosis, providing the preclinical framework for novel therapeutics targeting at both Mcl-1 and/or S1P to improve the patient outcome in MM.

IL-6 secreted from senescent mesenchymal stem cells promotes proliferation and migration of breast cancer cells.

Human mesenchymal stem cells (hMSCs) are currently investigated for a variety of therapeutic applications. However, MSCs isolated from primary tissue cannot meet clinical grade needs and should be expanded in vitro for several passages. Although hMSCs show low possibility for undergoing oncogenic transformation, they do, similar to other somatic cells, undergo cellular senescence and their therapeutic potential is diminished when cultured in vitro. However, the role of senescent MSCs in tumor progression remains largely elusive. In the current study, by establishing senescent human umbilical cord mesenchymal stem cells (s-UCMSCs) through the replicative senescence model and genotoxic stress induced premature senescence model, we show that s-UCMSCs significantly stimulate proliferation and migration of breast cancer cells in vitro and tumor progression in a co-transplant xenograft mouse model compared with ‘young’ counterparts (defined as MSCs at passage 5, in contrast to senescent MSCs at passage 45). In addition, we identified IL-6, a known pleiotropic cytokine, as a principal mediator for the tumor-promoting activity of s-UCMSCs by induction of STAT3 phosphorylation. Depletion of IL-6 from s-UCMSCs conditioned medium partially abrogated the stimulatory effect of s-UCMSCs on the proliferation and migration of breast tumor cells.

Activation of sphingosine kinase mediates suppressive effect of interleukin-6 on human multiple myeloma cell apoptosis

Interleukin 6 (IL‐6) influences the growth and survival of multiple myeloma (MM) cells via the activation of multiple signalling cascades. Although sphingosine kinase (SPHK) signalling is known to play important roles in the regulation of cell proliferation and apoptosis, the role of SPHK activation in IL‐6 signalling and in the pathology of MM remains unclear. This study found that IL‐6 activated SPHK in MM cells, which mediates the suppressive effects of IL‐6 on MM cell apoptosis. Both MM cell lines and primary MM cells constitutively expressed SPHK, and treatment of MM cells with IL‐6 resulted in activation of SPHK in a concentration‐dependent manner. Specific inhibitors of the phosphatidylinositol‐3 kinase and extracellular signal‐regulated kinase/mitogen‐activated protein kinase pathways blocked the IL‐6‐induced activation of SPHK. It was further demonstrated that IL‐6‐induced activation of SPHK inhibited dexamethasone‐induced apoptosis of MM cells. IL‐6 stimulation or retroviral‐mediated overexpression of SPHK1 in MM cells resulted in increased intracellular SPHK activity and upregulation of myeloid cell leukaemia‐1 (Mcl‐1), leading to increased cell proliferation and survival. Conversely, inhibition of SPHK1 by small interfering RNA reduced IL‐6‐induced upregulation of Mcl‐1 and blocked the suppressive effect of IL‐6 on MM cell apoptosis. Taken together, these results delineate a key role for SPHK activation in IL‐6‐induced proliferation and survival of MM cells, and suggest that SPHK may be a potential new therapeutic target in MM.

Isolation and functional identification of a novel human hepatic growth factor: Hepatopoietin Cn

Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion‐exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [3H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)‐based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30‐kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine‐rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. Conclusion: HPPCn is a novel hepatic growth factor that plays a role in liver regeneration. (HEPATOLOGY 2008;47:986–995.)

Adenoviral-mediated transfer of human wild-type p53, GM-CSF and B7-1 genes results in growth suppression and autologous anti-tumor cytotoxicity of multiple myeloma cells in vitro

Multiple myeloma (MM) remains incurable despite the use of high-dose chemotherapy and stem cell transplantation. However, immunotherapy is expected to offer long-term disease control, or even possibly a cure. We have previously demonstrated the suppressive effect of a recombinant adenovirus carrying human wild-type p53, granulocyte–macrophage colony-stimulating factor, and B7-1 genes (Ad-p53/GM-CSF/B7-1) on the growth of laryngeal cancer cells. In the present study, we evaluated the effects of an Ad-p53/GM-CSF/B7-1-modified myeloma cell vaccine strategy aimed to induce apoptosis and to augment the immunogenicity of MM cells. Both MM cell lines and purified primary myeloma cells were infected with Ad-p53/GM-CSF/B7-1. High expression levels of these three genes were confirmed separately by Western blot, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. When wild-type p53, GM-CSF and B7-1 genes were introduced, the growth of MM cells was inhibited via enhanced apoptosis and the immunogenicity of tumor cells was augmented. The combinatorial effect of these three genes on inducing cytotoxic T lymphocytes (CTLs) was more evident than that of p53 individually or any combinations of two (p53 plus GM-CSF or p53 plus B7-1). Furthermore, significant proliferation of autologous peripheral blood lymphocytes (PBLs) and specific cytotoxicity against autologous primary MM cells were induced in vitro. These results suggest that myeloma cell vaccination co-transferred with p53, GM-CSF and B7-1 genes may be a promising immunotherapeutic approach against MM.

The protective role of Hepatopoietin Cn on liver injury induced by carbon tetrachloride in rats

Background:  Hepatopoietin Cn (HPPCn) is a member of the leucine‐rich acidic nuclear protein family (LANP), and studies of partially hepatectomized (PH) mice show that levels of HPPCn mRNA increase following liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo.

SENP1 inhibition induces apoptosis and growth arrest of multiple myeloma cells through modulation of NF-κB signaling

SUMO/sentrin specific protease 1 (Senp1) is an important regulation protease in the protein sumoylation, which affects the cell cycle, proliferation and differentiation. The role of Senp1 mediated protein desumoylation in pathophysiological progression of multiple myeloma is unknown. In this study, we demonstrated that Senp1 is overexpressed and induced by IL-6 in multiple myeloma cells. Lentivirus-mediated Senp1 knockdown triggers apoptosis and reduces viability, proliferation and colony forming ability of MM cells. The NF-κB family members including P65 and inhibitor protein IkBα play important roles in regulation of MM cell survival and proliferation. We further demonstrated that Senp1 inhibition decreased IL-6-induced P65 and IkBα phosphorylation, leading to inactivation of NF-кB signaling in MM cells. These results delineate a key role for Senp1in IL-6 induced proliferation and survival of MM cells, suggesting it may be a potential new therapeutic target in MM.

SENP1 regulates hepatocyte growth factor-induced migration and epithelial-mesenchymal transition of hepatocellular carcinoma

The deregulation of HGF/c-Met signaling is implicated in epithelial-mesenchymal transition (EMT) and progress of hepatocellular carcinoma (HCC). However, the epigenetic mechanisms that HGF/c-Met regulates EMT and metastasis of HCC cells are less explored. In this study, we demonstrated that HCC cells express a high level of SUMO/sentrin-specific protease 1 (Senp1) which is induced by HGF/c-Met signals. Lentivirus-mediated small hairpin RNA (shRNA) transduction results in Senp1 silence in HCC cells. Senp1 silence reduces the HGF-induced proliferation and migration of HCC cells. Senp1 inhibition also induces HCC cell apoptosis and growth arrest. Furthermore, Senp1 knockdown inhibits epithelial-to-mesenchymal transition, with increase of E-cadherin and ZO-1 expression, decrease of fibronectin and N-cadherin expression. The EMT-related transcription factor Zeb1 was SUMO-modified and decreased in Senp1-silenced HCC cells. These results delineate that senp1 might play an important role in the regulation of HGF-induced invasion and migration of HCC cells.