Activity In Hepatocellular Carcinoma Cells Research Articles

SEPTIN10-mediated crosstalk between cytoskeletal networks controls mechanotransduction and oncogenic YAP/TAZ signaling

The transcriptional co-activators of the Hippo pathway, YAP and TAZ, are regulated by mechanotransduction, which depends on dynamic actin cytoskeleton remodeling. Here, we identified SEPTIN10 as a novel cytoskeletal protein, which is transcriptionally regulated by YAP/TAZ and whose overexpression correlates with poor survival and vascular invasion in hepatocellular carcinoma (HCC) patients. Functional characterization demonstrated that SEPTIN10 promotes YAP/TAZ-dependent cell viability, migration and invasion of liver cancer cells. Mechanistically, SEPTIN10 interacts with actin and microtubule filaments supporting actin stress fiber formation and intracellular tension through binding to CAPZA2 while concurrently inhibiting microtubule polymerization through the blockage of MAP4 function. This functional antagonism is important for cytoskeleton-dependent feedback activation of YAP/TAZ, as microtubule depolymerization induces actin stress fiber formation and subsequently YAP/TAZ activity. Importantly, the crosstalk between microfilaments and microtubules is mediated by SEPTIN10 as its loss abrogates actin stress fiber formation after microtubule disruption. Together, the YAP/TAZ target gene SEPTIN10 controls the dynamic interplay between actin and microtubule filaments, which feeds back on Hippo pathway activity in HCC cells and thus acts as molecular switch with impact on oncogenic signaling and cancer cell biology.

Lycorine inhibits migration and proliferation of hepatocellular carcinoma cells by reducing transketonase expression.

Background: Previous studies have showed that lycorine can restrain the development of multiple tumor types, containing hepatocellular carcinoma (HCC), but the underlying mechanisms remain unknown. Methods: We assessed the impact of lycorine on hepatocellular cancer cell proliferation, migration, colony formation, cell cycle, and apoptosis. The possible inhibitory effect of lycorine on the activity of HCC cells was analyzed by RNA-seq, and transketolase (TKT) expression in HCC and nontumorous tissues was detected using RT-PCR. The expression of TKT protein in HCC and tumor adjacent non-cancerous tissues was detected by immunohistochemistry. We evaluated the association of expression of TKT in HCC tissues with prognosis, and investigated the inhibitory effect of lycorine on tumor growth in vivo. Results: Lycorine significantly inhibited the proliferation, invasion, migration, colony formation, cell cycle of HCC cells, but had no obvious impact on apoptosis. Twenty-eight genes were found to be down-regulated in HuH7 and HepG2 cells after lycorine treatment, and the difference of TKT gene expression was significantly. The expression of TKT protein was significantly higher in HCC than in non-tumorous tissues. The expression of TKT was correlated with tumor size, Edmondson grade, AFP, and overall survival. Survival analysis suggested that high expression of TKT was associated with a poor survival. The average tumor volume and weight were significantly reduced in the lycorine injection group, but the body weights of the mice did not change significantly. Conclusion: Lycorine can restrict the migration and proliferation of HCC cells by down-regulating TKT expression, and it may be a potential meaningful drug for the prevention and treatment of HCC.

Norcantharidin-Encapsulated C60-Modified Nanomicelles: A Potential Approach to Mitigate Cytotoxicity in Renal Cells and Simultaneously Enhance Anti-Tumor Activity in Hepatocellular Carcinoma Cells.

The objective of this study was to examine the preparation process of DSPE-PEG-C60/NCTD micelles and assess the impact of fullerenol (C60)-modified micelles on the nephrotoxicity and antitumor activity of NCTD. The micelles containing NCTD were prepared using the ultrasonic method and subsequently optimized and characterized. The cytotoxicity of micelles loaded with NCTD was assessed using the CCK-8 method on human hepatoma cell lines HepG2 and BEL-7402, as well as normal cell lines HK-2 and L02. Acridine orange/ethidium bromide (AO/EB) double staining and flow cytometry were employed to assess the impact of NCTD-loaded micelles on the apoptosis of the HK-2 cells and the HepG2 cells. Additionally, JC-1 fluorescence was utilized to quantify the alterations in mitochondrial membrane potential. The generation of reactive oxygen species (ROS) following micelle treatment was determined through 2',7'-dichlorofluorescein diacetate (DCFDA) staining. The particle size distribution of the DSPE-PEG-C60/NCTD micelles was determined to be 91.57 nm (PDI = 0.231). The zeta potential of the micelles was found to be -13.8 mV. The encapsulation efficiency was measured to be 91.9%. The in vitro release behavior of the micelles followed the Higuchi equation. Cellular experiments demonstrated a notable decrease in the toxicity of the C60-modified micelles against the HK-2 cells, accompanied by an augmented inhibitory effect on cancer cells. Compared to the free NCTD group, the DSPE-PEG-C60 micelles exhibited a decreased apoptosis rate (12%) for the HK-2 cell line, lower than the apoptosis rate observed in the NCTD group (36%) at an NCTD concentration of 75 μM. The rate of apoptosis in the HepG2 cells exhibited a significant increase (49%), surpassing the apoptosis rate observed in the NCTD group (24%) at a concentration of 150 μM NCTD. The HK-2 cells exhibited a reduction in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM) upon exposure to C60-modified micelles compared to the NCTD group. The DSPE-PEG-C60/NCTD micelles, as prepared in this study, demonstrated the ability to decrease cytotoxicity and ROS levels in normal renal cells (HK-2) in vitro. Additionally, these micelles showed an enhanced antitumor activity against human hepatocellular carcinoma cells (HepG2, BEL-7402).

Tumor-derived exosomes promote macrophages M2 polarization through miR-1-3p and regulate the progression of liver cancer

Hepatic carcinoma is one of the most life-threatening malignancies in the world. In the clinic, it is urgent to establish a clear mechanism of hepatic carcinoma development as the basis for intervention and treatment. The purpose of this study was to explore the regulatory effect of tumor-derived exosomes on the progression of hepatocellular carcinoma.qPCR was used to detect the expression of miR-1–3p. CCk-8 and EdU staining were used to detect the proliferation and activity of hepatocellular carcinoma cells under different conditions. Transwell assay was used to detect migration and invasion of hepatocellular carcinoma cells. The morphology and size of exosomes were detected by transmission electron microscope and nanoparticle tracking analysis. Western blot was used to detect the expression of markers of exosomes. Immunofluorescence staining was used to explore the location of exosomes in hepatocellular carcinoma cells.The results showed that the expression of miR-1–3p was significantly reduced in hepatocellular carcinoma cells, and the exosomes transfected with miR-1–3p could enter macrophages and express miR-1–3p in large quantities. Macrophages polarized to M2 type under the action of miR-1–3p. Polarized M2 macrophages further down-regulated the proliferation, migration and invasion of Huh-7 cells.In summary, miR-1–3p can enter macrophages through exosomes and affect their polarization, thus affecting the growth of hepatic carcinoma cells. miR-1–3p may be a potentially effective target for regulating liver cancer progression.

Lipophilic statins inhibit YAP co-activator transcriptional activity in HCC cells through Rho-mediated modulation of the actin cytoskeleton.

Introduction: Hepatocellular carcinoma (HCC) is the third leading cause of liver-related death. Lipophilic statins have been associated with a decrease in HCC incidence, raising the possibility of their use as chemoprevention agents. The Yes associated protein (YAP) and Transcriptional co-Activator (TAZ) have emerged as an important pro-oncogenic mechanism in HCC. Statins modulate YAP/TAZ in other solid tumors but few studies have assessed their mechanisms in HCC. We aimed to delineate how lipophilic statins regulate YAP protein localization by interrogating the mevalonate pathway in a step-wise fashion using pharmacological and genetic approaches in HCC cells. Methods: Huh7 and Hep3B HCC cells were treated with the lipophilic statins cerivastatin and atorvastatin. YAP protein localization was determined using quantitative immunofluorescence (IF) imaging. The gene expression of CTGF and CYR61, known YAP/TEAD-regulated genes, were measured using quantitative real-time PCR. Rescue experiments were conducted using metabolites of the mevalonate pathway including mevalonic acid and geranylgeranyl pyrophosphate (GG-PP). The cellular cytoskeleton was assessed using F-actin IF staining. Results: YAP protein was extruded from the nucleus to the cytoplasm with statin treatment. Consistently, CTGF and CYR61 mRNA expression significantly decreased with statins. Cytoskeletal structure was also compromised with statins. Gene expression, YAP protein localization and cytoskeletal structure were all restored to baseline with exogenous GG-PP but not with other metabolites of the mevalonate pathway. Direct Rho GTPase inhibitor treatment mirrored the statin effects on YAP. Conclusions: YAP protein localization is regulated by lipophilic statins via Rho GTPases causing cytoskeletal structural changes and is independent of cholesterol metabolites.

USP8 positively regulates hepatocellular carcinoma tumorigenesis and confers ferroptosis resistance through β-catenin stabilization

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic carcinoma, which is a growing public health problem worldwide. One of the main genetic alterations in HCC is the deregulated Wnt/β-catenin signaling, activation of β-catenin is associated with the progression of HCC. In the present study, we aimed to identify novel modulators in controlling β-catenin ubiquitination and stability. USP8 was overexpressed in HCC tissues and correlated with β-catenin protein level. High expression of USP8 indicated poor prognosis of HCC patients. USP8 depletion significantly decreased β-catenin protein level, β-catenin target genes expression and TOP-luciferase activity in HCC cells. Further mechanistic study revealed that the USP domain of USP8 interacted with the ARM domain of β-catenin. USP8 stabilized β-catenin protein via inhibiting K48-specific poly-ubiquitination process on β-catenin protein. In addition, USP8 depletion inhibited the proliferation, invasion and stemness of HCC cells and conferred ferroptosis resistance, which effects could be further rescued by β-catenin overexpression. In addition, the USP8 inhibitor DUB-IN-3 inhibited the aggressive phenotype and promoted ferroptosis of HCC cells through degradation of β-catenin. Thus, our study demonstrated that USP8 activated the Wnt/beta-catenin signaling through a post-translational mechanism of β-catenin. High expression of USP8 promoted the progression and inhibited ferroptosis of HCC. Targeting the USP8 may serve as a promising strategy for patients with HCC.

Inhibition of CDK9 exhibits anticancer activity in hepatocellular carcinoma cells via targeting ribonucleotide reductase

Cyclin-dependent kinase 9 (CDK9) inhibitors are a novel category of anticancer treatment for cancers. However, their effects on hepatocellular carcinoma (HCC) are rarely investigated. Human ribonucleotide reductase (RR, which consists of RRM1 and RRM2 subunits) catalyzes the conversion of ribonucleoside diphosphate into 2′-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools, which play essential roles in DNA synthesis and DNA repair. In this study, we identified that CDK9 protein expression in adjacent non-tumor tissues predicted HCC patients' overall and progression-free survivals. The anticancer activity of a CDK9-selective inhibitor, LDC000067, on HCC cells was positively associated with its ability to inhibit the expression of RRM1 and RRM2. LDC000067 downregulated RRM1 and RRM2 expression through post-transcriptional pathway. Specifically, LDC000067 triggered RRM2 protein degradation via multiple pathways, including proteasome-, lysosome-, and calcium-dependent pathways. Furthermore, CDK9 positively correlates with RRM1 or RRM2 expression in HCC patients, and the expressions of these three genes were associated with the higher infiltration of immune cells in HCC. Taken together, this study identified the prognostic relevance of CDK9 in HCC and the molecular mechanism for the anticancer effect of CDK9 inhibitors on HCC.

HIGD2A silencing impairs hepatocellular carcinoma growth via inhibiting mitochondrial function and the MAPK/ERK pathway

BackgroundThe Hypoxia inducible gene domain family member 2A (HIGD2A) protein is indispensable for the assembly of the mitochondrial respiratory supercomplex, which has been implicated in cell proliferation and cell survival under hypoxic conditions. Because the liver has a naturally low oxygen microenvironment, the role of HIGD2A in the development of hepatocellular carcinoma (HCC) remains largely unknown.MethodsGene expression data and clinical information were obtained from multiple public databases. A lentivirus-mediated gene knockdown approach was conducted to explore the function and mechanism of HIGD2A activity in HCC cells. In vivo and in vitro assays were performed to investigate the biological roles of HIGD2A.ResultsHIGD2A was overexpressed in HCC tissues and cell lines and was associated with a worse prognosis. Silencing HIGD2A expression significantly attenuated cell proliferation and migration, caused S-phase cell cycle arrest, and decreased tumor formation in nude mice. Mechanistically, HIGD2A depletion greatly decreased cellular ATP levels by disrupting mitochondrial ATP production. Moreover, HIGD2A knockdown cells displayed impaired mitochondrial function, such as mitochondrial fusion, increased expression of the mitochondrial stress response protein, and decreased oxygen consumption. Furthermore, knockdown of HIGD2A markedly attenuated the activation of the MAPK/ERK pathway.ConclusionsHIGD2A promoted liver cancer cell growth by fueling mitochondrial ATP synthesis and activating the MAPK/ERK pathway, suggested that targeting HIGD2A may represent a new strategy for HCC therapy.

Sphingosine 1-phosphate receptor 2 promotes the onset and progression of non-alcoholic fatty liver disease-related hepatocellular carcinoma through the PI3K/AKT/mTOR pathway

PurposeRecent studies have revealed an increase in the incidence rate of non-alcoholic fatty liver disease-related hepatocellular carcinoma (NAFLD-HCC). Furthermore, the association of Sphingosine 1-phosphate receptor 2 (S1PR2) with various types of tumours is identified, and the metabolism of conjugated bile acids (CBAs) performs an essential function in the onset and development of HCC. However, the association of CBA and S1PR2 with NAFLD-HCC is unclear.MethodsThe relationship between the expression of S1PR2 and the prognosis of patients suffering from NAFLD-HCC was investigated by bioinformatics techniques. Subsequently, the relationship between S1PR2 and the biological behaviours of HCC cell lines Huh 7 and HepG2 was explored by conducting molecular biology assays. Additionally, several in vivo animal experiments were carried out for the elucidation of the biological impacts of S1PR2 inhibitors on HCC cells. Finally, We used Glycodeoxycholic acid (GCDA) of CBA to explore the biological effects of CBA on HCC cell and its potential mechanism.ResultsHigh S1PR2 expression was linked to poor prognosis of the NAFLD-HCC patients. According to cellular assay results, S1PR2 expression could affect the proliferation, invasion, migration, and apoptosis of Huh 7 and HepG2 cells, and was closely associated with the G1/G2 phase of the cell cycle. The experiments conducted in the In vivo conditions revealed that the overexpression of S1PR2 accelerated the growth of subcutaneous tumours. In addition, JTE-013, an antagonist of S1PR2, effectively inhibited the migration and proliferation of HCC cells. Furthermore, the bioinformatics analysis highlighted a correlation between S1PR2 and the PI3K/AKT/mTOR pathway.GCDA administration further enhanced the expression levels of p-AKT, p-mTOR, VEGF, SGK1, and PKCα. Moreover, both the presence and absence of GCDA did not reveal any significant change in the levels of S1PR2, p-AKT, p-mTOR, VEGF, SGK1, and PKCα proteins under S1PR2 knockdown, indicating that CBA may regulates the PI3K/AKT/mTOR pathway by mediating S1PR2 expression.ConclusionS1PR2 is a potential prognostic biomarker in NAFLD-HCC. In addition, We used GCDA in CBAs to treat HCC cell and found that the expression of S1PR2 was significantly increased, and the expression of PI3K/AKT/mTOR signalling pathway-related signal molecules was also significantly enhanced, indicating that GCDA may activate PI3K/AKT/mTOR signalling pathway by up-regulating the expression of S1PR2, and finally affect the activity of hepatocellular carcinoma cells. S1PR2 can be a candidate therapeutic target for NAFLD-HCC. Collectively, the findings of this research offer novel perspectives on the prevention and treatment of NAFLD-HCC.

Cyclin-Dependent Kinase 9 Inhibition as a Potential Treatment for Hepatocellular Carcinoma

Purpose: Composite cyclin-dependent kinase (CDK) inhibition has shown potential as a treatment for hepatocellular carcinoma (HCC) in preclinical studies. We tested whether the specific inhibition of CDK9 was effective against HCC. Methods: The effects of two specific CDK9 inhibitors, BAY1143572 and AZD4573, in HCC cell lines were examined. We tested the in vivo efficacy of CDK9 inhibition in mouse xenograft models of HuH7 human HCC cells and in an orthotopic model of BNL mouse HCC cells. Overexpression and knockdown of CDK9 were performed to confirm the efficacy of CDK9 inhibition. Results: CDK9 inhibitors exhibited potent antiproliferative activities in HCC cells regardless of the levels of c-myc expression while inhibiting the downstream signals of CDK9, such as the phosphorylation of RNA polymerase II. These 2 CDK9 inhibitors induced apoptosis in HCC cells and reduced the expression of antiapoptotic proteins such as myeloid cell leukemia-1 and survivin. In the xenograft studies, mice receiving either CDK9 inhibitor exhibited significantly slower tumor growth than did the mice receiving vehicles. In the orthotopic model, the HCC growth in mice receiving a CDK9 inhibitor also tended to be slower than that in the control group. Overexpression of CDK9 in HuH7 cells reduced the efficacy of both CDK9 inhibitors. Knockdown of CDK9 expression reduced the proliferative activities of HCC cells. Conclusion: We demonstrated the in vitro and in vivo activity of CDK9 inhibition on multiple HCC cell lines. Our data support further clinical development of CDK9 inhibitors as a treatment for HCC.

Downregulation of SETD5 Suppresses the Tumorigenicity of Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is an aggressive and incurable cancer. Although understanding of the molecular pathogenesis of HCC has greatly advanced, therapeutic options for the disease remain limited. In this study, we demonstrated that SETD5 expression is positively associated with poor prognosis of HCC and that SETD5 depletion decreased HCC cell proliferation and invasion while inducing cell death. Transcriptome analysis revealed that SETD5 loss downregulated the interferon-mediated inflammatory response in HCC cells. In addition, SETD5 depletion downregulated the expression of a critical glycolysis gene, PKM (pyruvate kinase M1/2), and decreased glycolysis activity in HCC cells. Finally, SETD5 knockdown inhibited tumor growth in xenograft mouse models. These results collectively suggest that SETD5 is involved in the tumorigenic features of HCC cells and that targeting SETD5 may suppress HCC progression.

VIPR1 promoter methylation promotes transcription factor AP-2α binding to inhibit VIPR1 expression and promote hepatocellular carcinoma cell growth in vitro

To explore the transcriptional regulation mechanism and biological function of low expression of vasoactive intestinal peptide receptor 1 (VIPR1) in hepatocellular carcinoma (HCC). We constructed plasmids carrying wild-type VIPR1 promoter or two mutant VIPR1 promoter sequences for transfection of the HCC cell lines Hep3B and Huh7, and examined the effect of AP-2α expression on VIPR1 promoter activity using dual-luciferase reporter assay. Pyrosequencing was performed to detect the changes in VIPR1 promoter methylation level in HCC cells treated with a DNA methyltransferase inhibitor (DAC). Chromatin immunoprecipitation was used to evaluate the binding ability of AP-2α to VIPR1 promoter. Western blotting was used to assess the effect of AP-2α knockdown on VIPR1 expression and examine the differential expression of VIPR1 in the two cell lines. The effects of VIPR1 overexpression and knockdown on the proliferation, cell cycle and apoptosis of HCC cells were analyzed using CCK8 assay and flow cytometry. We also observed the growth of HCC xenograft with lentivirus-mediated over-expression of VIPR1 in nude mice. Compared with the wild-type VIPR1 promoter group, co-transfection with the vector carrying two promoter mutations and the AP-2α-over-expressing plasmid obviously restored the luciferase activity in HCC cells (P < 0.05). DAC treatment of the cells significantly decreased the methylation level of VIPR1 promoter and inhibited the binding of AP-2α to VIPR1 promoter (P < 0.01). The HCC cells with AP-2α knockdown showed increased VIPR1 expression, which was lower in Huh7 cells than in Hep3B cells. VIPR1 overexpression in HCC cells caused significant cell cycle arrest in G2/M phase (P < 0.01), promoted cell apoptosis (P < 0.001), and inhibited cell proliferation (P < 0.001), while VIPR1 knockdown produced the opposite effects. In the tumor-bearing nude mice, VIPR1 overexpression in the HCC cells significantly suppressed the increase of tumor volume (P < 0.001) and weight (P < 0.05). VIPR1 promoter methylation in HCC promotes the binding of AP-2α and inhibits VIPR1 expression, while VIPR1 overexpression causes cell cycle arrest, promotes cell apoptosis, and inhibits cell proliferation and tumor growth.

The E3 ubiquitin ligase MG53 inhibits hepatocellular carcinoma by targeting RAC1 signaling

Ras-related C3 botulinum toxin substrate 1 (RAC1) overexpressiosn and hyperactivation are correlated with aggressive growth and other malignant characteristics in a wide variety of cancers including hepatocellular carcinoma (HCC). However, the regulatory mechanism of RAC1 expression and activation in HCC is not fully understood. Here, we demonstrated that E3 ubiquitin ligase MG53 (also known as tripartite motif 72, TRIM72) acted as a direct inhibitor of RAC1, and it catalyzed the ubiquitination of RAC1 and further inhibited RAC1 activity in HCC cells. Mechanistically, MG53 directly bound with RAC1 through its coiled-coil domain and suppressed RAC1 activity by catalyzing the Lys48 (K48)-linked polyubiquitination of RAC1 at Lys5 residue in HCC cells. We further demonstrated that MG53 significantly suppressed the malignant behaviors of HCC cells and enhanced the chemosensitivity of HCC cells to sorafenib treatment by inhibiting RAC1-MAPK signaling axis. In summary, we identified MG53 as a novel RAC1 inhibitor and tumor suppressor in HCC, and it suppressed HCC progression by inducing K48-linked polyubiquitination of RAC1 and further inhibiting the RAC1-MAPK signaling. Altogether, our investigation provided a new therapeutic strategy for RAC1 overactivated tumors by modulating MG53.

Iron chelator deferasirox inhibits NF-κB activity in hepatoma cells and changes sorafenib-induced programmed cell deaths

ObjectiveThe improvements of antitumor effects and tolerability on chemotherapy for advanced hepatocellular carcinoma (HCC) are warranted. Here, we aimed to elucidate the mechanism of the combining effect of tyrosine kinase inhibitor sorafenib (SOR) and iron chelator deferasirox (DFX) in human hepatoma cell lines, HepG2 and Huh-7. MethodsThe types of programmed cell deaths (PCDs); necrosis/necroptosis and apoptosis, were evaluated by flow cytometry and fluorescent microscopy. Human cleaved caspase-3 was analyzed by ELISA for apoptosis. GSH assay was used for ferroptosis. PCDs inhibition was analyzed by adding apoptosis inhibitor Z-VAD-FMK, ferroptosis inhibitor ferrostatin-1, necroptosis inhibitor necrosulfonamide, respectively. The expression of NF-κB was quantified by Western blotting. ResultsIn SOR monotherapy, cleaved caspase-3 expression was increased in all concentrations, confirming the result that SOR induces apoptosis. In SOR monotherapy, GSH/GSSG ratio was decreased on concentration-dependent, showing that SOR also induced ferroptosis. Lipid Peroxidation caused by SOR, corresponding to ferroptosis, was suppressed by DFX. In fluorescence microscopy of SOR monotherapy, apoptosis was observed at a constant rate on all concentrations, while necroptosis and ferroptosis were increased on high concentration. In sorafenib and deferasirox combinations, sub G1 phase increased additively. In SOR and DFX combinations, the cytotoxic effects were not suppressed by ferrostatin-1, but suppressed by Z-VAD-FMK and necrosulfonamide. In each monotherapy, and SOR + DFX combinations, the expression of NF-κB in nucleus was suppressed. Regarding PCD by SOR and DFX combination, ferroptosis was suppressed and both apoptosis and necroptosis became dominant. ConclusionSuppression of NF-κB is possibly involved in the effect of DFX. As a result, SOR and DFX combination showed additive antitumor effects for HCC through the mechanism of programed cell deaths and NF-kB signal modification.

Apigenin Increases Natural Killer Cytotoxicity to Human Hepatocellular Carcinoma Expressing HIF-1α through High Interaction of CD95/CD95L.

Natural killer (NK) cell activity is more attenuated in hepatocellular carcinoma (HCC) patients than normal. Hypoxic-inducible factor (HIF)-1α is highly expressed in tumors to maintain their metabolism in a hypoxic environment. The expression of HIF-1α in cancers can lead to cell growth, proliferation, invasion/metastasis and immune escape. Although apigenin, a flavonoid, is known to have various biological activities, it has not been demonstrated in NK cell immune activity in HCC cells. In this study, NK-92 cells were directly cocultured with HCC SK-Hep1 cells for 24 h to evaluate NK cell activity in HCC cells or HCC cells expressing HIF-1α by apigenin. NK cell cytotoxicity to HCC cells expressing HIF-1α was significantly increased, and NK cell-activating receptors, NKG2D, NKp30 and NKp44 were highly expressed. The activating effect of apigenin on NK cells substantially induced apoptosis in HCC cells expressing HIF-1α through high expression of CD95L on the surface of NK-92 cells. Moreover, apigenin excellently inhibited the level of TGF-β1 in a coculture of NK cells and HCC cells. In conclusion, apigenin seems to be a good compound that increases NK cell cytotoxicity to HCC cells by controlling HIF-1α expression.

Hydroxyacid Oxidase 2 (HAO2) Inhibits the Tumorigenicity of Hepatocellular Carcinoma and Is Negatively Regulated by miR-615-5p.

Background Hepatocellular carcinoma (HCC) is the sixth most common kind of cancer worldwide and the third leading cause of cancer mortality. Although a few studies have shown that hydroxyacid oxidase 2 (HAO2) may prevent HCC development, the molecular mechanism is unclear. Methods We examined the levels of HAO2 expression in 23 pairs of HCC/paracancerous tissues by quantitative real-time polymerase chain reaction (qRT-PCR) and evaluated HAO2's expression in The Cancer Genome Atlas (TCGA) database. Furthermore, we examined the biological activity of HAO2 utilizing cell-based functional assays. Additionally, we evaluated the relationship between miR-615-5p and HAO2 in Hep3B cells using a dual-luciferase reporter system and assessed the downstream regulatory mechanisms of miR-615-5p on HAO2. Finally, the nude mice tumor formation experiment was used to determine the impact of HAO2 on the tumorigenicity of HCC cells. Results HAO2 expression was considerably underexpression in HCC tissues and cells, and patients with low HAO2 expression had poorer disease-free survival. Inhibition of cell proliferation, migration, and invasion was observed when HAO2 was overexpressed. miR-615-5p had a negative relation with HAO2, and miR-615-5p restored HAO2's biological activity in HCC cells. Additionally, the tumor volume and weight were considerably reduced in the OV-HAO2 group compared to the OV-NC group. Conclusion HAO2 was found to be underexpressed in HCC tissues and cells, and HAO2 overexpression inhibited HCC cell motility, which was negatively regulated by miR-615-5p. Exogenous expression of HAO2 reduced the tumorigenicity of HCC cells in vivo in nude mice.

Inhibition of abnormally activated HIF-1α-GLUT1/3-glycolysis pathway enhances the sensitivity of hepatocellular carcinoma to 5-caffeoylquinic acid and its derivatives

Over the past few years, the antitumor activity exhibited by 5-caffeylquinic acid (5-CQA), especially its inhibitory effect on hepatocellular carcinoma (HCC) proliferation and metastasis, has been recognized as a new research hotspot. However, impacted by the weak antitumor toxicity of 5-CQA, its clinical application has been limited. In this study, the antitumor effect arising from 5-CQA on HCC cells was evaluated through cell viability assay. In addition, proteomics, flow cytometry, qRT-PCR and western blotting were adopted to investigate the drug resistance mechanism of HCC cells to 5-CQA. As indicated by the results, 5-CQA significantly inhibited the proliferation of HCC cell lines MHCC97H and HCCLM3 with IC5048 h of 546.8 μM and 452 μM, respectively. According to the in-depth studies, the abnormal activation of HIF-1α/glucose transporters/glycolysis pathway of 5-CQA could be a key molecular mechanism leading to drug resistance of HCC cells. Thus, this study found that glucose starvation, glucose analogue 2-DG, hexokinase inhibitor bromopyruvic acid and PKM2 inhibitor compound 3k inhibited HCC cell proliferation in synergy with 5-CQA. Furthermore, though the 5-CQA derivatives methyl chlorogenate (MCGA) and 3,5-dicaffeoylquinic acid (3,5-diCQA) exhibited more potent antiproliferation activity in HCC cells than 5-CQA, they also up-regulated the expression of GLUT1/3, whereas they had no effect on hepatocytes. To be specific, under low-glucose culture conditions, the order of sensitivity of HCC cells to CQAs was 3,5-diCQA > MCGA > 5-CQA. In brief, the above results revealed that intervention in glucose metabolism can facilitate the effects of 5-CQA and its derivatives for treating HCC.

Signaling by the tyrosine kinase Yes promotes liver cancer development.

Most patients with hepatocellular carcinoma (HCC) are diagnosed at a late stage and have few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or a dominant oncogene that can be targeted pharmacologically, unlike in other cancer types. Here, we report the identification of a previously uncharacterized oncogenic signaling pathway in HCC that is mediated by the tyrosine kinase Yes. Using genetic and pharmacological interventions in cellular and mouse models of HCC, we showed that Yes activity was necessary for HCC cell proliferation. Transgenic expression of activated Yes in mouse hepatocytes was sufficient to induce liver tumorigenesis. Yes phosphorylated the transcriptional coactivators YAP and TAZ (YAP/TAZ), promoting their nuclear accumulation and transcriptional activity in HCC cells and liver tumors. We also showed that YAP/TAZ were effectors of the Yes-dependent oncogenic transformation of hepatocytes. Src family kinase activation correlated with the tyrosine phosphorylation and nuclear localization of YAP in human HCC and was associated with increased tumor burden in mice. Specifically, high Yes activity predicted shorter overall survival in patients with HCC. Thus, our findings identify Yes as a potential therapeutic target in HCC.

Heat shock protein A12A activates migration of hepatocellular carcinoma cells in a monocarboxylate transporter 4-dependent manner.

Metastasis is responsible for most of the hepatocellular carcinoma (HCC)-associated death. However, its underlying mechanism has yet to be fully elucidated. Glycolysis-derived lactate has been shown to be a powerful regulator of cancer metastasis. Heat shock protein A12A (HSPA12A) encodes a novel member of HSP70 family. We have recently demonstrated that heat shock protein A12A (HSPA12A) inhibited renal cancer cell migration by suppressing lactate output and glycolytic activity, which were mediated by unstabilizing CD147 and promoting its degradation. By striking contrast, here we demonstrated that HSPA12A promoted migration of human HCC cells. Extracellular acidification, lactate export, and glycolytic activity in HCC cells were also promoted following HSPA12A overexpression. Further analysis revealed that HSPA12A interacted with MCT4 and increased its membrane localization, thereby promoting export of lactate generated from glycolysis; this led, ultimately, to HCC cell migration. Our results revealed the opposite effect of HSPA12A on migration of renal cancer cells and that of HCC cells. Of note, in contrast to the inhibitory effect on CD147 expression in renal cancer cells, we found that HSPA12A increased CD147 expression in HCC cells, indicating that the expression of CD147 might exist heterogeneity in different cancer cell types. Taken together, we identified HSPA12A as an activator of HCC migration, a role opposite to that of renal cancer cells. Inhibiting HSPA12A might be a potential therapeutic intervention for HCC metastasis.

UDCA Inhibits Hypoxic Hepatocellular Carcinoma Cell-Induced Angiogenesis Through Suppressing HIF-1α/VEGF/IL-8 Intercellular Signaling.

Background: A hypoxic microenvironment may induce angiogenesis and promote the development of hepatocellular carcinoma (HCC). The aim of this study was to evaluate whether ursodeoxycholic acid (UDCA) may inhibit hypoxic HCC cell–induced angiogenesis and the possible mechanisms. Methods: Tube formation and matrigel plug angiogenesis assays were used to evaluate angiogenesis in vitro and in vivo, respectively. Real-time PCR, enzyme-linked immunosorbent assay, and Western blot were used to evaluate the mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and IL-8, respectively. Dual-luciferase reporter assay was applied to assess the reporter gene expression of hypoxia-response element (HRE). Results: UDCA antagonized hypoxic Huh 7 cell-induced tube formation of EA.hy 926 cells. In HCC cells, UDCA inhibited hypoxia-induced upregulation of VEGF and IL-8 both in mRNA and protein levels. UDCA also inhibited IL-8–induced angiogenesis in vitro and in vivo through suppressing IL-8–induced phosphorylation of ERK. The levels of HIF-1α mRNA and protein and HRE-driven luciferase activity in HCC cells were upregulated by hypoxia and were all inhibited by UDCA. The proteasome inhibitor MG132 antagonized the effect of UDCA on HIF-1α degradation. In hypoxic condition, the phosphorylation of ERK and AKT was obviously increased in HCC cells, which was suppressed by UDCA. Transfection of the HIF-1α overexpression plasmid reversed the effects of UDCA on hypoxic HCC cell–induced angiogenesis, HRE activity, and expressions of IL-8 and VEGF. Conclusions: Our results demonstrated that UDCA could inhibit hypoxic HCC cell–induced angiogenesis through suppressing HIF-1α/VEGF/IL-8–mediated intercellular signaling between HCC cells and endothelial cells.