Qing-Fang Li

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.

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.

Hepatocyte growth factor protects endothelial cells against gamma ray irradiation-induced damage

AbstractAim:To investigate the effect of HGF on proliferation, apoptosis and migratory ability of human vascular endothelial cells against gamma ray irradiation.Methods:ECV304 cells derived from adult human umbilical vein endothelial cells (HUVEC) were irradiated with a single gamma ray dose of 20 Gy. Immunocytochemistry and Western blot analysis were used to detect c-Met protein expression and HGF/c-Met signal pathway. In the HGF-treated groups, ECV304 cells were incubated with HGF (20 or 40 ng/mL) 3 h prior to irradiation. At 48 h post-irradiation, the proliferation of ECV304 cells was measured by MTT assay, the apoptosis was assessed by flow cytometry, and the migratory ability of ECV304 cells was measured by transwell chamber assay.Results:c-Met protein is expressed in ECV304 cells and can be activated by HGF. Gamma ray irradiation inhibits proliferation and migration of ECV304 cells in a dose-dependent manner. HGF significantly promoted the proliferation of ECV304 cells, and flow cytometry revealed that HGF can inhibit apoptosis of ECV304 cells. Transwell chamber assay also showed that HGF increases migration activity of endothelial cells.Conclusion:HGF may afford protection to vascular endothelial cells against gamma ray irradiation-induced damage.

Bortezomib and sphingosine kinase inhibitor interact synergistically to induces apoptosis in BCR/ABl+ cells sensitive and resistant to STI571 through down-regulation Mcl-1

Interactions between the proteasome inhibitor, bortezomib, and the sphingosine kinase (SPK1) inhibitor, SKI, were examined in BCR/ABL human leukemia cells. Coexposure of K562 or chronic myeloid leukemia (CML) cells from patients to subtoxic concentrations of SKI (10 μM) and bortezomib (100 nM) resulted in a synergistic increase in caspase-3 cleavage and apoptosis. These events were associated with the downregulation of BCR-ABL and Mcl-1, and a marked reduction in SPK1 expression. In imatinib mesylate-resistant K562 cells that displayed decreased BCR-ABL expression, bortezomib/SKI treatment markedly increased apoptosis and inhibited colony-formation in association with the downregulation of Mcl-1. Finally, the bortezomib/SKI regimen also potently induced the downregulation of BCR/ABL and Mcl-1 in human leukemia cells. Collectively, these findings suggest that combining SKI and bortezomib may represent a novel strategy in leukemia, including apoptosis-resistant BCR-ABL(+) hematologic malignancies.

Effects of Adenovirus-Mediated Delivery of the Human Hepatocyte Growth Factor Gene in Experimental Radiation-Induced Heart Disease

PURPOSE Irradiation to the heart may lead to late cardiovascular complications. The purpose of this study was to investigate whether adenovirus-mediated delivery of the human hepatocyte growth factor gene could reduce post-irradiation damage of the rat heart and improve heart function. METHODS AND MATERIALS Twenty rats received single-dose irradiation of 20 Gy gamma ray locally to the heart and were randomized into two groups. Two weeks after irradiation, these two groups of rats received Ad-HGF or mock adenovirus vector intramyocardial injection, respectively. Another 10 rats served as sham-irradiated controls. At post-irradiation Day 120, myocardial perfusion was tested by myocardial contrast echocardiography with contrast agent injected intravenously. At post-irradiation Day 180, cardiac function was assessed using the Langendorff technique with an isolated working heart model, after which heart samples were collected for histological evaluation. RESULTS Myocardial blood flow was significantly improved in HGF-treated animals as measured by myocardial contrast echocardiography at post-irradiation Day 120 . At post-irradiation Day 180, cardiac function was significantly improved in the HGF group compared with mock vector group, as measured by left ventricular peak systolic pressure (58.80 +/- 9.01 vs. 41.94 +/- 6.65 mm Hg, p < 0.05), the maximum dP/dt (5634 +/- 1303 vs. 1667 +/- 304 mm Hg/s, p < 0.01), and the minimum dP/dt (3477 +/- 1084 vs. 1566 +/- 499 mm Hg/s, p < 0.05). Picrosirius red staining analysis also revealed a significant reduction of fibrosis in the HGF group. CONCLUSION Based on the study findings, hepatocyte growth factor gene transfer can attenuate radiation-induced cardiac injury and can preserve cardiac function.

Endocrine glands-derived vascular endothelial growth factor protects pancreatic cancer cells from apoptosis via upregulation of the myeloid cell leukemia-1 protein.

Endocrine glands-derived vascular endothelial growth factor (EG-VEGF, also termed as Prok1)--a novel cytokine that selectively acts on the endothelial cells of endocrine glands--was recently reported to be involved in the regulation of tumor cell growth and survival. However, its roles in the regulation of pancreatic cancer progression remain unclear. In this report, we investigated the suppressive effects of EG-VEGF on pancreatic cancer cell apoptosis and the relevant mechanisms. By using reverse-transcriptase polymerase chain reaction (RT-PCR) we found that the Mia PaCa II cells of the pancreatic cancer cell line express the mRNAs of both EG-VEGF (Prok1) and its receptors. EG-VEGF protects pancreatic cancer cells from apoptosis through upregulation of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein of the bcl-2 family. Treatment of pancreatic cancer cells with EG-VEGF results in the rapid phosphorylation of mitogen-activated protein kinase (MAPK), STAT3, and AKT, which are involved in the upregulation of Mcl-1 expression. EG-VEGF (Prok1) protects Mia PaCa II cells from apoptosis through G protein-coupled receptor (GPR)-induced activation of multiple signal pathways, and hence can be a novel target for pancreatic cancer therapy.

Methyl-β-cyclodextrin induces programmed cell death in chronic myeloid leukemia cells and, combined with imatinib, produces a synergistic downregulation of ERK/SPK1 signaling.

Lipid rafts mediate several survival signals in the development of chronic myeloid leukemia (CML). Methyl-&bgr;-cyclodextrin (M&bgr;CD) is an inhibitor specifically designed to disrupt lipid rafts in cells by depleting the cholesterol component. We hypothesize that treatment of CML cells with M&bgr;CD and imatinib could reduce imatinib resistance. Apoptotic and autophagic cell death was assayed using annexin V–propidium iodide double staining, immunoblotting, and immunocytochemistry. We next investigated whether M&bgr;CD could enhance the cytotoxicity of imatinib in imatinib-sensitive and imatinib-resistant K562 cells. Extracellular signal-regulated kinase/sphingosine kinase 1 signaling downstream of lipid raft-activated signaling pathways was significantly inhibited by treatment of cells with a combination of M&bgr;CD and imatinib compared with treatment with either agent alone. M&bgr;CD induces programmed cell death in CML cells, and its antileukemia action is synergistic with that of imatinib.

KAI1/CD82 suppresses hepatocyte growth factor-induced migration of hepatoma cells via upregulation of Sprouty2

We conducted a study concerning the suppressive mechanism of KAI1/CD82 on hepatoma cell metastasis. Hepatocyte growth factor (HGF) induces the migration of hepatoma cells through activation of cellular sphingosine kinase 1 (SphK1). Adenovirus-mediated gene transfer of KAI1 (Ad-KAI1) downregulates the SphK1 expression and suppresses the HGF-induced migration of SMMC-7721 human hepatocellcular carcinoma cells. Overexpression of KAI1/CD82 significantly elevates Sprouty2 at the protein level. Ablation of Sprouty2 with RNA interference can block the KAI1/CD82-induced suppression of hepatoma cell migration and downregulation of SphK1 expression. It is demonstrated that KAI1/CD82 suppresses HGF-induced migration of hepatoma cells via upregulation of Sprouty2.

Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

Regulation of human hepatocellular carcinoma cells by Spred2 and correlative studies on its mechanism

Members of the Spred gene family are negative regulators of the Ras/Raf-1/ERK pathway, which has been associated with several features of the tumor malignancy. However, the effect of Spred genes on hepatocellular carcinoma (HCC) remains uninvestigated. In the present work, we analyzed the in vitro and in vivo effects of Spred2 expression on the hepatic carcinoma cell line, SMMC-7721. In addition to attenuated ERK activation, which inhibited the proliferation and migration of unstimulated and HGF-stimulated SMMC-7721 cells. Adenovirus-mediated Spred2 overexpression induced the activation of caspase-3 and apoptosis, as well as reduced the expression level of Mcl-1. Most importantly, the knockdown of Spred2 markedly enhanced tumor growth in vivo. In conclusion, these results suggest that Spred2 could qualify as a potential therapeutic target in HCC.