Integrative transcriptomic and genomic analyses unveil the IFI16 variants and expression as MASLD progression markers.

BACKGROUND AND AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a broad and continuous spectrum of liver diseases ranging from fatty liver to steatohepatitis. The intricate interactions of genetic, epigenetic, and environmental factors in the development and progression of MASLD remain elusive. Here, we aimed to achieve an integrative understanding of the genomic and transcriptomic alterations throughout the progression of MASLD. APPROACH AND RESULTS: RNA-Seq profiling (n = 146) and whole-exome sequencing (n = 132) of MASLD liver tissue samples identified 3 transcriptomic subtypes (G1-G3) of MASLD, which were characterized by stepwise pathological and molecular progression of the disease. Macrophage-driven inflammatory activities were identified as a key feature for differentiating these subtypes. This subtype-discriminating macrophage interplay was significantly associated with both the expression and genetic variation of the dsDNA sensor IFI16 (rs6940, A>T, T779S), establishing it as a fundamental molecular factor in MASLD progression. The in vitro dsDNA-IFI16 binding experiments and structural modeling revealed that the IFI16 variant exhibited increased stability and stronger dsDNA binding affinity compared to the wild-type. Further downstream investigation suggested that the IFI16 variant exacerbated DNA sensing-mediated inflammatory signals through mitochondrial dysfunction-related signaling of the IFI16-PYCARD-CASP1 pathway. CONCLUSIONS: This study unveils a comprehensive understanding of MASLD progression through transcriptomic classification, highlighting the crucial roles of IFI16 variants. Targeting the IFI16-PYCARD-CASP1 pathway may pave the way for the development of novel diagnostics and therapeutics for MASLD.

The impact of alcohol consumption on hearing loss in male workers with a focus on alcohol flushing reaction: the Kangbuk Samsung Cohort Study.

Background: Despite hearing loss being a prevalent chronic condition, estimated to nearly 20% of the global population by the World Health Organization, the specific association with individual lifestyle factors, particularly alcohol consumption, remains unclear. In South Korea, approximately 80% of the population engages in alcohol consumption, with a notably high prevalence among males, indicating a high-risk drinking pattern. Therefore, this study aimed to assess the correlation between alcohol consumption and hearing loss in male workers, as well as to analyze additional variables such as alcohol flushing reaction, with the intention of improving worker health. Methods: The study was conducted from January 2012 to December 2019, targeting 114,114 participants who visited Kangbuk Samsung Hospital Total Healthcare Centers. Data were collected through pure-tone audiometry tests and alcohol-related questionnaire, and statistical analysis was performed using Cox regression analysis. Based on previous studies indicating a potential protective effect of light drinking on hearing loss, this group was designated as the reference. Additionally, stratified analyses were conducted based on the presence of alcohol flushing reaction and different working hours. Results: The hazard ratio (95% confidence interval) for hearing loss was higher in the heavy drinking group (1.23 [1.11-1.37]) compared to the moderate drinking group (1.09 [0.98-1.20]). Stratified analyses revealed a significantly elevated the hazard ratio of hearing loss in groups with alcohol flushing reaction compared to those without this factor. Conclusions: Our study demonstrated that moderate or heavy alcohol consumption in male workers can increase the risk of hearing loss, particularly in those with alcohol flushing reaction. These findings underscore the importance of addressing alcohol-related factors concerning hearing health among male workers.

Identification of signature gene set as highly accurate determination of metabolic dysfunction-associated steatotic liver disease progression.

BACKGROUND/AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by fat accumulation in the liver. MASLD encompasses both steatosis and MASH. Since MASH can lead to cirrhosis and liver cancer, steatosis and MASH must be distinguished during patient treatment. Here, we investigate the genomes, epigenomes, and transcriptomes of MASLD patients to identify signature gene set for more accurate tracking of MASLD progression. METHODS: Biopsy-tissue and blood samples from patients with 134 MASLD, comprising 60 steatosis and 74 MASH patients were performed omics analysis. SVM learning algorithm were used to calculate most predictive features. Linear regression was applied to find signature gene set that distinguish the stage of MASLD and to validate their application into independent cohort of MASLD. RESULTS: After performing WGS, WES, WGBS, and total RNA-seq on 134 biopsy samples from confirmed MASLD patients, we provided 1,955 MASLD-associated features, out of 3,176 somatic variant callings, 58 DMRs, and 1,393 DEGs that track MASLD progression. Then, we used a SVM learning algorithm to analyze the data and select the most predictive features. Using linear regression, we identified a signature gene set capable of differentiating the various stages of MASLD and verified it in different independent cohorts of MASLD and a liver cancer cohort. CONCLUSION: We identified a signature gene set (i.e., CAPG, HYAL3, WIPI1, TREM2, SPP1, and RNASE6) with strong potential as a panel of diagnostic genes of MASLD-associated disease.

Association between shift work and the risk of hypothyroidism in adult male workers in Korea: a cohort study.

Background: Shift work has been reported to have several harmful effects on the human body. However, a small number of studies have evaluated the association between shift work and adverse effects on the thyroid. In our longitudinal study, we examined the causal association between shift work and the risk of hypothyroidism. Methods: A Kangbuk Samsung Cohort Study was conducted on 112,648 men without thyroid disease at baseline who were followed up at least once between 2012 and 2019. Shift work status and shift schedule types were categorized using standardized questionnaires. Hypothyroidism was defined using the reference ranges of serum thyroid-stimulating hormones and free thyroxine levels. Hazard ratios (HRs) and 95% confidence intervals (CIs) for incident hypothyroidism were estimated using Cox proportional hazards regression analyses with the daytime work group as the reference. Results: During the 501,237 person-years of follow-up, there were 6,306 incident cases of hypothyroidism (incidence density, 1.26 per 100 person-years). The multivariable-adjusted HR of incident hypothyroidism for the shift work total group that included all shifts compared with the daytime work group was 1.27 (95% CI: 1.15-1.40). For the fixed evening, fixed night, rotating shift, and other shift workers, the multivariable-adjusted HRs (95% CI) were 1.11 (0.76-1.61), 2.18 (1.20-3.93), 1.39 (1.23-1.56), and 1.00 (0.82-1.22), respectively. In subgroup analyses by age, the association between shift work and hypothyroidism was more pronounced in younger participants (< 40 years; HR: 1.31; 95% CI: 1.16-1.47). Conclusions: Our large-scale cohort study showed an association between shift work and the incidence of hypothyroidism, especially in younger workers with night shifts.

Association between hearing loss and high-sensitivity C-reactive protein: the Kangbuk Samsung Cohort Study.

Background: Hearing loss (HL) is linked to an elevated risk of cardiovascular diseases (CVDs). The pathogeneses of HL and CVD commonly involve inflammatory responses. Previous studies investigated elevated levels of inflammatory biomarkers in subjects with HL, however, their findings did not demonstrate statistical significance. In our cross-sectional and longitudinal study, we investigated the correlation between HL and increased high-sensitivity C-reactive protein (hsCRP) levels to determine how HL is associated with CVDs. Methods: We conducted a cross-sectional study with workers aged over 18 years who underwent health check-ups at our institution between 2012 and 2018 (n = 566,507), followed by conducting a longitudinal study of workers aged > 18 who underwent health checkups at least twice at our institution between 2012 and 2018 (n = 173,794). The definition of HL was as an average threshold of ≥ 20 dB in pure-tone air conduction at 0.5, 1.0, and 2.0 kHz in both ears. The incidence of increased hsCRP levels throughout the follow-up period was defined as a level exceeding 3 mg/L. Logistic regression and generalized estimating equations were performed to estimate the risk of increased hsCRP levels according to the occurrence of HL in groups stratified by age. Results: In the cross-sectional study, the multivariate-adjusted odds ratio (OR) was 1.17 (95% confidence interval [CI]: 1.02-1.34); the OR was 0.99 (95% CI: 0.80-1.22) in those under 40 and 1.28 (1.08-1.53) in those over 40. In the longitudinal study, the multivariable-adjusted OR was 1.05 (95% CI: 0.92-1.19); the OR was 1.10 (95% CI: 0.90-1.35) in those under 40 and 1.20 (1.01-1.43) in those over 40. Conclusions: This cross-sectional and longitudinal study identified an association between HL and increased hsCRP levels in workers aged over 40 years.

Who has experienced better or worse health conditions since the outbreak of COVID-19?: results from a representative cross-sectional survey in Seoul.

BACKGROUND/OBJECTIVES: The coronavirus disease 2019 (COVID-19) pandemic has affected the entire world population in many ways. This study aimed to analyze the patterns of changes in eating, food purchasing and preparation, physical activity, and subjective health after COVID-19 outbreak by various sociodemographic factors and to understand the factors associated with changes in subjective health. SUBJECTS/METHODS: A cross-sectional survey using a representative sample from Seoul was used for the analysis. The data collection was conducted from September to October 2020. A total of 3,833 citizens aged more than 18 years old participated in the Seoul Food Survey. Descriptive statistics and generalized ordinal logistic regression models were used to understand the changes in health behaviors, health indicators, and subjective general health by various socioeconomic status. RESULTS: It was shown that the changes in household income, food expenditure, food consumption and physical activities differed significantly by age, education, occupation, income, weight, and food security status. Low-income and food-insecure households were affected more severely by the pandemic. Older age, household food insecurity, income reduction, increased home cooking and frequency of having instant foods, decreased physical activity and weight gain were significant factors explaining worse perceived health during the COVID-19 pandemic. CONCLUSIONS: The results suggest that focusing on older populations and low-income families with food insecurity should be prioritized during infectious disease outbreaks. In addition, the role of physical activities and instant food consumption in explaining perceived health should be investigated further in this prolonged battle with the pandemic situation.

Overall and cardiovascular mortality according to 10-year cardiovascular risk of the general health checkup: the Kangbuk Samsung Cohort Study.

Background: According to the occupational accident status analysis in 2020, of 1,180 occupational deaths, 463 were caused by cardiovascular disease (CVD). Workers should be assessed for CVD risk at regular intervals to prevent work-related CVD in accordance with the rules on occupational safety and health standards. However, no previous study has addressed risk and mortality. Therefore, this longitudinal study was conducted to evaluate the relationship between 10-year cardiovascular risk of the general health checkup and mortality. Methods: The study included 545,859 participants who visited Kangbuk Samsung Total Healthcare Centers from January 1, 2002, to December 31, 2017. We performed 10-year cardiovascular risk assessment for the participants and the risk was divided into 4 groups (low, moderate, high, and very high). The study used death data from the Korea National Statistical Office for survival status as an outcome variable by December 31, 2019, and the cause of death based on the International Classification of Diseases, 10th Revision (ICD-10) was identified. Statistical analysis was performed using Cox proportional hazards regression analysis, and the sum of the periods from the first visit to the date of death or December 31, 2019, was used as a time scale. We also performed a stratified analysis for age at baseline and sex. Results: During 5,253,627.9 person-years, 4,738 overall deaths and 654 cardiovascular deaths occurred. When the low-risk group was set as a reference, in the multivariable-adjusted model, the hazard ratios (HRs) (95% confidence interval [CI]) for overall mortality were 3.36 (2.87-3.95) in the moderate-risk group, 11.08 (9.27-13.25) in the high-risk group, and 21.20 (17.42-25.79) in the very-high-risk group, all of which were statistically significant. In cardiovascular deaths, the difference according to the risk classification was more pronounced. The HRs (95% CI) were 8.57 (4.95-14.83), 38.95 (21.77-69.69), and 78.81 (42.62-145.71) in each group. As a result of a subgroup analysis by age and sex, the HRs of all-cause mortality and cardiovascular mortality tended to be higher in the high-risk group. Conclusions: This large-scale longitudinal study confirmed that the risk of death increases with the 10-year cardiovascular risk of general health checkup.

Association between sitting-time at work and incidence of erosive esophagitis diagnosed by esophagogastroduodenoscopy: a Korean cohort study.

Background: Most previous longitudinal studies on lifestyle and gastroesophageal reflux disease (GERD) have focused on physical activity rather than sitting time. The main purpose of this study was to investigate the relationship between prolonged sitting time and the development of erosive esophagitis (EE). Methods: A self-report questionnaire was used for measuring sitting time in the Kangbuk Samsung Health Study. Sitting time was categorized into four groups: ≤ 6, 7-8, 9-10, and ≥ 11 hours/day. Esophagogastroduodenoscopy (EGD) was performed by experienced endoscopists who were unawared of the aims of this study. Hazard ratios (HRs) and 95% confidence intervals (CIs) for the development of EE were estimated using Cox proportional hazards analyses with ≤ 6 hours/day sitting time as the reference. Results: There were 6,524 participants included in the study. During a mean follow-up of 3.14 years, 2,048 incident cases developed EE. In age- and sex-adjusted models, the HR in the group sitting ≥ 11 hours per day compared ≤ 6 hours per day was 0.88 (95% CI: 0.76-0.99). After further adjusting for alcohol intake, smoking status, educational level, history of diabetes, and history of dyslipidemia, sitting time was still significantly related to the risk of EE (HR, 0.87; 95% CI: 0.76-0.98). After further adjustment for exercise frequency, this association persisted (HR, 0.86; 95% CI: 0.76-0.98). In subgroup analysis by obesity, the relationship between sitting time and EE was only significant among participants with body mass index < 25 kg/m2 (HR, 0.82; 95% CI: 0.71-0.95). Conclusions: Generally, prolonged sitting time is harmful to health, but with regard to EE, it is difficult to conclude that this is the case.

The effect of long working hours on developing type 2 diabetes in adults with prediabetes: The Kangbuk Samsung Cohort Study.

Background: Long working hours are known to account for approximately one-third of the total expected work-related diseases, and much interest and research on long working hours have recently been conducted. Additionally, as the prevalence of prediabetes and the high-risk group for diabetes are increasing worldwide, interest in prediabetes is also rising. However, few studies have addressed the development of type 2 diabetes and long working hours in prediabetes. Therefore, the aim of this longitudinal study was to evaluate the relationship between long working hours and the development of diabetes in prediabetes. Methods: We included 14,258 prediabetes participants with hemoglobinA1c (HbA1c) level of 5.7 to 6.4 in the Kangbuk Samsung Cohort Study. According to a self-reported questionnaire, we evaluated weekly working hours, which were categorized into 35-40, 41-52, and > 52 hours. Development of diabetes was defined as an HbA1c level ≥ 6.5%. Hazard ratios (HRs) and 95% confidence intervals (CIs) for the development of diabetes were estimated using Cox proportional hazards analyses with weekly working 35-40 hours as the reference. Results: During a median follow-up of 3.0 years, 776 participants developed diabetes (incidence density, 1.66 per 100 person-years). Multivariable-adjusted HRs of development of diabetes for weekly working > 52 hours compared with working 35-40 hours were 2.00 (95% CI: 1.50-2.67). In subgroup analyses by age (< 40 years old, ≥ 40 years old), sex (men, women), and household income (< 6 million KRW, ≥ 6 million KRW), consistent and significant positive associations were observed in all groups. Conclusions: In our large-scale longitudinal study, long working hours increases the risk of developing diabetes in prediabetes patients.

A case series of flight attendants at risk of COVID-19 in South Korea in 2020.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has infected many individuals worldwide. Individuals in contact with unspecified people during their work, may be at risk of occupational exposure. On June 22, 2020, 1,435 overseas patients were identified in the Republic of Korea. Considering the influx of overseas patients, aircraft-mediated COVID-19 transmission is a major concern. CASE PRESENTATION: We presented two flight attendants diagnosed with COVID-19 who shared the crew's resting area and ground transportation, and discussed the risks experienced by flight attendants. CONCLUSIONS: Biosafety guidelines for cabin crews should be intensified, and their COVID-19 risks must be further investigated. Policymakers must consider comprehensive surveillance systems for workers with high risks of occupational exposures and transmissions, such as flight attendants.

Factors associated with anti-hepatitis A virus immunoglobulin G seropositivity among Korean workers: a cross-sectional study.

OBJECTIVES: Hepatitis A incidence in Korea has dramatically increased in recent years. Individuals in their twenties and thirties, who account for majority of the workforce in Korea, are particularly susceptible to infection owing to a low seroprevalence of anti-hepatitis A virus (anti-HAV) immunoglobulin G (IgG). This study aimed to identify behavioural and occupational factors related to anti-HAV IgG seropositivity. DESIGN: Cross-sectional study. SETTING: A large university hospital in Seoul, Korea. PARTICIPANTS: Workers in formal employment having an annual routine health screening. PRIMARY OUTCOME MEASURE: Anti-HAV IgG seropositivity. RESULTS: Of 131 711 individuals who had an annual health screening at the study hospital in 2018, 68 612 met the inclusion criteria and were included in the analysis. Study participants were predominantly men (64.3%) and in their thirties (55.3%). The overall seroprevalence of anti-HAV IgG was 36.2%. In multivariate analyses, anti-HAV IgG seropositivity was independently associated with working in a workplace with ≥2 health managers (vs no health manager, adjusted OR 1.32, 95% CI 1.22 to 1.43); age 40-49 years (vs 20-29 years, OR 2.51, 95% CI 2.36 to 2.68); female sex (OR 1.54, 95% CI 1.48 to 1.59); experience of any general disease (vs no general disease history, OR 1.19, 95% CI 1.14 to 1.25), obesity (vs normal weight, OR 0.91, 95% CI 0.86 to 0.97); and hepatitis B antibody seropositivity (OR 2.39, 95% CI 2.31 to 2.49). CONCLUSIONS: The low prevalence of anti-HAV IgG seropositivity points to a need for implementation of workplace-based hepatitis A vaccine programmes. To promote workers' health and prevent hepatitis A outbreaks, occupational health managers, healthcare providers and policy-makers should focus on individuals who are susceptible to HAV, such as young men.

Peroxisome proliferator-activated receptor γ coactivator-1α is a predictor of lymph node metastasis and poor prognosis in human colorectal cancer.

Peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator-1α (PGC-1α) expression levels are correlated with clinical outcome in breast cancer. However, the potential biological and clinical significance of PPARγ and PGC-1α in colorectal cancer remains unknown. Here we investigated PPARγ and PGC-1α expression in colorectal cancer, and the associations of these expression levels with clinicopathological features. We also evaluated the roles of PPARγ and PGC-1α as prognostic factors in colorectal cancer. We performed immunohistochemical analysis to investigate PPARγ and PGC-1α expression in human colorectal cancer tissues and adjacent normal tissues from 108 primary colorectal cancer patients. We then examined how these expression levels correlated with clinicopathological features. Using the Kaplan-Meier method, we evaluated 3-year disease-free survival (DFS) and overall survival (OS) in patients with tumors expressing different levels of PPARγ and PGC-1α. Our results revealed that PPARγ expression was not significantly correlated with age at surgery, gender, differentiation, depth of infiltration, relapse, or TNM stage. Additionally, PGC-1α expression was not significantly correlated with age at surgery, differentiation, depth of infiltration, relapse, or TNM stage. However, PGC-1α expression was significantly correlated with nodal metastasis (p=0.020). Survival analysis demonstrated reduced OS in the PGC-1α-positive group compared to the PGC-1α-negative group (p=0.03). Our present findings suggest that PGC-1α may be useful for predicting nodal metastasis, and may represent a biomarker for poor prognosis in colorectal cancer.

In vitro and in vivo anti-leukemic effects of cladoloside C2 are mediated by activation of Fas/ceramide synthase 6/p38 kinase/c-Jun NH2-terminal kinase/caspase-8.

We previously demonstrated that the quinovose-containing hexaoside stichoposide C (STC) is a more potent anti-leukemic agent than the glucose-containing stichoposide D (STD), and that these substances have different molecular mechanisms of action. In the present study, we investigated the novel marine triterpene glycoside cladoloside C2 from Cladolabes schmeltzii, which has the same carbohydrate moiety as STC. We assessed whether cladoloside C2 could induce apoptosis in K562 and HL-60 cells. We also evaluated whether it showed antitumor action in mouse leukemia xenograft models, and its molecular mechanisms of action. We investigated the molecular mechanism behind cladoloside C2-induced apoptosis of human leukemia cells, and examined the antitumor effect of cladoloside C2 in a HL-60 and K562 leukemia xenograft model. Cladoloside C2 dose- and time-dependently induced apoptosis in the analyzed cells, and led to the activation of Fas/ceramide synthase 6 (CerS6)/p38 kinase/JNK/caspase-8. This cladoloside C2-induced apoptosis was partially blocked by specific inhibition by Fas, CerS6, and p38 siRNA transfection, and by specific inhibition of JNK by SP600125 or dominant negative-JNK transfection. Cladoloside C2 exerted antitumor activity through the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 without showing any toxicity in xenograft mouse models. The antitumor effect of cladoloside C2 was reversed in CerS6 shRNA-silenced xenograft models. Our results suggest that cladoloside C2 has in vitro and in vivo anti-leukemic effects due to the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 in lipid rafts. These findings support the therapeutic relevance of cladoloside C2 in the treatment of human leukemia.

Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement.

Mutations in the KRAS gene, which persistently activate RAS function, are most frequently found in many types of human cancers. Here, we proposed and verified a new approach against cancers harboring the KRAS mutation with high cancer selectivity and efficient anti-cancer effects based on targeted RNA replacement. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically target and reprogram the mutant KRAS G12V transcript to induce therapeutic gene activity in cells. Adenoviral vectors containing the specific ribozymes with downstream suicide gene were constructed and then infection with the adenoviruses specifically downregulated KRAS G12V expression and killed KRAS G12V-harboring cancer cells additively upon pro-drug treatment, but it did not affect the growth of wild-type KRAS-expressing cells. Minimal liver toxicity was noted when the adenoviruses were administered systemically in vivo. Importantly, intratumoral injection of the adenoviruses with pro-drug treatment specifically and significantly impeded the growth of xenografted tumors harboring KRAS G12V through a trans-splicing reaction with the target RNA. In contrast, xenografted tumors harboring wild-type KRAS were not affected by the adenoviruses. Therefore, RNA replacement with a mutant KRAS-targeting trans-splicing ribozyme is a potentially useful therapeutic strategy to combat tumors harboring KRAS mutation.

By activating Fas/ceramide synthase 6/p38 kinase in lipid rafts, stichoposide D inhibits growth of leukemia xenografts.

Stichoposide D (STD) is a marine triterpene glycoside isolated from sea cucumbers. We examined the molecular mechanisms underlying the antitumor activity of STD in human leukemia cells. The role of Fas (CD95), ceramide synthase 6 (CerS6) and p38 kinase during STD-induced apoptosis was examined in human leukemia cells. In addition, the antitumor effects of STD in K562 and HL-60 leukemia xenograft models were investigated. We found that STD induces Fas translocation to lipid rafts, and thus mediates cell apoptosis. We also observed the activation of CerS6 and p38 kinase during STD-induced apoptosis. The use of methyl-ß-cyclodextrin and nystatin to disrupt lipid rafts prevents the clustering of Fas and the activation of CerS6 and p38 kinase, and also inhibits STD-induced apoptosis. Specific inhibition by Fas, CerS6, and p38 kinase siRNA transfection partially blocked STD-induced apoptosis. In addition, STD has antitumor activity through the activation of CerS6 and p38 kinase without displaying any toxicity in HL-60 and K562 xenograft models. We observed that the anti-tumor effect of STD is partially prevented in CerS6 shRNA-silenced xenograft models. We first report that Fas/CerS6/p38 kinase activation in lipid rafts by STD is involved in its anti-leukemic activity. We also established that STD is able to enhance the chemosensitivity of K562 cells to etoposide or Ara-C. These data suggest that STD may be used alone or in combination with other chemotherapeutic agents to treat leukemia.

Targeted Regression of Hepatocellular Carcinoma by Cancer-Specific RNA Replacement through MicroRNA Regulation.

Hepatocellular carcinoma (HCC) has a high fatality rate and limited therapeutic options with side effects and low efficacy. Here, we proposed a new anti-HCC approach based on cancer-specific post-transcriptional targeting. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically induce therapeutic gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To circumvent side effects due to TERT expression in regenerating liver tissue, liver-specific microRNA-regulated ribozymes were constructed by incorporating complementary binding sites for the hepatocyte-selective microRNA-122a (miR-122a), which is down-regulated in HCC. The ribozyme activity in vivo was assessed in mouse models orthotopically implanted with HCC. Systemic administration of adenovirus encoding the developed ribozymes caused efficient anti-cancer effect and the least hepatotoxicity with regulation of ribozyme expression by miR-122a in both xenografted and syngeneic orthotopic murine model of multifocal HCC. Of note, the ribozyme induced local and systemic antitumor immunity, thereby completely suppressing secondary tumor challenge in the syngeneic mouse. The cancer specific trans-splicing ribozyme system, which mediates tissue-specific microRNA-regulated RNA replacement, provides a clinically relevant, safe, and efficient strategy for HCC treatment.

Tumor-to-muscle ratio of 8F-FDG PET for predicting histologic features and recurrence of HCC.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) recurrence is observed in up to 70-80% of patients despite a curative treatment. Microvascular invasion (MVI) and poor differentiation are strong risk factors for recurrence, but these cannot be known preoperatively. The aim of this study was to investigate the correlation of 18F-FDG PET with MVI and differentiation, and predictive role of tumor-to-background ratio of PET for recurrence in HCC. METHODOLOGY: Fifty-four patients had 18F-FDG PET/CT study before surgical resection as a first treatment of HCC between December 2008 and December 2012. We analyzed the predictive role of metabolic parameters of PET for recurrence of HCC. Maximal standardized uptake value, tumor-to-nontumor ratio, tumor-to-muscle ratio (TMR) and tumor-to-blood ratio were tested as metabolic index of 18F-FDG PET. RESULTS: Twenty-seven patients had increased uptake in preoperative PET and 14 (51.9%) of them experienced the recurrence. Increased uptake in PET and TMR were associated with MVI (p = 0.04, p = 0.005) and histologic differentiation (p = 0.018, p = 0.002). MVI was the only predictive factor for re- currence in multivariate analysis although TMR ≥ 6.36 showed a favorable result despite no statistical significance (p = 0.061). CONCLUSIONS: Increased 18F-FDG uptake of HCC, especially high TMR might be correlated with MVI and poor differentiation, and tends to have a risk for recurrence in HCC.

Overexpression of PGC­1α enhances cell proliferation and tumorigenesis of HEK293 cells through the upregulation of Sp1 and Acyl-CoA binding protein.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC­1α), a coactivator interacting with multiple transcription factors, regulates several metabolic processes. Although recent studies have focused on the role of PGC­1α in cancer, the underlying molecular mechanism has not been clarified. Therefore, we evaluated the role of PGC­1α in cell proliferation and tumorigenesis using human embryonic kidney (HEK)293 cells and colorectal cancer cells. We established stable HEK293 cell lines expressing PGC­1α and examined cell proliferation, anchorage-independent growth, and oncogenic potential compared to parental HEK293 cells. To identify the molecular PGC­1α targets for increased cell proliferation and tumorigenesis, the GeneFishing™ DEG (differentially expressed genes) screening system was used. Western blot analysis and immunofluorescence staining were performed for a regulated gene product to confirm the results. Forced expression of PGC­1α in HEK293 cells promoted cell proliferation and anchorage-independent growth in soft agar. In addition, HEK293 cells that highly expressed PGC­1α showed enhanced tumor formation when subcutaneously injected into the bilateral flanks of immunodeficient mice. The results of the GeneFishing DEG screening system identified one upregulated gene (Acyl-CoA binding protein; ACBP). Real-time RT-PCR, western blot analysis, and immunofluorescence staining showed that ACBP was markedly increased in HEK293 cells stably overexpressing PGC­1α (PGC­1α-HEK293 cells) compared to those expressing an empty vector. In PGC­1α, ACBP, and specificity protein 1 (Sp1) siRNA knockdown experiments in PGC­1α-HEK293 and SNU-C4 cells, we also observed inhibition of cell proliferation, reduced expression of antioxidant enzymes, and increased H2O2-induced reactive oxygen species production and apoptosis. These findings suggest that PGC­1α may promote cell proliferation and tumorigenesis through upregulation of ACBP. We provide evidence that increased Sp1 expression might contribute to enhanced ACBP expression by PGC­1α. The current results also suggest that PGC­1α, whose expression is related to enhanced cell proliferation and tumorigenesis, may be a good candidate molecular target for cancer therapy.

Targeted anticancer effect through microRNA-181a regulated tumor-specific hTERT replacement.

We previously generated a group I intron-based ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to stimulate transgene activity in cancer cells expressing the target RNA via an accurate and specific trans-splicing reaction. One of the major concerns of the hTERT RNA targeting anti-cancer approach is the potential side effects to hTERT(+) hematopoietic stem cell-derived blood cells. Thus, here we modified the ribozyme by inserting target sites against microRNA-181a, which is a blood cell-specific microRNA, downstream of its 3' exon. The specificity of transgene induction and anticancer activity in hTERT(+) cancer cells improved significantly with the modified ribozyme, resulting in selective targeting of hTERT(+) cancer cells, but not hematopoietic cells even if they are hTERT-positive. Importantly, the trans-splicing reaction of the microRNA-regulated ribozyme worked equally well in a nude mouse model of hepatocarcinoma-derived intrasplenic carcinomatosis, inducing highly specific expression of a therapeutic transgene and efficiently regressing hTERT-positive liver tumors with minimal liver toxicity when systemically delivered with an adenoviral vector encoding the ribozyme. These results suggest that a combined approach of microRNA regulation with targeted RNA replacement is more useful for effective anti-cancer treatment.

Doxorubicin-induced carbonylation and degradation of cardiac myosin binding protein C promote cardiotoxicity.

Dose-dependent oxidative stress by the anthracycline doxorubicin (Dox) and other chemotherapeutic agents causes irreversible cardiac damage, restricting their clinical effectiveness. We hypothesized that the resultant protein oxidation could be monitored and correlated with physiological functional impairment. We focused on protein carbonylation as an indicator of severe oxidative damage because it is irreversible and results in proteasomal degradation. We identified and investigated a specific high-molecular weight cardiac protein that showed a significant increase in carbonylation under Dox-induced cardiotoxic conditions in a spontaneously hypertensive rat model. We confirmed carbonylation and degradation of this protein under oxidative stress and prevention of such effect in the presence of the iron chelator dexrazoxane. Using MS, the Dox-induced carbonylated protein was identified as the 140-kDa cardiac myosin binding protein C (MyBPC). We confirmed the carbonylation and degradation of MyBPC using HL-1 cardiomyocytes and a purified recombinant untagged cardiac MyBPC under metal-catalyzed oxidative stress conditions. The carbonylation and degradation of MyBPC were time- and drug concentration-dependent. We demonstrated that carbonylated MyBPC undergoes proteasome-mediated degradation under Dox-induced oxidative stress. Cosedimentation, immunoprecipitation, and actin binding assays were used to study the functional consequences of carbonylated MyBPC. Carbonylation of MyBPC showed significant functional impairment associated with its actin binding properties. The dissociation constant of carbonylated recombinant MyBPC for actin was 7.35 ± 1.9 µM compared with 2.7 ± 0.6 µM for native MyBPC. Overall, our findings indicate that MyBPC carbonylation serves as a critical determinant of cardiotoxicity and could serve as a mechanistic indicator for Dox-induced cardiotoxicity.