Involvement of the ornithine decarboxylase/polyamine system in precondition-induced cardioprotection through an interaction with PKC in rat hearts

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.     

Anion exchanger inhibitor DIDS induces human poorly-differentiated malignant hepatocellular carcinoma HA22T cell apoptosis

Anion exchangers (AEs) of the Cl^-/HCO3^- exchanger family contribute to the regulation of intracellular acid-base balance. Recently, we found that anion exchanger 2 (AE2) was significantly expressed in human hepatocellular carcinoma (HCC) and in poorly-differentiated human HCC HA22T/VGH cells. In the present study, we further explored the pharmacological function of AE in four human HCC cell lines (SK-Hep-1, HA22T/VGH, HepG2, and Hep3B) following the treatment of 4,4’-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an AEs specific inhibitor. After administrations with 400–1000 μM of DIDS, cell proliferation was greatly inhibited in a dose-dependent manner from 47.5 to 65.0% in higher malignant HA22T/VGH cells, but not in other cell lines. The results of 4,6-diamidino-2-phenylindole (DAPI) staining, DNA fragmentation and flow cytometric analysis further revealed that cell apoptosis induced by DIDS was also observed in HA22T/VGH cells. Therefore, these findings suggested that AE may be involved, in part, in the proliferation and survival of HA22T cells and could be a new potential therapeutic target against specific human HCC. The authors Chih-Yang Huang and Jer-Yuh Lin contributed equally to this article.     

Differential expression of antioxidant enzymes in various hepatocellular carcinoma cell lines

Recent evidence suggests that reactive oxygen species (ROS) play an important role in the pathogenesis of various illnesses, and the ROS and antioxidant enzymes are highly associated with cell differentiation and diseases. In this study, we tested the hypothesis that specific antioxidant enzymes are differentially expressed in hepatocellular carcinoma (HCC) cell lines with various degrees of differentiation. We compared the expression of several antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GRx), and glutathione peroxidase (GPx) in five HCC cell lines with well (Hep G2 and Hep 3B) or poor (HA22T/VGH, HA55T/VGH, and SK-Hep-1) differentiation. Our results showed that both well-differentiated HCC cell lines expressed extremely higher CAT and GRx enzyme activities than all three poorly differentiated ones. Moreover, the protein and mRNA levels of CAT were much higher in two well-differentiated HCC cell lines than in all three poorly differentiated ones. Both well-differentiated HCC cell lines also showed a higher protein or mRNA expression of Cu/ZnSOD and MnSOD than three poorly differentiated ones. Our results demonstrate that specific antioxidant enzymes (especially, CAT and GRx) are differentially expressed in HCC cell lines with well or poor differentiation. These findings suggest that CAT and GRx are two potential differentiation markers for HCC.     

Absence of cytokeratin in human hepatoma cell lines

The immunofluorescence study revealed that both our established human hepatoma cell lines, HA22T/VGH and HA47T/VGH, were absent of cytokeratin. This observation was further confirmed by a western blot study. However, they as well as the other human hepatoma cells, Hep G2, Hep 3B, and SK-Hep-1 expressed vimentin.     

Increase of Bax/ Bcl-XL ratio and arrest of cell cycle by luteolin in immortalized human hepatoma cell line

Luteolin is a common constituent of many kinds of fruits and vegetables. It possesses the anti-neoplastic activities against several human cancers, but its activity against hepatocellular carcinoma (HCC) is seldom mentioned. To evaluate the activity against HCC and to provide information about the mechanism, we tested luteolin against five human hepatoma cell lines, namely HepG2, SK-Hep-1, PLC/PRF/5, Hep3B, and HA22T/VGH, with XTT assay and flow cytometry. The results showed that luteolin inhibited PLC/PRF/5, Hep3B and HA22T/VGH at a concentration of 1 microg/ml, but it needed 5 microg/ml to inhibit HepG2 and 10 microg/ml for SK-Hep1 (P <0.05). The inhibitive concentrations of 50% (IC50) of luteolin were between 7.29 microg/ml and 32.59 microg/ml, which were comparable with those of 5-FU (15.35 microg/ml to 32.84 microg/ml). The least effective cell line as affected by luteolin (SK-Hep1) was the most effective one when treating with 5-FU. The least effective cell line as affected by 5-FU (HA22T/VGH) was effectively affected by luteolin. It seemed that luteolin had some complementary activity to 5-FU against these HCC cell lines. The luteolin-treated PLC/PRF/5 cells exhibited typical changes of apoptosis with a characteristic DNA laddering pattern on gel electrophoresis. Luteolin also activated casepase-3, increased Bax protein with a concomitant decrease in Bcl-XL level. Increase in Bax/ Bcl-XL ratio and activation of caspase-3 supported the apoptotic finding on gel electrophoresis. Luteolin also induced cell cycle arrest at G0/G1 phase. We suggested that luteolin might exhibit anti-HCC activity as efficient as 5-FU by the mechanism of not only cell cycle arrest but also apoptosis.     

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 (PKC alpha) 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 microM of the antisense ODN Elk-1 suppressed both Elk-1 and PKC alpha 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 microM 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 microM 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.     

Induction of plasma protein secretion in a newly established human hepatoma cell line.

To study the expression and the regulation of hepatocyte markers, we have undertaken to establish human hepatoma cell lines of various phenotypes. We now report the establishment of a new human hepatoma cell line, HA22T/VGH. This cell line has many of the properties of human hepatocellular carcinoma. Only 5 of 15 plasma proteins investigated were detected in the medium of a 10-day-old HA22T/VGH culture. However, when the HA22T/VGH cells and a clonal derivative, C5, were cultured in an aggregated form, all 15 plasma proteins were found in the culture medium. These results indicate that hepatoma cell lines with different phenotypes can be established, and they provide a good experimental framework to investigate differentiation of human hepatocytes.     

Novel combination of Celecoxib and proteasome inhibitor MG132 provides synergistic antiproliferative and proapoptotic effects in human liver tumor cells

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Celecoxib (Celebrex®) exhibits antitumor effects in human HCC cells, and its mechanism of action is mediated either by its ability to inhibit cyclooxygenase 2 (COX-2) or by a number of various other COX-2 independent effects. Proteasome inhibitors (PIs) can exert cell growth inhibitory and apoptotic effects in different tumor cell types, including HCC cells. The present study examined the interaction between celecoxib and the PI MG132 in two human liver tumor cell lines HepG2 and HA22T/VGH. Our data showed that each inhibitor reduced proliferation and induced apoptosis in a dose-dependent manner in both cell lines. Moreover, the combination of celecoxib with MG132 synergistically inhibited cell viability and increased apoptosis, as documented by caspase 3 and 7 activation, PARP cleavage, and down-regulation of Bcl-2. Celecoxib and MG132, both alone and synergistically in combination, induced expression of the endoplasmic reticulum (ER) stress genes ATF4, CHOP, TRB3 and promoted the splicing of XBP1 mRNA. Knockdown of TRB3 mRNA expression by small interference RNA significantly decreased combination-induced cell death in HA22T/VGH cells, whereas it increased combination-induced cell death in HepG2 cells, suggesting that activation of the ER stress response might have either a detrimental or a protective role in liver tumor cell survival. In conclusion, our data indicate that combination treatment with celecoxib and MG132 resulted in synergistic antiproliferative and proapoptotic effects against liver cancer cells, providing a rational basis for the clinical use of this combination in the treatment of liver cancer.     

Combined arginine and ascorbic acid treatment induces apoptosis in the hepatoma cell line HA22T/VGH and changes in redox status involving the pentose phosphate pathway and reactive oxygen and nitrogen species

Arginine is a physiological substrate for nitric oxide synthase to generate nitric oxide (NO), which can influence tumor cell survival, while ascorbic acid is selectively toxic for cancer cells. This study explored the effect of an arginine/ascorbic acid combination on human cancer cell lines. The hepatoma cell line HA22T/VGH was the most sensitive of the tested cells to combination treatment. A combination of 5.74 mM of arginine and 0.57 mM of ascorbic acid induced HA22T/VGH cell death through apoptosis and an increase in levels of reactive oxygen species and NO, as well as its stable products NO₂⁻ and NO₃⁻. The combination also reduced the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase in the pentose phosphate pathway, a major mechanism for producing NADPH, resulting in a marked decrease in intracellular NADPH levels. A dramatic decrease in intracellular glutathione (GSH) levels, a decrease in the mitochondrial membrane potential, ATP depletion and release of cytochrome c were also seen. Caspase-9 and caspase-3 were activated, apoptotic protein Bax expression increased and the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL decreased. These results suggest that this combination induced HA22T/VGH cell death by interfering with redox state regulation by a reduction in pentose phosphate pathway activity and increasing oxidative and nitrosative stress.     

Antitumor effects of curcumin and structurally beta-diketone modified analogs on multidrug resistant cancer cells

Using concepts of bioisostery a series of curcumin analogs were synthesized: the diketonic system of the compound was elaborated into enaminones, oximes, and the isoxazole heterocycle. The cell growth inhibitory and apoptosis inducing effects of the new analogs were evaluated by in vitro assays in the hepatocellular carcinoma HA22T/VGH cells, as well as in the MCF-7 breast cancer cell line and in its multidrug resistant (MDR) variant MCF-7R. Increased antitumor activity on all cell lines was found with the isoxazole analog and especially with the benzyl oxime derivative; in the HA22T/VGH cell model, the latter compound inhibited constitutive NF-kappaB activation.     

Mutational analysis on the function of the SWAP-70 PH domain

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)Shares equally contribution Contract grant sponsor: National Science Council; Contract grant number: NSC 91-2314-B-075A-006, NSC 92-2314-B-075A-014.     

Suppression of tumorigenicity of human hepatocellular carcinoma cells by antisense oligonucleotide MZF-1

Our previous studies have found that reducing expression of myeloid zinc finger-1 (MZF-1) inhibited protein kinase C alpha (PKCalpha) expression and decreased cell migration and invasion in human hepatocellular carcinoma (HCC). In this study, we further investigated the role of MZF-1 in tumorigenesis. The SK-Hep-1 HCC cells were transfected with the antisense oligonucleotide (ODN) of MZF-1. The pretreated cells was then detected the mRNA and protein levels by RT-PCR and western blotting, and the cell growth was assayed by MTT. Meanwhile, the pretreated-SK-Hep-1 HCC cells were implanted subcutaneously into nude mice to observe the tumor growth and calculated tumor inhibitory rate. The results showed that, at the dosage of 5 microM, the antisense ODN MZF-1 suppressed both MZF-1 and PKCalpha productions by SK-Hep-1 HCC cells after cationic liposome-mediated transfection, to 15% and 30% in control cultures of 0 microM dosage, respectively. The growth of SK-Hep-1 HCC cells was inhibited by the 2-5 microM doses of the antisense ODN MZF-1, whereas the control reagent, the sense ODN MZF-1, showed no effects. In BALB/nude mice, SK-Hep-1 HCC cells pretreated with the 5 microM antisense ODN MZF-1 formed tumors much smaller than the cells with sense ODN. The mean of inhibitory rate of tumor growth was 71.2 +/- 8.6%, and the tumor formation time was prolonged from 14 days to 26 days. These findings suggested the usefulness of antisense ODN MZF-1 as a new reagent for cancer therapy.     

Expression of high- and low-affinity epidermal growth factor receptors in human hepatoma cell lines

Data are presented from a comparative research on expression of epidermal growth factor (EGF) receptors and response to EGF of six independently established cell lines derived from human hepatoma. These lines differ in terms of the degree of differentiation, presence of hepatitis B virus (HBV) DNA copies in integrated form and expression of HBV genes. Our results indicate differential expression of membrane EGF receptors and differential response to EGF under serum- and hormone-free culture conditions. Furthermore, a significant difference in affinity could be detected between EGF receptors of the two highly dedifferentiated cell lines (HA22T/VGH and Li7A) whose replication is inhibited by EGF concentrations capable of stimulating more differentiated phenotypes.     

Autocrine stimulation by insulin-like growth factor I is involved in the growth, tumorigenicity and chemoresistance of human esophageal carcinoma cells

Insulin-like growth factor I (IGF-I) receptor (IGF-IR)-mediated signals are known to be involved in cell growth and transformation and prevention of apoptosis. In this study, we demonstrated the coexpression of IGF-I and IGF-IR in human esophageal carcinoma tissues. We also demonstrated the IGF-I autocrine system in esophageal carcinoma cell lines. Both the CE48T/VGH and CE81T/VGH cell lines showed proliferative responses to IGF-I stimulation. Autokinase activity of IGF-IR in these cells can be triggered by the exogenous addition of IGF-I. In addition, an IGF-I peptide antagonist, JB1, specifically inhibited ligand-induced receptor autophosphorylation in a dose-dependent manner. Under serum-free conditions, JB1 also reduced the degree of IGF-IR phosphorylation and cell numbers. Furthermore, the addition of JB1 decreased the number of CE81T/VGH colonies formed in methyl cellulose agar and the size and the incidence of tumors which grew in mice with severe combined immunodeficiency. These results imply that an IGF-I autocrine system in human esophageal carcinoma cells could stimulate tumor growth. Finally, we found that IGF-I prevented the apoptosis of CE81T/VGH cells induced by chemotherapeutic drugs, such as cisplatin, 5-fluorouracil and camptothecin. Thus, interruption of IGF-IR function may provide a way to retard tumor growth and increase the sensitivity of esophageal carcinoma to chemotherapy.     

Differential response of the human hepatoma-derived cell line HA22T/VGH to polypeptide mitogens

Several human cell lines derived from primary cancer of the liver are able to grow under serum-free conditions and produce spreading and growth factors which are released into the culture medium. Since this autocrine growth under hormone-free conditions might play a basic role in malignant transformation, we studied the effect on cell replication and the presence of specific membrane receptors of epidermal growth factor (EGF) and insulin on a dedifferentiated human hepatoma cell line, named HA22T/VGH. Our results point to a similar inhibitory effect on cell replication in the presence of both EGF and insulin, in spite of detecting different affinities of binding.     

LINC01133 aggravates the progression of hepatocellular carcinoma by activating the PI3K/AKT pathway

LncRNAs exhibit crucial roles in various pathological diseases, including hepatocellular carcinoma (HCC). Therefore, it is significant to recognize the dysregulated lncRNAs in HCC progression. Recently, LINC01133 has been identified in several tumors. However, the biological role of LINC01133 in HCC remains poorly understood. Currently, we focused on the function of LINC01133 in HCC development. We observed that LINC01133 was significantly increased in HCC cells including HepG2, Hep3B, MHCC-97L, SK-Hep-1, and MHCC-97H cells compared with the normal human liver cell line HL-7702. In addition, PI3K/AKT signaling was highly activated in HCC cells. Knockdown of LINC01133 was able to inhibit HCC cell proliferation, cell colony formation, cell apoptosis, and blocked cell cycle arrest in the G1 phase. For another, downregulation of LINC01133 repressed HCC cell migration and invasion. Subsequently, the PI3K/AKT signaling pathway was strongly suppressed by silence of LINC01133 in Hep3B and HepG2 cells. Then, in vivo tumor xenografts models were established using Hep3B cells to explore the function of LINC01133 in HCC progression. Consistently, our study indicated that knockdown of LINC01133 dramatically repressed HCC tumor progression through targeting the PI3K/AKT pathway in vivo. Taken these together, we revealed that LINC01133 contributed to HCC progression by activating the PI3K/AKT pathway.