Sorafenib response in hepatocellular carcinoma: MicroRNAs as tuning forks.

Hepatocellular carcinoma (HCC) is the primary liver malignancy that contributes towards the second most common cause of cancer-related mortality. The targeted chemotherapeutic agent, sorafenib, is known to show a statistically significant but limited overall survival advantage in advanced HCC. However, the individual patient response towards sorafenib varies drastically, with most experiencing stable disease and few with partial response; complete response is very rare. Progressive disease despite the treatment is also evident in many patients, indicating drug resistance. These varied responses have been linked with the modulation of several intracellular signaling pathways. Notably, the regulation of these pathways through diverse operating biomolecules, including microRNAs (miRNAs), is the focus of recent studies. MicroRNAs are tiny, non-coding RNA molecules that regulate the expression of several target genes. In addition, miRNAs are known to play a role in the progression of HCC carcinogenesis. Interestingly, miRNAs have also been identified to play differential roles in terms of sorafenib response in HCC such as biomarkers and functional modulation of cellular response to sorafenib, hence, they are also being therapeutically evaluated. This review outlines the role of reported miRNAs in different aspects of sorafenib response in HCC.

Equilibrium dissociation and unfolding of human papillomavirus E2 transactivation domain.

Papillomavirus E2 protein that performs essential functions such as viral oncogene expression and replication represents specific target for therapeutic intervention. DNA-binding activity is associated with its C-terminal DNA-binding domain (DBD), while the N-terminal transactivation domain (TAD) is responsible for replication and transactivation functions. Although both demonstrate large dependence on dimerization for mediating their functions, KD for N-terminal dimerization is significantly high suggesting more dynamic role of this domain. However, unlike DBD, very little information is available on TAD dimerization, its folding and stability. Therefore, with an aim at delineating the regulatory switch of its dimerization, we have characterized high-risk HPV18 E2 TAD. Our studies demonstrate that E2 TAD is a weak but thermodynamically stable dimer (KD âˆ¼ 1.8 µM, [Formula: see text]  = 18.8 kcal mol(-1)) with α2-α3 helices forming the interface. It follows a three-state folding pathway, in which unfolding involves dissociation of a dimeric intermediate. Interestingly, 90% of the conformational free energy is associated with dimer dissociation (16.9 of 18.8 kcal mol(-1)) suggesting dimerization significantly contributes to its overall thermodynamic stability. These revelations might be important toward designing inhibitors for targeting dimerization or folding intermediates and hence multiple functions that E2 performs.

Circulating metabolite biomarkers: a game changer in the human prostate cancer diagnosis.

PURPOSE: Prostate cancer (PCa) is the second most commonly diagnosed cancer in men in Western and Asian countries. Serum prostate-specific antigen (PSA) test has been the routine diagnostic method despite the tremendous research in diagnostic markers for early detection of PCa. A shift towards a promising and potential biomarker for PCa detection is through metabolomic profiling of biofluids, particularly the blood and urine samples. Finding reliable, routinely usable circulating metabolite biomarkers may not be a distant reality. METHODS: We performed a PubMed-based literature search of metabolite biomarkers in blood and urine for the early detection of prostate cancer. The timeline of these searches was limited between 2007 and 2022 and the following keywords were used: 'metabolomics', 'liquid biopsy', 'circulating metabolites', 'serum metabolite', 'plasma metabolite', and 'urine metabolite' with respect to 'prostate cancer'. We focussed only on diagnosis-based studies with only the subject-relevant articles published in the English language and excluded all of the other irrelevant publications that included prostate tissue biomarkers and cell line biomarkers. RESULTS: We have consolidated all the blood and urine-based potential metabolite candidates in individual as well as panels, including lipid classes, fatty acids, amino acids, and volatile organic compounds which may become useful for PCa diagnosis. CONCLUSION: All these metabolome findings unveil the impact of different dimensions of PCa development, giving a promising strategy to diagnose the disease since suspected individuals can be subjected to repeated and largescale blood and urine testing.

Bacterial metabolite butyrate in modulating sorafenib-targeted microRNAs to curtail its resistance in hepatocellular carcinoma.

BACKGROUND AND AIM: The host dietary fibre is fermented into short-chain fatty acids (SCFA) by intestinal microbiota as bacterial metabolites like propionate, acetate and butyrate. Among these metabolites, the role of butyrate is well documented to provide energy to intestinal epithelial cells. Also, butyrate has anti-inflammatory and anti-tumour properties and decrease in its level by unbalanced diet can develops cancer. Lately, some research has suggested that sodium butyrate as an inhibitor of histone deacetylase (HDAC) may have anticancer potential for hepatocellular carcinoma (HCC), the most common type of liver cancer. Since, HCC is asymptomatic it is usually diagnosed at its advanced stage. Sorafenib with antiproliferative and antiangiogenic effects is the first line of treatment in advanced HCC. However, prolonged drug treatment to HCC patients develops adaptive resistance towards the sorafenib. Sorafenib resistance can also be enhanced by differentially expressed microRNAs. However, the significance of butyrate in HCC sorafenib resistance and its association with sorafenib-targeted microRNAs is yet to be unfurled. Here, an attempt has been made to explore the role of bacterial metabolite butyrate on sorafenib resistant HCC as well as on sorafenib-targeted microRNAs (miR-7641 and miR-199) to curtail sorafenib resistance in HCC. METHODS: Initially, in-silico analysis was performed using Human Metabolome Database (HMDB) so to identify specific butyrate producing faecal bacteria. Then, their specific 16s rRNA expression was compared between HCC patients and healthy individuals using qRT-PCR. Additionally, the cell viability (MTT) and apoptosis assays were performed in both parental and sorafenib resistant HepG2 cells to evaluate the role of sodium butyrate in sorafenib resistant HCC. Moreover, the association of sodium butyrate with sorafenib-targeted miR-7641 and miR-199 was also assessed using real time PCR, cell viability, cell apoptosis and transfection assays. RESULTS: In silico analysis demonstrated Roseburia cecical, Roseburia intestinalis, Eubacterium rectal, Faecalibacterium prausnitzii as specific butyrate producing faecal bacteria and their 16s rRNA expression was downregulated in HCC patients. In vitro study revealed the presence of sodium butyrate also decreased the cell viability as well as enhanced cell apoptosis of both parental and resistant HepG2 cells. Interestingly, sodium butyrate also decreased the expression of both sorafenib-targeted miR-7641 and miR-199. Further, combination of both sodium butyrate and antimiR-7641 or antimiR-199 also increased apoptosis and decreased viability of resistant cells. CONCLUSION: This is first study to unravel the association of butyrate producing bacteria with HCC patients and the significance of bacterial metabolite butyrate as anti-tumour in sorafenib resistant hepatocellular carcinoma. The study also demonstrated the plausible new aspects of bacterial metabolite butyrate association with sorafenib-targeted miRNAs (miR-7641 and miR-199). Hence, the study highlighted the therapeutic potential of bacterial metabolite butyrate that might improve the clinical management of hepatocellular carcinoma.

miR-23b-3p Modulating Cytoprotective Autophagy and Glutamine Addiction in Sorafenib Resistant HepG2, a Hepatocellular Carcinoma Cell Line.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second most common malignancy with increasing cancer deaths worldwide. HCC is mainly diagnosed at its advanced stage, and treatment with FDA-approved sorafenib, the multikinase inhibitor drug, is advised. Acquired resistance against sorafenib develops through several pathways involving hypoxia, autophagy, high glycolysis, or glutaminolysis. Small non-coding RNAs, similar to microRNAs (miRNAs), are also known to affect sorafenib resistance in HCC. However, there is a lack of information regarding the significance of differentially expressed miRNA (if any) on autophagy and glutamine regulation in sorafenib-resistant HCC. METHODS: The expression of autophagy and glutaminolysis genes was checked in both parental and sorafenib resistant HepG2 cell lines by real-time PCR. MTT and Annexin/PI assays were also performed in the presence of inhibitors such as chloroquine (autophagy inhibitor) and BPTES (glutaminolysis inhibitor). Next generation sequencing and in silico analysis were performed to select autophagy and glutamine addiction-specific microRNA. Selected miRNA were transfected into both HepG2 cells to examine its effect on autophagy and glutamine addiction in regulating sorafenib-resistant HCC. RESULTS: Our in vitro study depicted a higher expression of genes encoding autophagy and glutaminolysis in sorafenib-resistant HepG2 cells. Moreover, inhibitors for autophagy (chloroquine) and glutaminolysis (BPTES) showed a diminished level of cell viability and augmentation in cell apoptosis of sorafenib-resistant HepG2 cells. NGS and real-time PCR demonstrated the downregulated expression of miR-23b-3p in sorafenib-resistant cells compared to parental cells. In silico analysis showed that miR-23b-3p specifically targeted autophagy through ATG12 and glutaminolysis through GLS1. In transfection assays, mimics of miR-23b-3p demonstrated reduced gene expression for both ATG12 and GLS1, decreased cell viability, and increased cell apoptosis of sorafenib-resistant HepG2 cells, whereas the antimiRs of miR-23b-3p demonstrated contrasting results. CONCLUSION: Our study highlights the cytoprotective role of autophagy and glutamine addiction modulated by miR-23b-3p (tumor suppressor), suggesting new approaches to curb sorafenib resistance in HCC.

Genotyping of multi drug resistant Bacteroides fragilis group of clinical isolates from mangalore, south India.

INTRODUCTION: Bacteroides fragilis group, the most encountered anaerobic bacterium is emerging with resistance to antibiotics. This study explores the antibiogram and occurrence of resistance genes in isolates of B fragilis group from clinical samples. METHOD: In this study the antimicrobial susceptibility test was done using commercially available E strip test and the results were recorded according to CLSI guidelines. Genotypic investigations were performed by conventional PCR to detect the target resistant genes. RESULTS: Ceftriaxone, cefoxitin, clindamycin and imipenem were found to be the most resistant antimicrobials in E test method. Metronidazole has shown resistance in 7 strains in vitro while resistance nim genes were detected in 12 strains from 62 randomly selected isolates. Other resistance genes (cfiA, ermF and cepA) were expressed at 58%, 62.9% and 48.3% respectively, among these strains. CONCLUSION: B fragilis group harbouring the resistant genes may not be fully expressed phenotypically. Hence, detection of these genes by PCR might be necessary for a pertinent conclusion.

Repertoires of MicroRNA-30 family as gate-keepers in lung cancer.

Lung cancer is a prominent global health issue responsible for the highest fraction of cancer-related mortality. The disease burden has incited the investigation of associated molecular pathways, to explore better therapeutic possibilities. MicroRNAs are extensively studied in recent years for their pivotal role in the regulation of several tumorigenic pathways. MicroRNA-30 (miR-30) family is primarily investigated in case of non-small cell lung cancer (NSCLC) and has been found to play the role of a tumour suppressor. There are six members of miR-30 family: miR-30a, miR-30b, miR-30c-1, miR-30c-2, miR-30d and miR-30e. They regulate several imperative signalling pathways like p53, PI3K/AKT, resulting in the modulation of key carcinogenic events involving cell proliferation, apoptosis, metastasis, epithelial-mesenchymal transition, and drug resistance. Their altered levels are documented in NSCLC tissue and blood samples. They are suggested as biomarkers of disease progression and therapeutic outcomes in lung cancer. They possess immense therapeutic potential in the treatment of lung cancer and combat the emerging problem of drug resistance by modulating prime regulatory axes. However, there are many limitations in the existing studies, and additional research is required for the comprehensive understanding of pathways so that the tumour suppressive potential of miR-30 can be translated into clinical benefits. In this review, we present a deeper understanding of the regulatory role and clinical significance of miR-30 and have emphasized the emerging roles in lung cancer.

Vimentin as a potential therapeutic target in sorafenib resistant HepG2, a HCC model cell line.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is the most common liver cancer with high mortality rate in patients suffering from liver diseases. The drug of choice used in advanced-stage of HCC is sorafenib. However, adaptive resistance has been observed in HCC patients undergoing long-term sorafenib treatment, lowering its effectiveness. Hence, it is important to overcome drug resistance to improve overall management of HCC. Here, we have identified a candidate biomarker for sorafenib resistance in a HCC model cell line, HepG2. METHODS: Initially, comparative proteomic profiling of parental HepG2 [HepG2 (P)] and sorafenib-resistant HepG2 [HepG2 (R)] cells was performed via MALDI (matrix-assisted laser desorption/ionization) which revealed the deregulation of vimentin in HepG2 (R) cells. Gene and protein level expression of vimentin was also observed through quantitative real-time polymerase chain reaction (qRT PCR) and fluorescence-activated cell sorting (FACS), respectively. Furthermore, withaferin A was used to study regulation of vimentin expression and its significance in sorafenib resistance. RESULTS: Both gene and protein level of vimentin expression was found to be downregulated in HepG2 (R) in comparison to HepG2 (P). Interestingly, the study demonstrated that withaferin A further lowered the expression of vimentin in HepG2 (R) cells in a dose-dependent manner. Also, inhibition of vimentin lowered ABCG2 expression and decreased cell viability in parental as well as sorafenib resistant HepG2 cells. CONCLUSION: Hence, our study for the first time highlighted the probable therapeutic potential of vimentin in sorafenib resistant HepG2, a HCC model cell line.

Association of liver cirrhosis severity with type 2 diabetes mellitus in hepatocellular carcinoma.

Type 2 diabetes mellitus (T2DM) is a major risk factor associated with hepatocellular carcinoma (HCC). However, the association of T2DM with liver cirrhosis and therapy response in HCC patients is not clear. Hence, in this study, we have evaluated the influence of T2DM on liver cirrhosis severity of HCC and sorafenib response. HCC patients were divided in two groups: T2DM (n = 20) and non-T2DM (nT2DM; n = 50). We found significantly higher number of patients in T2DM group had decompensated liver disease with Child-Turcotte-Pugh score ≥ 7. Additionally, 71.4% patients were observed to be sorafenib sensitive in T2DM group which was significantly higher as compared to 30% in nT2DM group. This study has highlighted the predisposition of HCC patients with T2DM toward more severe liver disease who were found to be better respondents of sorafenib. Impact statement We have explored the association of type 2 diabetes mellitus (T2DM) on liver cirrhosis severity along with response toward sorafenib in hepatocellular carcinoma (HCC). Most HCC patients exhibit prior history of liver cirrhosis that results following long span of chronic liver disease. T2DM constitutes as an important risk factor for HCC development which is known to elevate its incidence. Further, sorafenib is the FDA approved therapy for HCC whose therapeutic outcome is not investigated in HCC patients with T2DM till date. This observation-based study has unveiled a positive association between T2DM and severity of liver cirrhosis as well as sorafenib response in HCC as examined in a clinical setting.

Crosstalk between microRNA-122 and FOX family genes in HepG2 cells.

MicroRNA-122 (miR-122) is liver specific and plays an important role in physiology as well as diseases including hepatocellular carcinoma (HCC). Downregulation of miR-122 in HCC modulates apoptosis. Similarly, the putative targets of miR-122, the forkhead box (FOX) family genes also play an important role in the regulation of apoptosis. Hence, an interplay between miR-122 and FOX family genes has been explored in this study. Initially, an augmentation of apoptosis was noticed in HepG2 cells after transfection with miR-122. Further, the predicted miR-122 targets, the FOX family genes ( FOXM1b, FOXP1, and FOXO4) were selected via in silico analysis based on their role in apoptosis. We checked the expression of all these genes at transcript level after the transfection of miR-122 and found that the relative expression of FOXP1 and FOXM1b was significantly downregulated (p < 0.005) and that of FOXO4 was upregulated (p < 0.005). Thus, the finding indicates deregulation of these FOX genes as a result of miR-122 augmentation might be involved in the modulation of apoptosis.