Jeong Moon

SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of β-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 β-catenin, we postulated that β-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target β-catenin in a colon cancer model, suppresses β-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 β-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced β-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of β-catenin. Treatment with MG132, a proteasomal inhibitor, restored β-catenin protein levels, suggesting that SIRT1-mediated degradation of β-catenin requires proteasomal activity. It was reported that inhibition of GSK-3β or Siah-1 stabilizes β-catenin in colon cancer cells, but suppression of GSK-3β or Siah-1 using siRNA in the presence of resveratrol instead diminished β-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3β and Siah-1 are not involved in SIRT1-mediated degradation of β-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 β-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 β-catenin.

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 β-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.

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.

Upregulation of Stat1-HDAC4 confers resistance to etoposide through enhanced multidrug resistance 1 expression in human A549 lung cancer cells

Despite efforts to develop efficient chemotherapeutic drug strategies to treat cancer, acquired drug resistance is a commonly encountered problem. In the present study, to investigate this phenomenon, human A549 lung cancer cells resistant to the topoisomerase inhibitor etoposide (A549RT‑eto) were used and compared with A549 parental cells. A549RT‑eto cells demonstrated increased resistance to etoposide‑induced apoptosis when compared with A549 parental cells. Notably, A549RT‑eto cells were observed to exhibit greater levels of histone deacetylase 4 (HDAC4), phospho‑Stat1 and P‑glycoprotein [P‑gp; encoded by the multidrug resistance 1 (MDR1) gene], compared with A549 cells. To address whether HDAC4 protein is involved in etoposide resistance in A549 cells, A549RT‑eto cells were treated with trichostatin A (TSA; an HDAC inhibitor) during etoposide treatment. The combined treatment was demonstrated to enhance etoposide‑induced apoptosis and reduce expression levels of HDAC4, P‑gp and phospho‑Stat1. In addition, the suppression of Stat1 with siRNA enhanced etoposide‑induced apoptosis and reduced the expression levels of HDAC4 and P‑gp, suggesting that Stat1 is essential in the regulation of resistance to etoposide, and in the upregulation of P‑gp. Notably, TSA treatment reduced P‑gp transcript levels but Stat1 siRNA treatment did not, suggesting that P‑gp is regulated by HDAC at the transcriptional level and by Stat1 at the post‑transcriptional level. These results suggest that the upregulation of Stat1 and HDAC4 determines etoposide resistance through P‑gp expression in human A549 lung cancer cells.

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.

Feroniellin A-induced autophagy causes apoptosis in multidrug-resistant human A549 lung cancer cells

During the screening of natural chemicals that can reverse multidrug resistance in human A549 lung cancer cells resistant to etoposide (A549RT-eto), we discovered that Feroniellin A (FERO), a novel furanocoumarin, shows toxicity toward A549RT-eto cells in a dose- and time-dependent manner. FERO reduced the expression of NF-κB, leading to downregulation of P-glycoprotein (P-gp), encoded by MDR1, which eventually sensitized A549RT-eto cells to apoptosis. FERO specifically diminished transcription and promoter activity of MDR1 but did not inhibit the expression of other multidrug resistance genes MRP2 and BCRP. Moreover, co-administration of FERO with Bay11-7802, an inhibitor of NF-κB, accelerated apoptosis of A549RT-eto cells through decreased expression of P-gp, indicating that NF-κB is involved in multidrug resistance. Conversely, addition of Z-VAD, a pan-caspase inhibitor, blocked FERO-induced apoptosis in A549RT-eto cells but did not block downregulation of P-gp, indicating that a decrease in P-gp expression is necessary but not sufficient for FERO-induced apoptosis. Interestingly, we found that FERO also induces autophagy, which is characterized by the conversion of LC3 I to LC3 II, induction of GFP-LC3 puncta, enhanced expression of Beclin-1 and ATG5, and inactivation of mTOR. Furthermore, suppression of Beclin-1 by siRNA reduced FERO-induced apoptosis in A549RT-eto cells and activation of autophagy by rapamycin accelerated FERO-induced apoptosis, suggesting that autophagy plays an active role in FERO-induced apoptosis. Herein, we report that FERO reverses multidrug resistance in A549RT-eto cells and exerts its cytotoxic effect by induction of both autophagy and apoptosis, which suggests that FERO can be a useful anticancer drug for multidrug-resistant lung cancer.

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.

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.