Mechanisms of Sorafenib Resistance in HCC Culture Relate to the Impaired Membrane Expression of Organic Cation Transporter 1 (OCT1).

Introduction: Sorafenib, an FDA-approved drug for advanced hepatocellular carcinoma (HCC) treatment, encounters resistance in many patients. Deciphering the mechanisms underlying sorafenib resistance is crucial for devising alternative strategies to overcome it. Aim: This study aimed to investigate sorafenib resistance mechanisms using a diverse panel of HCC cell lines. Methods: HCC cell lines were subjected to continuous sorafenib treatment, and stable cell lines (Huh 7.5 and Huh 7PX) exhibiting sustained growth in its presence were isolated. The investigation of drug resistance mechanisms involved a comparative analysis of drug-targeted signal transduction pathways (EGFR/RAF/MEK/ERK/Cyclin D), sorafenib uptake, and membrane expression of the drug uptake transporter. Results: HCC cell lines (Huh 7.5 and Huh 7PX) with a higher IC50 (10µM) displayed a more frequent development of sorafenib resistance compared to those with a lower IC50 (2-4.8µM), indicating a potential impact of IC50 variation on initial treatment response. Our findings reveal that activated overexpression of Raf1 kinases and impaired sorafenib uptake, mediated by reduced membrane expression of organic cation transporter-1 (OCT1), contribute to sorafenib resistance in HCC cultures. Stable expression of the drug transporter OCT1 through cDNA transfection or adenoviral delivery of OCT1 mRNA increased sorafenib uptake and successfully overcame sorafenib resistance. Additionally, consistent with sorafenib resistance in HCC cultures, cirrhotic liver-associated human HCC tumors often exhibited impaired membrane expression of OCT1 and OCT3. Conclusion: Intrinsic differences among HCC cell clones, affecting sorafenib sensitivity at the expression level of Raf kinases, drug uptake, and OCT1 transporters, were identified. This study underscores the potential of HCC tumor targeted OCT1 expression to enhance sorafenib treatment response.

A Single-Step Immunocapture Assay to Quantify HCC Exosomes Using the Highly Sensitive Fluorescence Nanoparticle-Tracking Analysis.

Introduction: Extracellular vesicles could serve as a non-invasive biomarker for early cancer detection. However, limited methods to quantitate cancer-derived vesicles in the native state remain a significant barrier to clinical translation. Aim: This research aims to develop a rapid, one-step immunoaffinity approach to quantify HCC exosomes directly from a small serum volume. Methods: HCC-derived exosomes in the serum were captured using fluorescent phycoerythrin (PE)-conjugated antibodies targeted to GPC3 and alpha-fetoprotein (AFP). Total and HCC-specific exosomes were then quantified in culture supernatant or patient-derived serums using fluorescence nanoparticle tracking analysis (F-NTA). The performance of HCC exosome quantification in the serum was compared with the tumor size determined by MRI. Results: Initially we tested the detection limits of the F-NTA using synthetic fluorescent and non-fluorescent beads. The assay showed an acceptable sensitivity with a detection range of 104-108 particles/mL. Additionally, the combination of immunocapture followed by size-exclusion column purification allows the isolation of smaller-size EVs and quantification by F-NTA. Our assay demonstrated that HCC cell culture releases a significantly higher quantity of GPC3 or GPC3+AFP positive EVs (100-200 particles/cell) compared to non-HCC culture (10-40 particles/cell) (p<0.01 and p<0.05 respectively). The F-NTA enables absolute counting of HCC-specific exosomes in the clinical samples with preserved biological immunoreactivity. The performance of F-NTA was clinically validated in serum from patients ± cirrhosis and with confirmed HCC. F-NTA quantification data show selective enrichment of AFP and GPC3 positive EVs in HCC serum compared to malignancy-free cirrhosis (AUC values for GPC3, AFP, and GPC3/AFP were found 0.79, 0.71, and 0.72 respectively). The MRI-confirmed patient cohort indicated that there was a positive correlation between total tumor size and GPC3-positive exosome concentration (r:0.78 and p<0.001). Conclusion: We developed an immunocapture assay that can be used for simultaneous isolation and quantification of HCC-derived exosomes from a small serum volume with high accuracy.

Effect of COVID-19 Pandemic on Hepatocellular Carcinoma Diagnosis: Results from a Single Turkey Center Study.

BACKGROUND AND AIMS: Currently malignancies of the liver are the sixth most frequently diagnosed cancers worldwide. The admission of patients to hospitals decreased due to the restriction of the Coronavirus disease 2019 (COVID-19) pandemic, especially patients suspected with cancer were delayed in their diagnosis and treatment. With this study, we aimed to investigate whether the Covid-19 pandemic caused a decrease in the number of hepatocellular cancers (HCC) or a delay in its diagnosis. METHODS: The study, which included newly diagnosed HCC patients, was conducted as a retrospective cross sectional study, in a single Turkey medical center. The patients were divided into pre-COVID-19 and post- COVID-19 two-year periods and compared in terms of tumor size, biochemical parameters, clinical and demographic features. RESULTS: A total of 63 HCC patients, 46 (73%) patients before the COVID-19 pandemic and 17 (27%) patients diagnosed during the COVID-19 pandemic were included. Maximum diameter of lesions and serum alpha- fetoprotein levels showed a statistically significant difference between the groups. Maximum tumor size in the pre-COVID-19 period was 4.58±3.77 mm, while in the COVID-19 period was 7.42±6.88 mm, the difference between two groups being statistically significant (p<0.05). HCC in the pre-COVID-19 period were detected mostly at Barcelona Clinic for Liver Cancer (BCLC) stage A (45.7%, n=21), while in the COVID-19 period most of HCC were detected at stage B (35.3%, n=6). CONCLUSIONS: The COVID-19 pandemic limited the access of patients to screening programs for HCC. The significant disruption in screening cirrhotic patients for HCC has led to a delay in diagnosis.

Artificial intelligence-based personalized diet: A pilot clinical study for irritable bowel syndrome.

We enrolled consecutive IBS-M patients (n = 25) according to Rome IV criteria. Fecal samples were obtained from all patients twice (pre-and post-intervention) and high-throughput 16S rRNA sequencing was performed. Six weeks of personalized nutrition diet (n = 14) for group 1 and a standard IBS diet (n = 11) for group 2 were followed. AI-based diet was designed based on optimizing a personalized nutritional strategy by an algorithm regarding individual gut microbiome features. The IBS-SSS evaluation for pre- and post-intervention exhibited significant improvement (p < .02 and p < .001 for the standard IBS diet and personalized nutrition groups, respectively). While the IBS-SSS evaluation changed to moderate from severe in 78% (11 out of 14) of the personalized nutrition group, no such change was observed in the standard IBS diet group. A statistically significant increase in the Faecalibacterium genus was observed in the personalized nutrition group (p = .04). Bacteroides and putatively probiotic genus Propionibacterium were increased in the personalized nutrition group. The change (delta) values in IBS-SSS scores (before-after) in personalized nutrition and standard IBS diet groups are significantly higher in the personalized nutrition group. AI-based personalized microbiome modulation through diet significantly improves IBS-related symptoms in patients with IBS-M. Further large-scale, randomized placebo-controlled trials with long-term follow-up (durability) are needed.

Evaluation of Non-invasive Fibrosis Markers in Predicting Esophageal Variceal Bleeding.

BACKGROUND/AIMS: Esophageal variceal bleeding (EVB) is an important cause of mortality and morbidity in liver cirrhosis. In this study, we aimed to predict the possibility of EVB in patients with cirrhosis using a non-invasive score. METHODS: A total of 359 patients with cirrhosis were divided into two groups based on the presence or absence of EVB. ChildTurcotte-Pugh (CTP) score, a model for end-stage liver disease, aspartate aminotransferase to alanine aminotransferase ratio, aspartate aminotransferase to platelet ratio index (APRI), fibrosis-4-index (FIB-4), aspartate aminotransferase to alanine aminotransferase ratio/platelet ratio index (AARPRI), and S-index were measured for all participants. Receiver operating characteristic curves were obtained for all parameters, and the optimal cut-off value was determined in predicting EVB. RESULTS: In patients with EVB, the number of platelets (PLT) were low (p<0.001) and APRI, AARPRI, FIB-4, and S-index were significantly higher than those in patients without EBV. APRI, AARPRI, FIB-4, PLT, and S-index were statistically significant predictors of EVB (p<0.05). CONCLUSION: FIB-4 and AARPRI, which are non-invasive markers of fibrosis, can be used to predict EVB. In addition, the 66.5 109/L cut-off value for PLT is important for EVB.

Preliminary examination of the relations between disease stage, illness perceptions, coping strategies, and psychological morbidity in chronic hepatitis B and C guided by the Common-Sense Model of Illness.

BACKGROUND AND AIMS: Chronic viral hepatitis (CVH) has a spectrum from asymptomatic disease to cirrhosis and hepatocellular carcinoma. In our study, we aimed to establish the relations between disease stage, illness perception, coping strategies and psychological morbidity in CVH. METHODS: A total of 182 patients with chronic hepatitis B (CHB) (n = 136) and chronic hepatitis C (CHC) (n = 46) were enrolled. Illness perceptions were measured with the Brief Illness Perceptions Questionnaire. Coping strategies were measured with the Carver Brief Coping Questionnaire. Anxiety and depression were measured with the Hospital Anxiety and Depression Scale. Relations were evaluated by structural equation modeling (SEM). RESULTS: In CHB, combining the questionnaire data using SEM resulted in a final model with an excellent fit [χ2 (2) = 0.00, P = 1.000, χ2/N = 0.00, root mean square error of approximation (RMSEA) < 0.001, comparative fit index (CFI) = 1.000, goodness-of-fit index (GFI) = 1.000]. Disease stage had a significant direct influence on illness perceptions (ß = 0.23, P = 0.006). Illness perceptions had a significant direct influence on emotional coping strategy, depression and anxiety (ß = 0.20, P = 0.019; ß = 0.33, P < 0.001; ß = 0.59, P < 0.001, respectively). While the use of emotional coping strategies was associated significantly (P = 0.01) with the presence of anxiety, problem-focused coping strategy was associated with depression (P = 0.004). In CHC, SEM resulted in a final model with an excellent fit [χ2 (2) = 0.078, P = 0.962, χ2/N = 0.039, RMSEA<0.001, CFI = 1.000, GFI = 0.999]. Disease stage did not have a significant direct influence on illness perceptions (P > 0.05). Illness perceptions had a significant direct influence on depression and anxiety (ß = 0.27, P = 0.023; ß = 0.44, P < 0.001, respectively). CONCLUSION: The psychological consequences of the disease vary depending on the person's perception of illness and coping strategies.

Evaluation of epicardial adipose tissue and carotid intima-media thickness as a marker of atherosclerosis in patients with inflammatory bowel disease.

BACKGROUND AND AIM: this study aimed to compare carotid intima media (CIMT) and epicardial adipose tissue (EAT) measurements, which are considered as markers for the detection of early atherosclerosis in healthy controls and inflammatory bowel disease (IBD) cases. METHODS: a total of 60 IBD patients (25 Crohn's disease and 35 ulcerative colitis) and 60 healthy patients (as a control group) were included in the study. The measurements of CIMT and EAT were performed using echocardiography and ultrasonography, respectively. Statistical analysis was used to determine the relationship between the parameters. RESULTS: the thickness of bilateral (right and left) CIMT and EAT were significantly higher in IBD than in the control group (p < 0.05). There was a positive correlation between EAT and bilateral (right and left) CIMT in IBD patients (p < 0.05). CONCLUSION: IBD is associated with an increased thickness of EAT and CIMT. Chronic inflammation in IBD may increase the risk of atherosclerotic heart disease. Thus, only measuring the thickness of EAT and CIMT can be used as an objective, easy, simple, affordable, non-invasive and accessible assessment method in order to screen for this risk.

Relationship between pruritus and autotaxin in intrahepatic cholestasis of pregnancy.

OBJECTIVE: Intrahepatic cholestasis of pregnancy is a temporary, pregnancy-specific disease that resolves with delivery, characterized by itching (pruritus), as well as high transaminase and serum bile acid levels in the third trimester of pregnancy. Due to the effects of Autotaxin on the physiology of pregnancy, we aimed to investigate Autotaxin activity in patients with intrahepatic cholestasis of pregnancy. PATIENTS AND METHODS: Sixty-nine patients diagnosed with intrahepatic cholestasis of pregnancy and 20 healthy pregnant women were enrolled in the study. Fasting serum bile acid, pruritus intensity, serum parameters, gestational week of the patients at the time of diagnosis were recorded, and birth week and birth weight were monitored. Autotaxin serum level was measured enzymatically. RESULTS: The mean serum bile acid level (n=69; 38.74±35.92µmol/L) in patients with intrahepatic cholestasis of pregnancy (n=69) was detected to be higher than healthy pregnant women (n=20; 5.05±1.88µmol/L) (p<0.001). Weak correlation was detected between serum bile acid level and itch intensity (p=0.014, r=0.295), while no relation was detected between Autotaxin and itch intensity (p=0.446, r=0.09). Although mean Autotaxin (intrahepatic cholestasis of pregnancy: 678.10±424.42pg/mL, control: 535.16±256.47pg/mL) levels were high in patients with intrahepatic cholestasis of pregnancy, it was not statistically significant (p=0.157). CONCLUSION: In our study, we observed that the serum Autotaxin level did not make a significant difference in patients with intrahepatic cholestasis of pregnancy compared to healthy pregnant women. These findings suggest that larger clinical studies are required to reveal the physio-pathological effects of Autotaxin on pregnancy.

Social stigmatization in Turkish patients with chronic hepatitis B and C.

BACKGROUND AND AIM: Viral hepatitis is the most important cause of chronic hepatitis worldwide. Stigmatization is defined as a feeling of rejection and isolation of patients by society due to illness. There are no studies on chronic viral hepatitis in the literature in English, which has its own religious and socio-cultural structure. In our study, we aimed to investigate the presence of social stigmatism and psychosocial effects on patients with different stages of chronic viral hepatitis B and C. METHODS: Forty-five patients with chronic hepatitis C and 114 patients with chronic hepatitis B were enrolled in the study. Berger's scale was used for stigmatization, composed of 40 four-point Likert items that have four subscales: personalized stigma, disclosure, negative self-image, and public attitude. Stigma score ranges between one and four. Stigma is accepted as present if the overall score is above two. RESULTS: Overall the mean stigma scores were 1.97±0.58 and 2.14±0.57 for chronic hepatitis B and C, respectively. There was stigma in 47.4% of the patients with chronic hepatitis B, and 60% of the patients with chronic hepatitis C. Being male was the risk factor on overall stigma, disclosure and public attitude in chronic hepatitis C. Living in an urban setting was the risk factor on negative self-image in chronic hepatitis C and on personalized stigma and disclosure in chronic hepatitis B. CONCLUSIONS: To the best of our knowledge, this is the first study that provides qualitative information about chronic hepatitis-related stigma. Stigmatization is a major problem in Turkey and worldwide. We believe that increasing the knowledge of the patients and society by teaching about the transmission routes of the disease and focusing on vaccination studies will prevent stigmatization.

The Role of e-NOS in Chronic Cholestasis-Induced Liver and Renal Injury in Rats: The Effect of N-Acetyl Cysteine.

Introduction. The role of chronic cholestasis (CC) in liver injury and fibrosis remains unclear. The aims of this study were to define the role of endothelial nitric oxide synthase (e-NOS) in CC and the protective effect of N-acetyl-L-cysteine (NAC) in liver and kidney injury. Materials and Methods. Group A (sham group); Group B (CBDL); and Group C (CBDL + NAC). Group C received daily dosage of NAC (100 mg/kg) intraperitoneally for up to 4 weeks. Results. The rate of bridging fibrosis was higher (100% versus 20%, P = .025), but the intensity of e-NOS in liver was lower in rats that received NAC (1.3 versus 2.7, P = .046). The necrotic area in the kidneys among rats that received NAC was lower at week 4 (48% versus 57%; P < .001). The numbers of e-NOS stained cells in kidney were similar in sham group and the two groups with CBDL. Discussion. NAC reduced the stimulus for liver fibrosis in this rat model of CC and attenuated liver and kidney injury. Our study showed that e-NOS expression increased in liver tissue of rats with CC and that this was reversed by NAC. Treatment with NAC might restore e-NOS protein expression and prevent liver injury in CC.