Inhibition of Dickkopf-1 enhances the anti-tumor efficacy of sorafenib via inhibition of the PI3K/Akt and Wnt/ß-catenin pathways in hepatocellular carcinoma.

BACKGROUND: Sorafenib improves the overall survival in patients with advanced hepatocellular carcinoma (HCC). Dickkopf-1 (DKK1) is commonly overexpressed in HCC. In this study, we investigated whether the inhibition of DKK1 enhances the anti-tumor efficacy of sorafenib in HCC. METHODS: HCC cells were treated with sorafenib and WAY-262611, which is an inhibitor of DKK1. Transgenic mouse models were also developed using hydrodynamic tail vein injection. Mice were orally administered with sorafenib (32 mg/kg), WAY-262611 (16 mg/kg), or sorafenib + WAY-262611 for 10 days. Mechanisms of sorafenib and WAY-262611 were explored via western blotting, immunostaining, and RNA sequencing. RESULTS: DKK1 was significantly overexpressed in patients with HCC than in the healthy controls and patients with liver diseases except HCC (all P < 0.05). Compared with sorafenib alone, sorafenib + WAY-262611 significantly inhibited the cell viability, invasion, migration, and colony formation by promoting apoptosis and altering the cell cycles in HCC cells (all P < 0.05). Moreover, sorafenib + WAY-262611 decreased the p110α, phospho-Akt (all P < 0.05), active ß-catenin (all P < 0.05) and phospho-GSK-3ß (Ser9) expression levels, while increasing the phospho-GSK-3ß (Tyr216) expression levels compared with those in the sorafenib alone in vitro and in vivo. In addition, sorafenib + WAY-262611 inhibited tumor progression by regulating cell proliferation and apoptosis, significantly better than sorafenib alone in mouse models. CONCLUSIONS: Our results indicate that DKK1 inhibition significantly enhances the anti-tumor efficacy of sorafenib by inhibiting the PI3K/Akt and Wnt/ß-catenin pathways via regulation of GSK3ß activity, suggesting a novel therapeutic strategy for HCC. Video Abstract.

Co-administration of ursodeoxycholic acid with rosuvastatin/ezetimibe in a non-alcoholic fatty liver disease model.

Background: Ursodeoxycholic acid (UDCA), statins, and ezetimibe (EZE) have demonstrated beneficial effects against non-alcoholic fatty liver disease (NAFLD). We investigated the efficacy of the combination of UDCA and the mix of rosuvastatin (RSV)/EZE in the treatment of NAFLD. Methods: NAFLD mouse models were developed by injecting thioacetamide, fasting, and high-carbohydrate refeeding, high-fat diet, and choline-deficient L-amino acid-defined high-fat diet (CDAHFD). Low-dose UDCA (L-UDCA; 15 mg/kg) or high-dose UDCA (H-UDCA; 30 mg/kg) was administered with RSV/EZE. We also employed an in vitro model of NAFLD developed using palmitic acid-treated Hepa1c1c7 cells. Results: Co-administration of RSV/EZE with UDCA significantly decreased the collagen accumulation, serum alanine aminotransferase (ALT) levels, and mRNA levels of fibrosis-related markers than those observed in the vehicle group in thioacetamide-treated mice (all P < 0.01). In addition, in the group fasted and refed with a high-carbohydrate diet, UDCA/RSV/EZE treatment decreased the number of apoptotic cells and serum ALT levels compared with those observed in the vehicle group (all P < 0.05). Subsequently, H-UDCA/RSV/EZE treatment decreased the number of ballooned hepatocytes and stearoyl-CoA desaturase 1 (SCD-1) mRNA levels (P = 0.027) in the liver of high-fat diet-fed mice compared with those observed in the vehicle group. In the CDAHFD-fed mouse model, UDCA/RSV/EZE significantly attenuated collagen accumulation and fibrosis-related markers compared to those observed in the vehicle group (all P < 0.05). In addition, UDCA/RSV/EZE treatment significantly restored cell survival and decreased the protein levels of apoptosis-related markers compared to RSV/EZE treatment in palmitic acid-treated Hepa1c1c7 cells (all P < 0.05). Conclusion: Combination therapy involving UDCA and RSV/EZE may be a novel strategy for potent inhibition of NAFLD progression.

Applications of molecular barcode sequencing for the detection of low-frequency variants in circulating tumour DNA from hepatocellular carcinoma.

PURPOSE: Liquid biopsy has emerged as a promising tool for minimally invasive and accurate detection of various malignancies. We aimed to apply molecular barcode sequencing to circulating tumour DNA (ctDNA) from liquid biopsies of hepatocellular carcinoma (HCC). STUDY DESIGN: Patients with HCC or benign liver disease were enrolled between 2017 and 2018. Matched tissue and serum samples were obtained from these patients. Plasma cell-free DNA was extracted and subjected to targeted sequencing with ultra-high coverage and molecular barcoding. RESULTS: The study included 143 patients: 102 with HCC, 7 with benign liver tumours and 34 with chronic liver disease. No tier 1/2 or oncogenic mutations were detected in patients with benign liver disease. Among the HCC patients, 49 (48%) had tier 1/2 mutations in at least one gene; detection rates were higher in advanced stages (75%) than in early stages (26%-33%). TERT was the most frequently mutated gene (30%), followed by TP53 (16%), CTNNB1 (14%), ARID2 (5%), ARID1A (4%), NFE2L2 (4%), AXIN1 (3%) and KRAS (1%). Survival among patients with TP53 mutations was significantly worse (p = 0.007) than among patients without these mutations, whereas CTNNB1 and TERT mutations did not affect survival. ctDNA testing combined with α-fetoprotein and prothrombin induced by vitamin K absence-II analyses improved HCC detection, even in early stages. CONCLUSIONS: ctDNA detection using molecular barcoding technology offers dynamic and personalized information concerning tumour biology, such information can guide clinical diagnosis and management. This detection also has the potential as a minimally invasive approach for prognostic stratification and post-therapeutic monitoring.

Dickkopf-1 promotes angiogenesis by upregulating VEGF receptor 2-mediated mTOR/p70S6K signaling in hepatocellular carcinoma.

The expression of Dickkopf-1 (DKK1), a negative regulator of the Wnt/ß-catenin signaling pathway, is upregulated in hepatocellular carcinoma (HCC). Here, we investigated the tumorigenic and angiogenic potential of DKK1 in HCC. Stable cell lines were established using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9)-based DKK1 knock-out system in Hep3B cells and the tetracycline-based DKK1 inducible system in Huh7 cells. Multicellular tumor spheroids (MCTSs) were cultured using Hep3B stable cells. We also employed xenografts generated using Hep3B stable cells and transgenic mouse models established using hydrodynamic tail vein injection. The angiogenic potential increased in HUVECs treated with CM from Huh7 stable cells with high DKK1 expression and Hep3B wild-type cells. DKK1 accelerated the downstream molecules of vascular endothelial growth factor receptor 2 (VEGFR2)-mediated mTOR/p70 S6 kinase (p70S6K) signaling. MCTSs generated using Hep3B wild-type cells promoted compact spheroid formation and increased the expression of CD31 and epithelial-mesenchymal transition (EMT) markers, and increased the VEGFR2-mediated mTOR/p70S6K signaling, compared to the controls (all P<0.01). Xenograft tumors generated using Hep3B cells with DKK1 knock-out (n=10) exhibited slower growth than, the controls (n=10) and the expression of Ki-67, VEGFR2, CD31 and EMT markers decreased (all P<0.05). In addition, forced DKK1 expression with HRAS in transgenic mouse livers (n=5) resulted in the formation of more tumors and increased expression of downstream molecules of VEGFR2-mediated mTOR/p70S6K signaling pathway as well as Ki67, CD31 and EMT markers (P<0.05), compared to that of the controls (n=5). Our findings indicate that DKK1 facilitates angiogenesis and tumorigenesis by upregulating VEGFR2-mediated mTOR/p70S6K signaling in HCC.

Knockdown of Atg7 suppresses Tumorigenesis in a murine model of liver cancer.

Hepatocellular Carcinoma (HCC) is the most common type of primary liver cancer in adults and a leading cause of cancer-related deaths worldwide. Studies have shown that autophagy is significantly involved in carcinogenesis, in particular, driven by activated RAS signaling. Autophagy related 7 (Atg7) is a critical component for the formation of autophagosome and required for autophagy processes. We investigated the role of autophagy in RAS-driven tumorigenesis in the liver, via the knockdown of Atg7 in the model. Transposon vectors encoding short hairpin RNAs targeting Atg7 (Atg7 shRNA) were constructed. Inhibition of autophagy via Atg7 knockdown was tested in Hep3B cells cultured in nutrient-starved medium. Formation of autophagosome was suppressed in nutrient-starved Hep3B cells expressing Atg7 shRNA, demonstrating that it efficiently inhibited autophagy in HCC cells. Transposons encoding Atg7 shRNA were mixed with those expressing HRASG12V and p53 shRNA, and subsequently used for hydrodynamic injection to 5-week-old C57BL/6 mice. Tumorigenesis in livers induced by HRASG12V and p53 shRNA was significantly suppressed by Atg7 knockdown. The inhibition of autophagy led to a decreased proliferation of cancer cells, as determined by Ki-67 staining. Our data indicate that knockdown of Atg7 led to a significant decrease in tumorigenesis in a murine HCC model induced by activated RAS. Inhibition of autophagosome formation is expected to be a therapeutic option for liver cancer.

Current Status and Future Direction of Immunotherapy in Hepatocellular Carcinoma: What Do the Data Suggest?

Most patients with hepatocellular carcinoma (HCC) are diagnosed at an advanced stage of disease. Until recently, systemic treatment options that showed survival benefits in HCC have been limited to tyrosine kinase inhibitors, antibodies targeting oncogenic signaling pathways or VEGF receptors. The HCC tumor microenvironment is characterized by a dysfunction of the immune system through multiple mechanisms, including accumulation of various immunosuppressive factors, recruitment of regulatory T cells and myeloid-derived suppressor cells, and induction of T cell exhaustion accompanied with the interaction between immune checkpoint ligands and receptors. Immune checkpoint inhibitors (ICIs) have been interfered this interaction and have altered therapeutic landscape of multiple cancer types including HCC. In this review, we discuss the use of anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies in the treatment of advanced HCC. However, ICIs as a single agent do not benefit a significant portion of patients. Therefore, various clinical trials are exploring possible synergistic effects of combinations of different ICIs (anti-PD-1/PD-L1 and anti-CTLA-4 antibodies) or ICIs and target agents. Combinations of ICIs with locoregional therapies may also improve therapeutic responses.

HKR3 regulates cell cycle through the inhibition of hTERT in hepatocellular carcinoma cell lines.

Hepatocellular carcinoma is a malignant disease with improved hepatic regeneration and survival, and is activated by human telomere transferase (hTERT). hTERT is expressed during early fetal development and switched off in most adult tissues, but it becomes reactivated in HCC. The exact mechanism regulating these expression changes remains unknown during HCC progress. We evaluated the relationship between hTERT expression and human kruppel-related 3 (HKR3) and cell cycle-related factors in HCC cell lines. Following transfection for hTERT knockdown and HKR3 overexpression, proteomic and transcriptomic analyses related to hTERT were performed using liquid chromatography/mass spectrometry (LC/MS) and RNA sequencing (RNAseq) in HCC cell lines. The expression levels of hTERT, HKR3, and cell cycle-related factors were measured using western blotting, and tumor growth were evaluated via cell proliferation and cell cycle assays. Transcriptomic and proteomic analyses showed that HKR3, hTERT and cyclin-dependent kinase inhibitor 2A (CDKN2A) were correlated. Up-regulation of HKR3 expression decreased hTERT and cyclin activation and suppressed the G1/S phase of the cell cycle through CDKN2A activation. Our results suggest that HKR3 induced regulation of cell cycle through hTERT inhibition and CDKN2A activation. Our results will facilitate further exploration of the pathways regulating human telomerase activity in HCC cell lines.

Effect of tibolone on the survival of early stage cervical adenocarcinoma patients.

OBJECTIVE: Gynecologic oncologists are uncertain about the safety of tibolone application in cervical adenocarcinoma (AC) patients. This study examined the possible adverse effects of tibolone on the survival of cervical AC patients. METHODS: Medical records of 70 cervical AC patients with International Federation of Gynecology and Obstetrics stages IA to IB were reviewed. A bilateral salpingo-oophorectomy was performed in all patients, and survival outcomes between tibolone users (n=38) and non-users (n=32) were compared. RESULTS: A comparison of the tibolone users with non-users revealed similar clinicopathological variables. Progression-free survival (P=0.34) and overall survival (P=0.22) were similar in the users and non-users. The risks of progression (hazard ratio [HR], 1.71; 95% confidence interval [CI], 0.46-6.37; P=0.43) and death (HR, 1.59; 95% CI, 0.06-45.66; P=0.79) were also similar in both groups. CONCLUSION: Tibolone has no adverse effect on the survival of cervical AC patients and can be administered safely to this population. These findings may be helpful in improving the quality of life of cervical AC patients.

Transforming Growth Factor-ß Promotes Liver Tumorigenesis in Mice via Up-regulation of Snail.

BACKGROUND & AIMS: Transforming growth factor beta (TGF-ß) suppresses early stages of tumorigenesis, but also contributes to migration and metastasis of cancer cells. A large number of human tumors contain mutations that inactivate its receptors, or downstream proteins such as Smad transcription factors, indicating that the TGF-ß signaling pathway prevents tumor growth. We investigated the effects of TGF-ß inhibition on liver tumorigenesis in mice. METHODS: C57BL/6 mice received hydrodynamic tail-vein injections of transposons encoding HRASG12V and a short hairpin RNA (shRNA) to down-regulate p53, or those encoding HRASG12V and MYC, or those encoding HRASG12V and TAZS89A, to induce liver tumor formation; mice were also given injections of transposons encoding SMAD7 or shRNA against SMAD2, SMAD3, SMAD4, or SNAI1 (Snail), with or without ectopic expression of Snail. Survival times were compared, and livers were weighted and examined for tumors. Liver tumor tissues were analyzed by quantitative reverse-transcription PCR, RNA sequencing, immunoblots, and immunohistochemistry. We analyzed gene expression levels in human hepatocellular carcinoma samples deposited in The Cancer Genome Atlas. A cell proliferation assay was performed using human liver cancer cell lines (HepG2 and Huh7) stably expressing Snail or shRNA against Snail. RESULTS: TGF-ß inhibition via overexpression of SMAD7 (or knockdown of SMAD2, SMAD3, or SMAD4) consistently reduced formation and growth of liver tumors in mice that expressed activated RAS plus shRNA against p53, or in mice that expressed activated RAS and TAZ. TGF-ß signaling activated transcription of the Snail gene in liver tumors induced by HRASG12V and shRNA against p53, and by activated RAS and TAZ. Knockdown of Snail reduced liver tumor formation in both tumor models. Ectopic expression of Snail restored liver tumorigenesis suppressed by disruption of TGF-ß signaling. In human hepatocellular carcinoma, Snail expression correlated with TGF-ß activation. Ectopic expression of Snail increased cellular proliferation, whereas Snail knockdown led to reduced proliferation in human hepatocellular carcinoma cells. CONCLUSIONS: In analyses of transgenic mice, we found TGF-ß signaling to be required for formation of liver tumors upon expression of activated RAS and shRNA down-regulating p53, and upon expression of activated RAS and TAZ. Snail is the TGF-ß target that is required for hepatic tumorigenesis in these models.

Development of a transgenic mouse model of hepatocellular carcinoma with a liver fibrosis background.

BACKGROUND: Liver fibrosis and its end-stage disease, cirrhosis, are major risk factors for hepatocellular carcinoma (HCC) and present in 80 to 90 % of patients with HCC. Current genetically engineered mouse models for HCC, however, generally do not feature liver fibrosis, which is a critical discrepancy between human HCC and murine models thereof. In this study, we developed a simple transgenic mouse model of HCC within the context of a fibrotic liver. METHODS: Employing hydrodynamic transfection (HT), coupled with the Sleeping Beauty (SB) transposon system, liver was stably transfected with transposons expressing cMyc and a short hairpin RNA down-regulating p53 (shp53). A chronic liver injury model, induced by hepatotoxic carbon tetrachloride (CCl4), was applied to the transgenic mice, allowing cells expressing cMyc plus shp53 to become malignant in the background of liver fibrosis. RESULTS: Livers harvested about 3 months after HT had excessive collagen deposition and activated hepatic stellate cells surrounding the tumors. Hepatocarcinogenesis was significantly accelerated in the fibrotic livers compared to those of the control, significantly decreasing the life span of the mice. The tumor incidence and average number of tumors per mouse were significantly higher in the group treated with CCl4 compared to the vehicle-treated control mice, following HT (p < 0.01). CONCLUSIONS: Considering the simplicity and efficiency in generating HCC for fibrotic livers, the transgenic HCC model has the potential to be effectively used in preclinical testing of HCC anticancer therapy and in studies of hepatocarcinogenesis in fibrotic livers.

Comparison of liver oncogenic potential among human RAS isoforms.

Mutation in one of three RAS genes (i.e., HRAS, KRAS, and NRAS) leading to constitutive activation of RAS signaling pathways is considered a key oncogenic event in human carcinogenesis. Whether activated RAS isoforms possess different oncogenic potentials remains an unresolved question. Here, we compared oncogenic properties among RAS isoforms using liver-specific transgenesis in mice. Hydrodynamic transfection was performed using transposons expressing short hairpin RNA downregulating p53 and an activated RAS isoform, and livers were harvested at 23 days after gene delivery. No differences were found in the hepatocarcinogenic potential among RAS isoforms, as determined by both gross examination of livers and liver weight per body weight ratio (LW/BW) of mice expressing HRASQ61L, KRAS4BG12V and NRASQ61K. However, the tumorigenic potential differed significantly between KRAS splicing variants. The LW/BW ratio in KRAS4AG12V mice was significantly lower than in KRAS4BG12V mice (p < 0.001), and KRAS4AG12V mice lived significantly longer than KRRAS4BG12V mice (p < 0.0001). Notably, tumors from KRAS4AG12V mice displayed higher expression of the p16INK4A tumor suppressor when compared with KRAS4BG12V tumors. Forced overexpression of p16INK4A significantly reduced tumor growth in KRAS4BG12V mice, suggesting that upregulation of p16INK4A by KRAS4AG12V presumably delays tumor development driven by the latter oncogene.

Hepatic expression of Sonic Hedgehog induces liver fibrosis and promotes hepatocarcinogenesis in a transgenic mouse model.

BACKGROUND & AIMS: Liver fibrosis is an increasing health concern worldwide and a major risk factor for hepatocellular carcinoma (HCC). Although the involvement of Hedgehog signaling in hepatic fibrosis has been known for some time, the causative role of activated Hedgehog signaling in liver fibrosis has not been verified in vivo. METHODS: Using hydrodynamics-based transfection, a transgenic mouse model has been developed that expresses Sonic Hedgehog (SHH), a ligand for Hedgehog signaling, in the liver. Levels of hepatic fibrosis and fibrosis-related gene expression were assessed in the model. Hepatic expression of SHH was induced in a murine model for hepatocellular adenoma (HCA) and tumor development was subsequently investigated. RESULTS: The transgenic mice revealed SHH expression in 2-5% of hepatocytes. Secreted SHH activated Hedgehog signaling in numerous cells of various types in the tissues. Hepatic expression of SHH led to fibrosis, activation of hepatic stellate cells, and an upregulation of various fibrogenic genes. Liver injury and hepatocyte apoptosis were observed in SHH mice. Persistent expression of SHH for up to 13months failed to induce tumors in the liver; however, it promoted liver tumor development induced by other oncogenes. By employing a HCA model induced by P53(R172H) and KRAS(G12D), we found that the SHH expression promoted the transition from HCA to HCC. CONCLUSIONS: SHH expression in the liver induces liver fibrosis with concurrent activation of hepatic stellate cells and fibrogenic genes. It can also enhance hepatocarcinogenesis induced by other oncogenes.

Analysis of miRNA expression patterns in human and mouse hepatocellular carcinoma cells.

AIM: Hepatocellular carcinoma (HCC), one of the most common malignancies in adults displays aberrant miRNA expression during its pathogenesis. We assessed expression of miRNA in surgically resected human HCC of an early stage and murine HCC with a high malignancy in order to find miRNA overexpressed in HCC regardless of tumor stage and underlying etiology. Further, the role of the deregulated miRNA in HCC pathogenesis was investigated. METHODS: miRNA were isolated from HCC tissues and surrounding non-tumorous tissues from HCC patients and a murine transgenic model of HCC. A quantitative reverse transcription polymerase chain reaction was performed to determine expression levels of miRNA. Human HCC cell lines stably expressing individual miRNA were generated to investigate the biological function of overexpressed miRNA. RESULTS: We found that levels of miR-221, -181b-1, -155-5p, -25 and -17-5p were significantly upregulated in both human and murine HCC regardless of tumor stage, underlying etiology or the presence of fibrosis. Using HCC cell lines stably expressing respective miRNA, we found that miR-221 increased the proliferation of hepatoma cells, while miR-17-5p induced cell migration. CONCLUSION: We identified miRNA that are consistently upregulated in HCC. The overexpressed miRNA could potentially be used as a bona fide biomarker for HCC.

Transgenic mouse model expressing P53(R172H), luciferase, EGFP, and KRAS(G12D) in a single open reading frame for live imaging of tumor.

Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53(R172H) and KRAS(G12D), are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53(R172H) and KRAS(G12D).

Signaling pathways implicated in alpha-melanocyte stimulating hormone-induced lipolysis in 3T3-L1 adipocytes.

Melanocortins, besides their central roles, have also recently been reported to regulate adipocyte metabolism. In this study, we attempted to characterize the mechanism underlying alpha-melanocyte-stimulating hormone (MSH)-induced lipolysis, and compared it with that of the adrenocorticotrophin hormone (ACTH) in 3T3-L1 adipocytes. Similar to ACTH, MSH treatment resulted in the release of glycerol into the cell supernatant. The activity of hormone-sensitive lipase, a rate-limiting enzyme, which is involved in lipolysis, was significantly increased by MSH treatment. In addition, a variety of kinases, including protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) were also phosphorylated as the result of MSH treatment, and their specific inhibitors caused a reduction in MSH-induced glycerol release and HSL activity, indicating that MSH-induced lipolysis was mediated by these kinases. These results suggest that PKA and ERK constitute the principal signaling pathways implicated in the MSH-induced lipolytic process via the regulation of HSL in 3T3-L1 adipocytes.

IL-18 E42A mutant is resistant to the inhibitory effects of HPV-16 E6 and E7 oncogenes on the IL-18-mediated immune response.

Our previous studies showed that the down-modulation of IL-18-induced immune response caused by oncoproteins E6 and E7 as one of the mechanisms underlying immune escape in HPV-induced cervical cancer cells. E42 residue of IL-18 also appears to be critical in the activity of IL-18. Single point mutation E42 in IL-18 show promise in the study of IL-18 binding motifs for HPV oncoproteins. We attempted to ascertain whether site-specific IL-18 mutant E42A would modulate the inhibitory effects of IL-18-induced immune responses via the HPV 16 E6 and E7 oncoproteins. Compared to wild type IL-18, E42A-induced IFN-gamma production was not inhibited by HPV 16 E6 and E7. In vitro and in vivo binding assays have also revealed that E6 and E7 do not result in the inhibition of the binding of E42A to its IL-18 receptor alpha chain. There were no effects on the E42A-induced phosphorylations of p38 and JNK observed in the presence of E6 or E7. The degradation of IkappaB by E42A was not affected by E6 or E7 in NK0 cells. Moreover, E42A-induced NF-kappaB activation was also not inhibited by these oncoproteins. These results suggest that E42A is a stronger activator than wild type IL-18, and is not susceptible to inhibition by the HPV oncoproteins E6 and E7. Thus, it is suggested that E42A could be used in immunotherapy for patients with cervical cancer.

E7-expressing HaCaT keratinocyte cells are resistant to oxidative stress-induced cell death via the induction of catalase.

Cervical carcinoma is one of the most prevalent cancers in women worldwide, and human papillomavirus (HPV) type 16 is the most common agent linked to human cervical carcinoma. In order to identify various relevant factors affected by the E7 oncogene, we established a stable cell line, which constitutively expressed E7 using the HaCaT human keratinocyte cell line. The increased expression and activity of catalase in the E7-expressing HaCaT cells (HaCaT/E7) were verified via matrix-assisted laser desorption/ionization-time of flight, Western blot, and reverse transcription-polymerase chain reaction analyses. The regulation of catalase by E7 was investigated by the detection of catalase promoter activity. E7 enhanced the activities of both the catalase promoter and nuclear factor-kappaB, one of the major transcription factors regulating the expression of the catalase gene. HaCaT/E7 cells produced lower quantities of intracellular reactive oxygen species (ROS), and appeared to be more resistant to H(2)O(2)-induced cell death. Moreover, in order to test the specific effects of E7 on catalase induction, the HaCaT/E7 cells were transiently transfected with E7 antisense vector, resulting in reductions in both the expression and activity of catalase, and a recovery of intracellular ROS levels, thus resulting in recovered sensitivity to H(2)O(2)-induced cell death. These results suggest that the HPV 16 E7 oncogene induces higher resistance to ROS-induced cell injury in the E7-infected cells, probably via the modulation of several anti-oxidant enzymes, including catalase.

Survivin reduces activation-induced T cell death in G1 phase.

A process termed activation-induced cell death (AICD) is responsible for peripheral T cell tolerance after negative selection of self-reactive T cells, and deletion of hyperactivated T cells following the immune response. Cells in G1 phase of the cell cycle are most susceptible to AICD. We have investigated the relationship between the induction of AICD by phorbol 12-myristate 13-acetate plus ionomycin during the cell cycle and the expression of survivin, an inhibitor of the apoptosis protein (LAP) family. AICD was highly induced in cells of the human T cell line Jurkat E6.1 arrested in G1 phase, whereas survivin was hardly expressed in G1 and instead it was highly expressed in G2/M. Moreover, transient over-expression of survivin in G1 partially blocked the induction of AICD. These results suggest that survivin inhibits the induction of AICD, especially in G1 phase.

Alpha-lipoic acid decreases thiol reactivity of the insulin receptor and protein tyrosine phosphatase 1B in 3T3-L1 adipocytes.

Alpha-lipoic acid is known to increase insulin sensitivity in vivo and to stimulate glucose uptake into adipose and muscle cells in vitro. In this study, alpha-lipoic acid was demonstrated to stimulate the autophosphorylation of insulin receptor and glucose uptake into 3T3-L1 adipocytes by reducing the thiol reactivity of intracellular proteins. To elucidate mechanism of this effect, role of protein thiol groups and H(2)O(2) in insulin receptor autophosphorylation and glucose uptake was investigated in 3T3-L1 adipocytes following stimulation with alpha-lipoic acid. Alpha-lipoic acid or insulin treatment of adipocytes increased intracellular level of oxidants, decreased thiol reactivity of the insulin receptor beta-subunit, increased tyrosine phosphorylation of the insulin receptor, and enhanced glucose uptake. Alpha-lipoic acid or insulin-stimulated glucose uptake was inhibited (i) by alkylation of intracellular, but not extracellular, thiol groups downstream of insulin receptor activation, and (ii) by diphenylene iodonium at the level of the insulin receptor autophosphorylation. alpha-Lipoic acid also inhibited protein tyrosine phosphatase activity and decreased thiol reactivity of protein tyrosine phosphatase 1B. These findings indicate that oxidants produced by alpha-lipoic acid or insulin are involved in activation of insulin receptor and in inactivation of protein tyrosine phosphatases, which eventually result in elevated glucose uptake into 3T3-L1 adipocytes.

Alpha-lipoic acid inhibits adipocyte differentiation by regulating pro-adipogenic transcription factors via mitogen-activated protein kinase pathways.

Obesity is associated with a number of pathological disorders such as non-insulin-dependent diabetes, hypertension, hyperlipidemia, and cardiovascular diseases. alpha-Lipoic acid (LA) has been demonstrated to activate the insulin signaling pathway and to exert insulin-like actions in adipose and muscle cells. Based on this similarity LA is expected to promote adipogenesis in pre-adipocytes. Here, however, we report that LA inhibited differentiation of 3T3-L1 pre-adipocytes induced by a hormonal mixture or troglitazone. Northern blot analysis of cells demonstrated that this inhibition was accompanied with attenuated expression of adipocyte-specific fatty acid-binding protein and lipoprotein lipase. Electrophoretic mobility shift assay and Western blot analysis of cells demonstrated that LA modulates transcriptional activity and/or expression of a set of anti- or pro-adipogenic transcription factors. LA treatment of 3T3-L1 pre-adipocytes also resulted in prolonged activation of major mitogen-activated protein kinase signaling pathways but showed little or no effect on the activity of the insulin receptor/Akt signaling pathway. These findings suggest that LA inhibits insulin or the hormonal mixture-induced differentiation of 3T3-L1 pre-adipocytes by modulating activity and/or expression of pro- or anti-adipogenic transcription factors mainly through activating the MAPK pathways.