COUP-TFII plays a role in cAMP-induced Schwann cell differentiation and in vitro myelination by up-regulating Krox20.

Schwann cells (SCs) are known to produce myelin for saltatory nerve conduction in the peripheral nervous system (PNS). Schwann cell differentiation and myelination processes are controlled by several transcription factors including Sox10, Oct6/Pou3f1, and Krox20/Egr2. Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII/NR2F2) is an orphan receptor that plays a role in the development and differentiation. However, the role of COUP-TFII in the transcriptional regulatory network of SC differentiation has not been fully identified yet. Thus, the objective of this study was to investigate the role and molecular hierarchy of COUP-TFII during cAMP-induced SC differentiation. Our results showed that dibutyryl-cAMP (db-cAMP) increased expression levels of COUP-TFII along with the expressions of Oct6, Krox20, and myelin-related genes known to be related to SC differentiation. Our mechanistic studies showed that COUP-TFII acted downstream of Hsp90/ErbB2/Gab1/ERK-AKT pathway during db-cAMP-induced SC differentiation. In addition, we found that COUP-TFII induced Krox20 expression by directly binding to Krox20-MSE8 as revealed by chromatin immunoprecipitation assay and promoter activity assay. In line with this, the expression of COUP-TFII was increased before up-regulation of Oct6, Krox20, and myelin-related genes in the sciatic nerves during early postnatal myelination period. Finally, COUP-TFII knockdown by COUP-TFII siRNA or via AAV-COUP-TFII shRNA in SCs inhibited db-cAMP-induced SC differentiation and in vitro myelination of sensory axons, respectively. Taken together, these findings indicate that COUP-TFII might be involved in postnatal myelination through induction of Krox20 in SCs. Our results present a new insight into the transcriptional regulatory mechanism in SC differentiation and myelination.

p53 regulates mitochondrial dynamics by inhibiting Drp1 translocation into mitochondria during cellular senescence.

Cellular senescence acts as an important barrier to tumorigenesis by eliminating precancerous cells. Previous studies have shown an essential role of the tumor suppressor p53 in cellular senescence, but how p53 induces cellular senescence is not fully understood. We found that p53 promoted the formation of highly interconnected and elongated mitochondria prior to the onset of cellular senescence. The inhibition of mitochondrial elongation upon p53 expression suppressed cellular senescence, suggesting that mitochondrial elongation is required for the induction of p53-dependent senescence. p53-induced mitochondrial elongation resulted in mitochondrial dysfunction and subsequent increases in intracellular reactive oxygen species (ROS) levels, an important mediator of cellular senescence. Mechanistically, the inhibitory phosphorylation of Drp1 Ser637 increased upon p53 expression, suppressing the translocation of Drp1 into mitochondria. The transcriptional function of p53 was crucial for controlling the inhibitory phosphorylation of Drp1, whereas p21 was nonessential. Protein kinase A (PKA) activity was responsible for p53-mediated Drp1 Ser637 phosphorylation and mitochondrial dysfunction. Taken together, these results suggest that p53 regulates mitochondrial dynamics through the PKA-Drp1 pathway to induce cellular senescence.

Evaluation of neutrophil extracellular traps as the circulating marker for patients with acute coronary syndrome and acute ischemic stroke.

INTRODUCTION: Neutrophil extracellular traps (NETs) are known to be induced by various factors. In this study, we tried to identify circulating levels of NETs in patients with acute coronary syndrome (ACS) and acute ischemic stroke (AIS) and to confirm its suitability as a new circulating marker in their detection. METHODS: We prospectively enrolled 95 patients with a diagnosis of ACS (N = 37) or AIS (N = 58) in Dong-A University Hospital, Busan, Korea. The control group was selected from healthy adults (N = 25) who visited the hospital for health screening. Circulating levels of NETs were evaluated by measuring plasma concentrations of double-stranded DNA (dsDNA) and DNA-histone complex. RESULTS: The concentrations of dsDNA were statistically higher in patients with ACS or AIS than those in the control group (both P < .001). In the univariable and multivariable analyses, statistically significant risk factors were troponin I (TnI) level and dsDNA concentration in the ACS group (P = .046 and P = .015, respectively) and only dsDNA concentration in the AIS group (P = .002). In the receiver operating characteristic curve analyses, the area under the curve values for TnI level and dsDNA concentration in the ACS group were 0.878 and 0.968, respectively, and the value for dsDNA concentration in the AIS group was 0.859. CONCLUSIONS: In this study, it was confirmed that the circulating level of NETs was increased in patients with ACS and AIS at initial presentation. Findings in this study show that NETs could be used as a new circulating marker for the initial diagnosis of ACS or AIS.

Heat Shock Protein 90 is Required for cAMP-Induced Differentiation in Rat Primary Schwann Cells.

Schwann cells (SCs) play an important role in producing myelin for rapid neurotransmission in the peripheral nervous system. Activation of the differentiation and myelination processes in SCs requires the expression of a series of transcriptional factors including Sox10, Oct6/Pou3f1, and Egr2/Krox20. However, functional interactions among several transcription factors are poorly defined and the important components of the regulatory network are still unknown. Until now, available evidence suggests that SCs require cAMP signaling to initiate the myelination program. Heat shock protein 90 (Hsp90) is known as a chaperone required to stabilize ErbB2 receptor. In recent years, it was reported that cAMP transactivated the ErbB2/ErbB3 signaling in SCs. However, the relationship between Hsp90 and cAMP-induced differentiation in SCs is undefined. Here we investigated the role of Hsp90 during cAMP-induced differentiation of SCs using Hsp90 inhibitor, geldanamycin and Hsp90 siRNA transfection. Our results showed that dibutyryl-cAMP (db-cAMP) treatment upregulated Hsp90 expression and led to nuclear translocation of Gab1/ERK, the downstream signaling pathway of the ErbB2 signaling mechanism in myelination. The expression of myelin-related genes and nuclear translocation of Gab1/ERK following db-cAMP treatment was inhibited by geldanamycin pretreatment and Hsp90 knockdown. These findings suggest that Hsp90 might play a role in cAMP-induced differentiation via stabilization of ErbB2 and nuclear translocation of Gab1/ERK in SCs.

Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells.

Marine triterpene glycosides are attractive candidates for the development of anticancer agents. Holotoxin A1 is a triterpene glycoside found in the edible sea cucumber, Apostichopus (Stichopus) japonicus. We previously showed that cladoloside C2, the 25(26)-dihydro derivative of holotoxin A1 induced apoptosis in human leukemia cells by activating ceramide synthase 6. Thus, we hypothesized that holotoxin A1, which is structurally similar to cladoloside C2, might induce apoptosis in human leukemia cells through the same molecular mechanism. In this paper, we compared holotoxin A1 and cladoloside C2 for killing potency and mechanism of action. We found that holotoxin A1 induced apoptosis more potently than cladoloside C2. Moreover, holotoxin A1-induced apoptosis in K562 cells by activating caspase-8 and caspase-3, but not by activating caspase-9. During holotoxin A1-induced apoptosis, acid sphingomyelinase (SMase) and neutral SMase were activated in both K562 cells and human primary leukemia cells. Specifically inhibiting acid SMase and neutral SMаse with chemical inhibitors or siRNAs significantly inhibited holotoxin A1-induced apoptosis. These results indicated that holotoxin A1 might induce apoptosis by activating acid SMase and neutral SMase. In conclusion, holotoxin A1 represents a potential anticancer agent for treating leukemia. Moreover, the aglycone structure of marine triterpene glycosides might affect the mechanism involved in inducing apoptosis.

Peroxisome proliferator-activated receptor γ coactivator-1α is a predictor of lymph node metastasis and poor prognosis in human colorectal cancer.

Peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator-1α (PGC-1α) expression levels are correlated with clinical outcome in breast cancer. However, the potential biological and clinical significance of PPARγ and PGC-1α in colorectal cancer remains unknown. Here we investigated PPARγ and PGC-1α expression in colorectal cancer, and the associations of these expression levels with clinicopathological features. We also evaluated the roles of PPARγ and PGC-1α as prognostic factors in colorectal cancer. We performed immunohistochemical analysis to investigate PPARγ and PGC-1α expression in human colorectal cancer tissues and adjacent normal tissues from 108 primary colorectal cancer patients. We then examined how these expression levels correlated with clinicopathological features. Using the Kaplan-Meier method, we evaluated 3-year disease-free survival (DFS) and overall survival (OS) in patients with tumors expressing different levels of PPARγ and PGC-1α. Our results revealed that PPARγ expression was not significantly correlated with age at surgery, gender, differentiation, depth of infiltration, relapse, or TNM stage. Additionally, PGC-1α expression was not significantly correlated with age at surgery, differentiation, depth of infiltration, relapse, or TNM stage. However, PGC-1α expression was significantly correlated with nodal metastasis (p=0.020). Survival analysis demonstrated reduced OS in the PGC-1α-positive group compared to the PGC-1α-negative group (p=0.03). Our present findings suggest that PGC-1α may be useful for predicting nodal metastasis, and may represent a biomarker for poor prognosis in colorectal cancer.

Cooperative interaction of hepatocyte growth factor and neuregulin regulates Schwann cell migration and proliferation through Grb2-associated binder-2 in peripheral nerve repair.

The sequential reactive changes in Schwann cell phenotypes in transected peripheral nerves, including dedifferentiation, proliferation and migration, are essential for nerve repair. Even though the injury-induced migratory and proliferative behaviors of Schwann cells resemble epithelial and mesenchymal transition (EMT) in tumors, the molecular mechanisms underlying this phenotypic change of Schwann cells are still unclear. Here we show that the reactive Schwann cells exhibit migratory features dependent on the expression of a scaffolding oncoprotein Grb2-associated binder-2 (Gab2), which was transcriptionally induced by neuregulin 1-ErbB2 signaling following nerve injury. Injury-induced Gab2 expression was dependent on c-Jun, a transcription factor critical to a Schwann cell reprograming into a repair-type cell. Interestingly, the injury-induced activation (tyrosine phosphorylation) of Gab2 in Schwann cells was regulated by an EMT signal, the hepatocyte growth factor-c-Met signaling, but not by neuregulin 1. Gab2 knockout mice exhibited a deficit in nerve repair after nerve transection due to limited Schwann cell migration. Furthermore, Gab2 was required for the proliferation of Schwann cells following nerve injury and in vitro, and was over-expressed in human Schwann cell-derived tumors. In contrast, the tyrosine phosphorylation of Gab1 after nerve injury was principally regulated by the neuregulin 1-ErbB2 signaling and was indispensable for remyelination after crush injury, but not for the proliferation and migration of Schwann cells. Our findings indicate that Gab1 and Gab2 in Schwann cells are nonredundant and play a crucial role in peripheral nerve repair.

Schwann cell dedifferentiation-associated demyelination leads to exocytotic myelin clearance in inflammatory segmental demyelination.

Schwann cells (SCs), which form the peripheral myelin sheath, have the unique ability to dedifferentiate and to destroy the myelin sheath under various demyelination conditions. During SC dedifferentiation-associated demyelination (SAD) in Wallerian degeneration (WD) after axonal injury, SCs exhibit myelin and junctional instability, down-regulation of myelin gene expression and autophagic myelin breakdown. However, in inflammatory demyelinating neuropathy (IDN), it is still unclear how SCs react and contribute to segmental demyelination before myelin scavengers, macrophages, are activated for phagocytotic myelin digestion. Here, we compared the initial SC demyelination mechanism of IDN to that of WD using microarray and histochemical analyses and found that SCs in IDN exhibited several typical characteristics of SAD, including actin-associated E-cadherin destruction, without obvious axonal degeneration. However, autophagolysosome activation in SAD did not appear to be involved in direct myelin lipid digestion by SCs but was required for the separation of SC body from destabilized myelin sheath in IDN. Thus, lysosome inhibition in SCs suppressed segmental demyelination by preventing the exocytotic myelin clearance of SCs. In addition, we found that myelin rejection, which might also require the separation of SC cytoplasm from destabilized myelin sheath, was delayed in SC-specific Atg7 knockout mice in WD, suggesting that autophagolysosome-dependent exocytotic myelin clearance by SCs in IDN and WD is a shared mechanism. Finally, autophagolysosome activation in SAD was mechanistically dissociated with the junctional destruction in both IDN and WD. Thus, our findings indicate that SAD could be a common myelin clearance mechanism of SCs in various demyelinating conditions.

Ceramide as a Target of Marine Triterpene Glycosides for Treatment of Human Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous myeloid clonal disorder exhibiting the accumulation of immature myeloid progenitors in the bone marrow and peripheral blood. Standard AML therapy requires intensive combination chemotherapy, which leads to significant treatment-related toxicity. The search for new, low toxic marine agents, inducing the generation of ceramide in leukemic cells is a new approach to improve the therapy of leukemia. This review focuses on the metabolism of sphingolipids, the role of ceramide in treating leukemia, and the antitumor activity, related to ceramide metabolism, of some marine metabolites, particularly stichoposides, triterpene glycosides extracted from sea cucumbers of the family Stichopodiidae.

An anthraquinone derivative, emodin sensitizes hepatocellular carcinoma cells to TRAIL induced apoptosis through the induction of death receptors and downregulation of cell survival proteins.

Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently under clinical trials for cancer, however many tumor cells, including hepatocellular carcinoma (HCC) develop resistance to TRAIL-induced apoptosis. Hence, novel agents that can alleviate TRAIL-induced resistance are urgently needed. In the present report, we investigated the potential of emodin to enhance apoptosis induced by TRAIL in HCC cells. As observed by MTT cytotoxicity assay and the externalization of the membrane phospholipid phosphatidylserine, we found that emodin can significantly potentiate TRAIL-induced apoptosis in HCC cells. When investigated for the mechanism(s), we observed that emodin can downregulate the expression of various cell survival proteins, and induce the cell surface expression of both TRAIL receptors, death receptors (DR) 4 as well as 5. In addition, emodin increased the expression of C/EBP homologous protein (CHOP) in a time-dependent manner. Knockdown of CHOP by siRNA decreased the induction of emodin-induced DR5 expression and apoptosis. Emodin-induced induction of DR5 was mediated through the generation of reactive oxygen species (ROS), as N-acetylcysteine blocked the induction of DR5 and the induction of apoptosis. Also, the knockdown of X-linked inhibitor of apoptosis protein by siRNA significantly reduced the sensitization effect of emodin on TRAIL-induced apoptosis. Overall, our experimental results clearly indicate that emodin can indeed potentiate TRAIL-induced apoptosis through the downregulation of antiapoptotic proteins, increased expression of apoptotic proteins, and ROS mediated upregulation of DR in HCC cells.

COUP-TFs regulate eye development by controlling factors essential for optic vesicle morphogenesis.

Transcriptional networks, which are initiated by secreted proteins, cooperate with each other to orchestrate eye development. The establishment of dorsal/ventral polarity, especially dorsal specification in the optic vesicle, is poorly understood at a molecular and cellular level. Here, we show that COUP-TFI (Nr2f1) and COUP-TFII (Nr2f2) are highly expressed in the progenitor cells in the developing murine eye. Phenotype analysis of COUP-TFI and COUP-TFII single-gene conditional knockout mouse models suggests that COUP-TFs compensate for each other to maintain morphogenesis of the eye. However, in eye-specific COUP-TFI/TFII double-knockout mice, progenitor cells at the dorso-distal optic vesicle fail to differentiate appropriately, causing the retinal pigmented epithelium cells to adopt a neural retina fate and abnormal differentiation of the dorsal optic stalk; the development of proximo-ventral identities, neural retina and ventral optic stalk is also compromised. These cellular defects in turn lead to congenital ocular colobomata and microphthalmia. Immunohistochemical and in situ hybridization assays reveal that the expression of several regulatory genes essential for early optic vesicle development, including Pax6, Otx2, Mitf, Pax2 and Vax1/2, is altered in the corresponding compartments of the mutant eye. Using ChIP assay, siRNA treatment and transient transfection in ARPE-19 cells in vitro, we demonstrate that Pax6 and Otx2 are directly regulated by COUP-TFs. Taken together, our findings reveal novel and distinct cell-intrinsic mechanisms mediated by COUP-TF genes to direct the specification and differentiation of progenitor cells, and that COUP-TFs are crucial for dorsalization of the eye.

Dickkopf 4 Alone and in Combination with Leucyl-tRNA Synthetase as a Good Prognostic Biomarker for Human Colorectal Cancer.

The prognosis of patients with colorectal cancer (CRC) is affected by invasion and metastasis. Leucyl-tRNA synthetase (LARS) was shown to be related to the growth and migration of lung cancer cells. Dickkopf 4 (DKK4) is known as a Wnt/ß-catenin pathway inhibitor, and its upregulation was reported in several cancers. However, the clinical significance of LARS and DKK4 in human CRC has not been clearly defined. We investigated the expression of LARS and DKK4 by immunohistochemical staining in tissue microarrays from 642 primary CRC patients and analyzed the relationship between their expression and the clinicopathological characteristics of CRC patients. LARS and DKK4 expressions were not related to gender, age at surgery, histologic grade, size, tumor location, tumor invasion, or metastasis, but LARS expression was significantly correlated with TNM stage, N stage, and lymph node metastasis. DKK4 expression was inversely related to the TNM stage and N stage. Survival analysis demonstrated that the OS and DFS in the LARS high expression group were not different compared to the LARS low expression group. OS and DFS in the DKK4 high expression group were significantly higher than in the DKK4 low expression group. In addition, OS and DFS in the group with the combination of the LARS high/DKK4 low expression were significantly lower than in the LARS high/DKK4 high expression group. The low expression of DKK4 alone can be used as a predictor of relapse in CRC patients. In addition, DKK4 low expression in the case of LARS high expression can be used as a poor prognostic factor in CRC patients. Thus, our findings suggest that DKK4 alone or in combination with LARS at diagnosis may be a useful prognostic factor for CRC.

PGC-1α Regulates Cell Proliferation, Migration, and Invasion by Modulating Leucyl-tRNA Synthetase 1 Expression in Human Colorectal Cancer Cells.

Although mounting evidence has demonstrated that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) can promote tumorigenesis, its role in cancer remains controversial. To find potential target molecules of PGC-1α, GeneFishingTM DEG (differentially expressed genes) screening was performed using stable HEK293 cell lines expressing PGC-1α (PGC-1α-HEK293). As results, leucyl-tRNA synthetase 1 (LARS1) was upregulated. Western blot analysis showed that LARS1 was increased in PGC-1α overexpressed SW480 cells but decreased in PGC-1α shRNA knockdown SW620 cells. Several studies have suggested that LARS1 can be a potential target of anticancer agents. However, the molecular network of PGC-1α and LARS1 in human colorectal cancer cells remains unclear. LARS1 overexpression enhanced cell proliferation, migration, and invasion, whereas LARS1 knockdown reduced them. We also observed that expression levels of cyclin D1, c-Myc, and vimentin were regulated by LARS1 expression. We aimed to investigate whether effects of PGC-1α on cell proliferation and invasion were mediated by LARS1. Our results showed that PGC-1α might modulate cell proliferation and invasion by regulating LARS1 expression. These results suggest that LARS1 inhibitors might be used as anticancer agents in PGC-1α-overexpressing colorectal cancer. Further studies are needed in the future to clarify the detailed molecular mechanism by which PGC-1α regulates LARS1 expression.

Ligand of scavenger receptor class A indirectly induces maturation of human blood dendritic cells via production of tumor necrosis factor-alpha.

Dendritic cells (DCs) are the most potent antigen-presenting cells for naive T cells. In this study, scavenger receptor class A type I and type II (SR-A) were shown to be expressed by peripheral blood DCs (PBDCs) and monocyte-derived DCs (MDDCs). In addition, the binding of anti-SR-A antibody to these cells was lower in the presence of fucoidan, an SR-A agonist. Treatment of these DCs with fucoidan or anti-SR-A antibody markedly increased the surface expression of costimulatory molecules CD83 and major histocompatibility complex class II on the CD11c(high)CD123(low) myeloid subset of PBDCs. Furthermore, fucoidan-treated PBDCs produced tumor necrosis factor-alpha (TNF-alpha) but not IL-12p70. In addition, fucoidan-induced maturation was eliminated by pretreatment with TNF-alpha-neutralizing antibody. Finally, interferon-gamma secretion and T-cell proliferation were enhanced by coculture of T cells with fucoidan-matured PBDCs. Specific inhibitors of p38 MAPK and glycogen synthase kinase 3 suppressed TNF-alpha production and maturation of fucoidan-treated PBDCs. Moreover, MDDCs lacking SR-A failed to up-regulate CD83 expression, TNF-alpha production, and phosphorylation of p38 MAPK and glycogen synthase kinase 3-beta in the presence of fucoidan. Taken together, these results suggest that ligation of SR-A leads to induction of TNF-alpha, which subsequently induces PBDC maturation, thereby leading to enhanced T-cell stimulatory capacity.

Doxycycline potentiates the anti-proliferation effects of gemcitabine in pancreatic cancer cells.

Gemcitabine is often recommended as a first-line treatment for patients with metastatic pancreatic cancer. However, gemcitabine resistance is a major challenge in the treatment of pancreatic ductal adenocarcinoma. Our group serendipitously identified the role of doxycycline as a potentiator of gemcitabine efficacy in pancreatic cancer cells. Doxycycline and gemcitabine co-treatment was significantly more cytotoxic to pancreatic cancer cells compared to gemcitabine alone. Interestingly, doxycycline only exerted synergistic effects when coupled with gemcitabine as opposed to other conventional chemotherapeutics including nucleoside analogs. The anti-clonogenic effects of gemcitabine on pancreatic cancer cells were also enhanced by doxycycline. According to cell cycle analyses, doxycycline prolonged gemcitabine-mediated S phase cell cycle arrest. Further, gene expression profiling analyses indicated that a small set of genes involved in cell cycle regulation were uniquely modulated by gemcitabine and doxycycline co-treatment compared to gemcitabine alone. Western blot analyses indicated that several cell cycle-related proteins, including cyclin D1, p21, and DNA damage inducible transcript 4 (DDIT4), were further modulated by doxycycline and gemcitabine co-treatment. Taken together, our findings indicate that doxycycline enhances the effects of gemcitabine on cell cycle progression, thus rendering pancreatic cancer cells more sensitive to gemcitabine. However, additional studies are required to assess the mechanisms of doxycycline and gemcitabine synergism, which might lead to novel treatment options for pancreatic cancer.

The mechanism of fucoidan-induced apoptosis in leukemic cells: involvement of ERK1/2, JNK, glutathione, and nitric oxide.

Fucoidan, a sulfated polysaccharide in brown seaweed, has various biological activities including anti-tumor activity. We investigated the effects of fucoidan on the apoptosis of human promyeloid leukemic cells and fucoidan-mediated signaling pathways. Fucoidan induced apoptosis of HL-60, NB4, and THP-1 cells, but not K562 cells. Fucoidan treatment of HL-60 cells induced activation of caspases-8, -9, and -3, the cleavage of Bid, and changed mitochondrial membrane permeability. Fucoidan-induced apoptosis, cleavage of procaspases, and changes in the mitochondrial membrane permeability were efficiently blocked by depletion of mitogen-activated protein kinase (MAPK) kinase kinase 1 (MEKK1), and inhibitors of MAPK kinase 1 (MEK1) and c Jun NH2-terminal kinase (JNK). The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and JNK was increased in fucoidan-treated HL-60, NB4, and THP-1 cells, but not K562 cells. ERK1/2 activation occurred at earlier times than JNK activation and JNK activation was blocked by MEK1 inhibitor. In addition, fucoidan-induced apoptosis was inhibited by addition of glutathione and/or L-NAME, and fucoidan decreased intracellular glutathione concentrations and stimulated nitric oxide (NO) production. Buthionine-[R,S]-sulfoximine rendered HL-60 cells more sensitive to fucoidan. Depletion of MEKK1 and inhibition of MEK1 restored the intracellular glutathione content and abrogated NO production, whereas inhibition of JNK activation by SP600125 restored intracellular glutathione content but failed to inhibit NO production in fucoidan-treated HL-60 cells. These results suggest that activation of MEKK1, MEK1, ERK1/2, and JNK, depletion of glutathione, and production of NO are important mediators in fucoidan-induced apoptosis of human leukemic cells.

The anticancer activity of 3- and 10-bromofascaplysins is mediated by caspase-8, -9, -3-dependent apoptosis.

3- and 10-Bromofascaplysins was previously found to possess cytotoxic activity. In this study, we investigated their cancer preventive and proapoptotic properties. These effects were tested on mouse skin epidermal JB6 P(+) Cl41 cell line, its stable transfectants, and human tumor HL-60, THP-1, SNU-C4, SK-MEL-28, DLD-1, MDA-MB-231, and HeLa cells using a variety of assessments, including a cell viability (MTS) assay, flow cytometry, anchorage-independent soft agar assay, luciferase assay, mitochondrial permeability assay, and Western blotting. 3- and 10-Bromofascaplysins were effective at submicromolar concentrations as the anticancer agents, which exerted their action, at least in part, through the induction of caspase-8, -9, -3-dependent apoptosis.

PGC-1α Regulates Cell Proliferation and Invasion via AKT/GSK-3ß/ß-catenin Pathway in Human Colorectal Cancer SW620 and SW480 Cells.

BACKGROUND/AIM: Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial biogenesis and metabolism. We investigated the effect of PGC-1α knockdown in the human colorectal cancer cell line SW620, which highly expresses PGC-1α. MATERIALS AND METHODS: We established the PGC-1α shRNA-silenced SW620 stable cell line (PGC-1α shRNA-SW620 cells) and examined cell proliferation by cell counts and carboxyfluorescein succinimidyl ester (CFSE) staining, migration by wound-healing and transwell migration assay, and invasion by transwell assays. RESULTS: PGC-1α knockdown inhibited cell proliferation, migration, and invasion in SW620 cells. Western blot analysis showed that p-AKT, p-GSK-3ß, ß-catenin, N-cadherin and vimentin expression were all reduced, but E-cadherin had increased expression in PGC-1α shRNA-SW620 cells. We also examined cell proliferation, migration, invasion and the expression of p-AKT, p-GSK-3ß, ß-catenin, N-cadherin, vimentin, and E-cadherin in PGC-1α overexpressing SW480 cells (a low PGC-1α expressing line). We observed a complete reversal of the results seen in the knockdown. CONCLUSION: PGC-1α might regulate cell proliferation and invasion via AKT/GSK-3ß/ß-catenin pathway in SW620 and SW480 cells.

COUP-TFII Overexpression Inhibits Cell Proliferation and Invasion via Increased Expression of p53 and PTEN and Decreased Akt Phosphorylation in Human Colorectal Cancer SNU-C4 Cells.

BACKGROUND/AIM: Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) plays an important role in cancer. We examined the effect of COUP-TFII overexpression on the proliferation and invasion of the human colorectal cancer SNU-C4 cells. MATERIALS AND METHODS: SNU-C4 cells were stably transfected with COUP-TFII expression plasmid to overexpress COUP-TFII (COUP-TFII-SNU-C4 cells). Cell proliferation, colony-forming ability and transwell invasion assays were performed. To elucidate the underlying molecular mechanism of COUP-TFII action, western blot analysis, p53 shRNA transfection, and Myr-Akt transfection were performed. RESULTS: Cell proliferation and colony-forming ability were significantly inhibited in COUP-TFII-SNU-C4 cells. Western blot analyses demonstrated that while the expression of p53 and PTEN was increased, the p-Akt levels were decreased in COUP-TFII-SNU-C4 cells. Knockdown of p53 partially restored the cell proliferation, but did not reverse the inhibition of invasion. Constitutive activation of Akt via Myr-Akt transfection reversed the inhibited cell proliferation and invasion by COUP-TFII. CONCLUSION: p53 is required for the inhibition of cell proliferation, and decreased phosphorylation of Akt may mediate the inhibition of cell proliferation and invasion by COUP-TFII.

COUP-TFII Knock-down Promotes Proliferation and Invasion in Colorectal Cancer Cells via Activation of Akt Pathway and Up-regulation of FOXC1.

BACKGROUND/AIM: The chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) regulates cancer cell proliferation and invasion via complex molecular mechanisms. We aimed to investigate whether COUP-TFII modulates proliferation and invasion of the colorectal adenocarcinoma cell line HT-29. MATERIALS AND METHODS: HT-29 cells were stably tranfected with COUP-TFII shRNA plasmid to knock-down COUP-TFII (COUP-TFII shRNA-HT-29 cells). Cell proliferation, colony formation assay, invasion assay, microarray assays and western blot analyses were performed. RESULTS: Cell proliferation and invasion were significantly enhanced in COUP-TFII shRNA-HT-29 cells. The protein levels of forkhead box C1 (FOXC1), p-Akt, p-glycogen synthase kinase-3ß (p-GSK-3ß), and ß-catenin, which are known to be involved in cell proliferation and invasion, were significantly increased in COUP-TFII shRNA-HT-29 cells. Akt inhibitor IV and dominant negative (DN)-Akt expression vector transfection reversed the increased proliferation and invasion, which was accompanied by decreased protein levels of p-Akt, p-GSK-3ß, ß-catenin and FOXC1. CONCLUSION: COUP-TFII knock-down promoted proliferation and invasion via activation of Akt/GSK-3ß/ß-catenin and up-regulation of FOXC1. Further studies on the molecular mechanism of interaction between ß-catenin and FOXC1 expression may reveal novel target molecules for metastatic colorectal cancer therapy.