Proliferation Of Human Hepatocellular Carcinoma Cells Research Articles

Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro.

Prohibitin 1 (PHB1) has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed, and it participates in a variety of essential cellular functions, including apoptosis, cell cycle regulation, proliferation, and survival. Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma (HCC). However, the role of PHB1 in HCC is controversial. To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro. HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria; then, PHB1 levels in the sera and liver tissues of these participates were determined using ELISA, RT-PCR, and immunohistochemistry. Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA (shRNA-PHB1) for 24-72 h. Cell proliferation was analysed with an MTT assay. Cell cycle progression and apoptosis were analysed using flow cytometry (FACS). The mRNA and protein expression levels of the cell cycle-related molecules p21, Cyclin A2, Cyclin E1, and CDK2 and the cell apoptosis-related molecules cytochrome C (Cyt C), p53, Bcl-2, Bax, caspase 3, and caspase 9 were measured by real-time PCR and Western blot, respectively. Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals, and decreased PHB1 was positively correlated with low differentiation, TNM stage III-IV, and alpha-fetoprotein ≥ 400 μg/L. Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner. FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis. The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells. The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41% ± 1.06%, which was significantly greater than that of apoptotic control cells (3.65% ± 0.85%, P < 0.01) and empty vector-transfected cells (4.21% ± 0.52%, P < 0.01). Similar results were obtained with SMMC-7721 cells. Furthermore, the mRNA and protein expression levels of p53, p21, Bax, caspase 3, and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2, Cyclin E1, CDK2, and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells. However, when PHB1 was upregulated in human HCC cells, Cyt C expression levels were increased in the cytosol and decreased in the mitochondria, which indicated that Cyt C had been released into the cytosol. Conversely, these effects were reversed when PHB1 was knocked down. PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.

Interfering with the AKT/mTOR/STAT3/ID1 signaling axis with usenamine A restrains the proliferative and invasive potential of human hepatocellular carcinoma cells

BackgroundUsenamine A, a novel natural compound initially isolated from the lichen Usnea longissima, has exhibited promising efficacy against hepatoma in prior investigation. Nevertheless, the underlying mechanisms responsible for its antihepatoma effects remain unclear. Furthermore, the role of the AKT/mechanistic target of the rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3)/inhibitor of differentiation/DNA binding 1 (ID1) signaling axis in hepatocellular carcinoma (HCC), and the potential anti-HCC effects of drugs targeting this pathway are not well understood.MethodsCCK-8 assay was used to investigate the effects of usenamine A on the proliferation of human HCC cells. Moreover, the effects of usenamine A on the invasion ability of human HCC cells were evaluated by transwell assay. In addition, expression profiling analysis, quantitative real-time PCR, immunoblotting, immunohistochemistry (IHC) analysis, RNAi, immunoprecipitation, and chromatin immunoprecipitation (ChIP) assay were used to explore the effects of usenamine A on the newly identified AKT/mTOR/STAT3/ID1 signaling axis in human HCC cells.ResultsUsenamine A inhibited the proliferation and invasion of human HCC cell lines (HepG2 and SK-HEP-1). Through the analysis of gene expression profiling, we identified that usenamine A suppressed the expression of ID1 in human HCC cells. Furthermore, immunoprecipitation experiments revealed that usenamine A facilitated the degradation of the ID1 protein via the ubiquitin–proteasome pathway. Moreover, usenamine A inhibited the activity of STAT3 in human HCC cells. ChIP analysis demonstrated that STAT3 positively regulated ID1 expression at the transcriptional level in human HCC cells. The STAT3/ID1 axis played a role in mediating the anti-proliferative and anti-invasive impacts of usenamine A on human HCC cells. Additionally, usenamine A suppressed the STAT3/ID1 axis through AKT/mTOR signaling in human HCC cells.ConclusionUsenamine A displayed robust anti-HCC potential, partly attributed to its capacity to downregulate the AKT/mTOR/STAT3/ID1 signaling pathway and promote ubiquitin–proteasome-mediated ID1 degradation. Usenamine A has the potential to be developed as a therapeutic agent for HCC cases characterized by abnormal AKT/mTOR/STAT3/ID1 signaling, and targeting the AKT/mTOR/STAT3 signaling pathway may be a viable option for treating patients with HCC exhibiting elevated ID1 expression.

Ubiquitin ligase NEDD4 promotes the proliferation of hepatocellular carcinoma cells through targeting PCDH17 protein for ubiquitination and degradation

Neural precursor cell expressed developmentally downregulated 4 (NEDD4), an E3 ubiquitin ligase, is commonly upregulated in human hepatocellular carcinoma (HCC) and functions as an oncogenic factor in the progression of HCC, but the molecular mechanism needs be further explored. In this study, we found that NEDD4 could facilitate the proliferation of HCC cells, which was associated with regulating the ERK signaling. Further investigation showed that protocadherin 17 (PCDH17) was a potential substrate of NEDD4, and restoration of PCDH17 could block the facilitation of ERK signaling and HCC cells proliferation induced by NEDD4 overexpression. Whereafter, we confirmed that NEDD4 interacted with PCDH17 and promoted the Lys33-linked polyubiquitination and degradation of it via the proteasome pathway. Finally, NEDD4 protein level was found to be inversely correlated with that of PCDH17 in human HCC tissues. In conclusion, these results suggest that NEDD4 acts as an E3 ubiquitin ligase for PCDH17 ubiquitination and degradation thereby promoting the proliferation of HCC cells through regulating the ERK signaling, which may provide novel evidence for NEDD4 to be a promising therapeutic target for HCC.

Schistosoma mansoni–Induced Oxidative Stress Triggers Hepatocellular Proliferation

Schistosomiasis is one of the most prominent parasite-induced infectious diseases, affecting more than 250 million people. Schistosoma mansoni causes metabolic exhaustion and a strong redox imbalance in the liver, causing parenchymal damage, and may predispose for cancer. We investigated whether oxidative stress provokes hepatocellular proliferation upon S. mansoni infection. The cell cycle, replication stress response, and proliferation were analyzed on transcriptional and protein levels in the livers of S. mansoni-infected hamsters and by mechanistic gain- and loss-of-function experiments in human hepatoma cells. Major results were validated in human biopsy specimens of S. mansoni-infected patients. S. mansoni infection induced licensing factors of DNA replication and cell-cycle checkpoint cyclins in parallel with a DNA damage response in hamster hepatocytes. Moreover, even unisexual infection without egg effects, as a reflection of a chronic inflammatory process, resulted in a moderate activation of several cell-cycle markers. S. mansoni soluble egg antigens induced proliferation of human hepatoma cells that could be abolished by reduced glutathione. Our data suggest that hepatocellular proliferation is triggered by S. mansoni egg-induced oxidative stress.

MicroRNA-188-5p inhibits hepatocellular carcinoma proliferation and migration by targeting forkhead box N2

BackgroundThis study aimed to identify the biological functions, expression modes, and possible mechanisms underlying the relationship between metastatic human hepatocellular carcinoma (HCC) and MicroRNA-188-5p (miR-188) dysregulation using cell lines.MethodsA decrease in miR-188 was detected in low and high metastatic HCC cells compared to that in normal hepatic cells and non-invasive cell lines. Gain- and loss-of-function experiments were performed in vitro to investigate the role of miR-188 in cancer cell (Hep3B, HepG2, HLF, and LM3) proliferation and migration.ResultsmiR-188 mimic transfection inhibited the proliferation of metastatic HLF and LM3 cells but not non-invasive HepG2 and Hep3B cells; nonetheless, miR-188 suppression promoted the growth of HLF and LM3 cells. miR-188 upregulation inhibited the migratory rate and invasive capacity of HLF and LM3, rather than HepG2 and Hep3B cells, whereas transfection of a miR-188 inhibitor in HLF and LM3 cells had the opposite effects. Dual-luciferase reporter assays and bioinformatics prediction confirmed that miR-188 could directly target forkhead box N2 (FOXN2) in HLF and LM3 cells. Transfection of miR-188 mimics reduced FOXN2 levels, whereas miR-188 inhibition resulted in the opposite result, in HLF and LM3 cells. Overexpression of FOXN2 in HLF and LM3 cells abrogated miR-188 mimic-induced downregulation of proliferation, migration, and invasion. In addition, we found that miR-188 upregulation impaired tumor growth in vivo.ConclusionsIn summary, this study showed thatmiR-188 inhibits the proliferation and migration of metastatic HCC cells by targeting FOXN2.

Polysaccharides from Hemerocallis citrina Baroni Inhibit the Growth of Hepatocellular Carcinoma Cells by Regulating the Wnt/β-Catenin Pathway

Hemerocallis citrina Baroni is an edible plant with anti-inflammatory, antidepressant, and anticancer activities. However, studies on H. citrina polysaccharides are limited. In this study, a polysaccharide named HcBPS2 was isolated and purified from H. citrina. Monosaccharide component analysis showed that HcBPS2 was composed of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid. Notably, HcBPS2 significantly inhibited human hepatoma cell proliferation, but had little effect on human normal liver cells (HL-7702). Mechanism investigations indicated HcBPS2 suppressed human hepatoma cell growth through the induction of G2/M phase arrest and mitochondria-dependent apoptosis in human hepatoma cells. In addition, the data revealed that HcBPS2 treatment led to the inactivation of Wnt/β-catenin signaling, which then gave rise to cell cycle arrest and apoptosis in human hepatoma cancer cells. Collectively, these findings suggested that HcBPS2 may serve as a therapeutic agent against liver cancer.

CDCA7 serves as a novel prognostic marker in human hepatocellular carcinoma

ABSTRACT c-Myc oncogene plays an important role in tumorigenesis, cell division cycle associated 7 (CDCA7), recently found that it is a direct target gene of c-Myc, is upregulated in many tumors, but its role in tumor progression is still poorly understood. CDCA7 expression and prognosis were analyzed in hepatocellular carcinoma using TIMER2.0 and Kaplan–Meier databases, while genomic changes were studied using cbioportal. LinkedOmics identified relevant genes and WebGestalt analyzed the associated pathways. Protein interaction networks were explored using the STRING database, and the core PPI network was analyzed with the MCODE plugin of Cytoscape. CDCA7 expression was detected in 30 paired HCC specimens by real-time PCR, and its effect on HCC cell proliferation was determined in vitro. CDCA7 expression was frequently up-regulated in human hepatocellular carcinoma (HCC), and its expression was positively correlated with prognosis. The TIMER2.0 database showed that CDCA7 was differentially expressed in hepatocellular carcinoma, with high expression in tumor tissues and low expression in normal tissues. The Kaplan-Meier database shows that high CDCA7 expression has a worse prognosis. The cBioportal database showed that the genomic change rate of CDCA7 in hepatocellular carcinoma was 2.15%, including mutations, amplifications, and deep deletions. Pathway analysis of related genes showed that CDCA7-related genes were mainly focused on cell division-related pathways. The experimental results also validate our study. CDCA7 could contribute to HCC progression and raise the possibility that CDCA7 is a potential new therapeutic target for HCC treatment.

Pharmacological inhibition of LSD1 suppresses growth of hepatocellular carcinoma by inducing GADD45B.

Lysine-specific histone demethylase 1 (LSD1) is an attractive target for malignancies therapy. Nevertheless, its role in hepatocellular carcinoma (HCC) progression and the potential of its inhibitor in HCC therapy remains unclear. Here, we show that LSD1 overexpression in human HCC tissues is associated with HCC progression and poor patient survival. ZY0511, a highly selective and potent inhibitor of LSD1, suppressed human HCC cell proliferation in vitro and tumor growth in cell-derived and patient-derived HCC xenograft models in vivo. Mechanistically, ZY0511 induced mRNA expression of growth arrest and DNA damage-inducible gene 45beta (GADD45B) by inducing histone H3 at lysine 4 (H3K4) methylation at the promoter of GADD45B, a novel target gene of LSD1. In human HCC tissues, LSD1 level was correlated with a decreased level of GADD45B, which was associated with HCC progression and predicted poor patient survival. Moreover, co-administration of ZY0511 and DTP3, which specifically enhanced the pro-apoptotic effect of GADD45B, effectively inhibited HCC cell proliferation both in vitro and in vivo. Collectively, our study revealed the potential value of LSD1 as a promising target of HCC therapy. ZY0511 is a promising candidate for HCC therapy through upregulating GADD45B, thereby providing a novel combinatorial strategy for treating HCC.

Comprehensive analysis of ferroptosis-related genes for clinical and biological significance in hepatocellular carcinoma

ObjectiveThis study aims to build a prognostic model of hepatocellular carcinoma (HCC) with ferroptosis-associated genes and explore their molecular function.MethodsGene expression data and clinical information were obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases and the International Cancer Genome Consortium (ICGC). A ferroptosis-associated gene set was obtained from the FerrDb database to identify differentially expressed genes. Then, we performed pathway enrichment analysis and immune infiltration analysis. A combined model based on ferroptosis-associated genes for predicting the overall survival of HCC was built by univariate and multivariate Cox regression analyses. Quantitative real-time polymerase chain reaction, Western blotting, colony formation, CCK-8, and EdU incorporation assays were performed to clarify the function of CAPG in the regulation of cell proliferation in human HCC. Ferroptosis was evaluated by glutathione (GSH), malondialdehyde (MDA), and total iron detection.ResultsForty-nine ferroptosis-related genes were significantly correlated with HCC, 19 of which had prognostic significance. CAPG, SLC7A11 and SQSTM1 were used to construct a novel risk model. The areas under the curves (AUCs) were 0.746 and 0.720 (1 year) in the training and validation groups, respectively. The survival analysis indicated that patients with high risk scores exhibited worse survival in the training and validation groups. The risk score was also identified as an independent prognostic factor of overall survival (OS), which established and validated the predictive abilities of the nomogram. The risk score was also significantly correlated with the expression of immune checkpoint genes. In vitro data showed that CAPG knockdown dramatically suppressed HCC cell proliferation, and the underlying molecular mechanisms might be that the silencing of CAPG reduced the expression of SLC7A11 and promoted ferroptosis.ConclusionThe established risk model can be used to predict the prognosis of HCC. At the mechanistic level, CAPG may drive HCC progression by regulating SLC7A11, and ferroptosis activation in HCC patients with high CAPG expression may serve as a potential therapeutic strategy.

Retracted] Alpinetin suppresses proliferation of human hepatoma cells by the activation of MKK7 and elevates sensitization to cis‑diammined dichloridoplatium.

Following the publication of the above paper, it was drawn to the Editors' attention by a concerned reader that western blot data shown in Fig.2B were strikingly similar to data that had appeared in different form in another article. Owing to the fact that the contentious data in the above article were already under consideration for publication elsewhere prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 27: 1090‑1096, 2012; DOI: 10.3892/or.2011.1580].

LncRNA miR4458HG modulates hepatocellular carcinoma progression by activating m6A-dependent glycolysis and promoting the polarization of tumor-associated macrophages

Long non-coding RNAs (lncRNAs) play significant roles in different biological functions of cancers. However, their function in the metabolism of glucose in patients with human hepatocellular carcinoma (HCC) remains largely unknown. In this study, HCC and paired intact liver tissues were utilized to examine the miR4458HG expression using qRT-PCR and human HCC cell lines to examine cell proliferation, colony formation, and glycolysis after transfection of siRNAs targeting miR4458HG or miR4458HG vectors. The molecular mechanism of miR4458HG was clarified through in situ hybridization, Western blotting, qRT-PCR, RNA pull-down, and RNA immunoprecipitation analysis. The results showed that the miR4458HG affected HCC cell proliferation, activated the glycolysis pathway, and promoted the polarization of tumor-associated macrophage in vitro and in vivo models. Mechanistically, miR4458HG bound IGF2BP2 (a key RNA m6A reader) and facilitated IGF2BP2-mediated target mRNA stability, including HK2 and SLC2A1 (GLUT1), and consequently altered HCC glycolysis and tumor cell physiology. At the same time, HCC-derived miR4458HG could be wrapped in the exosomes and promoted the polarization of tumor-associated macrophage by increasing ARG1 expression. Hence, miR4458HG is oncogenic in nature among patients with HCC. To develop an effective treatment strategy of HCC patients presenting with high glucose metabolism, physicians should focus on miR4458HG and its pathway.

Targeting Glutamine Metabolism with a Novel Na+/K+-ATPase Inhibitor RX108 in Hepatocellular Carcinoma.

The poor prognosis and limited therapeutic options for human hepatocellular carcinoma (HCC), the most common form of liver cancer, highlight the urgent need to identify novel therapeutic modalities. Here, we describe the antitumor activity and underlying molecular mechanisms of a novel Na+/K+-ATPase inhibitor RX108 in human HCC cells and its xenograft model. RX108 dose-dependently inhibited HCC cell proliferation in vitro and tumor growth in a xenograft mouse model, and that the inhibition was associated with induction of apoptosis. Mechanistically, RX108 significantly downregulated alanine serine cysteine transporter 2 (ASCT2) protein expression and reduced glutamine and glutamate concentration in HCC cells and tumors. In addition, RX108 exposure led to a significant decrease in cell energy metabolism in Huh7 and Hep3B cells, including decreased levels of glutathione, NADH, NADPH, and mitochondrial respiration oxygen consumption rate. Furthermore, HCC cells exhibited evidence of glutamine addiction; the antiproliferative effect of RX108 was dependent on glutamine transport. Clinically, elevated ASCT2 mRNA expression in HCCs was associated with unfavorable survival. Taken together, these findings reveal a novel approach to target glutamine metabolism through inhibiting Na+/K+-ATPase and provide a rationale for using RX108 to treat HCC in patients whose tumors express ASCT2 at high levels. RX108 is currently under clinical development.

Gut microbiota promote liver regeneration through hepatic membrane phospholipid biosynthesis

Hepatocyte growth and proliferation depends on membrane phospholipid biosynthesis. Short-chain fatty acids (SCFAs) generated by bacterial fermentation, delivered through the gut-liver axis, significantly contribute to lipid biosynthesis. We therefore hypothesized that dysbiotic insults like antibiotic treatment not only affect gut microbiota, but also impair hepatic lipid synthesis and liver regeneration. Stable isotope labeling and 70% partial hepatectomy (PHx) was carried out in C57Bl/6J wild-type mice, in mice treated with broad-spectrum antibiotics, in germ-free mice and mice colonized with minimal microbiota. The microbiome was analyzed by 16S rRNA gene sequencing and microbial culture. Gut content, liver, blood and primary hepatocyte organoids were tested by mass spectrometry-based lipidomics, quantitative reverse-transcription PCR (qRT-PCR), immunoblot and immunohistochemistry for expression of proliferative and lipogenic markers. Matched biopsies from hyperplastic and hypoplastic liver tissue of patients subjected to surgical intervention to induce hyperplasia were analyzed by qRT-PCR for lipogenic enzymes. Three days of antibiotic treatment induced persistent dysbiosis with significantly decreased beta-diversity and richness, but a massive increase of Proteobacteria, accompanied by decreased colonic SCFAs. After PHx, antibiotic-treated mice showed delayed liver regeneration, increased mortality, impaired hepatocyte proliferation and decreased hepatic phospholipid synthesis. Expression of the lipogenic enzyme SCD1 was upregulated after PHx but delayed by antibiotic treatment. Germ-free mice essentially recapitulated the phenotype of antibiotic treatment. Phospholipid biosynthesis, hepatocyte proliferation, liver regeneration and survival were rescued in gnotobiotic mice colonized with a minimal SCFA-producing microbial community. SCFAs induced the growth of murine hepatocyte organoids and hepatic SCD1 expression in mice. Further, SCD1 was required for proliferation of human hepatoma cells and was associated with liver regeneration in human patients. Gut microbiota are pivotal for hepatic membrane phospholipid biosynthesis and liver regeneration. Gut microbiota affect hepatic lipid metabolism through the gut-liver axis, but the underlying mechanisms are poorly understood. Perturbations of the gut microbiome, e.g. by antibiotics, impair the production of bacterial metabolites, which normally serve as building blocks for membrane lipids in liver cells. As a consequence, liver regeneration and survival after liver surgery is severely impaired. Even though this study is preclinical, its results might allow physicians in the future to improve patient outcomes after liver surgery, by modulation of gut microbiota or their metabolites.

Triptonide inhibits growth and metastasis in HCC by suppressing EGFR/PI3K/AKT signaling.

Liver cancer represents one of the deadliest cancers, with a rising incidence worldwide. Triptonide is found in the traditional Chinese medicinal plant Tripterygium wilfordii Hook. This study aimed to examine the anticancer properties of triptonide in human hepatocellular carcinoma (HCC). HCC cells were administered with triptonide at various levels, and CCK-8 and colony formation assays were carried out for detecting HCC cell proliferation. Then, cell apoptosis and cell cycle distribution were evaluated by flow cytometry. Tumor growth was monitored noninvasively by ultrasound imaging. Cell migration and invasion were quantitated by wound healing and Transwell assays. A metastasis model was established via tail vein injection of HCC cells in nude mice. Immunoblot was performed to quantitate the expression of proteins involved in the EGFR/PI3K/AKT signaling and its downstream effectors. Triptonide repressed cell proliferation and induced cell cycle arrest and apoptosis in cultured HCC cells, and suppressed tumor growth in vivo. In addition, triptonide inhibited EMT, migration and invasion in cultured HCC cells, and lung metastasis in nude mice. Mechanistically, triptonide acted by inhibiting the EGFR/PI3K/AKT signaling and regulated its downstream effectors, e.g., the cell cycle-associated protein cyclin D1, the apoptosis-related protein Bcl-2, the EMT marker E-cadherin, and the invasion-related protein MMP-9. Triptonide suppresses proliferation, EMT, migration and invasion, and promotes apoptosis and cell cycle arrest by repressing the EGFR/PI3K/AKT signaling. Therefore, triptonide might be considered for liver cancer treatment.

Quercetin, the Ingredient of Xihuang Pills, Inhibits Hepatocellular Carcinoma by Regulating Autophagy and Macrophage Polarization.

The key active component(s) in an anti-tumor preparation used in traditional Chinese medicine, Xihuang Pills, remains unclear. We used a network pharmacology analysis to construct a component-disease-target network diagram and used this to determine quercetin as a critical active ingredient in Xihuang Pills. Subsequently, human hepatocellular carcinoma (HCC) cell lines, H22 and HepG2 cells, were treated with quercetin, and BALB/c mice were injected with H22 cells and treated with different concentrations of quercetin. Tumor volume and weight were determined in these mice with and without quercetin administration. Immune and pro-inflammatory factors were measured using Enzyme Linked Immunosorbent Assay (ELISA). Macrophage polarization was assessed by western blot and flow cytometry. Finally, PD-L1, autophagy-related proteins, and the NF-κB pathway were also analyzed. Quercetin could significantly inhibit the proliferation, migration, and invasion characteristics of HCC cells and promote apoptosis in a concentration-dependent manner in vitro. After quercetin treatment, tumor volume and weight significantly decreased in vivo. Granulocyte-macrophage and granulocyte colony-stimulating factor (GM-CSF and G-CSF, respectively) levels were blunted in response to quercetin, as well as the PD-L1 level. CD86+ cell ratio was increased, while the CD206+ cell ratio was decreased, suggesting that macrophages tend to undergo M1 polarization in response to quercetin. The expression of LC3 II/I was increased, while the expression of p62 was down-regulated. The pro-inflammatory factors TNF-α, IL-6, and IL-17A, as well as NF-κB signaling were suppressed in a quercetin concentration-dependent manner. Quercetin is a key ingredient of anti-HCC activity in Xihuang Pills by regulating macrophage polarization and promoting autophagy via the NF-κB pathway.

MicroRNA 424-5p promotes the sensitivity of hepatocellular carcinoma cells to sorafenib by targeting Kinesin family member 23

Objective: To explore the effect of MicroRNA 424-5p/Kinesin family member 23(miR-424-5p/KIF23)axis on the malignant phenotype of hepatoma cells and its sensitivity of sorafenib. Methods: Real-time quantitative reverse PCR(qRT-PCR) and/or Western blot were used to detect the expression of miR-424-5p and KIF23 in liver cancer tissues and paracancerous tissues, human hepatocellular carcinoma(HCC) cells HepG2 and normal hepatocyte LO2. HepG2 cells transfected with miR-424-5p mimic and miR-424-5p mimic NC were respectively defined as miR-424-5p mimic group and mimic NC group, HepG2 cells transfected with KIF23 overexpression vector pcDNA3.1-KIF23 or empty vector pcDNA3.1 respectively were defined as OE-KIF23 group and Vector group, and HepG2 cells co-transfected with miR-424-5p mimic and overexpression vector pcDNA3.1-KIF23 were defined as mimic+OE-KIF23 group: The KIF23-3'UTR wild-type vector (KIF23-WT) and the mutant vector (KIF23-MT) were co-transfected with miR-424-5p micic and mimic NC, respectively, and the targeting relationship between miR-424-5p and KIF23 was verified by dual-luciferase reporting experiments. The cell counting Kit-8 (CCK-8) was used to detect HepG2 cell proliferation and sensitivity to sorafenib. Flow cytometry was used to assess apoptosis in HepG2 cells. Transwell and scratch experiments were used to detect HepG2 cell migration and invasion capabilities. Intergroup data were compared using t-tests or analysis of variance. Results: Compared with the paracancerous tissue and normal hepatocytes, miR-424-5p in the HCC tissue and hepatocellular cells was significantly down-regulated (the relative expression was 0.604±0.121, 0.585±0.064), and KIF23 was significantly up-regulated (the relative expression was 5.451±1.834, 2.482±0.545), P<0.05. miR-424-5p mimic can inhibit the proliferation, migration and invasion of HCC cells and promote apoptosis of HCC cells (P<0.05). Overexpression of KIF23 can promote the proliferation, migration and invasion of HCC cells and inhibit apoptosis of HCC cells (P<0.05). The luciferase activity of HepG2 cells in the mimic and KIF23-WT co-transfection groups was significantly reduced compared with HepG2 cells in the mimic NC and KIF23-WT co-transfection groups (the relative fluorescence intensities were 3.668±0.091 and 2.629±0.056, respectively, P<0.05),however, there was no significant comparison between the luciferase activity of cells in the mimic and KIF23-MT co-transfection groups compared with those in the mimic NC and KIF23-MT co-transfection groups. miR-424-5p mimic can reverse the role of overexpression of KIF23 in promoting the ability of HCC cells to proliferate, migrate and invade (P<0.05). The inhibition rates of sorafenib on HepG2 cells in the mimic+OE-KIF23 group and the OE-KIF23 group were 47.491%±3.863% and 36.246%±6.063% (t=3.027, P<0.05). Conclusion: miR-424-5p can inhibit the proliferation, migration and invasion of HCC cells and can increase the sensitivity of HCC cells to sorafenib by targeting the expression level of KIF23.

A novel nitidine chloride nanoparticle overcomes the stemness of CD133+EPCAM+ Huh7 hepatocellular carcinoma cells for liver cancer therapy

BackgroundStemness of CD133+EPCAM+ hepatocellular carcinoma cells ensures cancer resistance to apoptosis,which is a challenge to current liver cancer treatments. In this study, we evaluated the tumorcidal activity of a novel nanoparticle of nitidine chloride (TPGS-FA/NC, TPGS-FA: folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate, NC: nitidine chloride), against human hepatocellular carcinoma (HCC) cell line Huh7 growth in vitro and in vivo.MethodsHuh7 cells were treated with TPGS-FA/NC. Cell proliferation was assessed using MTT and colony assays. The expression of cell markers and signaling proteins was detected using western blot analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Huh7 cells. TPGS-FA/NC (7.5, 15, 30, 60, 120 μg/mL) dose-dependently inhibited the proliferation of HCC cells, which associated with a reduction in AQP3 and STAT3 expression. Importantly,TPGS-FA/NC (10, 20, and 40 μg/mL) significantly reduced the EpCAM+/CD133+cell numbers, suppressed the sphere formation. The in vivo antitumor efficacy of TPGS-FA/NC was proved in Huh7 cell xenograft model in BALB/c nude mice, which were administered TPGS-FA/NC(4 mg· kg − 1· d − 1, ig) for 2 weeks.ResultsTPGS-FA/NC dose-dependently suppressed the AQP3/STAT3/CD133 axis in Huh7 cells. In Huh7 xenograft bearing nude mice, TPGS-FA/NC administration markedly inhibited Huh7 xenograft tumor growth .ConclusionsTPGS-FA/NC inhibit HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the clinical therapy of hepatocellular carcinoma.

ZIC5 promotes human hepatocellular carcinoma cell proliferation through upregulating COL1A1.

Zinc finger of the cerebellum 5 (ZIC5) has been found to be abnormally expressed in a variety of tumors. This study aimed to reveal the expression and functional mechanism of ZIC5 in hepatocellular carcinoma (HCC). Analysis of ZIC5 expression in various tumors and its relationship with survival were derived from The Cancer Genome Atlas (TCGA) database. Cell counting kit-8 (CCK-8) and colony formation assays were performed for the detection of HCC cell proliferation. Differentially expressed genes (DEGs) after ZIC5 overexpression in Huh1 cells were determined by RNA sequencing. Western blot assays were conducted to detect the protein levels of c-Myc, Bcl2, p21, E-cadherin, N-cadherin, vimentin, and collagen type I alpha 1 (COL1A1). Dramatically increased expression of ZIC5 was observed in various tumor tissues, including HCC. Pearson's correlation analysis revealed that the mRNA levels of ZIC5 had a positive correlation with the mRNA levels of MKI67 in HCC tissues. Patients with high ZIC5 expression had a shorter overall survival time. Moreover, ZIC5 overexpression promoted HCC cell proliferation. Then, we found that COL1A1 was significantly upregulated by ZIC5 overexpression to promote the proliferation, migration, and invasion of HCC cells. ZIC5 could accelerate the proliferation, migration, and invasion of HCC cells through upregulating COL1A1. This is the first report that ZIC5 and COL1A1 are intrinsically linked, expanding new research ideas for subsequent HCC research.

KSR2-14\u20133-3\u03b6 complex serves as a biomarker and potential therapeutic target in sorafenib-resistant hepatocellular carcinoma

BackgroundKinase suppressor of Ras 2 (KSR2) is a regulator of MAPK signaling that is overactivated in most hepatocellular carcinoma (HCC). We sought to determine the role of KSR2 in HCC pathogenesis.MethodsWe tested the level of KSR2 in HCC tissues and cell lines by tissue microarray, qPCR, and western blotting. Functionally, we determined the effects of KSR2 on the proliferation, migration, and invasion of HCC cells through colony formation assays, scratch assays, transwell migration assays, and xenograft tumor models. Co-immunoprecipitation (co-IP) experiments were used to assess the interaction of phospho-serine binding protein 14–3-3ζ and KSR2, and the effects of this interaction on growth and proliferation of human HCC cells were tested by co-overexpression and knockdown experiments. Additionally, we used flow cytometry to examine whether the KSR2 and 14–3-3ζ interaction conveys HCC resistance to sorafenib.ResultsKSR2 was significantly upregulated in HCC tissues and cell lines, and high KSR2 expression associated with poor prognosis in HCC patients. KSR2 knockdown significantly suppressed HCC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, co-IP experiments identified that 14–3-3ζ complexed with KSR2, and elevated 14–3-3ζ increased KSR2 protein levels in HCC cells. Importantly, Kaplan–Meier survival analysis showed that patients with both high KSR2 and high 14–3-3ζ expression levels had the shortest survival times and poorest prognoses. Interestingly, HCC cells overexpressing both KSR2 and 14–3-3ζ, rather than either protein alone, showed hyperactivated MAPK signaling and resistance to sorafenib.ConclusionsOur results provide new insights into the pro-tumorigenic role of KSR2 and its regulation of the MAPK pathway in HCC. The KSR2–14–3-3ζ interaction may be a therapeutic target to enhance the sorafenib sensitivity of HCC.

Impaired degradation of YAP1 and IL6ST by chaperone-mediated autophagy promotes proliferation and migration of normal and hepatocellular carcinoma cells

ABSTRACT Impaired degradation of the transcriptional coactivator YAP1 and IL6ST (interleukin 6 cytokine family signal transducer), two proteins deregulated in liver cancer, has been shown to promote tumor growth. Here, we demonstrate that YAP1 and IL6ST are novel substrates of chaperone-mediated autophagy (CMA) in human hepatocellular carcinoma (HCC) and hepatocyte cell lines. Knockdown of the lysosomal CMA receptor LAMP2A increases protein levels of YAP1 and IL6ST, without changes in mRNA expression. Additionally, both proteins show KFERQ-dependent binding to the CMA chaperone HSPA8 and accumulate into isolated lysosomes after stimulation of CMA by prolonged starvation. We further show that LAMP2A downregulation promotes the proliferation and migration in HCC cells and a human hepatocyte cell line, and that it does so in a YAP1- and IL6ST-dependent manner. Finally, LAMP2A expression is downregulated, and YAP1 and IL6ST expression is upregulated, in human HCC biopsies. Taken together, our work reveals a novel mechanism that controls the turnover of two cancer-relevant proteins and suggests a tumor suppressor function of CMA in the liver, advocating for the exploitation of CMA activity for diagnostic and therapeutic purposes. Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; CMA: chaperone-mediated autophagy; eMI: endosomal microautophagy; HCC: hepatocellular carcinoma; HSPA8: heat shock protein family A (Hsp70) member 8; IL6ST: interleukin 6 cytokine family signal transducer; JAK: Janus kinase; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MAPK8: mitogen-activated protein kinase 8; P6: pyridine 6; SQSTM1: sequestosome 1; TUBA: tubulin alpha; VDAC1: voltage dependent anion channel 1; VP: verteporfin; YAP1: Yes1 associated transcriptional regulator.