Jer Yuh Liu

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.

Reduction of PKCα decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma

Protein kinase C (PKC) superfamily play key regulatory roles on the development of cancer. However, the exact role of these enzymes in human hepatocellular carcinoma (HCC) has not been well established. Using the RT‐PCR and Western blotting to analyze the levels of PKC isoforms mRNA and protein in the five different differentiated hepatoma cell lines, we found that PKCα was highly expressed in the poor‐differentiated HCC cell lines (SK‐Hep‐1 and HA22T/VGH) as compared with that in the well‐differentiated HCC cell lines (PLC/PRF/5, Hep3B, and HepG2). When treated with PKCα antisense oligonucleotides (ODN), both HA22T/VGH and SK‐Hep‐1 cells lines showed the reduction of PKCα expression, as well as a deceleration in the growth rate and in the level of cyclin D1, but the increase in the levels of p53 and p21WAF1/CIP1. Moreover, the reduction of PKCα expression also inhibited the migratory and invasive potential of both HA22T/VGH and SK‐Hep‐1 cells lines, and revealed a down‐regulation of several migration/invasion‐related genes (MMP‐1, u‐PA, u‐PAR, and FAK). These phenomenon were also confirmed by DNA‐based small interfering RNA (siRNA) PKCα and PKCα/β specific inhibitor Go6976. Thus, the results indicated that PKCα may be associated with regulation of cell proliferation/migration/invasion in human poorly differentiated HCC cells, suggesting a role for the PKCα in the malignant progression of human HCC. J. Cell. Biochem. 103: 9–20, 2008.

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.

Age-related differences in the release of luteinizing hormone and gonadotropin-releasing hormone in ovariectomized rats.

The effect of aging on the release of gonadotropin-releasing hormone (GnRH) in vitro and of luteinizing hormone (LH) both in vivo and in vitro in ovariectomized (Ovx) rats was studied. Old (21-24 months) and young (3-4 months) rats were Ovx before use. They were injected subcutaneously with estradiol benzoate (25 micrograms/kg) or sesame oil for 3 days and then challenged with GnRH (0.5, 2 or 10 micrograms/kg) via a jugular catheter. Blood samples were collected immediately before and at 5, 10, 20, 40 and 60 min following GnRH injection. For in vitro study, Ovx rats were decapitated. The anterior pituitary glands (APs) were incubated with GnRH (0.1 or 10 nM) and estradiol (0, 0.1, 1 or 10 nM) at 37 degrees C for 30 min. The mediobasal hypothalamus was superfused with Lockes solution at 37 degrees C for 210 min, and stimulated with 60 mM KCl at 90 and 150 min. The medium samples were collected at 10-min intervals. Concentrations of GnRH and LH in plasma and medium samples were measured by radioimmunoassay. In all rats, the basal and GnRH-stimulated levels of plasma LH were lower in old than in young rats. The spontaneous release of LH in vitro from APs of Ovx rats was increased by aging, whereas GnRH-stimulated release of LH in vitro was lower in old than in young animals. The potassium-stimulated, but not spontaneous, release of GnRH was lower in old than in young Ovx rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharide induces cellular hypertrophy through calcineurin/NFAT-3 signaling pathway in H9c2 myocardiac cells

Evidences suggest that lipopolysaccharide (LPS) participates in the inflammatory response in the cardiovascular system; however, it is unknown if LPS is sufficient to cause the cardiac hypertrophy. In the present study, we treated H9c2 myocardiac cells with LPS to explore whether LPS causes cardiac hypertrophy, and to identify the precise molecular and cellular mechanisms behind hypertrophic responses. Here we show that LPS challenge induces pathological hypertrophic responses such as the increase in cell size, the reorganization of actin filaments, and the upregulation of hypertrophy markers including atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in H9c2 cells. LPS treatment significantly promotes the activation of GATA-4 and the nuclear translocation of NFAT-3, which act as transcription factors mediating the development of cardiac hypertrophy. After administration of inhibitors including U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor), FK506 (calcineurin inhibitor), and QNZ (NFκB inhibitor), LPS-induced hypertrophic characteristic features, such as increases in cell size, actin fibers, and levels of ANP and BNP, and the nuclear localization of NFAT-3 are markedly inhibited only by calcineurin inhibitors, CsA and FK506. Collectively, these results suggest that LPS leads to myocardiac hypertrophy through calcineurin/NFAT-3 signaling pathway in H9c2 cells. Our findings further provide a link between the LPS-induced inflammatory response and the calcineurin/NFAT-3 signaling pathway that mediates the development of cardiac hypertrophy.

Cardiomyoblast apoptosis induced by insulin-like growth factor (IGF)-I resistance is IGF-II dependent and synergistically enhanced by angiotensin II

Objective: This study explores the synergistic effect of cardiomyoblast apoptosis induced by angiotensin II (Ang II) and Insulin-like growth factor (IGF)-I resistance, and elucidates the role of IGF-II via IGF-II receptor (R) and calcineurin pathways in apoptosis induced by Ang II and IGF-I resistance. Methods: Apoptosis of cultured cardiomyoblast H9c2 cells was assessed by DNA fragmentation on agarose gel electrophoresis, nuclear condensation stained with DAPI, and Western blot analysis of pro-apoptotic Bad and cytochrome c in various combinations of control, Ang II, antisense IGF (I or II), IGF (I or II) antibody, IGF (I or II) receptor (R) antibody, or calcineurin inhibitor (Cyclosporine A, (CsA)). Results: We found the following: (1) The combination of Ang II and IGF-I deficiencies had a synergistic effect on apoptosis, confirmed by DNA fragmentation, nuclei condensation, and increases in such pro-apoptotic proteins as Bad, cytochrome c, caspase 9, and caspase 3 in H9c2 cells. (2) IGF-II and IGF-IIR protein products were increased by antisense IGF-I and IGF-I resistance, but these IGF-II protein products were not affected by sense IGF-I and non-specific antibody IgG in H9c2 cells. (3) The alteration of Bad protein level and the release of cytochrome c, both induced by treatments containing combinations of Ang II and antisense IGF-I, IGF-I antibody or IGF-IR antibody, were inhibited by IGF-II antibody. (4) DNA fragmentation, Bad, and cytochrome c which was induced by treatments combining IGF-IR antibody with Ang II or combining IGF-IR antibody with IGF-II were remarkably attenuated by CsA. Conclusion: IGF-I deficiency and/or IGF-IR resistance induced apoptosis in cardiomyoblast cells. The apoptosis, which might have been caused by the upregulation of IGF-II and IGF-IIR genes possibly activated the downstream calcineurin pathway, was synergistically augmented by Ang II.

Age-related differences in the spontaneous and thyrotropin-releasing hormone-stimulated release of prolactin and thyrotropin in ovariectomized rats.

Effect of estradiol on the spontaneous and thyrotropin-releasing hormone (TRH)-stimulated release of prolactin (PRL) and thyrotropin (TSH) in young and aged ovariectomized (Ovx) rats was investigated. Old (22-26 months) and young (3 months) female rats were Ovx 3 weeks before use. They were injected subcutaneously with estradiol benzoate (EB, 25 micrograms/kg) or sesame oil for 3 days and were catheterized via the right jugular vein. Twenty hours after the last administration of EB, rats were injected with TRH (10 micrograms/kg) through the catheter. Blood samples were collected before and 5, 10, 20, 40 and 60 min after TRH injection. On the day following blood sampling, all rats were decapitated. The anterior pituitary glands (APs) were excised, and incubated with or without TRH (10 ng/ml) at 37 degrees C for 30 min. The basal level of PRL concentration in plasma samples was 5-fold higher in old Ovx rats than in young Ovx rats. Five min after TRH injection, the increase in plasma PRL was greater in old animals than in young animals. Plasma PRL remained higher in old animals than in young animals at 10, 20, 40 and 60 min following TRH challenge. Administration of EB to old and to young Ovx rats produced increases in both basal and TRH-stimulated secretions of PRL, but did not affect the difference in plasma PRL patterns between old and young animals. The release of PRL from APs was increased significantly in all rats after a 30-min incubation with TRH. In Ovx rats injected with oil, the basal release of PRL in vitro was increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisense oligonucleotide Elk-1 suppresses the tumorigenicity of human hepatocellular carcinoma cells

In previous studies, we showed that reducing Ets‐like protein‐1 (Elk‐1) expression inhibited protein kinase C alpha (PKCα) expression and decreased cell migration and invasion in human hepatocellular carcinoma (HCC). In this study, we have investigated the role of Elk‐1 in tumorigenesis. SK‐Hep‐1 HCC cells were transfected with the ElK‐1 antisense oligonucleotide (ODN). In the pretreated cells we detected a reduction of mRNA level using RT—PCR. The inhibitory rate of cell growth was measured by MTT assay. Pretreated‐SK‐Hep‐1 HCC cells were implanted subcutaneously into nude mice to observe the tumor growth and calculate tumor inhibitory rate. The results showed that 5 μM of the antisense ODN Elk‐1 suppressed both Elk‐1 and PKCα production by SK‐Hep‐1 HCC cells after cationic liposome‐mediated transfection, to 8% and 1% of control values, respectively, and the growth of SK‐Hep‐1 HCC cells was inhibited at 2–5 μM doses of the antisense ODN Elk‐1. The control reagent, sense ODN Elk‐1, showed no effects. In BALB/nude mice, SK‐Hep‐1 HCC cells transfected with the 5 μM antisense ODN Elk‐1 formed tumors much smaller than those of sense ODN Elk‐1 pretreated cells. The maximum inhibitory rate of tumor growth was 80.8 ± 12.6% and the tumor formation time was prolonged from 13 to 25 days. These findings suggested the usefulness of antisense ODN Elk‐1 as a new reagent for liver cancer therapy.

Apoptotic and anti-proliferative effects of 17β-estradiol and 17β-estradiol-like compounds in the Hep3B cell line

Since there is evidence for estrogen and estrogen-like compounds to have beneficial effect on the pathogenesis of hepatocellular carcinoma (HCC), this study was designed to investigative the apoptotic and anti-proliferative effects of these compounds on the human hepatoma Hep3B cell line. The Hep3B cells were treated with 17β-estradiol (E2), diethylstilbestrol (DES), tamoxifen, and genistein. After treatments of these compounds at the concentration of 10-6 or 10-8 M, the Hep3B cells were demonstrated to have significant DNA fragmentation, nucleus condensation, cytochrome-c leaking from the mitochondria and caspase-3 activation by DAPI and Western blotting. The cells were also observed to have declined proliferative potential by MTT assay, arrested cell cycle by flow-cytometry measurements. However, the cytochrome-c leaking from the mitochondria induced by E2 and E2-like compounds was blocked totally by ICI 182,780 treatment. These finding suggest that estrogen and the estrogen-like compounds may induce anti-proliferative and apoptotic effects in Hep3B cells, and the E2 and the E2-like compounds mediated apoptotic effect was estrogen receptor dependent. Among the drugs tested, E2, E2 agonists (DES and genistein) and partial antagonist (tamoxifen), all showed the stronger anti-tumor potential.

Opposing Action of Estrogen Receptors α and β on Tumor Necrosis Factor-α Gene Expression and Caspase-8-mediated Apoptotic Effects in HA22T Cells

Hepatocellular carcinoma (HCC), the major manifestation of primary liver cancer, is one of the most frequent and malignant cancers worldwide, especially in Taiwan. Estrogen receptors (ERs) have been reported to play either a proliferation- or apoptosis-enhancing role in the differentiation of cancers, including HCC. In a previous experiment, we showed that transient overexpressed estrogen receptor-α induced early stage HCC cell line Hep 3B cell apoptosis by increasing the hTNF-α gene expression in a ligand-independent manner. To further clarify if the apoptotic effect occurs in poorly differentiated HCC cell line, HA22T, and elucidate the roles of ERs and TNF-α, DNA fragmentation and caspase activity were measured in late stage HCC cell line, HA22T, by measuring the expression of hER-α and hER-β using a Tetracycline-induciable system (Tet-on). Increased DNA fragmentation and caspase-3 activity were found in hERβ-overexpressed HA22T cells treated with estrogen (10−8 M) but not in hERα-overexpressed HA22T cells. Using RT-PCR/PCR and western blotting in HA22T cells, overexpressed hER-β was also found to increase the expression of hTNF-α mRNA and induce hTNF-α-dependent luciferase activity in a ligand-dependent manner. Additionally, LPS treatment and hER-β overexpression both enhance caspase-8 activities, whereas neither hER-β nor E2 treatment affected caspase-9 activities. In addition, the overexpressed hER-β plus E2 enhanced DNA fragmentation and caspase-8 activities were only partially reduced by anti-hTNF-α (0.1ng/ml), which was possibly due to the involvement of P53 and TGF-β. Taken together, our data indicates that overexpressed hER-β but not hER-α may induce caspase-8-mediated apoptosis by increasing the hTNF-α gene expression in a ligand-dependent manner in poorly differentiated HA22T cells. (Mol Cell Biochem xxx: 1–9, 2005)