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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.

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.

Abstract. 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 doxorubicininduced 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. IntroductionTo discover new genes that play a crucial role in common tumorigenesis regardless of tissue origin, we analyzed commonly upregulated unknown genes in 242 normal and 300 tumor samples originating from 11 different tissues using the Affymetrix gene chip system. An Affymetrix fragment, later named cancer upregulated gene (CUG) 2 was identified as a candidate gene that is commonly upregulated in various tumor tissues such as ovary, liver, lung and colon. This CUG2 was mapped to chromosome 6q22.32; it spans ~8.5 kb with a three-exon structure and encodes a 88-amino acid polypeptide (1). Further study has revealed that CUG2 is a new component of centromere required for a proper kinetochore function during cell division (2). Of interest, CUG2 has been shown to harbor an oncogenic effect in a transplanted model using NIH3T3 cells expressing CUG2, in a manner similar to Ras (1). Although CUG2 overexpression leads to activated Ras and MAPKs including p38 MAPK, which eventually facilitates oncolytic reoviral replication (3), CUG2 contrastingly provides resistance to oncolytic vesicular stomatitis virus infection through activation of STAT1 as shown in our recent study (4).Although STAT1 is well known as a master transcription factor for IFN-related intracellular signaling, leading to antiviral activity, several lines of evidence hav...

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Abstract.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. IntroductionThe type I interferon (IFN) receptor consists of 2 subunits, IFN-α receptor 1 (IFNAR1) and IFNAR2, which belong to the type II cytokine receptor superfamily (1). This heterodimeric complex is able to interact with IFN-α and IFN-β, resulting in the phosphorylation of Tyk2 and Jak1 that are bound to IFNAR1 and IFNAR2, respectively (2). Subsequently, the Stat proteins, Stat1 and Stat2 are phosphorylated at specific tyrosine residues, which allows the 2 proteins to form a Stat1/2 heterodimer based on SH2/phosphotyrosine interactions. The formation of this heterodimer facilitates its association with IFN regulatory factor (IRF)9 to form an active heterotrimeric transcription factor called IFN-stimulated gene factor (ISGF)3. ISGF3 targets specific sequences such as IFN-stimulated response element and IFN-γ-activated sequence in the promoters of IFN-stimulated genes, leading to the establishment of antiviral status (3).After type I IFN binds to its cognate type I IFN receptor, the receptor is downregulated by endocytosis mediated by cargospecific clathrin machinery, and degraded via the lysosomal and proteosomal pathways in order to limit the magnitude and duration of IFN signaling (4,5). The adaptin protein 2 complex, a component of the cargo-specific clathrin machinery recognizes the Tyr-based endocytic motif of IFNAR1, leading to the efficient endocytosis of IFNAR1 (5). The Skip, Cullin, F-box containing complex β-TrCP E3 ubiquitin ligase mediates the ubiquitination of IFNAR1 in a phosphorylation-dependent manner, eventually designating IFNAR1 for lysosomal degradation (5). Catalytic activation of Tyk2 is required for these events but is not essential for IFNAR1 internalization (6). Conversely, it ...

Abstract. Hepatitis B virus X (HBX) protein has been reported to induce upregulation of β-catenin, a known protooncogene, in p53-knockout and p53-mutant hepatic cell lines both in a GSK-3β-dependent manner and via interaction with adenomatous polyposis coli, which results in protection from β-catenin degradation. In this study, we describe a novel mechanism for HBX-mediated upregulation of β-catenin. We observed that HBX interacts with SIRT1, a class III histone deacetylase. Furthermore, the presence of HBX attenuated the interaction between SIRT1 and β-catenin, leading to protection of β-catenin from the inhibitory action of SIRT1. Reduction of SIRT1 with siRNA or suppression of SIRT1 activity with nicotinamide upregulated β-catenin protein levels. In contrast, enhancement of SIRT1 activity with resveratrol reduced β-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 β-catenin by sequestering SIRT1, which leads to anticancer drug treatment resistance.