N-benzyl-N-methyldecan-1-amine and its derivative mitigate 2,4- dinitrobenzenesulfonic acid-induced colitis and collagen-induced rheumatoid arthritis.

As our previous study revealed that N-benzyl-N-methyldecan-1-amine (BMDA), a new molecule originated from Allium sativum, exhibits anti-neoplastic activities, we herein explored other functions of the compound and its derivative [decyl-(4-methoxy-benzyl)-methyl-amine; DMMA] including anti-inflammatory and anti-oxidative activities. Pretreatment of THP-1 cells with BMDA or DMMA inhibited tumor necrosis factor (TNF)-α and interleukin (IL)-1ß production, and blocked c-jun terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), MAPKAP kinase (MK)2 and NF-κΒ inflammatory signaling during LPS stimulation. Rectal treatment with BMDA or DMMA reduced the severity of colitis in 2,4-dinitrobenzenesulfonic acid (DNBS)-treated rat. Consistently, administration of the compounds decreased myeloperoxidase (MPO) activity (representing neutrophil infiltration in colonic mucosa), production of inflammatory mediators such as cytokine-induced neutrophil chemoattractant (CINC)-3 and TNF-α, and activation of JNK and p38 MAPK in the colon tissues. In addition, oral administration of these compounds ameliorated collagen-induced rheumatoid arthritis (RA) in mice. The treatment diminished the levels of inflammatory cytokine transcripts, and protected connective tissues through the expression of anti-oxidation proteins such as nuclear factor erythroid-related factor (Nrf)2 and heme oxygenase (HO)1. Additionally, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels did not differ between the BMDA- or DMMA-treated and control animals, indicating that the compounds do not possess liver toxicity. Taken together, these findings propose that BMDA and DMMA could be used as new drugs for curing inflammatory bowel disease (IBD) and RA.

Elevated Expression of JMJD5 Protein Due to Decreased miR-3656 Levels Contributes to Cancer Stem Cell-Like Phenotypes under Overexpression of Cancer Upregulated Gene 2.

Overexpression of cancer upregulated gene (CUG) 2 induces cancer stem cell-like phenotypes, such as enhanced epithelial-mesenchymal transition, sphere formation, and doxorubicin resistance. However, the precise mechanism of CUG2-induced oncogenesis remains unknown. We evaluated the effects of overexpression of CUG2 on microRNA levels using a microRNA microarray. Levels of miR-3656 were decreased when CUG2 was overexpressed; on the basis of this result, we further examined the target proteins of this microRNA. We focused on Jumonji C domain-containing protein 5 (JMJD5), as it has not been previously reported to be targeted by miR-3656. When CUG2 was overexpressed, JMJD5 expression was upregulated compared to that in control cells. A 3' untranslated region (UTR) assay revealed that an miR-3656 mimic targeted the JMJD5 3'UTR, but the miR-3656 mimic failed to target a mutant JMJD5 3'UTR, indicating that miR-3656 targets the JMJD5 transcript. Administration of the miR-3656 mimic decreased the protein levels of JMD5 according to Western blotting. Additionally, the miR-3656 mimic decreased CUG2-induced cell migration, evasion, and sphere formation and sensitized the cells to doxorubicin. Suppression of JMJD5, with its small interfering RNA, impeded CUG2-induced cancer stem cell-like phenotypes. Thus, overexpression of CUG2 decreases miR-3656 levels, leading to upregulation of JMJD5, eventually contributing to cancer stem cell-like phenotypes.

Translocalization of enhanced PKM2 protein into the nucleus induced by cancer upregulated gene 2 confers cancer stem cell-like phenotypes.

Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of ß-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator. [BMB Reports 2022;55(2): 98-103].

Overexpression of Cancer Upregulated Gene 2 (CUG2) Decreases Spry2 Through c-Cbl, Leading to Activation of EGFR and ß-Catenin Signaling.

PURPOSE: The mechanism by which cancer upregulated gene 2 (CUG2) overexpression induces cancer stem cell-like phenotypes is not fully understood. Because the increased activity and expression of epidermal growth factor receptor (EGFR) kinase have been reported in A549 cancer cells overexpressing CUG2 (A549-CUG2) compared with control cells (A549-Vec), the Sprouty2 (Spry2) protein has gained attention as the downstream molecule of EGFR signaling. Therefore, we aim to identify the role of Spry2 in CUG2-overexpressing lung cancer cells. MATERIALS AND METHODS: Spry2 expression levels were examined in A549-CUG2 and A549-Vec cells by Western blotting and qRT-PCR. Cell migration, invasion, and sphere formation were examined after Spry2 suppression and overexpression. EGFR-Stat1 and Akt-ERK protein phosphorylation levels were detected via immunoblotting. NEK2 kinase and ß-catenin reporter assay were performed for downstream of Spry2 signaling. RESULTS: Although A549-CUG2 cells showed lower levels of the Spry2 protein than A549-Vec cells, no difference in levels of Spry2 transcript was observed between both cells via qRT-PCR. Furthermore, MG132 treatment enhanced the protein levels and ubiquitination of Spry2, suggesting that Spry2 protein expression can be regulated via the ubiquitin-proteasome pathway. The enforced expression of c-Cbl, known as the binding partner of Spry2, decreased the Spry2 protein levels, whereas its knockdown oppositely increased them. Epithelial-mesenchymal transition (EMT) and sphere formation were increased in A549-Vec cells during Spry2 siRNA treatment, confirming the role of Spry2 in CUG2-induced oncogenesis. Furthermore, EMT and sphere formation were determined by the Spry2 protein levels through the regulation of EGFR-Stat1 and ß-catenin-NEK2-Yap1 signaling pathways. CONCLUSION: CUG2 reduces Spry2 protein levels, the negative signaling molecule of cell proliferation, via c-Cbl, possibly activating the EGFR and ß-catenin signaling pathways and, in turn, contributing to the induction of cancer stem cell-like phenotypes.

Hepatitis C virus core protein enhances hepatocellular carcinoma cells to be susceptible to oncolytic vesicular stomatitis virus through down-regulation of HDAC4.

Since hepatitis C virus (HCV) core protein is known to possess potential oncogenic activity, we explored whether oncolytic vesicular stomatitis virus (VSV) could efficiently induce cytolysis in hepatocellular carcinoma cells stably expressing HCV core protein (Hep3B-Core). We found that Hep3B-Core cells were more susceptible to VSV as compared to control (Hep3B-Vec) cells owing to core-mediated inactivation of STAT1 and STAT2 proteins. Core expression induced lower phosphorylation levels of type I IFN signaling proteins such as Tyk2 and Jak1, and a reduced response to exogenous IFN-α, which resulted in susceptibility to VSV. Furthermore, as STAT1 acetylation by switching phosphorylation regulated its activity, the role of STAT1 acetylation in susceptibility of Hep3B-Core cells to VSV was investigated. Treatment with trichostatin A, an inhibitor of histone deacetylase (HDAC), increased STAT1 acetylation but blocked IFN-α-induced phosphorylation of STAT1, leading to increase of susceptibility to VSV. Interestingly, the core protein decreased HDCA4 transcript levels, leading to down-regulation of HDAC4 protein. However, ectopic expression of HDAC4 conversely enforced phosphorylation of STAT1 and hindered VSV replication, indicating that core-mediated reduction of HDAC4 provides a suitable intracellular circumstance for VSV replication. Collectively, we suggest that VSV treatment will be a useful therapeutic strategy for HCV-infected hepatocellular carcinoma cells because HCV core protein suppresses the anti-viral threshold by down-regulation of the STAT1-HDAC4 signaling axis.

Oncotropic H-1 parvovirus infection degrades HIF-1α protein in human pancreatic cancer cells independently of VHL and RACK1.

Overexpression of HIF-1α, a transcription factor responsive to hypoxia, is frequently observed in malignant tumors, which sometimes show resistance to chemotherapy and radiation therapy. Consequently, decrease of HIF-1α through virotherapy offers a logical strategy for the treatment of aggressive tumors. In this study, we found that infection with the oncolytic H-1 parvovirus decreased HIF-1α protein levels in pancreatic cancer cells under CoCl2 or hypoxia. The H-1 virus-induced decrease of HIF-1α was regulated by a proteasome-mediated pathway. Suppression of VHL, an E3 ligase and a critical regulator of HIF-1α, or enforced expression of UCP, an E2 ubiquitin-conjugating enzyme, failed to inhibit the H-1 virus-induced decrease of HIF-1α. Furthermore, siRNA-mediated suppression of RACK1, another regulator of HIF-1α, did not prevent H-1 viral infection from lowering HIF-1α protein levels. Although decrease of HIF-1α was observed after H-1 viral infection, constitutive expression of HIF-1α limited H-1 viral replication. After combined treatment with H-1 parvovirus and YC-1, an inhibitor of HIF-1α, the apoptosis of pancreatic cancer cells was greater than after treatment with H-1 virus alone or YC-1 alone. Accordingly, we propose that H-1 parvovirus could be used with YC-1 as a potential therapeutic agent against aggressive tumors exhibiting hypoxia and increased levels of HIF-1α.

Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling.

Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling.

Acetylshikonin induces apoptosis of hepatitis B virus X protein-expressing human hepatocellular carcinoma cells via endoplasmic reticulum stress.

Since it has been known that shikonin derived from a medicinal plant possesses anti-cancer activity, we wonder whether acetylshikonin (ASK), a derivate of shikonin, can be used to treat hepatocellular carcinoma cells expressing hepatitis B virus X protein (HBX), an oncoprotein from hepatitis B virus. When ASK was added to Hep3B cells stably expressing HBX, it induced apoptosis in a dose-dependent manner. ASK induced upregulation and export of Nur77 to the cytoplasm and activation of JNK. Likewise, suppression of Nur77 and JNK inactivation protected the cells from ASK-induced apoptosis, indicating that Nur77 upregulation and JNK activation were required for ASK-mediated apoptosis. Furthermore, ASK increased the expression of Bip and ubiquitination levels of cellular proteins, features of endoplasmic reticulum (ER) stress, via the production of reactive oxygen species in a dose-dependent manner. Suppression of reactive oxygen species with N-acetylcysteine reduced levels of Bip protein and ubiquitination levels of cellular proteins during ASK treatment, leading to protection of cells from apoptosis. Cycloheximide treatment reduced ASK-induced ER stress, suggesting that protein synthesis is involved in ASK-induced ER stress. Moreover, we showed using salubrinal, an ER stress inhibitor that reactive oxygen species production, JNK activation, and Nur77 upregulation and its translocation to cytoplasm are necessary for ER-induced stress. Interestingly, we found that JNK inactivation suppresses ASK-induced ER stress, whereas Nur77 siRNA treatment does not, indicating that JNK is required for ASK-induced ER stress. Accordingly, we report that ASK induces ER stress, which is prerequisite for apoptosis of HBX-expressing hepatocellular carcinoma cells.

Suppression of autophagic genes sensitizes CUG2-overexpressing A549 human lung cancer cells to oncolytic vesicular stomatitis virus-induced apoptosis.

We showed in our previous study that cancer upregulated gene (CUG) 2, a novel oncogene, confers resistance to infection of oncolytic vesicular stomatitis virus (VSV) by activating Stat1-mediated signal transduction. Since many studies have reported that autophagy is involved in virus replication, we investigated whether autophagy also plays a role in the antiviral activity in A549 cells overexpressing CUG2 (A549-CUG2). We suppressed Atg5 or Beclin 1 expression using siRNA and examined its effect on the susceptibility of cells to infection by oncolytic VSV. We found that A549-CUG2 cells treated with Atg5 or Beclin 1 siRNA became susceptible to VSV infection, whereas A549-CUG2 cells treated with control siRNA were resistant. This result suggests that autophagy is involved in the antiviral response of A549-CUG2 cells. Further investigation revealed that autophagy impairment enhanced the generation of reactive oxygen species (ROS), which resulted in inactivation of S6 kinase. Under these conditions, the levels of ISG15 transcript and protein decreased, which conferred on A549-CUG2 cell susceptibility to VSV infection. Finally, we found that overloading of H2O2 sensitized control A549-CUG2 cells to VSV-induced apoptosis. Taken together, these results indicate that autophagy impairment induces excessive ROS formation, which decreases S6 kinase activity and ISG15 expression, ultimately rendering the A549-CUG2 cells susceptible to VSV infection. We propose that autophagy impairment is a potential strategy for successful VSV virotherapy of CUG2-overexpressing tumors.

Extract of Bryophyllum laetivirens reverses etoposide resistance in human lung A549 cancer cells by downregulation of NF-κB.

Since multidrug resistance (MDR) is one of the main reasons for failure in cancer treatment, its suppression may increase the efficacy of cancer therapy. In the present study we attempted to identify a new and effective anticancer drug against MDR cancer cells. We first found that lung cancer A549 cells resistant to etoposide (A549RT-eto) exhibit upregulation of NF-κB and SIRT1 in comparison to A549 parental cells. During a search for anticancer drug candidates from medicinal plant sources, we found that an extract fraction (F14) of Bryophyllum laetivirens leaves downregulated expression of NF-κB and SIRT1, sensitizing the levels of A549RT-eto cells to apoptosis through downregulation of P-glycoprotein (P-gp), which is encoded by the MDR1 gene. To address whether NF-κB is involved in resistance to etoposide through P-gp, we treated A549RT-eto cells with Bay11-7802, an inhibitor of NF-κB. We then observed that Bay11-7802 treatment reduced P-gp expression levels, and furthermore combined treatment with the F14 extract and Bay11-7802 accelerated apoptosis through a decrease in P-gp levels, suggesting that NF-κB is involved in MDR. To address whether upregulation of SIRT1 is involved in resistance to etoposide through P-gp, we treated A549RT-eto cells with SIRT1 siRNA or nicotinamide (NAM), an inhibitor of SIRT1. we found that suppression of SIRT1 did not reduce P-gp levels. furthermore, the combined treatment with the F14 extract, and SIRT1 siRNA or NAM did not accelerate apoptosis, indicating that SIRT1 is not involved in the regulation of P-gp levels in A549RT-eto cells. Taken together, we suggest that upregulation of NF-κB determines etoposide resistance through P-gp expression in human A549 lung cancer cells. We herein demonstrated that B. laetivirens extract reverses etoposide resistance in human A549 lung cancer cells through downregulation of NF-κB.

Cancer upregulated gene 2, a novel oncogene, enhances migration and drug resistance of colon cancer cells via STAT1 activation.

Cancer upregulated gene (CUG) 2, as a novel oncogene, has been predominantly detected in various cancer tissues, such as ovary, liver, lung and colon. We recently showed that CUG2 elevates STAT1 activity, leading to resistance to infection by oncolytic vesicular stomatitis virus. To investigate a possible role for CUG2-induced activation of STAT1 in oncogenesis, we first established a colon cancer cell line stably expressing CUG2 (Colon26L5-CUG2). Colon26L5-CUG2 exhibited higher levels not only in phosphorylation of STAT1, but also phosphorylation of Jak1/Tyk2 compared to that of the control (Colon26L5-Vec) cell line. Inhibition of Akt or ERK activity reduced phosphorylation of STAT1 in Colon26L5-CUG2 cells whereas inhibition of p38 MAPK did not significantly decrease levels of STAT1 phosphorylation, indicating that cell proliferation signals may be involved in CUG2-mediated activation of STAT1. Suppression of STAT1 expression diminished cell migration and wound healing compared to the control cells. In addition, since CUG2 expression conferred resistance to DNA damage caused by doxorubicin treatment, we investigated whether STAT1 is involved in resistance to doxorubicin-induced cell death. We found that STAT1 was not activated in Colon26L5-Vec cells while phosphorylated STAT1 was maintained in Colon26L5-CUG2 cells during doxorubicin treatment. Furthermore, suppression of STAT1 expression sensitized Colon26L5-CUG2 cells to doxorubicin-induced apoptosis whereas the control cells exhibited resistance to doxorubicin. Taken together, our results suggest that CUG2 enhances metastasis and drug resistance through STAT1 activation, which eventually contributes to tumor progression.

SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis.

We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of ß-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of ß-catenin.

Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of ß-catenin, we postulated that ß-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target ß-catenin in a colon cancer model, suppresses ß-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of ß-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced ß-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of ß-catenin. Treatment with MG132, a proteasomal inhibitor, restored ß-catenin protein levels, suggesting that SIRT1-mediated degradation of ß-catenin requires proteasomal activity. It was reported that inhibition of GSK-3ß or Siah-1 stabilizes ß-catenin in colon cancer cells, but suppression of GSK-3ß or Siah-1 using siRNA in the presence of resveratrol instead diminished ß-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3ß and Siah-1 are not involved in SIRT1-mediated degradation of ß-catenin in the cells. Finally, activation of SIRT1 inhibited the proliferation of Panc-PAUF cells by down-regulation of cyclin-D1, a target molecule of ß-catenin. These results suggest that SIRT1 activation may be a therapeutic strategy for treatment of pancreatic cancer cells that express PAUF via the down-regulation of ß-catenin.

Hepatitis B virus X (HBX) protein upregulates ß-catenin in a human hepatic cell line by sequestering SIRT1 deacetylase.

Hepatitis B virus X (HBX) protein has been reported to induce upregulation of ß-catenin, a known proto-oncogene, in p53-knockout and p53-mutant hepatic cell lines both in a GSK-3ß-dependent manner and via interaction with adenomatous polyposis coli, which results in protection from ß-catenin degradation. In this study, we describe a novel mechanism for HBX-mediated upregulation of ß-catenin. We observed that HBX interacts with SIRT1, a class III histone deacetylase. Furthermore, the presence of HBX attenuated the interaction between SIRT1 and ß-catenin, leading to protection of ß-catenin from the inhibitory action of SIRT1. Reduction of SIRT1 with siRNA or suppression of SIRT1 activity with nicotinamide upregulated ß-catenin protein levels. In contrast, enhancement of SIRT1 activity with resveratrol reduced ß-catenin protein levels. Furthermore, in Hep3B cells stably expressing HBX, overexpression of SIRT1 or treatment with resveratrol enhanced sensitivity to doxorubicin-induced apoptosis, indicating that upregulation of SIRT1 could be a therapeutic strategy for HBV-related hepatocellular carcinoma. Based on these results, we propose that HBX upregulates ß-catenin by sequestering SIRT1, which leads to anticancer drug treatment resistance.

Hepatitis B virus X protein inhibits extracellular IFN-α-mediated signal transduction by downregulation of type I IFN receptor.

We have previously shown that hepatitis B virus (HBV) protein X (HBX), a regulatory protein of HBV, activates Stat1, leading to type I interferon (IFN) production. Type I IFN secreted from HBX-expressing hepatic cells enforces antiviral signals through its binding to the cognate type I IFN receptor. We therefore investigated how cells handle this detrimental situation. Interestingly, compared to Chang cells stably expressing an empty vector (Chang-Vec), Chang cells stably expressing HBX (Chang-HBX) showed lower levels of IFN-α receptor 1 (IFNAR1) protein, a subunit of type I IFN receptor. The levels of IFNAR1 transcripts detected in Chang-HBX cells were lower than the levels in Chang-Vec cells, indicating that HBX regulates IFNAR1 at the transcriptional level. Moreover, we observed that HBX induced the translocation of IFNAR1 to the cytoplasm. Consistent with these observations, HBX also downregulated Tyk2, which is required for the stable expression of IFNAR1 on the cell surface. Eventually, Chang-HBX cells consistently maintained a lower level of IFNAR1 expression and displayed no proper response to IFN-α, while Chang-Vec cells exhibited a proper response to IFN-α treatment. Taken together, we propose that HBX downregulates IFNAR1, leading to the avoidance of extracellular IFN-α signal transduction.

Up-regulation of Foxo4 mediated by hepatitis B virus X protein confers resistance to oxidative stress-induced cell death.

The hepatitis B virus X (HBX) protein, a regulatory protein of the hepatitis B virus (HBV), has been shown to generate reactive oxygen species (ROS) in human liver cell lines; however, the mechanism by which cells protect themselves under this oxidative stress is poorly understood. Here, we show that HBX induces the up-regulation of Forkhead box class O 4 (Foxo4) not only in Chang cells stably expressing HBX (Chang-HBX) but also in primary hepatic tissues from HBX-transgenic mice. HBX also increased ROS, but reduction of the abundance of ROS using N-acetylcystein (NAC) diminished the levels of Foxo4. Elevated Foxo4 was also detected in nuclei of Chang-HBX cells but not in Chang cells stably expressing the vector (Chang-Vec), suggesting that HBX activates the transcriptional activity of Foxo4. When we examined whether HBX bypasses JNK signaling that targets Foxo4, we found that the activity of JNK but not of ERK is required for the up-regulation of Foxo4 even in the presence of HBX. Furthermore, the reduction of Foxo4 levels using siRNA or a JNK inhibitor rendered Chang-HBX cells sensitive to apoptosis under oxidative stress, suggesting that up-regulation of Foxo4 mediated by HBX enhances resistances to oxidative stress-induced cell death. Accordingly, we propose that Foxo4 may be a useful target for suppression in the treatment of HBV-associated hepatocellular carcinoma cells.

Pancreatic adenocarcinoma up-regulated factor (PAUF) enhances the expression of ß-catenin, leading to a rapid proliferation of pancreatic cells.

It is not yet understood how the enhanced expression of pancreatic adenocarcinoma up-regulated factor (PAUF; a novel oncogene identified in our recent studies), contributes to the oncogenesis of pancreatic cells. We herein report that PAUF up-regulates the expression and transcriptional activity of ß-catenin while the suppression of PAUF by shRNA down-regulates ß-catenin. The induction of b-catenin by PAUF is mediated by the activities of Akt and GSK-3ß, but inhibition of downstream ERK does not reduce ß-catenin expression. To test whether PAUF emulates either the Wnt3a-mediated or the protein kinase A-mediated signaling pathway for the stabilization of ß-catenin, we examined the phosphorylation status of ß-catenin in the presence of PAUF compared with that of ß-catenin during treatment with Wnt3a or dibutyryl cAMP, a cell permeable cyclic AMP analogue. PAUF expression induces phosphorylation at Ser-33/37/Thr-41 and Ser-675 of ß-catenin but no phosphorylation at Ser-45, indicating that a unique phosphorylation pattern of b-catenin is caused by PAUF. Finally, the expression of PAUF up-regulates both cyclin-D1 and c-Jun, target genes of ß-catenin, leading to a rapid proliferation of pancreatic cells; conversely decreased PAUF expression (by shRNA) results in the reduced proliferation of pancreatic cells. Treatment with hexachlorophene (an inhibitor of ß-catenin) reduces the proliferation of pancreatic cells despite the presence of PAUF. Taken together, we propose that PAUF can up-regulate and stabilize ß-catenin via a novel pattern of phosphorylation, thereby contributing to the rapid proliferation of pancreatic cancer cells.

Reovirus infection induces apoptosis of TRAIL-resistant gastric cancer cells by down-regulation of Akt activation.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been used to treat a variety of cancer cells. However, since some gastric cancer cells are resistant to TRAIL, we explored whether reovirus induces cytolysis in TRAIL-resistant gastric cancer cells. We found that TRAIL-resistant SNU-216 gastric cancer cells were susceptible to apoptosis by reovirus infection. Furthermore, co-treatment with reovirus and TRAIL accelerated apoptosis of SNU-216 cells by down-regulation of Akt activation as assessed by a very low activation of Akt in TRAIL-sensitive SNU-668 gastric cancer cells. Inhibition of Akt signaling with wortmannin or suppression of Akt expression with sh-Akt lentivirus promoted reovirus-mediated apoptosis of SNU-216 gastric cancer cells. Reovirus infection also down-regulates the activation of signaling molecules such as Ras and ERK involved in cell proliferation and survival but not the activation of p38 MAPK involved in cellular stress. In addition, the co-treatment with reovirus and TRAIL resulted in cleavage of caspase-8, caspase-9 and Bid, leading to a decrease in the mitochondrial membrane potential, indicating that reovirus may utilize the mitochondrial intrinsic apoptotic pathway in TRAIL-resistant SNU-216 gastric cancer cells. Accordingly, we first demonstrate that reovirus infection down-regulates Akt activation, leading to apoptosis of TRAIL-resistant gastric cancer cells.

Inhibition of GSK-3beta enhances reovirus-induced apoptosis in colon cancer cells.

Reovirus functions as an oncolytic agent for many types of cancer including colon cancer. Although most studies have emphasized the role of activated Ras signaling in enhancing reoviral oncolysis in susceptible cells, we note that many colon cancers also display elevated beta-catenin. Thus, it is possible that enhanced beta-catenin may augment reoviral susceptibility in colon cancer cells. To explore this hypothesis, HEK293 cells were treated with the glycogen synthase kinase (GSK)-3beta inhibitor LiCl, thereby inducing beta-catenin, followed by reoviral infection. Co-administration with LiCl indeed enhanced cell death compared to reovirus infection alone, but this was not associated with elevated reoviral replication. Similarly, HEK293 cells expressing the Frizzled-1 receptor in Wnt3a-conditioned medium also showed reovirus replication equivalent to that in cells in control medium, further suggesting that up-regulation of beta-catenin does not enhance the replication of reovirus. Instead, we observed that inhibition of GSK-3beta with LiCl decreased reovirus-induced NF-kappaB activation, leading to accelerated apoptosis via caspase 8 activation. We further found that colon cancer HCT116 cells were sensitized to apoptosis by co-treatment with reovirus and a GSK-3beta inhibitor, AR-A014418. Finally, we identified that inhibition of NF-kappaB sensitized apoptosis of HEK293 or HCT 116 cells during reovirus infection. Taken together, we propose that inhibition of GSK-3beta sensitizes reovirus-induced apoptosis of colon cancer cells by down-regulation of NF-kappaB activity, offering a potentially improved therapeutic strategy for the treatment of colon cancer.

Hexachlorophene suppresses beta-catenin expression by up-regulation of Siah-1 in EBV-infected B lymphoma cells.

Many studies have shown that the activation of beta-catenin signaling can promote oncogenesis, and it is therefore of interest to find agents that modulate this pathway. Recent work has shown using B lymphoma cells that infection by Epstein-Barr virus (EBV) and expression of its latent membrane protein (LMP)-1, cause increases in the expression of beta-catenin and cellular transformation. Conversely, results from cell-based small molecule screening studies have shown that the antibiotic hexachlorophene can down-regulate beta-catenin in colon cancer cells. Here we report that hexachlorophene also counteracts the elevated beta-catenin levels in EBV-infected B lymphomas. This is associated with restoration in levels of Siah-1 (an E3 ubiquitin ligase that is active in beta-catenin regulation) which had been diminished by LMP-1. Our results suggest that Siah-1 is targeted by both LMP-1 and hexachlorophene with opposite effects. The hexachlorophene modulation of Siah-1 and beta-catenin is independent of p53 and results in reduced expression of cyclin-D1 and c-Myc (target genes of beta-catenin), leading to the growth arrest of B lymphoma cells. From these results we propose that hexachlorophene may provide a novel therapeutic strategy for EBV-infected B lymphoma cells by reducing beta-catenin levels via the restoration of Siah-1.