Jin Ming Hwang

p38 Mitogen-Activated Protein Kinase Pathway Is Involved in Protein Kinase Cα–Regulated Invasion in Human Hepatocellular Carcinoma Cells

Protein kinase Calpha (PKCalpha) has been suggested to play an important role in tumorigenesis, invasion, and metastasis. In this study, we investigated the signal pathways selectively activated by PKCalpha in human hepatocellular carcinoma (HCC) cells to determine the role of mitogen-activated protein kinases (MAPK) in PKCalpha-mediated HCC migration and invasion. A stable SK-Hep-1 cell clone (siPKCalpha-SK) expressing DNA-based small interfering RNA (siRNA) PKCalpha was established and was then characterized by cell growth, migration, and invasion. The expression of PKCalpha was decreased in siPKCalpha-SK, and cell growth, migration, and invasion were reduced. These changes were associated with the decrease in p38 MAPK phosphorylation level, but not in c-jun-NH(2)-kinase-1/2 (JNK-1/2) and extracellular signal-regulated kinase-1/2 (ERK-1/2). This phenomenon was confirmed in the SK-Hep-1 cells treated with antisense PKCalpha olignucleotide. The p38 MAPK inhibitor SB203580 or dominant negative p38 mutant plasmid (DN-p38) was used to evaluate the dependency of p38 MAPK in PKCalpha-regulated migration and invasion. Attenuation of cell migration and invasion was revealed in the SK-Hep-1 cells treated with the SB203580 or DN-p38, but not with ERK-1/2 inhibitor PD98059 or JNK-1/2 inhibitor SP600125. Overexpression of constitutively active MKK6 or PKCalpha may restore the inactivation of p38 and the attenuation of cell migration and invasion in siPKCalpha-SK. Similar findings were observed in the stable HA22T/VGH cell clone expressing siRNA PKCalpha. This study provides new insight into the role of p38 MAPK in PKCalpha-mediated malignant phenotypes, especially in PKCalpha-mediated cancer cell invasion, which may have valuable implications for developing new therapies for some PKCalpha-overexpressing cancers.

Baicalein inhibits the migration and invasive properties of human hepatoma cells

Flavonoids have been demonstrated to exert health benefits in humans. We investigated whether the flavonoid baicalein would inhibit the adhesion, migration, invasion, and growth of human hepatoma cell lines, and we also investigated its mechanism of action. The separate effects of baicalein and baicalin on the viability of HA22T/VGH and SK-Hep1 cells were investigated for 24h. To evaluate their invasive properties, cells were incubated on matrigel-coated transwell membranes in the presence or absence of baicalein. We examined the effect of baicalein on the adhesion of cells, on the activation of matrix metalloproteinases (MMPs), protein kinase C (PKC), and p38 mitogen-activated protein kinase (MAPK), and on tumor growth in vivo. We observed that baicalein suppresses hepatoma cell growth by 55%, baicalein-treated cells showed lower levels of migration than untreated cells, and cell invasion was significantly reduced to 28%. Incubation of hepatoma cells with baicalein also significantly inhibited cell adhesion to matrigel, collagen I, and gelatin-coated substrate. Baicalein also decreased the gelatinolytic activities of the matrix metalloproteinases MMP-2, MMP-9, and uPA, decreased p50 and p65 nuclear translocation, and decreased phosphorylated I-kappa-B (IKB)-β. In addition, baicalein reduced the phosphorylation levels of PKCα and p38 proteins, which regulate invasion in poorly differentiated hepatoma cells. Finally, when SK-Hep1 cells were grown as xenografts in nude mice, intraperitoneal (i.p.) injection of baicalein induced a significant dose-dependent decrease in tumor growth. These results demonstrate the anticancer properties of baicalein, which include the inhibition of adhesion, invasion, migration, and proliferation of human hepatoma cells in vivo.

Cardiac Fas-dependent and mitochondria-dependent apoptosis in ovariectomized rats

BACKGROUND Very limited information has been published regarding cardiac apoptosis in menopausal women or in those after bilateral oophorectomy. The purpose of this study was to evaluate whether cardiac Fas-dependent (type I) and mitochondria-dependent (type II) apoptotic pathways are activated in ovariectomized rats. METHODS Thirty-two female Wistar rats at 6-7 months of age were randomly divided into sham-operated group (Sham) and bilateral ovariectomized group (OVX). Two months after the operation, the cardiac characteristics, myocardial architecture, and two major apoptotic pathways in the excised left ventricle from rats were measured by histopathological analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR), and positive TUNEL assays. RESULTS The whole heart weight, the left ventricular weight, the ratios of whole heart weight to tibia length, and the ratios of left ventricle to tibia length were significantly increased in OVX relative to Sham. Abnormal myocardial architecture, enlarged interstitial spaces, more minor cardiac fibrosis, and more cardiac TUNEL-positive apoptotic cells were observed in OVX. The key components of Fas-dependent apoptosis (TNF-alpha, Fas ligand, Fas death receptors, Fas-associated death domain (FADD), activated caspase 8, and activated caspase 3) and key components of mitochondria-dependent apoptosis (Bad, Bax, Bax-to-Bcl2 ratio, cytosolic cytochrome c, activated caspase 9, and activated caspase 3) were significantly increased in OVX hearts. CONCLUSIONS The absence of female ovaries will activate the cardiac Fas-dependent and mitochondria-dependent apoptotic pathways, which might provide one of possible mechanism for developing heart failure in post-menopause women.

Hypoxia-induced compensatory effect as related to Shh and HIF-1α in ischemia embryo rat heart

Chronic cardiac ischemia/hypoxia induces coronary collateral formation and cardiomyocyte proliferation. Hypoxia can induce cellular adaptive responses, such as synthesis of VEGF for angiogenesis and IGF-2 for proliferation. Both reduce apoptotic effects to minimize injury or damage. To investigate the mechanism of neoangiogenesis and proliferation of fetal heart under umbilical cord compression situation, we used H9c2 cardiomyoblast cell culture, and in vivo embryonic hearts as our study models. Results showed hypoxia induced not only the increase of IGF-2 and VEGF expression but also the activation of their upstream regulatory genes, HIF-1α and Shh. The relationship between HIF-1α and Shh was further studied by using cyclopamine and 2-ME2, inhibitor of Shh and HIF-1α signaling, respectively, in the cardiomyoblast cell culture under hypoxia. We found that the two inhibitors not only blocked their own signal pathway, but also inhibited each other. The observations revealed when fetal heart under hypoxia that HIF-1α and Shh pathways maybe involve in cell proliferation and neoangiogenesis to minimize injury or damage, whereas the complex cross-talk between the two pathways remains unknown.

Second-hand smoke-induced cardiac fibrosis is related to the Fas death receptor apoptotic pathway without mitochondria-dependent pathway involvement in rats.

Exposure to environmental tobacco smoke has been epidemiologically linked to heart disease among nonsmokers. However, the molecular mechanism behind the pathogenesis of cardiac disease is unknown. In this study, we found that Wistar rats, exposed to tobacco cigarette smoke at doses of 5, 10, or 15 cigarettes for 30 min twice a day for 1 month, had a dose-dependently reduced heart weight to body weight ratio and enhanced interstitial fibrosis as identified by histopathologic analysis. The mRNA and activity of matrix metalloprotease-2 (MMP-2), representing the progress of cardiac remodeling, were also elevated in the heart. In addition, we used reverse-transcriptase polymerase chain reaction and Western blotting to demonstrate significantly increased levels of the apoptotic effecter caspase-3 in treated animal hearts. Dose-dependently elevated mRNA and protein levels of Fas, and promoted apoptotic initiator caspase-8 (active form), a molecule of a death-receptor–dependent pathway, coupled with unaltered or decreased levels of cytosolic cytochrome c and the apoptotic initiator caspase-9 (active form), molecules of mitochondria-dependent pathways, may be indicative of cardiac apoptosis, which is Fas death-receptor apoptotic-signaling dependent, but not mitochondria pathway dependent in rats exposed to second-hand smoke (SHS). With regard to the regulation of survival pathway, using dot blotting, we found cardiac insulin-like growth factor-1 (IGF-1) and IGF-1 receptor mRNA levels to be significantly increased, indicating that compensative effects of IGF-1 survival signaling could occur. In conclusion, we found that the effects of SHS on cardiomyocyte are mediated by the Fas death-receptor–dependent apoptotic pathway and might be related to the epidemiologic incidence of cardiac disease of SHS-exposed non-smokers.

Proliferation- and migration-enhancing effects of ginseng and ginsenoside Rg1 through IGF-I- and FGF-2-signaling pathways on RSC96 Schwann cells

The aim of the present study is to evaluate the proliferation‐ and migration‐enhancing effects of ginseng and its component, ginsenoside (Rg1) on RSC96 Schwann cells. We investigated the molecular signaling pathways, which include: (1) survival signaling, IGFs‐IGFIR‐Akt‐Bcl2 and proliferative signaling, cell cycle factors and mitogen‐activated protein kinase (MAPK) pathways, (2) migrating and anti‐scar signaling, FGF‐2‐uPA‐MMPs.We treated RSC96 cells with different concentrations (100, 200, 300, 400, 500 µg ml−1) of ginseng and its constituent, Rg1 (5, 10, 15, 20, 25 µg ml−1). We observed a proliferative effect in a dose‐dependent manner by PCNA western blotting assay, MTT assay, and wound healing test. Furthermore, we also found in the results of western blotting assay, ginseng and Rg1 enhance protein expression of IGF‐I pathway regulators, cell cycle controlling proteins, and MAPK signaling pathways to promote the cell proliferation. In addition, ginseng and Rg1 also stimulated the FGF‐2‐uPA‐MMP 9 migrating pathway to enhance the migration of RSC96 Schwann cells. Using MAPK chemical inhibitors, U0126, SB203580, and SP600125, the proliferative effects of ginseng and Rg1 on RSC96 cells were identified to be MAPK signaling‐dependent. On the basis of the results, applying appropriate doses of ginseng and Rg1 with biomedical materials would be a potential approach for enhancing neuron regeneration. Copyright

Reduction of anion exchanger 2 expression induces apoptosis of human hepatocellular carcinoma cells

Anion exchanger (AE) 2, belonging to the chloride–bicarbonate transporter family, has been reported to involve cell survival for hepatocellular carcinoma (HCC) cells. Our previous findings showed that AE2 gene was highly expressed in a poorly differentiated HCC cell line, HA22T/VGH. Additionally, treatment with 4,4′-diisothiocyanatostilbene-2,20-disulfonic acid (DIDS), an AE-specific inhibitor, significantly inhibited cell proliferation and induced cell apoptosis for the HA22T/VGH. To further investigate the biological functions of AE2 in human HCC, suppression of AE2 expression by the antisense oligonucleotide-AE2 (AS-AE2) was performed, and the cell viability, cell cycle regulation, and cell apoptosis for HCC cell lines were monitored. The results showed that AS-AE2 treatment could efficiently suppress the mRNA expression of AE2 for various differentiated HCC cells, including HA22T/VGH, SK-Hep-1, PLC/PRF/5, Hep3B, and HepG2. Moreover, AS-AE2 treatment significantly reduced cell viability, arrested cell cycle at sub-G1 phase, and induced cell apoptosis for the poorly differentiated HA22T/VGH, but not for other moderately or well-differentiated HCC cell lines. The findings indicated that AE2 may play an important role in the progression of HCC cells, and provide a new strategy for the development of therapeutic treatment against human HCC.

Leu27IGF2 plays an opposite role to IGF1 to induce H9c2 cardiomyoblast cell apoptosis via Gaq signaling

This study examines the role of IGF2/mannose 6-phosphate receptor (IGF2R) signaling in the signaling transduction regulation and cell apoptosis in H9c2 cardiomyoblast cells. However, it is difficult to recognize the distinct activation of IGF2 signaling without interfacing with IGFI receptor (IGF1R) after exposure to IGF2. Leu27IGF2, an analog of IGF2 that interacts selectively with the IGF2R, was used to specifically activate IGF2R signaling in this study. DNA fragmentation and TUNEL assay revealed that in contrast to IGF1 treatment preventing angiotensin II and AG1024-induced cell apoptosis, Leu27IGF2 appears to synergistically increase apoptosis in those cells. We further found cell apoptosis induction and an increase in the active form of caspase 3 in the treatment of cells with Leu27IGF2, but not IGF1. To detect the interaction between IGF2R and Galphaq using the immunoprecipitation assay, we found that IGF2R could directly interact with Galphaq and after treatment with Leu27IGF2 the binding ability of Galphaq to IGF2R had increased. This sequentially resulted in the phosphorylation of phospholipase C-beta, a key downstream modulator of Galphaq, on serine 537. Moreover, disruption of the Galphaq protein by small interferon RNA reduced the cell apoptosis induced by Leu27IGF2. Our findings demonstrate that IGF2R activation appears to induce cell apoptosis via Galphaq-deriving signaling cascades and its effect is completely different from IGF1R survival signaling.

JNK suppression is essential for 17β-Estradiol inhibits prostaglandin E2-Induced uPA and MMP-9 expressions and cell migration in human LoVo colon cancer cells

BackgroundEpidemiological studies demonstrate that the incidence and mortality rates of colorectal cancer in women are lower than in men. However, it is unknown if 17β-estradiol treatment is sufficient to inhibit prostaglandin E2 (PGE2)-induced cellular motility in human colon cancer cells.MethodsWe analyzed the protein expression of urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), matrix metallopeptidases (MMPs), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinases (TIMPs), and the cellular motility in PGE2-stimulated human LoVo cells. 17β-Estradiol and the inhibitors including LY294002 (Akt activation inhibitor), U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), QNZ (NFκB inhibitor) and ICI 182 780 were further used to explore the inhibitory effects of 17β-estradiol on PGE2-induced LoVo cell motility. Students t-test was used to analyze the difference between the two groups.ResultsUpregulation of urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA) and matrix metallopeptidases (MMPs) is reported to associate with the development of cancer cell mobility, metastasis, and subsequent malignant tumor. After administration of inhibitors including LY294002, U0126, SB203580, SP600125 or QNZ, we found that PGE2 treatment up-regulated uPA and MMP-9 expression via JNK1/2 signaling pathway, thus promoting cellular motility in human LoVo cancer cells. However, PGE2 treatment showed no effects on regulating expression of tPA, MMP-2, plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1, -2, -3 and -4 (TIMP-1, -2, -3 and -4). We further observed that 17β-estradiol treatment inhibited PGE2-induced uPA, MMP-9 and cellular motility by suppressing activation of JNK1/2 in human LoVo cancer cells.ConclusionsCollectively, these results suggest that 17β-estradiol treatment significantly inhibits PGE2-induced motility of human LoVo colon cancer cells.

BNIP3 induces IL6 and calcineurin/NFAT3 hypertrophic-related pathways in H9c2 cardiomyoblast cells

Ischemia/reperfusion injury causes cardiomyocyte apoptosis, ventricular remodeling, leading to a dilated heart. Hypoxia is one of the causes involved in ischemia damage, and BNIP3 is a hypoxia-inducible marker and also a sensor to induce mitochondria-dependent apoptosis. Recent reports discussed ablating BNIP3 can restrain cardiomyocytes apoptosis and post-infarction remodeling. BNIP3 is a crucial therapeutic target. However, the BNIP3-induced hypertrophy aspect is rarely investigated. Here, we transiently transfected BNIP3 plasmids into H9c2 cardiomyoblast cells to evaluate the molecular signaling and hypertrophy markers using Western blot. We measured the cell size change using actin staining. We disclose that BNIP3 overexpression induced an increase in cell size, activated the pathological-related hypertrophy signaling pathways, such as IL6-MEK5-ERK5, IL6-JAK2-STAT1/3, calcineurin/NFAT3 and p38β MAPK resulting in the fetal genes, ANP and BNP expressing. Concluding above, BNIP3 acts as a pathological hypertrophy inducer, which might be a potential therapeutic target for heart damage prevention.