Andrographolide targets EGFR to impede epithelial-mesenchymal transition in human breast cancer cells.

Despite the primary surgical treatment for breast cancer patients, malignant invasiveness and metastasis remain threatening factors for women with breast cancer. As chemotherapy yields unsatisfactory results, it prompted us to search for effective natural agents with few side-effects. Although andrographolide (ADGL), a natural diterpenoid lactone isolated from Andrographis paniculata, presents anticancer effects, the molecular mechanism remains unknown. Initially, on comparing the expression of proteins related to epithelial-mesenchymal transition (EMT) between nonmetastatic cancer MCF7 cells and highly metastatic cancer MDA-MB-231 cells, we found that MDA-MB-231 cells exhibit higher protein levels of N-cadherin and vimentin and lower protein levels of E-cadherin when compared to MCF7 cells. Moreover, MDA-MB-231 cells also exhibited higher EGFR expression and activity, higher STAT1 activity and abundant HDAC4 expression. To elucidate whether these proteins are closely associated with EMT, EGFR, STAT1 or HDAC4, the proteins were silenced in MDA-MB-231 breast cancer cells by their specific siRNAs. We found that silencing these proteins reduced EMT, indicating an important role of EGFR, STAT1 and HDAC4 in EMT progression. When we treated MDA-MB-231 cells with ADGL as a potential therapeutic drug, we found that ADGL treatment inhibited cell migration and invasion. Furthermore, it also recovered E-cadherin expression and decreased N-cadherin and vimentin protein levels. ADGL treatment reduced EGFR expression at a lower concentration (1 µg/mL); however, STAT1 activity and HDAC4 expression was reduced by a higher concentration (5 µg/mL) of ADGL. Moreover, we observed that the combined treatment with ADGL and siRNAs against these proteins highly sensitized the MDA-MB-231 cells to apoptosis compared to that with ADGL and control siRNA. Collectively, our results suggest that ADGL targets EGFR, thereby inhibiting EMT in human breast cancer cells.

N-benzyl-N-methyldecan-1-amine, derived from garlic, and its derivative alleviate 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice.

Given the intricate etiology and pathogenesis of atopic dermatitis (AD), the complete cure of AD remains challenging. This study aimed to investigate if topically applying N-benzyl-N-methyldecan-1-amine (BMDA), derived from garlic, and its derivative [decyl-(4-methoxy-benzyl)-methyl-1-amine] (DMMA) could effectively alleviate AD-like skin lesions in 2,4-dinitrochlorobenzene (DNCB)-treated mice. Administering these compounds to the irritated skin of DNCB-treated mice significantly reduced swelling, rash, and excoriation severity, alongside a corresponding decrease in inflamed epidermis and dermis. Moreover, they inhibited spleen and lymph node enlargement and showed fewer infiltrated mast cells in the epidermis and dermis through toluidine-blue staining. Additionally, they led to a lower IgE titer in mouse sera as determined by ELISA, compared to vehicle treatment. Analyzing skin tissue from the mice revealed decreased transcript levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6), IL-4, iNOS, and COX-2, compared to control mice. Simultaneously, the compounds impeded the activation of inflammation-related signaling molecules such as JNK, p38 MAPK, and NF-κB in the mouse skin. In summary, these findings suggest that BMDA and DMMA hold the potential to be developed as a novel treatment for healing inflammatory AD.

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.

Formoxanthone C Inhibits Malignant Tumor Phenotypes of Human A549 Multidrug Resistant-cancer Cells through Signal Transducer and Activator of Transcription 1-Histone Deacetylase 4 Signaling.

Considering that presence of cancer stem cell (CSC) subpopulation in tumor tissues confers anticancer drug resistance, we investigated whether human A549 lung cancer cells resistant to etoposide possess CSC-like phenotypes. Furthermore, it is known that these malignant tumor features are the leading cause of treatment failure in cancer. We have thus attempted to explore new therapeutic agents from natural products targeting these malignancies. We found that formoxanthone C (XanX), a 1,3,5,6-tetraoxygenated xanthone from Cratoxylum formosum ssp. pruniflorum, at a non-cytotoxic concentration reduced the expression of the signal transducer and activator of transcription 1 (STAT1) and histone deacetylase 4 (HDAC4) proteins, leading to inhibition of CSC-like phenotypes such as cell migration, invasion, and sphere-forming ability. Moreover, we found that treatment with STAT1 or HDAC4 small interfering RNAs significantly hindered these CSC-like phenotypes, indicating that STAT1 and HDAC4 play a role in the malignant tumor features. Taken together, our findings suggest that XanX may be a potential new therapeutic agent targeting malignant lung tumors.

The Korean Hepatitis C Virus Care Cascade in a Tertiary Institution: Current Status and Changes in Testing, Link to Care, and Treatment.

Background/Aims: The care cascade for hepatitis C virus (HCV) infection is impeded by multiple barriers, including suboptimal anti-HCV testing, link to care, and diagnosis. We explored the changes in the care cascade of HCV for the past 20 years and its current status in a large cohort from a tertiary referral center. Methods: We analyzed 1,144,468 patients who had anti-HCV testing between January 2001 and June 2020. Metrics related to the care cascade of HCV infection and the long-term prognosis of patients were explored. Results: The seroprevalence of anti-HCV positivity was 1.8%, with a recent decreasing trend. In all, 69.9% of anti-HCV positive patients performed HCV RNA testing, with a 65.7% positivity. Patients who did not have HCV RNA testing were older and more likely to have a non-hepatocellular carcinoma malignancy, normal alanine aminotransferase level, and good liver function. Linkage times for HCV RNA testing from the anti-HCV positivity and for antiviral treatment from HCV diagnosis decreased, notably after 2015, when highly efficacious oral antiviral treatment was introduced to Korea. The average treatment uptake rate was 35.4%, which increased to 38.9% after 2015. Of the 5,302 patients analyzed for long-term prognosis, the annual incidences of hepatocellular carcinoma were 1.02 or 2.14 per 100 person-years in patients with or without a sustained virological response, respectively. Conclusions: The care cascade of HCV infection has been suboptimal for the past 20 years, despite the recent changes. More effort should be made to increase HCV RNA testing and treatment uptake.

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].

Safety of direct oral anticoagulants compared to warfarin in cirrhotic patients with atrial fibrillation.

BACKGROUND/AIMS: The safety of direct oral anticoagulants (DOACs) compared with warfarin in patients with both nonvalvular atrial fibrillation (AF) and clinically confirmed liver cirrhosis (LC) has not been well studied. We compared the risk of a major bleeding event between DOAC and warfarin treatments in this patient population. METHODS: A total of 238 cirrhotic patients with AF were retrospectively analyzed. The major bleeding event risk was compared between DOAC- and warfarin-treated groups. The median follow-up duration was 5.6 years. RESULTS: Among the 238 study patients with LC and AF, 128 (53.8%) received DOACs and 110 (46.2%) received warfarin. The mean patient age was 68.8 years, and 78.2% were men. A major bleeding event occurred in 10 and 20 patients in the DOAC and warfarin groups, respectively, most commonly caused by gastrointestinal bleeding (70.0%). The cumulative risk of major bleeding did not differ between the groups by log-rank test (p = 0.12). This finding did not change when using 60 propensity score-matched pairs. A multivariable Cox regression model indicated that the concomitant use of antiplatelet agents (adjusted hazard ratio [aHR], 2.06; 95% confidence interval [CI], 1.00 to 4.30; p = 0.048) and presence of esophageal or gastric varices confirmed by endoscopic examination (aHR, 2.31; 95% CI, 1.03 to 5.17; p = 0.04) were associated with major bleeding in the entire cohort. CONCLUSION: A major bleeding event risk is not increased by DOAC compared with warfarin treatment. Antiplatelet agent use and varices are independently associated with a higher risk of major bleeding during anticoagulation.

The role of muscle depletion and visceral adiposity in HCC patients aged 65 and over undergoing TACE.

BACKGROUND: The incidence of hepatocellular carcinoma (HCC) has been increasing among the elderly populations. Trans-arterial chemoembolization (TACE), a widely used first-line non-curative therapy for HCCs is an issue in geriatrics. We investigated the prognosis of elderly HCC patients treated with TACE and determined the factors that affect the overall survival. METHODS: We included 266 patients who were older than 65 years and had received TACE as initial treatment for HCC. We analyzed the skeletal muscle index (SMI) and visceral-to-subcutaneous fat ratio (VSR) around the third lumbar vertebrae using computed tomography scans. Muscle depletion with visceral adiposity (MDVA) was defined by falling below the median SMI and above the median VSR value sex-specifically. We evaluated the overall survival in association with MDVA and other clinical factors. RESULTS: The mean age was 69.9 ± 4.5 years, and 70.3% of the patients were men. According to the Barcelona Clinic Liver Cancer (BCLC) staging system, 29, 136, and 101 patients were classified as BCLC 0, A, and B stages, respectively, and 79 (29.7%) had MDVA. During the median follow-up of 4.1 years, patients with MDVA had a shorter life expectancy than those without MDVA (P = 0.007) even though MDVA group had a higher objective response rate after the first TACE (82.3% vs. 75.9%, P = 0.035). Multivariate analysis revealed that MDVA (Hazard ratio [HR] 1.515) age (HR 1.057), liver function (HR 1.078), tumor size (HR 1.083), serum albumin level (HR 0.523), platelet count (HR 0.996), tumor stage (stage A, HR 1.711; stage B, HR 2.003), and treatment response after the first TACE treatment (HR 0.680) were associated with overall survival. CONCLUSIONS: MDVA is a critical prognostic factor for predicting survival in the elderly patients with HCC who have undergone TACE.

A simple and clinically applicable model to predict liver-related morbidity after hepatic resection for hepatocellular carcinoma.

BACKGROUND & AIM: Hepatic resection is a treatment option for patients with hepatocellular carcinoma (HCC). However, factors associated with candidacy for resection and predictive of liver-related morbidity after resection for HCC remain unclear. This study aimed to assess candidacy for liver resection in patients with HCC and to design a model predictive of liver-related morbidity after resection. METHODS: A retrospective analysis of 1,565 patients who underwent liver resection for HCC between January 2016 and December 2017 was performed. The primary outcome was liver-related morbidity, including post-hepatectomy biochemical dysfunction (PHBD), ascites, hepatic encephalopathy, rescue liver transplantation, and death from any cause within 90 days. PHBD was defined as international normalized ratio (INR) > 1.5 or hyperbilirubinemia (> 2.9 mg/dL) on postoperative day ≥ 5. RESULTS: The 1,565 patients included 1,258 (80.4%) males and 307 (19.6%) females with a mean age of 58.3 years. Of these patients, 646 (41.3%) and 919 (58.7%) patients underwent major and minor liver resection, respectively. Liver-related morbidity was observed in 133 (8.5%) patients, including 77 and 56 patients who underwent major and minor resection, respectively. A total of 83 (5.3%) patients developed PHBD. Multivariate analysis identified cut-off values of the platelet count, serum albumin concentration, and ICG R15 value for predicting liver-related morbidity after resection. A model predicting postoperative liver-related morbidity was developed, which included seven factors: male sex, age ≥ 55 years, ICG R15 value ≥ 15%, major resection, platelet count < 150,000/mm3, serum albumin concentration < 3.5 g/dL, and INR > 1.1. CONCLUSION: Hepatic resection for HCC was safe with 90-day liver-related morbidity and mortality rates of 8.5% and 0.8%, respectively. The developed point-based scoring system with seven factors could allow the prediction of the risk of liver-related morbidity after resection for HCC.

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.

Development of machine learning-based clinical decision support system for hepatocellular carcinoma.

There is a significant discrepancy between the actual choice for initial treatment option for hepatocellular carcinoma (HCC) and recommendations from the currently used BCLC staging system. We develop a machine learning-based clinical decision support system (CDSS) for recommending initial treatment option in HCC and predicting overall survival (OS). From hospital records of 1,021 consecutive patients with HCC treated at a single centre in Korea between January 2010 and October 2010, we collected information on 61 pretreatment variables, initial treatment, and survival status. Twenty pretreatment key variables were finally selected. We developed the CDSS from the derivation set (N = 813) using random forest method and validated it in the validation set (N = 208). Among the 1,021 patients (mean age: 56.9 years), 81.8% were male and 77.0% had positive hepatitis B BCLC stages 0, A, B, C, and D were observed in 13.4%, 26.0%, 18.0%, 36.6%, and 6.3% of patients, respectively. The six multi-step classifier model was developed for treatment decision in a hierarchical manner, and showed good performance with 81.0% of accuracy for radiofrequency ablation (RFA) or resection versus not, 88.4% for RFA versus resection, and 76.8% for TACE or not. We also developed seven survival prediction models for each treatment option. Our newly developed HCC-CDSS model showed good performance in terms of treatment recommendation and OS prediction and may be used as a guidance in deciding the initial treatment option for HCC.

Regorafenib Versus Nivolumab After Sorafenib Failure: Real-World Data in Patients With Hepatocellular Carcinoma.

Regorafenib and nivolumab are drugs approved for second-line treatment of patients with hepatocellular carcinoma (HCC) after sorafenib failure. However, the effectiveness of regorafenib and nivolumab following sorafenib has not been directly compared. This study retrospectively evaluated 373 patients with HCC who were treated with regorafenib (n = 223) or nivolumab (n = 150) after sorafenib failure between July 2017 and February 2019. Progression-free survival (PFS; hazard ratio [HR], 0.85; 95% confidence interval [CI], 0.69-1.06; P = 0.150), time to progression (TTP; HR, 0.95; 95% CI, 0.77-1.19; P = 0.680), and overall survival (OS; HR, 0.83; 95% CI, 0.64-1.07; P = 0.154) did not differ significantly between groups of patients treated with regorafenib and nivolumab, findings consistently observed by multivariable-adjusted, propensity score-matched, and inverse probability treatment weighting (IPTW) analyses. However, the objective response rate was significantly higher in the nivolumab than in the regorafenib group (13.3% vs. 4.0%; P = 0.002). When the effectiveness of regorafenib and nivolumab was compared in nonprogressors to treatment, defined as patients who achieved complete response, partial response, or stable disease after first response evaluation, PFS (HR, 0.50; 95% CI, 0.33-0.75; P = 0.001), TTP (HR, 0.48; 95% CI, 0.31-0.73; P < 0.001), and OS (HR, 0.51; 95% CI, 0.31-0.87; P = 0.013) were significantly longer in the 59 nonprogressors to nivolumab than in the 104 nonprogressors to regorafenib, findings also observed by multivariable-adjusted and IPTW analyses. Conclusion: Survival outcomes in patients treated with regorafenib and nivolumab after sorafenib failure did not differ significantly. However, nivolumab may be more effective than regorafenib in nonprogressors.

Cancer upregulated gene 2 (CUG2), a novel oncogene, promotes stemness-like properties via the NPM1-TGF-ß signaling axis.

Our previous study reported that cancer upregulated gene (CUG)2, a novel oncogene, induces both faster cell migration and anti-cancer drug resistance. We thus wonder whether CUG2 also induces stemness, a characteristic of cancer stem cells (CSCs) and further examine the molecular mechanism of this phenotype. To test that CUG2 induces stemness, we examined expression of stemness-related factors. Overexpression of CUG2 enhanced expression levels of stemness-related factors in human lung carcinoma A549 and immortalized bronchial BEAS-2B cells. Consequently, CUG2 increased cellular spherical cluster forming ability. Overexpression of CUG2 also induced tumor formation in xenotransplanted nude mice whereas transplantation of control cells failed to, implying that CUG2 possesses malignant tumorigenic potential. We paid attention to nucleophosmin (NPM1) for its known interaction with CUG2. Suppression of NPM1 hindered the CUG2-mediated stemness-like phenotypes and diminished TGF-ß transcriptional activity and signaling. TGF-ß increased stemness-like phenotypes in the control cells whereas TGF-ß inhibitor blocked induction of the phenotypes, indicating that NPM1 is required for CUG2-mediated stemness-like phenotypes through TGF-ß signaling. Furthermore, the suppression of Smad- and non-Smad-dependent TGF-ß signaling pathways also prevented CUG2 from inducing stemness-like phenotypes. Altogether, we suggest that the novel CUG2 oncogene promotes cellular transformation and stemness, mediated by nuclear NPM1 protein and TGF-ß signaling.

CGK062, a small chemical molecule, inhibits cancer upregulated gene 2­induced oncogenesis through NEK2 and ß­catenin.

The mechanisms through which cancer­upregulated gene 2 (CUG2), a novel oncogene, affects Wnt/ß­catenin signaling, essential for tumorigenesis, are unclear. In this study, we aimed to elucidate some of these mechanisms in A549 lung cancer cells. Under the overexpression of CUG2, the protein levels and activity of ß­catenin were evaluated by western blot analysis and luciferase assay. To examine a biological consequence of ß­catenin under CUG2 overexpression, cell migration, invasion and sphere formation assay were performed. The upregulation of ß­catenin induced by CUG2 overexpression was also accessed by xenotransplantation in mice. We first found that CUG2 overexpression increased ß­catenin expression and activity. The suppression of ß­catenin decreased cancer stem cell (CSC)­like phenotypes, indicating that ß­catenin is involved in CUG2­mediated CSC­like phenotypes. Notably, CUG2 overexpression increased the phosphorylation of ß­catenin at Ser33/Ser37, which is known to recruit E3 ligase for ß­catenin degradation. Moreover, CUG2 interacted with and enhanced the expression and kinase activity of never in mitosis gene A­related kinase 2 (NEK2). Recombinant NEK2 phosphorylated ß­catenin at Ser33/Ser37, while NEK2 knockdown decreased the phosphorylation of ß­catenin, suggesting that NEK2 is involved in the phosphorylation of ß­catenin at Ser33/Ser37. Treatment with CGK062, a small chemical molecule, which promotes the phosphorylation of ß­catenin at Ser33/Ser37 through protein kinase C (PKC)α to induce its degradation, reduced ß­catenin levels and inhibited the CUG2­induced features of malignant tumors, including increased cell migration, invasion and sphere formation. Furthermore, CGK062 treatment suppressed CUG2­mediated tumor formation in nude mice. Taken together, the findings of this study suggest that CUG2 enhances the phosphorylation of ß­catenin at Ser33/Ser37 by activating NEK2, thus stabilizing ß­catenin. CGK062 may thus have potential for use as a therapeutic drug against CUG2­overexpressing lung cancer cells.

Protopine isolated from Nandina domestica induces apoptosis and autophagy in colon cancer cells by stabilizing p53.

The tumor suppressor p53 plays essential roles in cellular protection mechanisms against a variety of stress stimuli and its activation induces apoptosis or autophagy in certain cancer cells. Here, we identified protopine, an isoquinoline alkaloid isolated from Nandina domestica, as an activator of the p53 pathway from cell-based natural compound screening based on p53-responsive transcription. Protopine increased the p53-mediated transcriptional activity and promoted p53 phosphorylation at the Ser15 residue, resulting in stabilization of p53 protein. Moreover, protopine up-regulated the expression of p21WAF1/CIP1 and BAX, downstream genes of p53, and inhibited the proliferation of HCT116 colon cancer cells. Apoptosis was elicited by protopine as indicated by caspase-3/7 activation, poly ADP ribose polymerase cleavage, and increased population of Annexin V-FITC-positive cells. Furthermore, protopine induced the formation of microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3-II turnover, typical biochemical markers of autophagy, in HCT116 cells. Our findings suggest that protopine exerts its antiproliferative activity by stimulating the p53 pathway and may have potential as a chemopreventive agent for human colon cancer.

Long-term clinical outcomes in patients with autoimmune hepatitis according to treatment response in Asian country.

BACKGROUND & AIMS: As surrogate markers for autoimmune hepatitis (AIH), serum alanine aminotransferase (ALT) and immunoglobulin G (IgG) are convenient to measure under immunosuppression. However, the long-term prognosis of patients who achieve complete biochemical remission (CBR) in comparison with patients who achieve only biochemical remission (BR) is uncertain. METHODS: A total of 291 patients (89.7% female) diagnosed with AIH were retrospectively reviewed. CBR was defined as normal ALT and IgG levels with immunosuppression, while BR was defined as normal ALT levels. CBR was further divided into early CBR (<1year) and late CBR (≥1year) by the timing of remission. Liver-related adverse outcomes including liver-related death, liver transplantation and hepatocellular carcinoma were evaluated. RESULTS: With immunosuppressive treatment, 222 (76.3%) patients achieved CBR (early CBR: 168 and late CBR: 54). BR was achieved in 55 (18.9%) patients and 14 (4.8%) patients remained non-remission. With a median follow-up duration of 6.6 years, the risk of liver-related mortality was the lowest in patients with CBR, followed by patients with late CBR, BR and non-response. The cumulative risk of liver-related adverse outcomes was the highest in patients with non-response (8.51/100 person-years [PYs]), followed by BR (1.95/100 PYs), late CBR (1.89/100 PYs) and early CBR (0.75/100 PYs). By multivariable analysis, age, cirrhosis and treatment responses were independently associated with liver-related adverse outcomes. CONCLUSIONS: Patients with CBR within 1 year after treatment initiation had the lowest risk of liver-related adverse outcomes. Patients with late CBR and those with only BR had a comparable risk of long-term outcomes.

Pink1 regulates FKBP5 interaction with AKT/PHLPP and protects neurons from neurotoxin stress induced by MPP.

Loss of function mutations in the PTEN-induced putative kinase 1 (Pink1) gene have been linked with an autosomal recessive familial form of early onset Parkinson's disease (PD). However, the underlying mechanism(s) responsible for degeneration remains elusive. Presently, using co-immunoprecipitation in HEK (Human embryonic kidney) 293 cells, we show that Pink1 endogenously interacts with FK506-binding protein 51 (FKBP51 or FKBP5), FKBP5 and directly phosphorylates FKBP5 at Serine in an in vitro kinase assay. Both FKBP5 and Pink1 have been previously associated with protein kinase B (AKT) regulation. We provide evidence using primary cortical cultured neurons from Pink1-deficient mice that Pink1 increases AKT phosphorylation at Serine 473 (Ser473) challenged by 1-methyl-4-phenylpyridinium (MPP+ ) and that over-expression of FKBP5 using an adeno-associated virus delivery system negatively regulates AKT phosphorylation at Ser473 in murine-cultured cortical neurons. Interestingly, FKBP5 over-expression promotes death in response to MPP+ in the absence of Pink1. Conversely, shRNA-mediated knockdown of FKBP5 in cultured cortical neurons is protective and this effect is reversed with inhibition of AKT signaling. In addition, shRNA down-regulation of PH domain leucine-rich repeat protein phosphatase (PHLPP) in Pink1 WT neurons increases neuronal survival, while down-regulation of PHLPP in Pink1 KO rescues neuronal death in response to MPP+ . Finally, using co-immunoprecipitation, we show that FKBP5 interacts with the kinase AKT and phosphatase PHLPP. This interaction is increased in the absence of Pink1, both in Mouse Embryonic Fibroblasts (MEF) and in mouse brain tissue. Expression of kinase dead Pink1 (K219M) enhances FKBP5 interaction with both AKT and PHLPP. Overall, our results suggest a testable model by which Pink1 could regulate AKT through phosphorylation of FKBP5 and interaction of AKT with PHLPP. Our results suggest a potential mechanism by which PINK1-FKBP5 pathway contributes to neuronal death in PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Cancer upregulated gene (CUG)2 elevates YAP1 expression, leading to enhancement of epithelial-mesenchymal transition in human lung cancer cells.

Although our previous studies have showed that a novel oncogene, cancer upregulated gene (CUG)2 induced epithelial-mesenchymal transition (EMT), the detailed molecular mechanism remains unknown. Because several lines of evidence documented that Yes-Associated Protein (YAP)1 is closely associated with cancer stem cell (CSC)-like phenotypes including EMT, stemness, and drug resistance, we wondered if YAP1 is involved in CUG2-induced EMT. We herein found that the overexpression of CUG2 increased YAP1 expression at the transcriptional as well as protein levels. Chromatin immunoprecipitation assay revealed that the elevated YAP1 transcripts are attributed to c-Jun and AP2 bindings to the YAP1 promoter. Akt and MAPK kinases including ERK, JNK, and p38 MAPK enhanced the level of YAP1 protein. In spite of a close relationship between ß-catenin and YAP1, not ß-catenin but NEK2 played the role in increasing YAP1 expression. Silencing YAP1 inhibited CUG2-induced cell migration and invasion. N-cadherin and vimentin expressions were decreased during YAP1 knockdown. The suppression of YAP1 diminished TGF-ß transcriptional activity and expression as well as phosphorylation level of Smad2 and Twist protein. Conversely, LY2109761 or Smad2 siRNA treatment reduced YAP1 protein levels, indicating a close interplay between YAP1 and TGF-ß signaling. Taken together, we suggest that CUG2 induces up-regulation of YAP1 expression, leading to enhancing CUG2-induced EMT via a close crosstalk between YAP1 and TGF-ß signaling.

Caged-xanthone from Cratoxylum formosum ssp. pruniflorum inhibits malignant cancer phenotypes in multidrug-resistant human A549 lung cancer cells through down-regulation of NF-κB.

Our recent study reported that multidrug-resistant (MDR) human A549 lung cancer cells (A549RT-eto) with the elevated expression of NF-κB showed epithelial-mesenchymal transition (EMT), increasing spheroid formation and elevating the expression levels of stemness-related factors, including Oct4, Nanog, Sox2, Bmi1, and Klf4. Therefore, when new therapeutic agents targeting these malignant cancer cells were explored, we found that caged-xanthone (CX) isolated from the roots of Cratoxylum formosum ssp. pruniflorum diminished the expression of NF-κB, P-glycoprotein (P-gp) protein levels, cell migration and invasion, and sphere-forming ability of A549RT-eto cells. To address the role of NF-κB in these malignant cancer features, we treated A549RT-eto cells with NF-κB siRNAs in the present work. We found that the knockdown of NF-κB inhibited EMT and sphere formation. Furthermore, co-treatment with CX and NF-κB siRNA accelerated the death of apoptotic cells through the decrease of P-gp protein levels. These results suggest that NF-κB was involved in malignant cancer phenotypes and MDR in A549RT-eto cells. Taken together, our findings suggest that CX can be a potential therapeutic agent for the treatment of malignant tumor cells.