Aqueous Ocimum gratissimum extract induces cell apoptosis in human hepatocellular carcinoma cells.

Treatment of advanced hepatocellular carcinoma (HCC) has exhibited a poor overall survival rate of only six to ten months, and the urgency of the development of more effective novel agents is ever present. In this line of research, we aimed to investigate the effects and inhibitive mechanisms of aqueous Ocimum gratissimum leaf extract (OGE), the extract of Ocimum gratissimum, which is commonly used as a therapeutic herb for its numerous pharmacological properties, on malignant HCC cells. Our results showed that OGE decreased the cell viability of HCC SK-Hep1 and HA22T cells in a dose-dependent manner (from 400 to 800 µg/mL), while there is little effect on Chang liver cells. Moreover, cell-cycle analysis shows increased Sub-G1 cell count in SK-Hep1 and HA22T cells which is not observed in Chang liver cells. These findings raise suspicion that the OGE-induced cell death may be mediated through proteins that regulate cell cycle and apoptosis in SK-Hep1 and HA22T cells, and further experimentation revealed that OGE treatment resulted in a dose-dependent decrease in caspase 3 and PARP expressions and in CDK4and p-ERK1/2expressions. Moreover, animal tests also exhibited decreased HCC tumor growth by OGE treatment. We therefore suggest that the inhibition of cell viability and tumor growth induced by OGE may be correlated to the alteration of apoptosis-related proteins.

MZF-1/Elk-1/PKCα is Associated with Poor Prognosis in Patients with Hepatocellular Carcinoma.

Background: Protein kinase C alpha (PKCα) is a key signaling molecule in human cancer development. As a therapeutic strategy, targeting PKCα is difficult because the molecule is ubiquitously expressed in non-malignant cells. PKCα is regulated by the cooperative interaction of the transcription factors myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) in human cancer cells. Methods: By conducting tissue array analysis, herein, we determined the protein expression of MZF-1/Elk-1/PKCα in various cancers. Results: The data show that the expression of MZF-1/Elk-1 is correlated with that of PKCα in hepatocellular carcinoma (HCC), but not in bladder and lung cancers. In addition, the PKCα down-regulation by shRNA Elk-1 was only observed in the HCC SK-Hep-1 cells. Blocking the interaction between MZF-1 and Elk-1 through the transfection of their binding domain MZF-160-72 decreased PKCα expression. This step ultimately depressed the epithelial-mesenchymal transition potential of the HCC cells. Conclusion: These findings could be used to develop an alternative therapeutic strategy against patients with the PKCα-derived HCC.

MZF-1/Elk-1 Complex Binds to Protein Kinase Cα Promoter and Is Involved in Hepatocellular Carcinoma.

In this study, the molecular mechanism of protein kinase C alpha (PKCα) gene regulation in hepatocellular carcinoma (HCC) involving Ets-like protein-1 (Elk-1) and myeloid zinc finger-1 (MZF-1) was investigated. The luciferase reporter assay results revealed that the presence of both MZF-1 and Elk-1 significantly contributed to the upregulation of PKCα gene transcription activity, and the transcriptional activity decreased when the transfection included a DNA-binding-deficient (∆DBD) gene vector of either MZF-1 or Elk-1 DNA-binding deficiency (MZF-1∆DBD or Elk-1∆DBD), thereby indicating that the enhanced expression of PKCα was caused by the binding of MZF-1 and/or Elk-1 with the PKCα promoter. We investigated MZF-1 and Elk-1 to determine whether they bind to each other. The results of immunoprecipitation (IP), Co-IP, chromatin IP (ChIP), and Re-ChIP analyses indicated that Elk-1 can directly bind to the N-terminal region of MZF-1 and MZF-1 can directly bind to the C-terminal region of Elk-1 to form a complex before attaching to the PKCα promoter. Furthermore, when MZF-1∆DBD or Elk-1∆DBD was added to the cells, PKCα expression decreased, and cell proliferation, migration, invasion, and tumorigenicity also decreased. These findings suggest that PKCα expression in HCC could be stimulated by the formation of MZF-1/Elk-1 complex, which directly binds to the PKCα promoter.

Lipopolysaccharide-stimulated leukocytes contribute to platelet aggregative dysfunction, which is attenuated by catalase in rats.

Endotoxemia causes several hematological dysfunctions, including platelet degranulation or disseminated intravascular coagulation, which lead to thrombotic and hemorrhagic events. Here, we tested the hypothesis that bacterial lipopolysaccharide (LPS)-stimulated leukocytes contribute to platelet aggregative dysfunction, and this function is attenuated by antioxidants. Platelet-rich plasma (PRP) was prepared from whole blood of normal and endotoxemic rats. The ability of platelet aggregation was measured by an aggregometer. LPS (50-100 µg/mL) was incubated with PRP, whole blood and PRP with polymorphonuclear leukocytes (PMNs) for 30 minutes, 60 minutes and 90 minutes, and platelet aggregation was detected. LPS-induced platelet aggregative dysfunction was undetectable in intact PRP which was isolated from normal whole blood, whereas it was detected in PRP isolated from endotoxemic rats and LPS-treated whole blood. Moreover, the effect of LPS-induced platelet aggregative dysfunction on intact PRP was observed when the PMNs were added. LPS-induced platelet aggregative dysfunction was significantly attenuated by catalase alone and in combination with N(G)-nitro-L-arginine methyl ester, but not by N(G)-nitro-L-arginine methyl ester alone. These results indicate that LPS-stimulated PMNs modulate platelet aggregation during LPS treatment and the effects are reversed by antioxidants. PMNs serve as an approach to understand LPS-induced platelet aggregative dysfunction during endotoxemia. During this process, the generation of reactive oxygen species, hydrogen peroxide especially, from LPS-stimulated PMNs could be an important potential factor in LPS-induced platelet aggregative dysfunction. Catalase contributes to the prevention of platelet dysfunction during LPS-induced sepsis.

The protection of hepatocyte cells from the effects of oxidative stress by treatment with vitamin E in conjunction with DTT.

We investigated the effect of vitamin E on membrane protein thiols under oxidative stress, which we induced by treating hepatocytes with tert-butyl hydroperoxide (TBH) for 60 mins. Those cells which we pretreated with vitamin E formed fewer blebs (22.3% compared to 60.0% in nonvitamin E-treated cells) and maintained cytosolic calcium concentration and the number of membrane protein thiols instead of showing the usual symptoms in cells undergoing oxidative stress. Dithiothreitol (DTT) also commonly reduces bleb formation in hepatocytes affected by TBH. However, our experiments clearly demonstrate that DTT does not prevent the changes in cytosolic calcium and membrane protein thiols in the blebbing cells. Consequently, we decided to pretreat cells with both DTT and vitamin E and found that the influence of TBH was entirely prevented. These findings may provide us with a new aspect for investigating the mechanism of bleb formation under oxidative stress.

Effect of carbon dioxide on pulmonary vascular tone at various pulmonary arterial pressure levels induced by endothelin-1.

There have been contradictory reports suggesting that CO(2) may constrict, dilate, or have no effect on pulmonary vessels. Permissive hypercapnia has become a widely adopted ventilatory technique used to avoid ventilator-induced lung injury, particularly in patients with acute respiratory distress syndrome (ARDS). On the other hand, respiratory alkalosis produced by mechanically induced hyperventilation is the mainstay of treatment for newborn infants with persistent pulmonary hypertension. It is important to clarify the vasomotor effect of CO(2) on pulmonary circulation in order to better evaluate the strategies of mechanical ventilation in intensive care. In the present study, pulmonary vascular responses to CO(2) were observed in isolated rat lungs (n = 32) under different levels of pulmonary arterial pressure (PAP) induced by various doses of endothelin-1 (ET-1). The purposes of this study were to investigate (1) the vasodilatory effect of 5% CO(2) in either N(2) (hypoxic-hypercapnia) or air (normoxic-hypercapnia) at different PAP levels induced by various doses of endothelin-1, and (2) the role of nitric oxide (NO) in mediating the pulmonary vascular response to hypercapnia, hypoxia, and ET-1. The results indicated that (1) CO(2) produces pulmonary vasodilatation at high PAP under ET-1 and hypoxic vasoconstriction; (2) the vasodilatory effect of CO(2) at different pressure levels varies in accordance with the levels of PAP, the dilatory effect tends to be more evident at higher PAP; and (3) endogenous NO attenuates ET-1 and hypoxic pulmonary vasoconstriction but does not augment the CO(2)-induced vasodilatation.

The activation of matrix metalloproteinase-2 induced by protein kinase C alpha in decidualization.

This study investigated the protein kinase C (PKC) and matrix metalloproteinase-2 (MMP-2) in the development of deciduomata in pseudo-pregnant and pregnant rats. The results showed that the expression of MMP-2 was significantly increased from day 2 to day 5 in pseudo-pregnancy and from day 7 to day 9 in pregnancy. To further investigate the correlation between MMP-2 and protein kinase C alpha (PKC alpha), the expression of MMP-2 in the 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated organotypic culture of decidual tissue was determined. The results showed that the active form of MMP-2 was significantly increased in the TPA-treated cultures. Moreover, this response was inhibited by the PKC inhibitor H7, the PKC alpha specific inhibitor Gö-6976 and the translation inhibitor cycloheximide, but not by the transcription inhibitor actinomycin D or the replication inhibitor mitomycin C. In addition, TPA also reversed the MMP-2 expression after by progesterone pretreatment in the primary decidual cells. These findings indicate that PKC alpha may play an important role in the regulation of the MMP-2 expression during decidualization.

Immunochemical localization of protein kinase Calpha in the biopsies of human hepatocellular carcinoma.

The purpose of this study was to elucidate the protein kinase C (PKC) alpha distribution in human hepatocellular carcinoma (HCC). The histoimmunopathologic technique was used to determine the localization and expression of PKCalpha in HCC biopsies. The HCC tissues were classified as cytosolic type (PKCalpha deposited in the cytoplasm in > 50% of cells) and membranous type for the remaining ones. There was a significant association of the membranous type with non-hepatitis C virus (HCV) infected patients. Moreover, the expression of PKCalpha in this type was significantly higher in HCC cells than that in the adjacent non-tumor liver cells. The result indicated that PKCalpha may play an important role in carcinogenesis of HCC patients with HBV infection and/or non-HCV infection.

Inhibition of intermediate-conductance Ca2+-activated K+ channel and cytoprotective properties of 4-piperidinomethyl-2-isopropyl-5-methylphenol.

The ionic mechanisms and cytoprotective activities of 4-piperidinomethyl-2-isopropyl-5-methylphenol (THPI), an analogue of thymol, were investigated in HL-60 granulocytes and in human erythrocytes, respectively. THPI inhibited K+ outward current (I(K)) in a concentration-dependent manner in HL-60 leukocytes, with an IC50 value of 4 microM. Neither iberiotoxin (200 nM) nor paxilline (1 microM) suppressed the amplitude of I(K), whereas clotrimazole (5 microM) significantly inhibited it. In the inside-out configuration of single channel recordings, application of THPI (5 microM) into the bath medium did not alter the single-channel conductance of intermediate-conductance Ca2+-activated K+ (IK(Ca)) channels (i.e K(Ca)3.1 channels), but it suppressed the channel activity significantly. THPI-induced inhibition of IK(Ca) channels was reversed by a further application of 1-ethyl-2-benzimidazolinone (10 microM). THPI-induced reduction in IK(Ca)-channel activity in these cells was primarily due to a decrease in mean open time. These results provide direct evidence that THPI is capable of suppressing the activity of IK(Ca) channels in HL-60 cells. The antioxidant action of THPI also revealed a beneficial cytoprotective effect against mitomycin C-mediated haemolytic effect in human erythrocytes. The results of this study suggest that blockade of IK(Ca) channels and the membrane-protecting activity of THPI would combine to have beneficial effects in lessening the severity of haemolytic crisis and reducing anaemia in sickle cell disease.

Overexpression of anion exchanger 2 in human hepatocellular carcinoma.

To compare gene expression patterns between the poorly-differentiated (HA22T/VGH) and well-differentiated (HepG2) hepatocellular carcinoma cells, messenger RNA was isolated form both kinds of cells and subjected to differential displays reversing transcriptase-polymerase chain reaction (DDRT-PCR) technology. Gene fragments showing difference in the expression were recovered, reamplified, cloned and sequenced. Anion exchanger 2 (AE2), an isoform of band 3 protein, was identified and chosen for further characterization. AE2 was strongly expressed in HA22T/VGH cells, while it was little expressed in the well-differentiated hepatocellular carcinoma cells (PLC/PRF5 and HepG2) or little in the normal liver cell (Chang liver). In the 28 pairs of HCC and adjacent non-tumor tissues, levels in the cancer tissue (32.7 +/- 5.0) were significantly higher than that in the adjacent non-tumor tissue (9.1 +/- 2.4) (P < 0.01). Moreover, 19 cases (68%) showed over-expression of AE2 in HCC tissues, 3 cases were similar in both tissues, and 6 cases exhibited little or undetectable signals. Twenty cases (71%) of adjacent normal tissue showed little or undetectable signals. The results indicated that overexpression of AE2 may be involved in the development of human HCC.

PKCalpha expression regulated by Elk-1 and MZF-1 in human HCC cells.

Our previous study found that PKCalpha was highly expressed in the poor-differentiated human HCC cells and associated with cell migration and invasion. In this study, we further investigated the gene regulation of this enzyme. We showed that PKCalpha expression enhancement in the poor-differentiated human HCC cells was found neither by DNA amplification nor by increasing mRNA stability using differential PCR and mRNA decay assays. After screening seven transcription factors in the putative cis-acting regulatory elements of human PKCalpha promoters, only Elk-1 and MZF-1 antisense oligonucleotide showed a significant reduction in the PKCalpha mRNA level. They also reduced cell proliferation, cell migratory and invasive capabilities, and DNA binding activities in the PKCalpha promoter region. Over-expression assay confirmed that the PKCalpha expression may be modulated by these two factors at the transcriptional level. Therefore, these results may provide a novel mechanism for PKCalpha expression regulation in human HCC cells.

Expression of protein kinase C alpha in biopsies and surgical specimens of human hepatocellular carcinoma.

Variations in the activity of protein kinase C (PKC) have been observed in different types of tumors. Although these inconsistent findings may be attributed to the alterations of individual PKC isoforms, the effects of general anesthetic may not be neglected. In this study, biopsies and surgical specimens were obtained from patients with HCC, and the levels of PKCalpha were analyzed by immunohistochemistry. PKCalpha expression in biopsies was mainly revealed on the cell membrane of hepatocytes whereas that in the surgical specimens was in the cytosol and on the membrane. In both types of specimens, the PKCalpha level in HCC was significantly higher than that in the adjacent non-tumorous tissue. Moreover, the level of PKCalpha in biopsies was significantly higher than that in surgical specimens of the corresponding tissue type. These findings suggested that general anesthetics may significantly affect the expression of PKCalpha, and the effects of anesthetics should not be neglected in observations which were made only based on surgical specimens.

17beta-estradiol reduces cardiac hypertrophy mediated through the up-regulation of PI3K/Akt and the suppression of calcineurin/NF-AT3 signaling pathways in rats.

This study was designed to determine the effects of 17beta-estradiol (E2) in overcoming the cardiac over-loading and cardiac fibrosis in rats. E2 (100 ng/kg) or oil was applied in female Sprague-Dawley rats with or without bilateral ovariectomy and with or without coarctation of the abdominal aorta after 4 or 8 days. By post-operative day 4, the heart weight, the left ventricular weight, the latent form of MMP-2 in rat hearts with or without the ovary intact had significantly increased while these changes were reversed after E2 treatment. Although animals with the ovaries intact overcame the hypertrophic effects and the consumption of MMP-2, these effects were not restored in ovariectomized animals in which more fibrosis could be found by day 8. Among the IGF-I signaling, the levels of IGF-I, the activities of PI3K-Akt for cardiomyocyte survival, and MEK-ERKs for non-cardiomyocyte proliferation pathways had significantly increased by day 4. These increasing trends were enhanced by E2 treatment. However, down-regulation was only observed on day 8 in ovariectomized animals. Similarly, elevated expressions of the steady-state mRNA of IGF-I, IGF-IR, and Cox vb were observed on day 4 in animals with the ovaries intact and these expressions were enhanced by E2 treatment. In contrast, down-regulation on day 8 in ovariectomized animals was not enhanced by E2. The calcineurin/NFAT-3 pathway was suppressed on day 4 but was elevated on day 8 in ovariectomized animals. These findings indicate that signaling pathways may be plausible mechanisms for the cardiac protective effects of E2 administration.

Augmented release of matrix metalloproteinase-9 by PKC activation in organotypic cultures of human breast cancer and adjacent normal breast tissue and fibroadenoma.

The organotypic culture technique and quantitative gelatin zymography were used to determine the expression of matrix metalloproteinase (MMP)-9 and MMP-2 in human breast cancer and adjacent normal breast tissue and fibroadenoma. MMP-9 and MMP2 were constitutively expressed in all cultures. The release of these two enzymes in breast cancer was higher than that in adjacent normal breast tissue and fibroadenoma. Administration of 12-o-tetradecanoyl- phorbol-13-acetate (TPA) increased the release of MMP-9 but not of MMP-2. This response was inhibited by protein kinase C (PKC) inhibitor (H7), transcription inhibitor (actinomycin D) and translation inhibitor (cycloheximide). Moreover, the increased level of MMP-9 by TPA in breast cancer was also higher than that in adjacent normal breast tissue and fibroadenoma. These phenomena were also observed in the DAG-treated culture. These findings suggested that the MMP-9 expression in the breast cancer tissue may be more sensitive for the PKC activation.