Hepatocellular Carcinoma Cell Lines Huh7 Research Articles

Murine double minute gene 2 (MDM2) promoted hepatocellular carcinoma (HCC) cell growth by targeting fructose-1,6-bisphosphatase (FBP1) for degradation

To study the roles and association of murine double minute gene 2 (MDM2) and fructose-1,6-biphosphatase (FBP1) in human hepatocellular carcinoma (HCC), growth response of human HCC cells was assessed using proliferation and apoptosis assay. Pro-survival AKT signaling associated proteins (p-AKT, survivin and cleaved caspase 3) were assessed using western blotting. The correlation between MDM2 and FBP1 was assessed using co-immunoprecipitation combined with ubiquitination assay. Our data suggested that low expression of FBP1 was correlated with high levels of MDM2 in HCC cell lines (Huh7 and Hep3B). Overexpression of FBP1 resulted in anti-proliferation, pro-apoptosis, the up-regulation of cleaved caspase 3 while the downregulation of survivin and phosphor (p)-AKT, however, knockdown of FBP1 led to the opposite. Furthermore, overexpression of MDM2 potently reversed FBP1-induced proliferation inhibition and apoptosis, while Nutlin-3 (an MDM2 inhibitor) reversed the change trends induced by FBP1 knockdown in the aforementioned events. Lastly, but not least importantly, our data elucidated that MDM2 binds directly to FBP1 and promotes FBP1 ubiquitination. In conclusion, our data firstly suggested the involvement of FBP1 and its association with MDM2 in HCC cell growth. MDM2-FBP1-regulated HCC cell growth and the activation of AKT were mediated, at least in part, through FBP1 degradation.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

ISOC1 is a novel potential tumor suppressor in hepatocellular carcinoma.

Isochorismatase domain-containing 1 (ISOC1) plays a carcinogenic role in various tumors. However, its expression and role in hepatocellular carcinoma (HCC) have not been elucidated. This is the first study to investigate the involvement of ISOC1 in HCC growth and migration. ISOC1 expression was analyzed using public databases and clinical samples, and clinical specimens were analyzed by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. ISOC1 was also overexpressed in two HCC cell lines (Huh7 and HepG2) to explore how ISOC1 affects HCC cells. Finally, a nude mouse xenograft tumor model was used to investigate the role of ISOC1 in HCC cell tumorigenicity. ISOC1 was downregulated in HCC tissues compared to that in matched paracancerous tissues, and low ISOC1 expression was associated with a poor prognosis. The proliferation and single-cell colony-forming ability of the ISOC1-overexpressing cell lines Huh7 and HepG2 were significantly inhibited. Moreover, ISOC1 overexpression suppressed the migration and invasion abilities of HCC cells in vitro, and ISOC1 upregulation hindered tumor growth in the xenograft tumor model in vivo. Therefore, ISOC1 is a potential HCC suppressor protein.

CXCR4 promotes gefitinib resistance of Huh7 cells by activating the c-Met signaling pathway.

C‐X‐C chemokine receptor type 4 (CXCR4) expression is associated with poor prognosis of hepatocellular carcinoma (HCC). The aim of this study was to explore the biological role of CXCR4 in gefitinib resistance of HCC. Compared with a normal, non‐gefitinib‐resistant, human HCC cell line (Huh7), CXCR4 mRNA and protein were highly expressed in gefitinib‐resistant Huh7 cells (Huh7‐R). Cell proliferation was decreased, and apoptosis was enhanced in Huh7 cells in the presence of gefitinib. These influences conferred by gefitinib treatment on proliferation and apoptosis of Huh7 cells were abolished by CXCR4 overexpression. CXCR4 knockdown reduced the proliferation ability of HuH‐7R cells after gefitinib treatment. Importantly, CXCR4 overexpression had no influence on caveolin 1 (Cav‐1) expression; similarly, Cav‐1 silencing did not cause a substantive change in CXCR4 expression. However, CXCR4 activated Cav‐1, c‐Met, and Raf‐1 in Huh7 cells, whereas Cav‐1 silencing repressed the expression of Raf‐1 and phosphorylated c‐Met in Huh7 cells. CXCR4 overexpression promoted proliferation and repressed apoptosis in gefitinib‐treated Huh7 cells, which was partly rescued by PHA‐665752 (a c‐Met inhibitor) treatment or c‐Met deficiency. Finally, we constructed a tumor xenograft model to determine the influence of CXCR4 overexpression on tumor growth of HCC. CXCR4 overexpression accelerated tumor growth of HCC, which was abrogated by c‐Met deficiency. These findings demonstrate that CXCR4 overexpression activates c‐Met via the Cav‐1 signaling pathway, thereby promoting gefitinib resistance of Huh7 cells. Thus, this study highlights novel insights into the mechanism of gefitinib resistance of HCC and CXCR4 may become a potential target for HCC treatment.

Synergistic antitumor effect of resveratrol and sorafenib on hepatocellular carcinoma through PKA/AMPK/eEF2K pathway.

Although sorafenib (Sor) is the only effective drug for hepatocellular carcinoma (HCC), its therapeutic potential to date is mainly limited to the low tumor response. This study was designed to explore whether resveratrol (Res) could potentiate the anticancerous activity of Sor. We used HepG2 and Huh7 HCC cell lines and BALB/c nude mice for in vitro and in vivo studies, respectively. The cultured cell lines and tumor induction in the mice were treated with different concentrations of Res and Sor alone, and the combination of Res and Sor to observe the antitumor effects. Significant inhibitory effects were observed in the combined treatment of Res and Sor compared to Res and Sor alone treatments both in vitro and in vivo as demonstrated by significantly high number of S phase cells and apoptotic cells. Moreover, these findings were accompanied by the reduction of CDK2, CDC25A, PKA, p-AMPK, and eEF2K protein levels and the increment of cyclin A, cleavage caspase-3, caspase-8, and caspase-9 protein levels. The combinational treatment exhibited more significant anticancerous effect than the Res and Sor alone treatments in mice-bearing HepG2 xenograft. Overall, our results suggest that PKA/AMPK/eEF2K pathway is involved in the synergistic anticancerous activity of Res and Sor combination treatment in HCC cells. Thus, Res and Sor combination therapy may be promising in increasing the tumor response of Sor in the future.

Catalpol synergistically potentiates the anti-tumour effects of regorafenib against hepatocellular carcinoma via dual inhibition of PI3K/Akt/mTOR/NF-κB and VEGF/VEGFR2 signaling pathways.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer characterized by dysregulation of several crucial cellular signaling pathways such as PI3K/p-Akt/mTOR/NF-κB and VEGF/VEGFR2 pathways. Novel therapies targeting these pathways have been discovered such as regorafenib which is small molecular multi-kinase inhibitor mainly targets VEGF/VEGFR2. Catalpol is an iridoid glycoside richly found in rehmannia glutinosa which is a fundamental herb used extensively in traditional Chinese medicine. It is evidenced that catalpol has many pharmacological effects on nervous and cardiovascular systems, in addition to exhibiting hypoglycemic, anti-inflammatory, anti-proliferative and anti-tumour activities. However, its effect on HCC isn't clear enough. So, this study aimed to investigate the anti-tumour effects of catalpol either alone or in combination with regorafenib on HCC. In vitro experiments were performed using HepG2 and HUH-7 hepatocellular carcinoma cell lines. MTT assays evaluated anti-proliferative effects of catalpol and/or regorafenib. Combination index was calculated via compusyn software to detect synergism. Tumour biomarkers were measured using ELISA technique. Results showed that catalpol has anti-tumour effects against HCC via targeting PI3K/p-Akt/mTOR/NF-κB and VEGF/VEGFR2 pathways. In addition, results revealed that our novel combination of catalpol and regorafenib showed potent synergistic anti-tumour effect via suppressing both of PI3K/p-Akt/mTOR/NF-κB and VEGF/VEGFR2 signaling pathways and their downstreams. Catalpol and/or regorafenib markedly suppressed PI3K/p-Akt/mTOR/NF-κB and VEGF/VEGFR2 signaling pathways and consequently showed potent anti-tumour effects against HCC. Results encourage further pre-clinical and clinical studies of this novel combination as a promising targeted therapy for HCC management.

Development of a Hybrid Polymer-Based Microfluidic Platform for Culturing Hepatocytes towards Liver-on-a-Chip Applications.

The drug development process can greatly benefit from liver-on-a-chip platforms aiming to recapitulate the physiology, mechanisms, and functionalities of liver cells in an in vitro environment. The liver is the most important organ in drug metabolism investigation. Here, we report the development of a hybrid cyclic olefin copolymer (COC) and polydimethylsiloxane (PDMS) microfluidic (HCP) platform to culture a Huh7 hepatoma cell line in dynamic conditions towards the development of a liver-on-a-chip system. The microfluidic platform is comprised of a COC bottom layer with a microchannel and PDMS-based flat top layer sandwiched together. The HCP device was applied for culturing Huh7 cells grown on a collagen-coated microchannel. A computational fluid dynamics modeling study was conducted for the HCP device design revealing the presence of air volume fraction in the chamber and methods for optimizing experimental handling of the device. The functionality and metabolic activity of perfusion culture were assessed by the secretion rates of albumin, urea, and cell viability visualization. The HCP device hepatic culture remained functional and intact for 24 h, as assessed by resulting levels of biomarkers similar to published studies on other in vitro and 2D cell models. The present results provide a proof-of-concept demonstration of the hybrid COC–PDMS microfluidic chip for successfully culturing a Huh7 hepatoma cell line, thus paving the path towards developing a liver-on-a-chip platform.

Role for calcium-activated potassium channels (BK) in migration control of human hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a leading cause of cancer‐related death worldwide. Its high metastasis rate is significantly correlated with poor patient prognosis. Elucidating the molecular mechanism underlying HCC metastasis is essential for HCC treatment. Owing to their high conductance, large‐conductance calcium‐activated potassium channels (BK channels) play a critical role in the control of membrane potential and have repeatedly been proposed as potential targets for cancer therapy. Emerging evidence suggests that BK channels are involved in the progression of cancer malignancies. The present study investigated the role of BK channels in mediating the hypoxia‐stimulated migration of HCC cells both in vitro and in vivo in the absence and presence of various BK channels modulators. We found that BK channels were functionally expressed on the membranes of the SMMC‐7721 and Huh7 HCC cell lines. Furthermore, blockage or activation of BK channels on the surface of HCC cells correspondingly inhibited or promoted HCC cell proliferation, migration and invasion in hypoxia conditions, with altered expression and distribution of cell‐cell adhesion molecule E‐cadherin and typical marker of mesenchymal cells, Vimentin, but not N‐cadherin. Hypoxia conditions did not alter BK channels expression but increased its open probability. Moreover, BK channels blocker IbTX significantly inhibited HCC cell remote colonization in HCC cell xenografted mice. In conclusion, the results of this study suggest that blocking BK channels offers an attractive strategy for treating HCC.

Novel interferon-sensitive genes unveiled by correlation-driven gene selection and systems biology

Interferons (IFNs) are key cytokines involved in alerting the immune system to viral infection. After IFN stimulation, cellular transcriptional profile critically changes, leading to the expression of several IFN stimulated genes (ISGs) that exert a wide variety of antiviral activities. Despite many ISGs have been already identified, a comprehensive network of coding and non-coding genes with a central role in IFN-response still needs to be elucidated. We performed a global RNA-Seq transcriptome profile of the HCV permissive human hepatoma cell line Huh7.5 and its parental cell line Huh7, upon IFN treatment, to define a network of genes whose coordinated modulation plays a central role in IFN-response. Our study adds molecular actors, coding and non-coding genes, to the complex molecular network underlying IFN-response and shows how systems biology approaches, such as correlation networks, network’s topology and gene ontology analyses can be leveraged to this aim.

Two New Purification Methods of Hepatitis C Virus Particles from Serum-Free Culture System

Background: The traditional ultracentrifugation purification method of hepatitis C virus (HCV) particles requires special equipment, limiting its wide application. Therefore, more effective and convenient methods for HCV are needed. Objectives: The present study aimed to establish simple and effective purification methods for HCV. Methods: The infectious clone of the HCV genome (JFH-1) was transfected to the human hepatoma cell line (Huh7.5.1) and cultured in Dulbecco’s modified eagle medium/nutrient mixture F-12. The infectivity of JFH-1 culture was determined by reverse transcription-quantitative polymerase chain reaction and immunofluorescence. After concentration by centrifugal filter devices, HCV particles were purified by heparin-affinity chromatography and magnetic separation technique. The purified viruses were detected by the western blot and immune-electron microscopy. Results: The infectious titer of JFH-1 transfected Huh7.5.1 in the serum-free culture medium was 4.5 × 104 FFU/mL, and HCV ribonucleic acid load was 3.946 × 106 IU/mL in 30 days of cell culture post-transfection. After purification by heparin-affinity chromatography or magnetic separation method, viral particles were visualized with spherical morphology and an average diameter of 55 nm assessed by electron microscopy. The viruses were confirmed by the western blot and immune-electron microscopy with specific antibodies to HCV. Conclusions: The heparin-affinity chromatography and magnetic separation methods were established for the purification of HCV, which were simple and efficient methods for the stable purification of HCV particles on a large scale.

Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant hepatocellular carcinoma cells.

As a multiple tyrosine kinase inhibitor, sorafenib is widely used to treat hepatocellular carcinoma (HCC), but patients frequently face resistance problems. Because the mechanism controlling sorafenib-resistance is not well understood, this study focused on the connection between tumor characteristics and the Nrf2 signaling pathway in a sorafenib-resistant HCC cell line. A sorafenib-resistant HCC cell line (Huh7) was developed by increasing the dose of sorafenib in the culture medium until the target concentration was reached. Cell morphology, migration/invasion rates, and expression of stemness-related and ATP-binding cassette (ABC) transporter genes were compared between sorafenib-resistant Huh7 cells and parental Huh7 cells. Next, a small interfering RNA was used to knock down Nrf2 expression in sorafenib-resistant Huh7 cells, after which cell viability, stemness, migration, and ABC transporter gene expression were examined again. Proliferation, migration, and invasion rates of sorafenib-resistant Huh7 cells were significantly increased relative to the parental cells with or without sorafenib added to the medium. The expression levels of stemness markers and ABC transporter genes were up-regulated in sorafenib-resistant cells. After Nrf2 was knocked down in sorafenib-resistant cells, cell migration and invasion rates were reduced, and expression levels of stemness markers and ABC transporter genes were reduced. Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant HCC cells.

Actinomycin D Arrests Cell Cycle of Hepatocellular Carcinoma Cell Lines and Induces p53-Dependent Cell Death: A Study of the Molecular Mechanism Involved in the Protective Effect of IRS-4.

Actinomycin D (ActD) is an FDA-approved NCI oncology drug that specifically targets and downregulates stem cell transcription factors, which leads to a depletion of stem cells within the tumor bulk. Recently, our research group demonstrated the importance of IRS-4 in the development of liver cancer. In this study, we evaluated the protective effects of IRS-4 against ActD. For this study, three hepatocellular carcinoma cell lines (HepG2, Huh7, and Chang cells) were used to study the mechanism of actinomycin D. Most assays were carried out in the Hep G2 cell line, due to the high expression of stem cell biomarkers. We found that ActD caused HepG2 cell necroptosis characterized by DNA fragmentation, decreased mitochondrial membrane potential, cytochrome c depletion, and decreased the levels of reduced glutathione. However, we did not observe a clear increase in apoptosis markers such as annexin V presence, caspase 3 activation, or PARP fragmentation. ActD produced an activation of MAP kinases (ERK, p38, and JNK) and AKT. ActD-induced activation of AKT and MAP kinases produced an activation of the Rb-E2F cascade together with a blockage of cell cycle transitions, due to c-jun depletion. ActD led to the inhibition of pCdK1 and pH3 along with DNA fragmentation resulting in cell cycle arrest and the subsequent activation of p53-dependent cell death in the HepG2 cell line. Only JNK and AKT inhibitors were protective against the effects of ActD. N-Acetyl-L-cysteine also had a protective effect as it restored GSH levels. A likely mechanism for this is IRS-4 stimulating GCL-GSH and inhibiting the Brk-CHK1-p53 pathway. The assessment of the IRS-4 in cancer biopsies could be of interest to carry out a personalized treatment with ActD.

CKS1B promotes the progression of hepatocellular carcinoma by activating JAK/STAT3 signal pathway

ABSTRACT Hepatocellular carcinoma (HCC) is a malignancy of considerable concern due to its continuous increase in morbidity and mortality. This study attempts to identify the molecules that play a key role in the progression of HCC, explore its potential mechanism, and provide more target choices for targeted therapy. Using overexpression plasmid and shRNA, CKS1B was respectively overexpressed and knocked down to explore its biological function roles in HCC progression and development. MTT and colony formation assays showed that knockdown of CKS1B inhibited the survival and proliferation of HCC cell lines (Hep3B and Huh7). The flow cytometry and western blot analysis showed that knockdown of CKS1B significantly induced the apoptosis of Hep3B and Huh7 cells. The wound healing and transwell invasion assays showed that knockdown of CKS1B had a significant inhibitory effect on the migration and invasion of Hep3B and Huh7 cells. These functional tests confirmed that CKS1B acts as an oncogene that regulates the malignant progression of HCC. Moreover, this study also demonstrated that knockdown of CKS1B inhibited the activation of JAK/STAT3 pathway, evidenced by the significantly downregulated p-STAT3 protein expression. Furthermore, knockdown of CKS1B also downregulated STAT3 target genes TIMP-1, Bcl-2 and VEGF, which were involved in controlling cell apoptosis and migration. On the contrary, overexpression of CKS1B caused the completely opposite results. Taken together, CKS1B acts as an oncogene to promote the proliferation and metastasis of HCC cells by activating JAK/STAT3 signaling pathway.

Integrative proteomics reveals the role of E3 ubiquitin ligase SYVN1 in hepatocellular carcinoma metastasis.

BackgroundTumor metastasis is a major factor for poor prognosis of hepatocellular carcinoma (HCC), but the relationship between ubiquitination and metastasis need to be studied more systematically. We analyzed the ubiquitinome of HCC in this study to have a more comprehensive insight into human HCC metastasis.MethodsThe protein ubiquitination levels in 15 HCC specimens with vascular invasion and 15 without vascular invasion were detected by ubiquitinome. Proteins with significantly different ubiquitination levels between HCCs with and without vascular invasion were used to predict E3 ubiquitin ligases associated with tumor metastasis. The topological network of protein substrates and corresponding E3 ubiquitin ligases was constructed to identify the key E3 ubiquitin ligase. Besides, the growth, migration and invasion ability of LM3 and HUH7 hepatoma cell lines with and without SYVN1 expression interference were measured by cell proliferation assay, subcutaneous tumor assay, umphal vein endothelium tube formation assay, transwell migration and invasion assays. Finally, the interacting proteins of SYVN1 were screened and verified by protein interaction omics, immunofluorescence, and immunoprecipitation. Ubiquitin levels of related protein substrates in LM3 and HUH7 cells were compared in negative control, SYVN1 knockdown, and SYVN1 overexpression groups.ResultsIn this study, our whole‐cell proteomic dataset and ubiquitinomic dataset contained approximately 5600 proteins and 12,000 ubiquitinated sites. We discovered increased ubiquitinated sites with shorter ubiquitin chains during the progression of HCC metastasis. In addition, proteomic and ubiquitinomic analyses revealed that high expression of E3 ubiquitin‐protein ligase SYVN1 is related with tumor metastasis. Furthermore, we found that SYVN1 interacted with heat shock protein 90 (HSP90) and impacted the ubiquitination of eukaryotic elongation factor 2 kinase (EEF2K).ConclusionsThe ubiquitination profiles of HCC with and without vascular invasion were significantly different. SYVN1 was the most important E3 ubiquitin‐protein ligase responsible for this phenomenon, and it was related with tumor metastasis and growth. Therefore, SYVN1 might be a potential therapeutic target for HCC.

The Modulation of PCSK9 and LDLR by Supercritical CO2 Extracts of Mentha longifolia and Isolated Piperitone Oxide, an In Vitro Study.

In the present study the ability of supercritical carbon dioxide (SCO2) extracts of M. longifolia L. leaves to modulate low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression was evaluated in cultured human hepatoma cell lines Huh7 and HepG2. Two SCO2 extracts, one oil (ML-SCO2) and a semisolid (MW-SCO2), were subjected to detailed chemical characterization by mono- and bidimensional nuclear magnetic resonance (1D, 2D-NMR), gas chromatography coupled with mass spectrometry (GC-MS) and liquid chromatography coupled with mass spectrometry (LC-MS). Chemical analysis revealed significant amounts of fatty acids, phytosterols and terpenoids. ML-SCO2 was able to induce LDLR expression at a dose of 60 µg/mL in HuH7 and HepG2 cell lines. Furthermore, ML-SCO2 reduced PCSK9 secretion in a concentration-dependent manner in both cell lines. Piperitone oxide, the most abundant compound of the volatile constituent of ML-SCO2 (27% w/w), was isolated and tested for the same targets, showing a very effective reduction of PCSK9 expression. The overall results revealed the opportunity to obtain a new nutraceutical ingredient with a high amount of phytosterols and terpenoids using the SCO2 extraction of M. longifolia L., a very well-known botanical species used as food. Furthermore, for the first time we report the high activity of piperitone oxide in the reduction of PCSK9 expression.

Inhibiting p21-activated kinase (PAK7) enhances radiosensitivity in hepatocellular carcinoma.

In light of the upregulation of p21-activated kinase (PAK7) in a variety of cancers, including hepatocellular carcinoma (HCC), we aimed to investigate the effect of PAK7 on the sensitivity of HCC cells to radiotherapy. PAK7 expression was determined in normal adult liver epithelial THLE-2 and human HCC cell lines. The effect of ionizing radiation (IR) on the HCC cell viability was evaluated by Sulforhodamine B (SRB) assay. HCC cell lines Mahlavu and Huh7 were chosen to assess the effect of PAK7 shRNAs on the viability, clone formation, apoptosis, cycle distribution and γ-H2AX expression after exposure to IR. As compared to THLE-2 cells, PAK7 was upregulated in poorly differentiated Mahlavu and SK-Hep-1 cells, but moderately or lowly expressed in well-differentiated Huh7 and HepG2 cells. HCC cells with moderate or low expression of PAK7 presented a decreased viability at 2 Gy IR, which had no significant effect on PAK7high HCC cells. Mahlavu and Huh7 cells transfected with PAK7 shRNAs showed increased inhibitory effect of IR on viability. In addition, PAK7 shRNAs reduced clone formation, facilitated the cell apoptosis, arrested cells at G2/M phase, and increased γ-H2AX expression. Moreover, changes above were more evident in the HCC cells co-treated with IR and PAK7 shRNAs. PAK7 downregulation could inhibit the viability, promote the apoptosis, arrest cells in G2/M phase, and induce the DNA damage in HCC cells, thereby enhancing the radiosensitivity in HCC.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Sec62 promotes pro-angiogenesis of hepatocellular carcinoma cells under hypoxia.

This study aimed to investigate the underlying molecular pathogenic mechanism of Sec62 in hepatocellular carcinoma (HCC). Microarray analysis was conducted to profile the global gene expression in the HCC cell line Huh7 cells transfected with Sec62high vs. NC and Sec62low vs. NC. Ingenuity pathway analysis and gene set enrichment analysis were used to perform Sec62-related signaling pathway analysis from screened differentially expressed genes (DEGs). A protein-protein interaction network was constructed. Experimental validation of the expression of key DEGs was conducted. Hypoxia-induced tube formation was undertaken to investigate the role of Sec62 in angiogenesis. A total of 74 intersected DEGs were identified from Huh7 cells with Sec62high vs. NC and Sec62low vs. NC. Among them, 65 DEGs were correlated with the expression of Sec62. The P53 signaling pathway was found to be enriched in Huh7 cells with Sec62high vs. NC, while the acute phase response signaling pathway was enriched in Huh7 cells with Sec62low vs. NC. DEGs, such as serine protease inhibitor E (SERPINE) and tumor necrosis factor receptor superfamily, member 11B (TNFRSF11B), were not only identified as the lead genes of these enriched pathways, but were also found to be closely related to Sec62. Moreover, knockdown of Sec62 decreased the expression of SERPINE1 (plasminogen activator inhibitor type 1 (PAI-1)) and TNFRSF11B, whereas overexpression of Sec62 had the opposite effects. In addition, knockdown of Sec62 inhibited hypoxia-induced tube formation via PAI-1. Sec62 promoted pro-angiogenesis of HCC under hypoxia by regulating PAI-1, and it may be a crucial angiogenic switch in HCC.

Artematrovirenins A–P, guaiane-type sesquiterpenoids with cytotoxicities against two hepatoma cell lines from Artemisia atrovirens

Random screening revealed that the EtOH extract of Artemisia atrovirens showed significant cytotoxicity against two human hepatoma cell lines (HepG2 and Huh7) with the inhibitory ratio of 98.9% and 99.7% at the concentration of 100 μg/mL. Further bioactivity-guided isolation of active fraction led to 16 new guaiane-type sesquiterpenoids, artematrovirenins A–P (1–16). Their structures were elucidated by extensive spectroscopic data. The absolute stereochemistry of compounds 1 and 14 was determined by single-crystal X-ray diffraction analyses. Pharmacological evaluation suggested that five compounds (3, 5, 8, 10, and 15) exhibited cytotoxicity, compounds 3 and 5 displayed cytotoxicity against HepG2 cell line with an IC50 values of 8.0 and 16.0 μM, as well as against Huh7 cell line with values of 18.2 and 32.2 μM.

Evaluation of pro-apoptotic potential of taxifolin against liver cancer.

Liver cancer is the second most common cause of cancer-induced deaths worldwide. Liver cirrhosis and cancer are a consequence of the abnormal angio-architecture formation of liver and formation of new blood vessels. This angiogenesis is driven by overexpression of hypoxia-inducible factor 1-alpha (Hif1-α) and vascular endothelial growth factor (VEGF). Apart from this, protein kinase B (Akt) is also impaired in liver cancer. Despite the advancement in conventional treatments, liver cancer remains largely incurable. Nowadays, the use of naturally occurring anticancer agents particularly flavonoids is subject to more attention due to their enhanced physicochemical properties. Therefore, this study underlines the use of a natural anticancer agent taxifolin in the treatment of liver cancer using hepatocellular carcinoma cell line HepG2 and Huh7. The aim of our study is to devise a natural and efficient solution for the disease prevalent in Pakistan. The study involved the assessment of binding of ligand taxifolin using molecular docking. The binding of taxifolin with the proteins (Hif1-α, VEGF and Akt) was calculated by docking using Vina and Chimera. Further evaluation was performed by cell viability assay (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay), colony formation assay, cell migration assay, DNA ladder assay and flow cytometry. To see whether taxifolin directly affected expression levels, analysis of gene expression of Hif1-α, VEGF and Akt was performed using real-time polymerase chain reaction (qPCR) and western blotting. In silico docking experiments revealed that these proteins showed favorable docking scores with taxifolin. Treatment with taxifolin resulted in the inhibition of the liver cancer growth and migration, and induced apoptosis in HepG2 and Huh7 cell lines at an inhibitory concentration (IC50) value of 0.15 µM and 0.22 µM, respectively. The expression of HIF1-α, VEGF and Akt was significantly reduced in a dose- dependent manner. The inhibitory effect of taxifolin on hepatic cells suggested its chemopreventive and therapeutic potential. The studied compound taxifolin exhibited pronounced pro-apoptotic and hepatoprotective potential. Our study has confirmed the pro-apoptotic potential of taxifolin in liver cancer cell lines and will pave a way to the use of taxifolin as a chemotherapeutic agent after its further validation on the animal models and humans based epidemiological studies.