Targeting Lin28 axis enhances glypican-3-CAR T cell efficacy against hepatic tumor initiating cell population.

Overexpression of Lin28 is detected in various cancers with involvement in the self-renewal process and cancer stem cell generation. In the present study, we evaluated how the Lin28 axis plays an immune-protective role for tumor-initiating cancer cells in hepatocellular carcinoma (HCC). Our result using HCC patient samples showed a positive correlation between indoleamine 2,3-dioxygenase-1 (IDO1), a kynurenine-producing enzyme with effects on tumor immune escape, and Lin28B. Using in silico prediction, we identified a Sox2/Oct4 transcriptional motif acting as an enhancer for IDO1. Knockdown of Lin28B reduced Sox2/Oct4 and downregulated IDO1 in tumor-initiating hepatic cancer cells. We further observed that inhibition of Lin28 by a small-molecule inhibitor (C1632) suppressed IDO1 expression. Suppression of IDO1 resulted in a decline in kynurenine production from tumor-initiating cells. Inhibition of the Lin28 axis also impaired PD-L1 expression in HCC cells. Consequently, modulating Lin28B enhanced in vitro cytotoxicity of glypican-3 (GPC3)-chimeric antigen receptor (CAR) T and NK cells. Next, we observed that GPC3-CAR T cell treatment together with C1632 in a HCC xenograft mouse model led to enhanced anti-tumor activity. In conclusion, our results suggest that inhibition of Lin28B reduces IDO1 and PD-L1 expression and enhances immunotherapeutic potential of GPC3-CART cells against HCC.

Modified E2 Glycoprotein of Hepatitis C Virus Enhances Proinflammatory Cytokines and Protective Immune Response.

Hepatitis C virus (HCV) is characterized by a high number of chronic cases owing to an impairment of innate and adaptive immune responses. CD81 on the cell surface facilitates HCV entry by interacting with the E2 envelope glycoprotein. In addition, CD81/E2 binding on immunity-related cells may also influence host response outcome to HCV infection. Here, we performed site-specific amino acid substitution in the front layer of E2 sequence to reduce CD81 binding and evaluate the potential of the resulting immunogen as an HCV vaccine candidate. The modified sE2 protein (F442NYT), unlike unmodified sE2, exhibited a significant reduction in CD81 binding, induced higher levels of proinflammatory cytokines, repressed anti-inflammatory response in primary monocyte-derived macrophages as antigen-presenting cells, and stimulated CD4+ T cell proliferation. Immunization of BALB/c mice with an E1/sE2F442NYT nucleoside-modified mRNA-lipid nanoparticle (mRNA-LNP) vaccine resulted in improved IgG1-to-IgG2a isotype switching, an increase in neutralizing antibodies against HCV pseudotype virus, a B and T cell proliferative response to antigens, and improved protection against infection with a surrogate recombinant vaccinia virus-expressing HCV E1-E2-NS2aa134-966 challenge model compared to E1/unmodified sE2 mRNA-LNP vaccine. Further investigation of the modified E2 antigen may provide helpful information for HCV vaccine development. IMPORTANCE Hepatitis C virus (HCV) E2-CD81 binding dampens protective immune response. We have identified that an alteration of amino acids in the front layer of soluble E2 (sE2) disrupts CD81 interaction and alters the cytokine response. Immunization with modified sE2F442NYT (includes an added potential N-linked glycosylation site and reduces CD81 binding activity)-mRNA-LNP candidate vaccine generates improved proinflammatory response and protective efficacy against a surrogate HCV vaccinia challenge model in mice. The results clearly suggested that HCV E2 exhibits immunoregulatory activity that inhibits induction of robust protective immune responses. Selection of engineered E2 antigen in an mRNA-LNP platform amenable to nucleic acid sequence alterations may open a novel approach for multigenotype HCV vaccine development.

SARS-CoV-2 Spike Protein Induces Paracrine Senescence and Leukocyte Adhesion in Endothelial Cells.

Increased mortality in COVID-19 cases is often associated with microvascular complications. We have recently shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein promotes an inflammatory cytokine interleukin 6 (IL-6)/IL-6R-induced trans signaling response and alarmin secretion. Virus-infected or spike-transfected human epithelial cells exhibited an increase in senescence, with a release of senescence-associated secretory phenotype (SASP)-related inflammatory molecules. Introduction of the bromodomain-containing protein 4 (BRD4) inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP-related inflammatory molecule release in TMNK-1 or EAhy926 (representative human endothelial cell lines), when cells were exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein. Cells also exhibited a senescence phenotype with enhanced p16, p21, and senescence-associated ß-galactosidase (SA-ß-Gal) expression and triggered SASP pathways. Inhibition of IL-6 trans signaling by tocilizumab and inhibition of inflammatory receptor signaling by the Bruton's tyrosine kinase (BTK) inhibitor zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike-transfected and endothelial cells exposed to spike-transfected CM. ROS generation in endothelial cell lines was reduced after treatment with tocilizumab and zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which have in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that human endothelial cells exposed to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. IMPORTANCE The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein-associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture medium (CM). Endothelial cell lines (TMNK-1 or EAhy926) treated with CM exhibited an increase in cellular senescence markers by a paracrine mode and led to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence and a potential contribution to microvascular complication in productively SARS-CoV-2-infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6, and BRD4) showed a reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2-associated pathogenesis.

SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells.

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response.

Sub-chronic cadmium and lead compound exposure induces reproductive toxicity and development of testicular germ cell neoplasia in situ in murine model: Attenuative effects of resveratrol.

Cadmium and lead are widespread, nonbiodegradable heavy metals of perpetual environmental concerns. The present study aimed to evaluate whether sub-chronic exposure to cadmium chloride (CdCl2 ) and lead acetate [Pb(CH3 COO)2 ] induces reproductive toxicity and development of testicular germ cell neoplasia in situ (GCNIS) in swiss albino mice. The effects of resveratrol to reverse the metal-induced toxicity were also analyzed. The mice were randomly divided into four groups for metal treatments and two groups received two different doses of each metal, CdCl2 (0.25 and 0.5 mg/kg) and Pb(CH3 COO)2 (3 and 6 mg/kg). The fourth group received oral doses of 20 mg/kg resveratrol in combination with 0.5 mg/kg CdCl2 or 6 mg/kg Pb(CH3 COO)2 for 16 weeks. Toxic effects of both metals were estimated qualitatively and quantitatively by the alterations in sperm parameters, oxidative stress markers, testicular histology, and protein expressions of the treated mice. Pronounced perturbation of sperm parameters, cellular redox balance were observed with severe distortion of testicular histo-architecture in metal exposed mice. Significant overexpression of Akt cascade and testicular GCNIS marker proteins were recorded in tissues treated with CdCl2 . Notable improvements were observed in all the evaluated parameters of resveratrol cotreated mice groups. Taken together, the findings of this study showed that long-term exposure to Cd and Pb compounds, induced acute reproductive toxicity and initiation of GCNIS development in mice. Conversely, resveratrol consumption abrogated metal-induced perturbation of spermatogenesis, testicular morphology, and the upregulation of Akt cascade proteins along with GCNIS markers, which could have induced the development of testicular cancer.

Hepatitis C Virus Mediated Inhibition of miR-181c Activates ATM Signaling and Promotes Hepatocyte Growth.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection promotes hepatocyte growth and progress to hepatocellular carcinoma. We previously observed that HCV infection of hepatocytes transcriptionally down-regulates miR-181c expression through CCAAT/enhancer binding protein ß (C/EBP-ß). Here, we examined the role of miR-181c in the regulation of cell cycle progression in relation to HCV infection. In silico analysis suggested that ataxia-telangiectasia mutated (ATM) protein, a protein kinase, is a direct target of miR-181c. ATM is a central mediator of response for cellular DNA double-strand break. APPROACH AND RESULTS: Our results demonstrated that ATM expression is higher in HCV-infected hepatocytes and chronic HCV-infected liver biopsy specimens. We have shown a direct interaction of miR-181c with the 3' untranslated region of ATM, and the presence of ATM in miR-181c-associated RNA-induced silencing complex. Exogenous expression of miR-181c inhibited ATM expression and activation of its downstream molecules, Chk2 and Akt. On the other hand, introduction of anti-miR-181c restored ATM and phosphorylated Akt. Furthermore, introduction of miR-181c significantly inhibited phospho-cyclin-dependent kinase 2 (CDK2) and cyclin-A expression, arresting cell cycle progression, whereas overexpression of miR-181c promoted apoptosis of HCV-infected hepatocytes and can be inhibited by overexpression of ATM from a clone lacking miR-181c binding sites. In addition, miR-181c significantly regressed tumor growth in the xenograft human hepatocellular carcinoma mouse model. CONCLUSIONS: Together, our results suggest that HCV infection suppresses miR-181c in hepatocytes, resulting in ATM activation and apoptosis inhibition for promotion of cell cycle progression. The results provide mechanistic insight into understanding the role of miR-181c in HCV-associated hepatocyte growth promotion, and may have the potential for therapeutic intervention.

Establishment of a Patient-Derived Xenograft Tumor From Hepatitis C-Associated Liver Cancer and Evaluation of Imatinib Treatment Efficacy.

BACKGROUND AND AIMS: Chronic hepatitis C virus (HCV) infection is one of the major causal factors for hepatocellular carcinoma (HCC). The treatment options for HCC are limited for lack of a convenient animal model for study in HCV infection and liver pathogenesis. This study aimed to develop a patient-derived xenograft (PDX) tumor in mice by using a tumor from a patient with HCV-associated HCC and evaluating this model's therapeutic potential. APPROACH AND RESULTS: After resection of the primary tumor from the patient liver, excess viable tumor was implanted into highly immunodeficient mice. A mouse xenograft tumor line was developed, and the tumor was successfully passaged for at least three rounds in immunodeficient mice. The patient's primary tumor and the mouse xenografts were histologically similar. Genetic profiling by short-tandem-repeat analysis verified that the HCC-PDX model was derived from the HCC clinical specimen. HCV RNA present in the patient liver specimen was undetectable after passage as xenograft tumors in mice. Human albumin, α1 -antitrypsin, glypican-3, α-smooth muscle actin, and collagen type 1A2 markers were detected in human original tumor tissues and xenograft tumors. Both the patient primary tumor and the xenograft tumors had a significantly higher level of receptor tyrosine kinase (c-Kit) mRNA. Treatment of HCC-PDX xenograft tumor-bearing mice with the c-Kit inhibitor imatinib significantly reduced tumor growth and phospho-Akt and cyclin D1 expression, as compared with untreated control tumors. CONCLUSIONS: Our results demonstrated establishment of an HCV-associated HCC-PDX model as a powerful tool for evaluating candidate drugs. Information on molecular changes in cancer-specific gene expression facilitates efficient targeted therapies and treatment strategies.

Transforming Growth Factor ß Acts as a Regulatory Molecule for Lipogenic Pathways among Hepatitis C Virus Genotype-Specific Infections.

Hepatitis C virus (HCV) infection promotes metabolic disorders, and the severity of lipogenic disease depends upon the infecting virus genotype. Here, we have examined HCV genotype 1-, 2-, or 3-specific regulation of lipid metabolism, involving transforming growth factor ß (TGF-ß)-regulated phospho-Akt (p-Akt) and peroxisome proliferator-activated receptor alpha (PPARα) axes. Since HCV core protein is one of the key players in metabolic regulation, we also examined its contribution in lipid metabolic pathways. The expression of regulatory molecules, TGF-ß1/2, phospho-Akt (Ser473), PPARα, sterol regulatory element-binding protein 1 (SREBP-1), fatty acid synthase (FASN), hormone-sensitive lipase (HSL), and acyl dehydrogenases was analyzed in virus-infected hepatocytes. Interestingly, HCV genotype 3a exhibited much higher activation of TGF-ß and p-Akt, with a concurrent decrease in PPARα expression and fatty acid oxidation. A significant and similar decrease in HSL, unlike in HCV genotype 1a, was observed with both genotypes 2a and 3a. Similar observations were made from ectopic expression of the core genomic region from each genotype. The key role of TGF-ß was further verified using specific small interfering RNA (siRNA). Together, our results highlight a significant difference in TGF-ß-induced activity for the HCV genotype 2a- or 3a-induced lipogenic pathway, exhibiting higher triglyceride synthesis and a decreased lipolytic mechanism. These results may help in therapeutic modalities for early treatment of HCV genotype-associated lipid metabolic disorders.IMPORTANCE Hepatic steatosis is a frequent complication associated with chronic hepatitis C virus (HCV) infection and is a key prognostic indicator for progression to fibrosis and cirrhosis. Several mechanisms are proposed for the development of steatosis, especially with HCV genotype 3a. Our observations suggest that transforming growth factor ß (TGF-ß) and peroxisome proliferator-activated receptor alpha (PPARα)-associated mechanistic pathways in hepatocytes infected with HCV genotype 2a and 3a differ from those in cells infected with genotype 1a. The results suggest that a targeted therapeutic approach for enhanced PPARα and lipolysis may reduce HCV genotype-associated lipid metabolic disorder in liver disease.

A genome-wide screen in Escherichia coli reveals that ubiquinone is a key antioxidant for metabolism of long-chain fatty acids.

Long-chain fatty acids (LCFAs) are used as a rich source of metabolic energy by several bacteria including important pathogens. Because LCFAs also induce oxidative stress, which may be detrimental to bacterial growth, it is imperative to understand the strategies employed by bacteria to counteract such stresses. Here, we performed a genetic screen in Escherichia coli on the LCFA, oleate, and compared our results with published genome-wide screens of multiple non-fermentable carbon sources. This large-scale analysis revealed that among components of the aerobic electron transport chain (ETC), only genes involved in the biosynthesis of ubiquinone, an electron carrier in the ETC, are highly required for growth in LCFAs when compared with other carbon sources. Using genetic and biochemical approaches, we show that this increased requirement of ubiquinone is to mitigate elevated levels of reactive oxygen species generated by LCFA degradation. Intriguingly, we find that unlike other ETC components whose requirement for growth is inversely correlated with the energy yield of non-fermentable carbon sources, the requirement of ubiquinone correlates with oxidative stress. Our results therefore suggest that a mechanism in addition to the known electron carrier function of ubiquinone is required to explain its antioxidant role in LCFA metabolism. Importantly, among the various oxidative stress combat players in E. coli, ubiquinone acts as the cell's first line of defense against LCFA-induced oxidative stress. Taken together, our results emphasize that ubiquinone is a key antioxidant during LCFA metabolism and therefore provides a rationale for investigating its role in LCFA-utilizing pathogenic bacteria.

A comparative analysis indicates SLC7A11 expression regulate the prognostic value of KEAP1-NFE2L2-CUL3 mutations in human uterine corpus endometrial carcinoma.

Uterine corpus endometrial cancer (UCEC) is a third most common malignancy in women with a poor prognosis in advanced stages. In this study, we performed an integrated comparative analysis of exome and transcriptome data from The Cancer Genome Atlas (TCGA) of Lung Adenocarcinoma (LUAD), and UCEC patients. Our multi-omics analysis shows that the UCEC patients carrying mutations in the KEAP1-NFE2L2-CUL3 genes were associated with better progression-free survival (PFS), whereas the KEAP1-NFE2L2-CUL3 mutation in LUAD showed poor outcomes. Functional annotations and correlative expression studies show that genes, particularly GCLC and GCLM related to glutathione synthesis are expressed at lower levels in the KEAP1-NFE2L2-CUL3 mutant UCEC compared to LUAD. This events result in glutathione deficiency and it may compromise to combat intracellular reactive oxygen species (ROS). However, the expression of genes involved in the glutathione recycling process was not affected. On the other hand, cellular import of cystine is high due to increased SLC7A11 expression in UCEC. Because glutathione synthesis is impaired, the unconverted cysteine accumulates in cells, leading to di-sulfite stress. Apart from NRF2, ARID1A is one of the positive regulators of SLC7A11. In support, UCEC patients with co-occurrence of KEAP1-NFE2L2-CUL3 and ARID1A mutation shows significantly decreased PFS with decline of SLC7A11 expression as compared to patients carrying only KEAP1-NFE2L2-CUL3 mutation. Thus, we hypothesize that the KEAP1-NFE2L2-CUL3 mutation in UCEC leads to uncontrollable ROS with di-sulfite stress, reflecting a favorable clinical outcome.

Hepatitis C virus E1 and modified E2 delivered from an mRNA vaccine induces protective immunity.

Hepatitis C virus (HCV) is characterized by a high number of chronic cases due to an impairment of protective innate and adaptive immune responses. Here, we examined the contribution of the individual ectodomains of E1, E2, or a modified E2 with reduced CD81 binding and an inserted N-linked glycosylation site in combination as vaccine antigen mRNA-lipid nanoparticles (LNPs). The induction of a protective immune response to surrogate recombinant vaccinia virus (VV) expressing homologous HCV glycoprotein(s) challenge infection in a BALB/c mouse model was observed. Vaccination with a mRNA-LNP expressing soluble E1 (sE1) significantly reduced vv/HCV titer in the mouse ovary. However, the addition of sE2 mRNA-LNP for immunization impaired the efficacy of the sE1 construct. Further analysis showed that Th1 related cytokine responses to the sE1 mRNA-LNP were significantly altered in the presence of sE2 following co-immunization. Evaluation of immunogenicity revealed that the use of modified sE2F442NYT nucleoside mRNA-LNP vaccine results in an improved cellular immune response, IgG2a isotype switching, enhanced total IgG, and an increase in the neutralizing antibody response against HCV pseudotype virus. HCV cross genotype specific reactivity to peptides representing conserved E2 specific linear epitopes were enhanced in modified E2 vaccinated animal sera. In the absence of a suitable immunocompetent small animal model for HCV infection, protection from surrogate HCV vaccinia challenge infection model was observed in the immunized mice as compared to sE1 alone or an unmodified sE2 mRNA-LNP vaccine. Inclusion of sE1 with modified sE2F442NYT as mRNA-LNP vaccine candidate appeared to be beneficial for protection.

The Entner-Doudoroff pathway is obligatory for gluconate utilization and contributes to the pathogenicity of Vibrio cholerae.

The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in Vibrio cholerae, the causative agent of cholera. In this study, activation of the genes edd and eda, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in V. cholerae. Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in V. cholerae and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes (ctxA and tcpA) and their regulator (toxT). Further, lowering of these transcript levels and cholera toxin production in vitro by an ED pathway-defective mutant (strain N16961 with a Δedd mutation [Δedd(N16961) strain]) suggested the importance of this pathway in regulating V. cholerae virulence. The in vivo relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in V. cholerae was shown to inhibit biofilm formation in vitro that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported in vivo expressed genes. All of these results suggest the importance of the ED pathway in V. cholerae pathogenesis.

Bystander effect of SARS-CoV-2 spike protein on human monocytic THP-1 cell activation and initiation of prothrombogenic stimulus representing severe COVID-19.

BACKGROUND: Hypercoagulable state and thromboembolic complications are potential life-threatening events in COVID-19 patients. Our previous studies demonstrated that SARS-CoV-2 infection as well as viral spike protein expressed epithelial cells exhibit senescence with the release of inflammatory molecules, including alarmins. FINDINGS: We observed extracellular alarmins present in the culture media of SARS-CoV-2 spike expressing cells activate human THP-1 monocytes to secrete pro-inflammatory cytokines to a significant level. The release of THP-1 derived pro-inflammatory cytokine signature correlated with the serum of acute COVID-19 patient, but not in post-COVID-19 state. Our study suggested that the alarmins secreted by spike expressing cells, initiated phagocytosis property of THP-1 cells. The phagocytic monocytes secreted complement component C5a and generated an autocrine signal via C5aR1 receptor. The C5a-C5aR1 signal induced formation of monocyte mediated extracellular trap resulted in the generation of a prothrombogenic stimulus with activating platelets and increased tissue factor activity. We also observed an enhanced C5a level, platelet activating factor, and high tissue factor activity in the serum of acute COVID-19 patients, but not in recovered patients. CONCLUSION: Our present study demonstrated that SARS-CoV-2 spike protein modulates monocyte responses in a paracrine manner for prothrombogenic stimulus by the generation of C5a complement component.

Akt inhibitor augments anti-proliferative efficacy of a dual mTORC1/2 inhibitor by FOXO3a activation in p53 mutated hepatocarcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most common malignancy-related deaths. p53 mutation in HCC associates with worse clinicopathologic features including therapeutic limitation. A combination of targeted therapy may have some advantages. Akt/mTOR signaling contributes to the regulation of cell proliferation and cell death. Akt inhibitor (AZD5363) and mTORC1/2 dual inhibitor (AZD8055) are in a clinical trial for HCC and other cancers. In this study, we examined whether these inhibitors successfully induce antiproliferative activity in p53 mutant HCC cells, and the underlying mechanisms. We observed that a combination of AZD5363 and AZD8055 treatment synergizes antiproliferative activity on p53 mutated or wild-type HCC cell lines and induces apoptotic cell death. Mechanistic insights indicate that a combination of AZD5363 and AZD8055 activated FOXO3a to induce Bim-associated apoptosis in p53 mutated HCC cells, whereas cells retaining functional p53 enhanced Bax. siRNA-mediated knock-down of Bim or Bax prevented apoptosis in inhibitor-treated cells. We further observed a combination of treatment inhibits phosphorylation of FOXO3a and protects FOXO3a from MDM2 mediated degradation by preventing the phosphorylation of Akt and SGK1. FOXO3a accumulates in the nucleus under these conditions and induces Bim transcription in p53 mutant HCC cells. Combination treatment in the HCC cells expressing wild-type p53 causes interference of FOXO3a function for direct interaction with functional p53 and unable to induce Bim-associated cell death. On the other hand, Bim-associated cell death occurs in p53 mutant cells due to uninterrupted FOXO3a function. Overall, our findings suggested that a combined regimen of dual mTORC1/2 and Akt inhibitors may be an effective therapeutic strategy for HCC patients harboring p53 mutation.

Inhibition of p70 isoforms of S6K1 induces anoikis to prevent transformed human hepatocyte growth.

AIMS: The mTOR/S6K1 signaling axis, known for cell growth regulation, is hyper-activated in multiple cancers. In this study, we have examined the mechanisms for ribosomal protein p70-S6 kinase 1 (S6K1) associated transformed human hepatocyte (THH) growth regulation. MAIN METHODS: THH were treated with p70-S6K1 inhibitor and analyzed for cell viability, cell cycle distribution, specific marker protein expression by western blot, and tumor inhibition in a xenograft mouse model. We validated our results by knockdown of p70-S6K1 using specific siRNA. KEY FINDINGS: p70-S6K1 inhibitor treatment caused impairment of in vitro hepatocyte growth, and arrested cell cycle progression at the G1 phase. Further, p70-S6K1 inhibitor treatment exhibited a decrease in FAK and Erk activation, followed by altered integrin-ß1 expression, caspase 8, and PARP cleavage appeared to be anoikis like growth inhibition. p70-S6K1 inhibitor also depolymerized actin microfilaments and diminished active Rac1/Cdc42 complex formation for loss of cellular attachment. Similar results were obtained with other transformed human hepatocyte cell lines. p70-S6K1 inhibition also resulted in a reduced phospho-EGFR, Slug and Twist; implicating an inhibition of epithelial-mesenchymal transition (EMT) state. A xenograft tumor model, generated from implanted THH in nude mice, following intraperitoneal injection of S6K1 inhibitor prevented further tumor growth. SIGNIFICANCE: Our results suggested that p70-S6K1 inhibition alters orchestration of cell cycle progression, induces cell detachment, and sensitizes hepatocyte growth impairment. Targeting p70 isoform of S6K1 by inhibitor may prove to be a promising approach together with other therapies for hepatocellular carcinoma (HCC) treatment.

A combination of AZD5363 and FH5363 induces lethal autophagy in transformed hepatocytes.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death worldwide. High Akt activation and aberrant ß-catenin expression contribute to HCC cell proliferation, stem cell generation, and metastasis. Several signaling pathway-specific inhibitors are in clinical trials and display different efficacies against HCC. In this study, we observed that a ß-catenin inhibitor (FH535) displays antiproliferative effect on transformed human hepatocytes (THH). A combination treatment of these cells with FH535 and Akt inhibitor (AZD5363) exerted a stronger effect on cell death. Treatment of THH with AZD5363 and FH535 inhibited cell-cycle progression, enhanced autophagy marker protein expression, and autophagy-associated death, while FH535 treatment alone induced apoptosis. The use of chloroquine or z-VAD further verified these observations. Autophagy flux was evident from lowering marker proteins LAMP2, LAPTM4B, and autophagic protein expression by confocal microscopy using mCherry-EGFP-LC3 reporter construct. A combination treatment with AZD5363 and FH535 enhanced p53 expression, by modulating MDM2 activation; however, AZD5363 treatment alone restricted p53 to the nucleus by inhibiting dynamin-related protein activation. Nuclear p53 plays a crucial role for activation of autophagy by regulating the AMPK-mTOR-ULK1 pathway. Hep3B cells with null p53 did not modulate autophagy-dependent death from combination treatment. Together, our results strongly suggested that a combination treatment of Akt and ß-catenin inhibitors exhibits efficient therapeutic potential for HCC.

Vibrio cholerae O1 clinical strains isolated in 1992 in Kolkata with progenitor traits of the 2004 Mozambique variant.

Retrospective analysis led to the detection of two Vibrio cholerae variant O1 strains (VC51 and VC53), which were isolated in 1992 in Kolkata from clinical cases, with identical traits to 2004 Mozambique variant O1 strains. The Mozambique O1 strains that caused a huge outbreak in 2004 have been shown to have phenotypic traits of both classical and El Tor biotypes, and thereby have been reported as variant. Our study demonstrated that two O1 strains isolated in Kolkata during 1992 were of the El Tor background as evidenced by polymyxin B (50 U ml(-1)) resistance, positivity in Voges-Proskauer reactions and sensitivity to biotype-specific vibrio phages. With the features of classical CTX prophage, localization in the small chromosome, and an absence of RS1 and pTLC, both Mozambique and Kolkata strains appeared to be identical. Furthermore, two Kolkata strains exhibited an identical ribotype to that of the Mozambique variant, displaying ribotype pattern RI that had been assigned to Kolkata V. cholerae O1 strains isolated on or before 1992. NotI pulsotype analysis indicated that these 1992 Kolkata strains along with the Mozambique variant O1 belonged to very closely related clones. Considering the chronological events, and the typical identity at the phenotypic and the genotypic level between the two O1 strains isolated during 1992 from Kolkata and during 2004 from Mozambique, we propose that some of the 1992 Kolkata O1 strains might have acted as progenitors for Mozambique variant O1 strains.

Strategies to Circumvent Host Innate Immune Response by Hepatitis C Virus.

Innate immune responses generate interferons, proinflammatory cytokines, complement activation, and natural killer (NK) cell response. Ultimately, this leads to the induction of a robust virus-specific adaptive immunity. Although the host innate immune system senses and responds to eliminate virus infection, hepatitis C virus (HCV) evades immune attack and establishes persistent infection within the liver. Spontaneous clearance of HCV infection is associated with a prompt induction of innate immunity generated in an infected host. In this review, we have highlighted the current knowledge of our understanding of host⁻HCV interactions, especially for endogenous interferon production, proinflammatory response, NK cell response, and complement activation, which may impair the generation of a strong adaptive immune response for establishment of chronicity. The information may provide novel strategies in augmenting therapeutic intervention against HCV.

Supra-physiological concentration of glyoxylate inhibits proliferation of human colon cancer cells through oxidative stress.

AIMS: The cytotoxic response of an intermediate metabolite glyoxylate (Glx) on colon carcinoma has been evaluated in vitro. MAIN METHODS: The anti-proliferative effect of Glx was assessed on HT-29 and HCT-116 cells by performing MTT assay as well as beta-hexosaminidase assay. Evaluation of apoptotic event of Glx treated cells was measured by flow cytometry using annexin-V/PI staining. The mitochondrial membrane potential and level of ROS were estimated using DiOC6(3)/CCCP and DCFH-DA method, respectively. The assessment of catalase, LDH and IDH were performed. KEY FINDINGS: The results of MTT assay indicated that treatment with Glx significantly inhibited the proliferation of HT-29 and HCT-116 cells. Beta-hexosaminidase assay also confirmed the inhibition of cellular viability. The dose-dependent Glx treatment indicated lowering the colony forming ability of HT-29 and HCT-116 cells. Flow cytometric data demonstrated the significant increment of late apoptotic event after Glx treatment. In addition, substantial LDH activity was noticed in both the colon cancer cells whereas the IDH activity was unaltered after extra-cellular addition of Glx. Further, dissipation of mitochondrial membrane potential and subsequently elevated ROS generation was also detected in the Glx treated colon cancer cells. However, gradual elevation of catalase activities indicated that Glx treatment on colon cancer cells exhibit oxidative stress. SIGNIFICANCE: This study depicts that supra-physiological concentration of Glx inhibits the proliferation of colon cancer cells due to oxidative stress.