Dual role of ERK2/NF-κB signaling in TRAIL sensitivity.

Targeting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling is a promising approach in cancer treatment. Although ERK and/or NF-κB signaling is involved in the expression of TRAIL receptors (TRAIL-R), the exact underlying mechanisms remain unknown. In this study, we evaluated the role of ERK2 and NF-κB in the cytotoxicity of TRAIL during cisplatin treatment. Cisplatin treatment of neuroepithelioma cells (SK-N-MC) significantly induced ERK2 activation and increased TRAIL cytotoxicity via the upregulation of death receptor 5 (DR5) expression. In partial ERK2 knockdown cell lines that maintained only basal levels of ERK2 activity, cisplatin treatment did not increase ERK2 activity or DR5 expression. These findings indicate that induced (rather than basal) ERK2 activity enhances TRAIL susceptibility via DR5 expression. In complete ERK2 knockdown cell lines with no basal ERK2 activity, DR4, DR5, and DcRs expression levels were increased, and additional treatment with cisplatin did not further increase TRAIL-R expression. Chemical inhibition of ERK2 also enhanced TRAIL cytotoxicity by upregulating DR4 and DR5 expression. These findings indicate that basal ERK2 activity suppresses TRAIL-R expression. Both basal and inducible ERK2 activities regulate TRAIL-R expression via the NF-κB signaling pathway. Overall, our findings suggest that the ERK2/NF-κB signaling pathway has a dual role in TRAIL susceptibility by differentially regulating TRAIL-R expression in the same cellular system.

Hepatocyte-specific Prominin-1 protects against liver injury-induced fibrosis by stabilizing SMAD7.

Prominin-1 (PROM1), also known as CD133, is expressed in hepatic progenitor cells (HPCs) and cholangiocytes of the fibrotic liver. In this study, we show that PROM1 is upregulated in the plasma membrane of fibrotic hepatocytes. Hepatocellular expression of PROM1 was also demonstrated in mice (Prom1CreER; R26TdTom) in which cells expressed TdTom under control of the Prom1 promoter. To understand the role of hepatocellular PROM1 in liver fibrosis, global and liver-specific Prom1-deficient mice were analyzed after bile duct ligation (BDL). BDL-induced liver fibrosis was aggravated with increased phosphorylation of SMAD2/3 and decreased levels of SMAD7 by global or liver-specific Prom1 deficiency but not by cholangiocyte-specific Prom1 deficiency. Indeed, PROM1 prevented SMURF2-induced SMAD7 ubiquitination and degradation by interfering with the molecular association of SMAD7 with SMURF2. We also demonstrated that hepatocyte-specific overexpression of SMAD7 ameliorated BDL-induced liver fibrosis in liver-specific Prom1-deficient mice. Thus, we conclude that PROM1 is necessary for the negative regulation of TGFß signaling during liver fibrosis.

Cytokines secreted by mesenchymal stem cells reduce demyelination in an animal model of Charcot-Marie-Tooth disease.

INTRODUCTION: Demyelinating Charcot-Marie-Tooth disease (CMT) is caused by mutations in the genes that encode myelinating proteins or their transcription factors. Our study thus sought to assess the therapeutic effects of cytokines secreted from mesenchymal stem cells (MSCs) on this disease. METHODS: The therapeutic potential of Wharton's jelly MSCs (WJ-MSCs) and cytokines secreted by WJ-MSCs was evaluated on Schwann cells (SCs) exhibiting demyelination features, as well as a mouse model of demyelinating CMT. RESULTS: Co-culture with WJ-MSC protected PMP22-overexpressing SCs from apoptotic cell death. Using a cytokine array, the secretion of growth differentiation factor-15 (GDF-15) and amphiregulin (AREG) was found to be elevated in WJ-MSCs when co-incubated with the PMP22-overexpressing SCs. Administration of both cytokines into trembler-J (Tr-J) mice, an animal model of CMT, significantly enhanced motor nerve conduction velocity compared to the control group. More importantly, this treatment alleviated the demyelinating phenotype of Tr-J mice, as demonstrated by an improvement in the mean diameter and g-ratio of the myelinated axons. CONCLUSIONS: Our findings demonstrated that WJ-MSCs alleviate the demyelinating phenotype of CMT via the secretion of several cytokines. Further elucidation of the underlying mechanisms of GDF-15 and AREG in myelination might provide a robust basis for the development of effective therapies against demyelinating CMT.

Inhibition of N-myc expression sensitizes human neuroblastoma IMR-32 cells expressing caspase-8 to TRAIL.

OBJECTIVES: This study aims to explore the roles of N-myc and caspase-8 in TRAIL-resistant IMR-32 cells which exhibit MYCN oncogene amplification and lack caspase-8 expression. MATERIALS AND METHODS: We established N-myc-downregulated IMR-32 cells using shRNA lentiviral particles targeting N-myc and examined the effect the N-myc inhibition on TRAIL susceptibility in human neuroblastoma IMR-32 cells expressing caspase-8. RESULTS: Cisplatin treatment in IMR-32 cells increased the expression of death receptor 5 (DR5; TRAIL-R2), but not other receptors, via downregulation of NF-κB activity. However, the cisplatin-mediated increase in DR5 failed to induce cell death following TRAIL treatment. Furthermore, interferon (IFN)-γ pretreatment increased caspase-8 expression in IMR-32 cells, but cisplatin failed to trigger TRAIL cytotoxicity. We downregulated N-myc expression in IMR-32 cells using N-myc-targeting shRNA. These cells showed decreased growth rate and Bcl-2 expression accompanied by a mild collapse in the mitochondrial membrane potential as compared with those treated with scrambled shRNA. TRAIL treatment in N-myc-negative cells expressing caspase-8 following IFN-γ treatment significantly triggered apoptotic cell death. Concurrent treatment with cisplatin enhanced TRAIL-mediated cytotoxicity, which was abrogated by an additional pretreatment with DR5:Fc chimera protein. CONCLUSIONS: N-myc and caspase-8 expressions are involved in TRAIL susceptibility in IMR-32 cells, and the combination of treatment with cisplatin and TRAIL may serve as a promising strategy for the development of therapeutics against neuroblastoma that is controlled by N-myc and caspase-8 expression.

Mesenchymal stem cells in suppression or progression of hematologic malignancy: current status and challenges.

Mesenchymal stem cells (MSCs) are known for being multi-potent. However, they also possess anticancer properties, which has prompted efforts to adapt MSCs for anticancer therapies. However, MSCs have also been widely implicated in pathways that contribute to tumor growth. Numerous studies have been conducted to adapt MSCs for further clinical use; however, the results have been inconclusive, possibly due to the heterogeneity of MSC populations. Moreover, the conflicting roles of MSCs in tumor inhibition and tumor growth impede their adaptation for anticancer therapies. Antitumorigenic and protumorigenic properties of MSCs in hematologic malignancies are not as well established as they are for solid malignancies, and data comparing them are still limited. Herein the effect of MSCs on hematologic malignancies, such as leukemia and lymphoma, their mechanisms, sources of MSCs, and their effects on different types of cancer, have been discussed. This review describes how MSCs preserve both antitumorigenic and protumorigenic effects, as they tend to not only inhibit tumor growth by suppressing tumor cell proliferation but also promote tumor growth by suppressing tumor cell apoptosis. Thus clinical studies trying to adapt MSCs for anticancer therapies should consider that MSCs could actually promote hematologic cancer progression. It is necessary to take extreme care while developing MSC-based cell therapies in order to boost anticancer properties while eliminating tumor-favoring effects. This review emphasizes that research on the therapeutic applications of MSCs must consider that they exert both antitumorigenic and protumorigenic effects on hematologic malignancies.

Human Adipose Tissue Stem Cells Promote the Growth of Acute Lymphoblastic Leukemia Cells in NOD/SCID Mice.

In this study, the effect of adipose tissue stem cells (ASCs) on the growth of acute lymphoblastic leukemia (ALL) cells was examined in an in vivo model. We established ALL cell lines expressing firefly luciferase (ALL/fLuc) by lentiviral infection that were injected intraperitoneally to NOD/SCID mice. The luciferase activities were significantly higher in mice co-injected with 105 ALL/fLuc cells and ASCs than in those injected with ALL/fLuc cells alone. Co-injection of 105 ALL/fLuc cells and ASCs in differing ratios into mice gradually increased the bioluminescence intensity in all groups, and mice co-injected with 1 or 2 × 106 ASCs showed higher bioluminescence intensity than those receiving lower numbers. Interestingly, in the mice injected with 105 or 107 ALL/fLuc cells alone, the formation of tumor masses was not observed for at least five weeks. Moreover, co-injection of 107 ALL/fLuc cells and 5 × 105 ASCs into mice increased the bioluminescence intensity in all groups, and showed significantly higher bioluminescence intensity compared to mice co-injected with human normal fibroblast HS68 cells. Overall, ASCs promote the growth of ALL cells in vivo, suggesting that ASCs negatively influence hematologic malignancy, which should be considered in developing cell therapy using ASCs.

Anti-leukemic effects of PPARγ ligands.

The peroxisome proliferator-activated receptor (PPAR) γ, a subtype of PPARs, is a member of the nuclear receptor family. PPARγ and its ligands contribute to various types of diseases including cancer. Given that currently developed therapies against leukemia are not very effective or safe, PPARγ ligands have been shown to be a new class of compounds with the potential to treat hematologic malignancies, particularly leukemia. The capability of PPARγ ligands to induce apoptosis, inhibit proliferation, and promote differentiation of leukemia cells suggests it has significant potential as a drug against leukemia. However, the specific mechanisms and molecules involved are not well-understood, although a number of PPARγ ligands with anti-leukemic effects have been identified. This may explain why PPARγ ligands have not been widely evaluated in clinical trials. To fill the gaps in the lack of understanding of specific anti-leukemic processes of PPARγ ligands and further adapt these molecules as anti-leukemic agents, this review describes previous studies of the anti-leukemic effects of PPARγ ligands.

Enhanced Immunosuppressive Properties of Human Mesenchymal Stem Cells Primed by Interferon-γ.

Mesenchymal stem cells (MSCs) are of particular interest for the treatment of immune-related diseases owing to their immunosuppressive properties. In this study, we aimed to identify the effect of interferon (IFN)-γ priming on immunomodulation by MSCs and elucidate the possible mechanism underlying their properties for the clinical treatment of allogeneic conflicts. Infusion of MSCs primed with IFN-γ significantly reduced the symptoms of graft-versus-host disease (GVHD) in NOD-SCID mice, thereby increasing survival rate when compared with naïve MSC-infused mice. However, infusion of IFN-γ-primed MSCs in which indoleamine 2,3-dioxygenase (IDO) was downregulated did not elicit this effect. The IDO gene was expressed in MSCs via the IFN-γ-Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) pathway, and the infusion of IDO-over-expressing MSCs increased survival rate in an in vivo GVHD model, similar to infusion of IFN-γ-primed MSCs. These data indicate that IFN-γ production by activated T-cells is correlated with the induction of IDO expression in MSCs via the IFN-γ-JAK-STAT1 pathway, which in turn results in the suppression of T-cell proliferation. Our findings also suggest that cell therapy based on MSCs primed with IFN-γ can be used for the clinical treatment of allogeneic conflicts, including GVHD.

Involvement of TLR3-Dependent PGES Expression in Immunosuppression by Human Bone Marrow Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) are known for their prostaglandin E2 (PGE2)-mediated immunosuppressive function but the precise molecular mechanisms underlying PGE2 biosynthesis during inflammation have not been completely elucidated. In this study, we have investigated the involvement of PGE2 pathway members in PGE2 production by bone marrow (BM)-MSCs in response to inflammatory stimuli, and their role in immunosuppression mediated by BM-MSCs. We found that IFN-γ and TNF-α increased cyclooxygenase (COX)-2 expression but not that of prostaglandin E synthase (PGES), or PGE2 production. On the other hand, the toll like receptor 3 (TLR3) stimulant poly(I:C) increased expression of both COX-2 and PGES, resulting in a significant increase in PGE2 levels. This effect was reversed upon COX-2 inhibition with indomethacin or PGES downregulation by siRNA. Reduced PGE2 levels decreased MSC's capacity to inhibit hPBMC proliferation. In addition, administration of MSCs with inhibited PGES expression into mice with graft-versus-host disease (GVHD) did not reduce mortality. In summary, the present study reveals that upregulation of PGES via TLR3 is critical for BM-MSCs-mediated immunosuppression by PGE2 secretion via the COX-2/PGE2 pathway. These results provide a basis for understanding the molecular mechanisms underlying the PGE2-mediated immunosuppressive properties of MSCs.

In situ analysis of chemical components induced by steaming between fresh ginseng, steamed ginseng, and red ginseng.

BACKGROUND: The chemical constituents of Panax ginseng are changed by processing methods such as steaming or sun drying. In the present study, the chemical change of Panax ginseng induced by steaming was monitored in situ. METHODS: Samples were separated from the same ginseng root by incision during the steaming process, for in situ monitoring. Sampling was sequentially performed in three stages; FG (fresh ginseng) → SG (steamed ginseng) → RG (red ginseng) and 60 samples were prepared and freeze dried. The samples were then analyzed to determine 43 constituents among three stages of P. ginseng. RESULTS: The results showed that six malonyl-ginsenoside (Rg1, Rb1, Rb3, Rc, Rd, Rb2) and 15 amino acids were decreased in concentration during the steaming process. In contrast, ginsenoside-Rh1, 20(S)-Rg2, 20(S, R)-Rg3 and Maillard reaction product such as AF (arginine-fructose), AFG (arginine-fructose-glucose), and maltol were newly generated or their concentrations were increased. CONCLUSION: This study elucidates the dynamic changes in the chemical components of P. ginseng when the steaming process was induced. These results are thought to be helpful for quality control and standardization of herbal drugs using P. ginseng and they also provide a scientific basis for pharmacological research of processed ginseng (Red ginseng).

Cell culture density affects the stemness gene expression of adipose tissue-derived mesenchymal stem cells.

The results of clinical trials using mesenchymal stem cells (MSCs) are controversial due to the heterogeneity of human MSCs and differences in culture conditions. In this regard, it is important to identify gene expression patterns according to culture conditions, and to determine how the cells are expanded and when they should be clinically used. In the current study, stemness gene expression was investigated in adipose tissue-derived MSCs (AT-MSCs) harvested following culture at different densities. AT-MSCs were plated at a density of 200 or 5,000 cells/cm2. After 7 days of culture, stemness gene expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The proliferation rate of AT-MSCs harvested at a low density (~50% confluent) was higher than that of AT-MSCs harvested at a high density (~90% confluent). Although there were differences in the expression levels of stemness gene, such as octamer-binding transcription factor 4, nanog homeobox (Nanog), SRY-box 2, Kruppel like factor 4, v-myc avian myelocytomatosis viral oncogene homolog (c-Myc), and lin-28 homolog A, in the AT-MSCs obtained from different donors, RT-qPCR analysis demonstrated differential gene expression patterns according to the cell culture density. Expression levels of stemness genes, particularly Nanog and c-Myc, were upregulated in AT-MSCs harvested at a low density (~50% confluent) in comparison to AT-MSCs from the same donor harvested at a high density (~90% confluent). These results imply that culture conditions, such as the cell density at harvesting, modulate the stemness gene expression and proliferation of MSCs.

Inhibition of ERK activity enhances the cytotoxic effect of peroxisome proliferator-activated receptor γ (PPARγ) agonists in HeLa cells.

In this study, we examined whether the peroxisome proliferator-activated receptor γ (PPARγ) agonists, ciglitazone (CGZ) and troglitazone (TGZ), induce cell death in human cervical cancer HeLa cells. The cells were treated with a range of CGZ or TGZ doses for 24 or 48 h. Low concentrations of CGZ (≤10 µM) or TGZ (≤20 µM) had no effect on cell viability whereas higher doses induced cell death in a time- and dose-dependent manner as evidenced by the detection of activated caspase-3 and PARP cleavage. Treatment with the PPARγ antagonist GW9662 followed by PPARγ agonists did not increase CGZ- or TGZ-induced cell death, indicating that PPARγ agonists induced HeLa cell death independently of PPARγ. Moreover, ERK1/2 activation was observed at a CGZ concentration of 25 µM and a TGZ concentration of 35 µM, both of which induced cell death. To elucidate the role of ERK1/2 activated by the two PPARγ agonists, the effect of U0126, an inhibitor of ERK1/2, on PPARγ-agonist-induced cell death was examined. Treatment with 10 or 20 µM U0126 followed by CGZ or TGZ induced the down-regulation of ERK1/2 activity and a decrease in Bcl-2 expression accompanied by the collapse of mitochondrial membrane potential, which in turn significantly enhanced CGZ- or TGZ-induced apoptotic cell death. Our results suggest that PPARγ agonists are capable of inducing apoptotic cell death in HeLa cells independently of PPARγ and that inhibition of ERK1/2 activity offers a strategy to enhance the cytotoxicity of PPARγ agonists in the treatment of cervical cancer.

Application of human mesenchymal stem cells cultured in different oxygen concentrations for treatment of graft-versus-host disease in mice.

Human mesenchymal stem cell (MSC) heterogeneity and problems associated with the ex vivo expansion of MSC are linked with the failure of MSC clinical trials. In this study, we compared the effect of MSCs cultured in different oxygen concentrations on GVHD in mice to elucidate whether hypoxia improves the immunosuppressive capacity of MSCs. Hypoxia increased the proliferative activity and the expression of several stemness and chemokine genes, such as KLF4, OCT4, C-MYC, CCL2, and CXCL10. Mice that received MSCs cultured in normoxia or hypoxia showed alleviated symptoms of GVHD and increased survival times. However, there was no significant difference in survival rates between mice that received MSCs cultured in normoxia and hypoxia. These data suggest that hypoxic culture is a useful method for maintaining and obtaining MSCs used for cell therapy and that the therapeutic potential of MSCs cultured in hypoxia warrants further investigation.

Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells.

Culture of mesenchymal stem cells (MSCs) under ambient conditions does not replicate the low oxygen environment of normal physiological or pathological states and can result in cellular impairment during culture. To overcome these limitations, we explored the effect of hypoxia (1 % O2) on the biological characteristics of MSCs over the course of different culture periods. The following biological characteristics were examined in human bone marrow-derived MSCs cultured under hypoxia for 8 weeks: proliferation rate, morphology, cell size, senescence, immunophenotypic characteristics, and the expression levels of stemness-associated factors and cytokine and chemokine genes. MSCs cultured under hypoxia for approximately 2 weeks showed increased proliferation and viability. During long-term culture, hypoxia delayed phenotypic changes in MSCs, such as increased cell volume, altered morphology, and the expression of senescence-associated-ß-gal, without altering their characteristic immunophenotypic characteristics. Furthermore, hypoxia increased the expression of stemness and chemokine-related genes, including OCT4 and CXCR7, and did not decrease the expression of KLF4, C-MYC, CCL2, CXCL9, CXCL10, and CXCR4 compared with levels in cells cultured under normoxia. In conclusion, low oxygen tension improved the biological characteristics of MSCs during ex vivo expansion. These data suggest that hypoxic culture could be a useful method for increasing the efficacy of MSC cell therapies.

Cell culture density affects the proliferation activity of human adipose tissue stem cells.

In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT-MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT-MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm(-2) . After 7 days of incubation, P4 and P12 AT-MSCs cultured in CC1 were thin and spindle-shaped, whereas those cultured in CC2 had extensive cell-to-cell contacts and an expanded cell volume. In addition, P4 and P12 AT-MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester dye showed that the fluorescence intensity of AT-MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation-associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT-MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT-MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions.

Strategies to improve the immunosuppressive properties of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are of particular interest for the treatment of immune-related diseases because of their immunosuppressive capacities. However, few clinical trials of MSCs have yielded satisfactory results. A number of clinical trials using MSCs are currently in progress worldwide. Unfortunately, protocols and methods, including optimized culture conditions for the harvest of MSCs, have not been standardized. In this regard, complications in the ex vivo expansion of MSCs and MSC heterogeneity have been implicated in the failure of clinical trials. In this review, potential strategies to obtain MSCs with improved immunosuppressive properties and the potential roles of specific immunomodulatory genes, which are differentially upregulated in certain culture conditions, will be discussed.

RAD001 (everolimus) enhances TRAIL cytotoxicity in human leukemic Jurkat T cells by upregulating DR5.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anti-cancer agents, is a promising new strategy for the treatment of cancer. However, aberrant PI3K/Akt/mTOR survival signaling may confer TRAIL resistance by altering the balance between pro- and anti-apoptotic proteins. In the present study, we showed that the Akt/mTOR inhibitor RAD001 (everolimus) induced cell death in a dose-dependent manner and enhanced TRAIL-induced apoptosis in human leukemic Jurkat T cells, which show PI3K/Akt/mTOR pathway activation and basal expression levels of death receptor (DR) 5 (TRAIL-R2). Investigation of the effect of RAD001 treatment on the expression of TRAIL receptors (TRAIL-Rs) in Jurkat T cells showed that RAD001 significantly upregulated DR5 by up to 51.22%, but not other TRAIL-Rs such as DR4 (TRAIL-R1), decoy receptor (DcR) 1 (TRAIL-R3), and DcR2 (TRAIL-R4). Pretreatment with DR5:Fc chimera abrogated the RAD001-induced increase of TRAIL cytotoxicity, indicating that the upregulation of DR5 by RAD001 plays a role in enhancing the susceptibility of Jurkat T cells to TRAIL. Our results indicate that combination treatment with RAD001 and TRAIL may be a novel therapeutic strategy in leukemia.

Gene expression profiles of human adipose tissue-derived mesenchymal stem cells are modified by cell culture density.

Previous studies conducted cell expansion ex vivo using low initial plating densities for optimal expansion and subsequent differentiation of mesenchymal stem cells (MSCs). However, MSC populations are heterogeneous and culture conditions can affect the characteristics of MSCs. In this study, differences in gene expression profiles of adipose tissue (AT)-derived MSCs were examined after harvesting cells cultured at different densities. AT-MSCs from three different donors were plated at a density of 200 or 5,000 cells/cm(2). After 7 days in culture, detailed gene expression profiles were investigated using a DNA chip microarray, and subsequently validated using a reverse transcription polymerase chain reaction (RT-PCR) analysis. Gene expression profiles were influenced primarily by the level of cell confluence at harvest. In MSCs harvested at ∼90% confluence, 177 genes were up-regulated and 102 genes down-regulated relative to cells harvested at ∼50% confluence (P<0.05, FC>2). Proliferation-related genes were highly expressed in MSCs harvested at low density, while genes that were highly expressed in MSCs harvested at high density (∼90% confluent) were linked to immunity and defense, cell communication, signal transduction and cell motility. Several cytokine, chemokine and growth factor genes involved in immunosuppression, migration, and reconstitution of damaged tissues were up-regulated in MSCs harvested at high density compared with MSCs harvested at low density. These results imply that cell density at harvest is a critical factor for modulating the specific gene-expression patterns of heterogeneous MSCs.