miRNA-17-92 protects endothelial cells from erastin-induced ferroptosis through targeting the A20-ACSL4 axis.

Endothelial cell death is linked to vascular diseases such as atherosclerosis and tissue ischemia. miRNA-17-92 (miR-17-92) is a multiple functional oncogenic miRNA cluster which plays vital roles in tumor angiogenesis and tissue development. However, its role in regulation of endothelial cell ferroptosis remains unclear. In this study, we revealed that miR-17-92 protects endothelial HUVEC cells from erastin-induced ferroptosis. miR-17-92 overexpression significantly reduced erastin-induced growth inhibition and ROS generation of HUVEC cells. Furthermore, Zinc lipoprotein A20, a validated target of miR-17-92, was identified as a novel regulator of endothelial cell ferroptosis. Lentivirus mediated A20 overexpression increased ROS generation and enhanced erastin-induced ferroptosis, whereas A20 knockdown inhibited erastin-induced ferroptosis. Mechanistic studies revealed that erastin-induced ferroptosis is associated with GPX4 downregulation and ACSL4 upregulation. miR-17-92 overexpression or A20 inhibition increased the ACSL4 expression in HUVEC cells. A20 was identified to directly with and regulate ACSL4 expression by immunoprecipitation. It suggests that the A20-ACSL4 axis plays important roles in erastin-induced endothelial ferroptosis. In conclusion, this study revealed a novel mechanism through which miR-17-92 protects endothelial cells from erastin-induced ferroptosis by targeting the A20-ACSL4 axis.

[MiR-486 Regulates the Glycometabolism of Hematopoietic Cells by Targeting Sirt1].

OBJECTIVE: To investigate the effect and mechanism of miR-486 on glycometabolism of hematopoietic cells. METHODS: qRT-PCR was applied to detect the expression of miR-486 or Sirt1 on TF-1 cells under hypoxia. Lentivirus was used to mediate the overexpression or inhibition of miR-486 on TF-1 cells and qRT-PCR was used to detect the expressions of Sirt1, glucose transporter 1(Glut1) and glucose transporter 4(Glut4). Lentivirus-mediated Sirt1-shRNA transduction was used to knockdown Sirt1 expression which was detected by qRT-PCR and Western blot. The expressions of Glut1 and Glut4 were determined by qRT-PCR. RESULTS: Hypoxia promoted the expression of miR-486 and inhibited the expression of Sirt1. MiR-486 overexpression could inhibit the expression of Sirt1 and promote the expressions of Glut1 and Glut4, whereas miR-486 silencing upregulated the sirt1 expression and inhibited the expressions of Glut1 and Glut4. And inhibition of Sirt1 expression increased the expressions of Glut1 and Glut4. CONCLUSION: MiR-486 can regulate the glycometabolism of hematopoietic cells by targeting Sirt1.

Exosomal miR-486 regulates hypoxia-induced erythroid differentiation of erythroleukemia cells through targeting Sirt1.

MicroRNAs (miRNAs) regulate the hypoxia-induced erythroid differentiation of hematopoietic cells. In this study, we identified that miR-486 was a rapid response miRNA to hypoxia in erythroleukemia TF-1 cells. Hypoxia exposure increased both intracellular and miR-486 levels of TF-1 cells. Ectopic miR-486 expression enhanced the growth and erythroid differentiation of TF-1 cells, whereas miR-486 inhibition suppressed their growth and erythroid differentiation. Treatment of TF-1 and cord blood CD34+ cells with exogenous containing miR-486 resulted in an increase of intracellular miR-486 level and enhanced erythroid differentiation. Furthermore, we identified that Sirt1 is a miR-486 target gene which modulates hypoxia-induced erythroid differentiation of TF-1 cells. Thus we identified a novel miRNA regulatory network that contributes to hypoxia-induced erythroid differentiation of hematopoietic cells.

Ptpmt1 induced by HIF-2α regulates the proliferation and glucose metabolism in erythroleukemia cells.

Hypoxia provokes metabolism misbalance, mitochondrial dysfunction and oxidative stress in both human and animal cells. However, the mechanisms which hypoxia causes mitochondrial dysfunction and energy metabolism misbalance still remain unclear. In this study, we presented evidence that mitochondrial phosphatase Ptpmt1 is a hypoxia response molecule that regulates cell proliferation, survival and glucose metabolism in human erythroleukemia TF-1 cells. Exposure to hypoxia or DFO treatment results in upregulation of HIF1-α, HIF-2α and Ptpmt1. Only inhibition of HIF-2α by shRNA transduction reduces Ptpmt1 expression in TF-1 cells under hypoxia. Ptpmt1 inhibitor suppresses the growth and induces apoptosis of TF-1 cells. Furthermore, we demonstrated that Ptpmt1 inhibition reduces the Glut1 and Glut3 expression and decreases the glucose consumption in TF-1 cells. In additional, Ptpmt1 knockdown also results in the mitochondrial dysfunction determined by JC1 staining. These results delineate a key role for HIF-2α-induced Ptpmt1 upregulation in proliferation, survival and glucose metabolism of erythroleukemia cells. It is indicated that Ptpmt1 plays important roles in hypoxia-induced cell metabolism and mitochondrial dysfunction.

Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment.

Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation.

HGF Gene Modification in Mesenchymal Stem Cells Reduces Radiation-Induced Intestinal Injury by Modulating Immunity.

BACKGROUND: Effective therapeutic strategies to address intestinal complications after radiation exposure are currently lacking. Mesenchymal stem cells (MSCs), which display the ability to repair the injured intestine, have been considered as delivery vehicles for repair genes. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF)-gene-modified MSCs on radiation-induced intestinal injury (RIII). METHODS: Female 6- to 8-week-old mice were radiated locally at the abdomen with a single 13-Gy dose of radiation and then treated with saline control, Ad-HGF or Ad-Null-modified MSCs therapy. The transient engraftment of human MSCs was detected via real-time PCR and immunostaining. The therapeutic effects of non- and HGF-modified MSCs were evaluated via FACS to determine the lymphocyte immunophenotypes; via ELISA to measure cytokine expression; via immunostaining to determine tight junction protein expression; via PCNA staining to examine intestinal epithelial cell proliferation; and via TUNEL staining to detect intestinal epithelial cell apoptosis. RESULTS: The histopathological recovery of the radiation-injured intestine was significantly enhanced following non- or HGF-modified MSCs treatment. Importantly, the radiation-induced immunophenotypic disorders of the mesenteric lymph nodes and Peyer's patches were attenuated in both MSCs-treated groups. Treatment with HGF-modified MSCs reduced the expression and secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ), increased the expression of the anti-inflammatory cytokine IL-10 and the tight junction protein ZO-1, and promoted the proliferation and reduced the apoptosis of intestinal epithelial cells. CONCLUSIONS: Treatment of RIII with HGF-gene-modified MSCs reduces local inflammation and promotes the recovery of small intestinal histopathology in a mouse model. These findings might provide an effective therapeutic strategy for RIII.

MiR-222 overexpression promotes proliferation of human hepatocellular carcinoma HepG2 cells by downregulating p27.

OBJECTIVE: Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related death worldwide. Increasing evidence suggests that microRNAs, a novel class of non-coding RNAs that function as endogenous suppressors of gene expression, are deregulated in HCC. Although microRNA-222 (miR-222) overexpression has been described in HCC, the role of miR-222 and its target genes in the proliferation of hepatocellular carcinoma cells remain poorly elucidated. METHODS: HepG2 cells were transfected with miR-222 mimic, inhibitor or their negative controls. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) and EdU incorporation assay. Flow cytometry was performed to assess the effects of miR-222 on HepG2 cell cycle progression. MiR-222 and putative targets genes (p27 and p57) expression levels were determined using qRT-PCR and/or Western blot. RESULTS: MiR-222 overexpression induced an enhancement of HepG2 cell proliferation in vitro, paralleling with an altered cell cycle progression via increased cell population in S phase. P27 expression, other than p57, was negatively regulated by miR-222 overexpression at post-transcriptional level in HepG2 cells. Transfection of either small interfering RNA (siRNA) for p27 or miR-222 mimic increased HepG2 cell proliferation rate, whereas co-transfection of p27 siRNA and miR-222 mimic did not further enhance HepG2 cell proliferation in comparison with the cells transfected with p27 siRNA or miR-222 mimic alone, validating that p27 is a target gene of miR-222 during HepG2 cell proliferation. CONCLUSION: This study suggests that miR-222 overexpression promotes HepG2 cell proliferation by downregulating p27.

Upregulation of HIF-1α via activation of ERK and PI3K pathway mediated protective response to microwave-induced mitochondrial injury in neuron-like cells.

Microwave-induced learning and memory deficits in animal models have been gaining attention in recent years, largely because of increasing public concerns on growing environmental influences. The data from our group and others have showed that the injury of mitochondria, the major source of cellular adenosine triphosphate (ATP) in primary neurons, could be detected in the neuron cells of microwave-exposed rats. In this study, we provided some insights into the cellular and molecular mechanisms behind mitochondrial injury in PC12 cell-derived neuron-like cells. PC12 cell-derived neuron-like cells were exposed to 30 mW/cm(2) microwave for 5 min, and damages of mitochondrial ultrastructure could be observed by using transmission electron microscopy. Impairments of mitochondrial function, indicated by decrease of ATP content, reduction of succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) activities, decrease of mitochondrial membrane potential (MMP), and increase of reactive oxygen species (ROS) production, could be detected. We also found that hypoxia-inducible factor-1 (HIF-1α), a key regulator responsible for hypoxic response of the mammalian cells, was upregulated in microwave-exposed neuron-like cells. Furthermore, HIF-1α overexpression protected mitochondria from injury by increasing the ATP contents and MMP, while HIF-1α silence promoted microwave-induced mitochondrial damage. Finally, we demonstrated that both ERK and PI3K signaling activation are required in microwave-induced HIF-1α activation and protective response. In conclusion, we elucidated a regulatory connection between impairments of mitochondrial function and HIF-1α activation in microwave-exposed neuron-like cells. By modulating mitochondrial function and protecting neuron-like cells against microwave-induced mitochondrial injury, HIF-1α represents a promising therapeutic target for microwave radiation injury.

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-ß, 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.

Methyl-ß-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-ß-cyclodextrin (MßCD) is an inhibitor specifically designed to disrupt lipid rafts in cells by depleting the cholesterol component. We hypothesize that treatment of CML cells with MßCD 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 MßCD 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 MßCD and imatinib compared with treatment with either agent alone. MßCD induces programmed cell death in CML cells, and its antileukemia action is synergistic with that of imatinib.

Overexpression of microRNA-29b induces apoptosis of multiple myeloma cells through down regulating Mcl-1.

MicroRNAs (miRNAs) are small, noncoding ribonucleic acids (ncRNAs), which regulate gene expression by targeting mRNAs for translational repression and degradation. Several lines of evidences have indicated that miRNAs act as tumor suppressors and oncogenes. However, the role of miRNAs in pathogenesis of multiple myeloma (MM) remains unclear. In this study, we examined the profile of miRNA expression of primary MM cells, using miRNA microarray and quantitative real-time polymerase chain reaction (qPCR) techniques. These results showed that in the bone marrow specimens analyzed, miRNA-29b was significantly downregulated. Similar results were also observed in human myeloma cell lines (HMCLs). Adenovirus-mediated overexpression of miR-29b induced apoptosis and elevated caspase-3 activation in HMCLs. Using a bioinformatics approach, we found a perfect complementarity between miRNA-29b and the 3'UTR of myeloid-cell-leukemia 1(Mcl-1). It is further confirmed that miRNA-29b downregulated the level of Mcl-1 without effect on the mRNA level using both qRT-PCR assays and Western blot analyses. Moreover, we observed that enforced miR-29b expression by using a retarget miRNA-29b expression vector (Ad5F11p-miR-29b) could induce apoptosis and elevate caspase-3 activation in HMCLs. Our results also indicated that miRNA-29b-induced apoptosis acted antagonistically with IL-6 in HMCLs. These findings suggest that miRNA-29b may play an important role in MM as a tumor suppressor.

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.

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.

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.

Prokineticin-1/endocrine gland-derived vascular endothelial growth factor is a survival factor for human multiple myeloma cells.

Prokineticin-1 (PK1) has been identified as a mitogen-specific protein for the endothelium of steroidogenic glands. Here we report a novel function of PK1 in the regulation of multiple myeloma (MM) cells. PK1 activates multiple signals including mitogen-activated protein kinase (MAPK), PI3K-AKT, and Jak-STAT3, sphingosine kinase-1 (SPK1) in MM cells. Treatment of MM cells with PK1 causes a time- and dose-dependent phosphorylation of MAPK, AKT and STAT3 and upregulation of SPK1 expression and cellular activity. We also show that PK1 upregulates Mcl-1 expression in a time- and dose-dependent manner in human MM cell lines and in the cells of patients with MM. Pertussis toxin, a pan-PK1 receptor inhibitor, can block PK1-induced upregulation of Mcl-1, indicating it relates to a G-protein-coupled receptor. We also show that PK1 protects MM cells against apoptosis induced by starvation for fetal calf serum (FBS), or for FBS and IL-6. Taken together, PK1 activates multiple signaling pathways and, upregulates Mcl-1 expression, leading to proliferation and survival of MM cells.

Spred2 is involved in imatinib-induced cytotoxicity in chronic myeloid leukemia cells.

Spreds, a recently established class of negative regulators of the Ras-ERK (extracellular signal-regulated kinase) pathway, are involved in hematogenesises, allergic disorders and tumourigenesis. However, their role in hematologic neoplasms is largely unknown. Possible effects of Spreds on other signal pathways closely related to Ras-ERK have been poorly investigated. In this study, we investigated the in vitro effects of Spred2 on chronic myeloid leukemia (CML) cells. In addition to inhibiting the well-established Ras-ERK cascade, adenovirus-mediated Spred2 over-expression inhibits constitutive and stem cell factor (SCF)-stimulated sphingosine kinase-1 (SPHK1) and Mcl-1 expression, as well as inhibiting proliferation and inducing apoptosis in CML cells. In K562 cells and primary CML cells, imatinib induces endogenous Spred2 expression. Spred2 silencing by stable RNA interference partly protects K562 cells against imatinib-induced apoptosis. Together, these data implicate Spred2 in imatinib-induced cytotoxicity in CML cells, possibly by inhibiting the Ras-ERK cascade and the pro-survival signaling molecules SPHK1 and Mcl-1. These findings reveal potential targets for selective therapy of CML.

[In vivo tracing of transplanted bone marrow mesenchymal stem cells with bioluminescence imaging].

Mesenchymal stem cell (MSC)-based cell therapy has shifted into clinical trials to repair the damage of various tissues. In this setting, the survival of the transplanted cells contributes critically to the therapeutic effectiveness. To investigate the in vivo tracing of MSCs, a recombinant retroviral vector carrying firefly-luciferase reporter gene [pL (FLUC) SN] was constructed and several GPE+86 cell clones that stably expressed fluc were selected. The retroviral supernatants were collected and used to transfect MSC derived from C57 mice. The cells were then screened with G418 and the expression of the exogenous gene was identified by luciferase enzyme activity analysis. Labeled mouse MSCs (2x10(6)) were injected into skeletal muscles, and the in situ expression was noninvasively tracked by in vivo bioluminescence imaging for 1, 3 and 6 days after transplantation. The results showed that the survival rates of the grafted cells dropped sharply with time, they were 57.2+/-11.7%, 8.6+/-2.5% and 5.4+/-3.1% on day 1, 3 and 6 after transplantation, and no fluorescent signals above background were detected on day 10. It is concluded that the method described above could be used for in vivo tracing of grafted cells. Furthermore, MSCs could not survive even transplanted into the none-ischemic skeletal muscles.

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

AIM: 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.

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.

In vitro and in vivo immunosuppressive characteristics of hepatocyte growth factor-modified murine mesenchymal stem cells.

BACKGROUND: Previous data have proven that hepatocyte growth factor (HGF) is able to maintain the survival of mesenchymal stem cells (MSCs), rendering HGF gene modification as an optional strategy for MSC therapy. However, the question about whether gene-transferred MSCs (MSC/HGFs) exhibit more potent immunosuppressive activity remains elusive. MATERIALS AND METHODS: Murine MSCs were isolated, culture-expanded and transfected by an adenovirus carrying human HGF cDNA (Ad-HGF). The transfection efficiency was evaluated by measuring HGF concentrations in the culture supernatants. An MHC-incompatible skin grafting model (C57BL-->BALB/c) was used to observe if MSC/HGF transfusion could prolong the survival time of skin transplants compared to MSCs. Furthermore, their inhibitory effects on the proliferation of T lymphocytes elicited by Con A and the activation of CD11b+ cells in mixed lymphocyte reaction were compared with carboxyfluorescein diacetate succinmidyl ester labeling and flow cytometric techniques. RESULTS: Ad-HGF was able to transfect mouse MSCs at high efficiency and administration of MSC/HGFs remarkably prolonged the mean survival time of skin grafts (16.73 +/- 0.57 days, p<0.01), compared with mice receiving MSCs (14.27 +/- 0.63 days), or saline (10.92 +/- 0.73 days). However, the presence of MSC/HGFs exhibited little additive impact on the suppression of T lymphocyte proliferation and activation of CD11b-positive and -negative cells in comparison with MSCs, though the inhibitory effects were evidently greater than with NIH3T3 cells and their Ad-HGF-modified counterparts. CONCLUSION: MSC/HGFs inhibit in vitro immune responses in a pattern similar to MSCs, but this gene modification might have beneficial effects for transplanted cells and damaged tissue.