Hepatocellular Carcinoma Hep3B Cells Research Articles

Differential effects of choline on TLR2/4 mediated signaling through possible regulation of toll-interacting protein in hepatocellular carcinoma cell lines

Abstract Objectives Toll-like receptor (TLR) mediated inflammatory status plays an important role in development and progression of hepatocellular carcinoma (HCC). Toll-interacting protein (TOLLIP) has an inhibitory effect on TLR-mediated inflammatory signalling and expression profile of TOLLIP varies between malignancies including HCC. Cholinergic anti-inflammatory pathway (CAP) is an endogenous mechanism that controls inflammatory status via α7nicotinic acetylcholine receptors (α7nAChR). This study aims to investigate the effect of CAP-acting agent choline on TOLLIP and its related TLR-mediated inflammatory response in HCC cells with distinct differentiation stages. Methods The expression patterns of α7nAChR, TLR2/4, TOLLIP, IL6,NFkB genes were evaluated by RT-PCR and ELISA in the presence of choline, along with the real-time cell proliferation and migration in HEP3B and SNU449 HCC cell lines. The interaction between choline and TOLLIP assessed by using in-silico analyses. Results Choline downregulated TOLLIP in Hep3B and SNU449 cells. However, the expressions of α7nAChR, NF-κB, IL-6, TLR2 and TLR4 showed a decreased pattern in well differentiated HEP3B cells, while an increased pattern in poorly differentiated SNU449 cells. Conclusions Choline might exert differential effects in TLR2/4-dependent signalling based on the differentiation stages of the HCC cells, suggesting its potential therapeutic effects in earlier stages of HCC which might be result of its partial modulation of TOLLIP.

ULK1 Mediated Autophagy-Promoting Effects of Rutin-Loaded Chitosan Nanoparticles Contribute to the Activation of NF-κB Signaling Besides Inhibiting EMT in Hep3B Hepatoma Cells.

Liver cancer remains to be one of the leading causes of cancer worldwide. The treatment options face several challenges and nanomaterials have proven to improve the bioavailability of several drug candidates and their applications in nanomedicine. Specifically, chitosan nanoparticles (CNPs) are extremely biodegradable, pose enhanced biocompatibility and are considered safe for use in medicine. CNPs were synthesized by ionic gelation, loaded with rutin (rCNPs) and characterized by ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) and transmission electron microscopy (TEM). The rCNPs were tested for their cytotoxic effects on human hepatoma Hep3B cells, and experiments were conducted to determine the mechanism of such effects. Further, the biocompatibility of the rCNPs was tested on L929 fibroblasts, and their hemocompatibility was determined. Initially, UV-vis and FTIR analyses indicated the possible loading of rutin on rCNPs. Further, the rutin load was quantitatively measured using Ultra-Performance Liquid Chromatography (UPLC) and the concentration was 88 µg/mL for 0.22 micron filtered rCNPs. The drug loading capacity (LC%) of the rCNPs was observed to be 13.29 ± 0.68%, and encapsulation efficiency (EE%) was 19.55 ± 1.01%. The drug release was pH-responsive as 88.58% of the drug was released after 24 hrs at the lysosomal pH 5.5, whereas 91.44% of the drug was released at physiological pH 7.4 after 102 hrs. The cytotoxic effects were prominent in 0.22 micron filtered samples of 5 mg/mL rutin precursor. The particle size for the rCNPs at this concentration was 144.1 nm and the polydispersity index (PDI) was 0.244, which is deemed to be ideal for tumor targeting. A zeta potential (ζ-potential) value of 16.4 mV indicated rCNPs with good stability. The IC50 value for the cytotoxic effects of rCNPs on human hepatoma Hep3B cells was 9.7 ± 0.19 μg/mL of rutin load. In addition, the increased production of reactive oxygen species (ROS) and changes in mitochondrial membrane potential (MMP) were observed. Gene expression studies indicated that the mechanism for cytotoxic effects of rCNPs on Hep3B cells was due to the activation of Unc-51-like autophagy-activating kinase (ULK1) mediated autophagy and nuclear factor kappa B (NF-κB) signaling besides inhibiting the epithelial-mesenchymal Transition (EMT). In addition, the rCNPs were less toxic on NCTC clone 929 (L929) fibroblasts in comparison to the Hep3B cells and possessed excellent hemocompatibility (less than 2% of hemolysis). The synthesized rCNPs were pH-responsive and possessed the physicochemical properties suitable for tumor targeting. The particles were effectively cytotoxic on Hep3B cells in comparison to normal cells and possessed excellent hemocompatibility. The very low hemolytic profile of rCNPs indicates that the drug could be administered intravenously for cancer therapy.

Discovery of 6-Formylpyridyl Urea Derivatives as Potent Reversible-Covalent Fibroblast Growth Factor Receptor 4 Inhibitors with Improved Anti-Hepatocellular Carcinoma Activity.

Fibroblast growth factor receptor 4 (FGFR4) has been considered as a potential anticancer target due to FGF19/FGFR4 mediated aberrant signaling in hepatocellular carcinoma (HCC). Several FGFR4 inhibitors have been reported, but none have gained approval. Herein, a series of 5-formyl-pyrrolo[3,2-b]pyridine-3-carboxamides and a series of 6-formylpyridyl ureas were characterized as selective reversible-covalent FGFR4 inhibitors. The representative 6-formylpyridyl urea 8z exhibited excellent potency against FGFR4WT, FGFR4V550L, and FGFR4V550M with IC50 values of 16.3, 12.6, and 57.3 nM, respectively. It also potently suppressed proliferation of Ba/F3 cells driven by FGFR4WT, FGFR4V550L, and FGFR4V550M, and FGFR4-dependent Hep3B and Huh7 HCC cells, with IC50 values of 1.2, 13.5, 64.5, 15.0, and 20.4 nM, respectively. Furthermore, 8z displayed desirable microsomal stability and significant in vivo efficacy in the Huh7 HCC cancer xenograft model in nude mice. The study provides a promising new lead for anticancer drug discovery directed toward overcoming FGFR4 gatekeeper mutation mediated resistance in HCC patients.

Nrf2 Regulates the Expression of CYP2D6 by Inhibiting the Activity of Krüppel-Like Factor 9 (KLF9).

The aim of the present study is to gain insight into the biology of Parkinson's disease (PD) and cancer to drive translational advances enabling more effective prevention and/or potential treatments. The expression of Cytochrome P450 2D6 (CYP2D6) is correlated with various diseases such as PD and cancer; therefore, exploring its regulatory mechanism at transcriptional levels is of interest. NF-E2-related factor 2 (Nrf2) has been known to be responsible for regulating phase II and phase III drug-metabolizing genes. The objectives of this study are to investigate the transcriptional regulation of CYP2D6 by Nrf2 and to analyze its role in PD and cancer. Nrf2 was transiently expressed in human hepatoma Hep3B cells, and the expression of CYP2D6 was examined by RT-qPCR. The promoter activity of CYP2D6 and the DNA binding of Nrf2 were examined by luciferase and ChIP assay, respectively. We then investigated the expression and correlation of Nrf2 and CYP2D6 in the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. In the present study, we demonstrated that Nrf2 down-regulated CYP2D6 mRNA expression in hepatoma Hep3B cells. Mechanistically, Nrf2 binds to the antioxidant responsive element (ARE) in the proximity of krüppel- like factor 9 (KLF9)-binding site within the -550/+51 of CYP2D6 promoter. The inhibition and activation of Nrf2 enhanced and suppressed KLF9 effects on CYP2D6 expression, respectively. The expression levels of Nrf2 and CYP2D6 were upregulated and downregulated in the PD patient GEO datasets compared to the healthy control tissues, and Nrf2 was negatively correlated with CYP2D6. In liver cancer patients, decreased CYP2D6 levels were apparent and associated with a lower probability of survival. Our work revealed the inhibitory role of Nrf2 in regulating CYP2D6 expression. Moreover, Nrf2- dependent regulation of CYP2D6 can be used as a prognostic factor and therapeutic strategy in PD and liver cancer.

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.

Acylated iridoid glucoside and xanthones from Canscora lucidissima: Their structures and ferroptosis inhibitory activity

Phytochemical study on the whole plants of a Gentianaceous medicinal plant, Canscora lucidissima, gave one new acylated iridoid glucoside, canscorin A (1), and two new xanthone glycosides (2 and 3) together with 17 known compounds including five xanthones, eight xanthone glycosides, two benzophenone glucosides, caffeic acid, and loganic acid. Canscorin A (1) was assigned as a loganic acid derivative having a hydroxyterephthalic acid moiety by spectroscopic analysis together with chemical evidence, while 2 and 3 were elucidated to be a rutinosylxanthone and a glucosylxanthone, respectively. The absolute configurations of the sugar moieties of 2 and 3 were determined by HPLC analysis. The isolated compounds were evaluated for their inhibitory activities against erastin-induced ferroptosis on human hepatoma Hep3B cells and LPS-stimulated IL-1β production from murine microglial cells.

Auranofin accelerates spermidine-induced apoptosis via reactive oxygen species generation and suppression of PI3K/Akt signaling pathway in hepatocellular carcinoma

Auranofin is a US Food and Drug Administration (FDA)-approved anti-arthritis medication that functions as a thioredoxin reductase inhibitor. Spermidine, a polyamine present in marine algae, can exert various physiological functions. Herein, we examined the synergistic anticancer activity of auranofin and spermidine in hepatocellular carcinoma (HCC). Combined treatment with auranofin and spermidine suppressed cell viability more efficiently than either treatment alone in HCC Hep3B cells. The isobologram plotted by calculating the half maximal inhibitory concentration (IC50) values of each drug indicated that the two drugs exhibited a synergistic effect. Based on the analysis of annexin V and cell cycle distribution, auranofin and spermidine markedly induced apoptosis in Hep3B cells. Moreover, auranofin and spermidine increased mitochondria-mediated apoptosis by promoting mitochondrial membrane potential (Δψm) loss. Auranofin and spermidine significantly increased reactive oxygen species (ROS) production in Hep3B cells, and the blocking ROS suppressed apoptosis induced by spermidine and auranofin. In addition, auranofin and spermidine reduced the expression of phosphorylated phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt), and PI3K inhibitor accelerated auranofin- and spermidine-induced apoptosis. Using ROS scavenger and PI3K inhibitor, we revealed that ROS acts upstream of auranofin- and spermidine-induced apoptosis. Collectively, our study suggests that combination treatment with auranofin and spermidine could afford synergistic anticancer activity via ROS overproduction and reduced PI3K/Akt signaling pathway.

Circadian regulator BMAL1::CLOCK promotes cell proliferation in hepatocellular carcinoma by controlling apoptosis and cell cycle

Hepatocellular carcinoma (HCC) remains a global health challenge whose incidence is growing worldwide. Previous evidence strongly supported the notion that the circadian clock controls physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Despite the progress, cellular and molecular mechanisms underpinning this HCC-clock crosstalk remain unknown. Addressing this knowledge gap, we show here that although the human HCC cells Hep3B, HepG2, and Huh7 displayed variations in circadian rhythm profiles, all cells relied on the master circadian clock transcription factors, BMAL1 and CLOCK, for sustained cell growth. Down-regulating Bmal1 or Clock in the HCC cells induced apoptosis and arrested cell cycle at the G2/M phase. Mechanistically, we found that inhibiting Bmal1/Clock induced dysregulation of the cell cycle regulators Wee1 and p21 which cooperatively contribute to tumor cell death. Bmal1/Clock knockdown caused downregulation of Wee1 that led to apoptosis activation and upregulation of p21 which arrested the cell cycle at the G2/M phase. Collectively, our results suggest that the circadian clock regulators BMAL1 and CLOCK promote HCC cell proliferation by controlling Wee1 and p21 levels, thereby preventing apoptosis and cell cycle arrest. Our findings shed light on cellular impact of the clock proteins for maintaining HCC oncogenesis and provide proof-of-principle for developing cancer therapy based on modulation of the circadian clock.

Green synthesis of fluorescent carbon nanodots from sage leaves for selective anticancer activity on 2D liver cancer cells and 3D multicellular tumor spheroids.

Carbon nanodots, a family of carbon-based nanomaterials, have been synthesized through different methods from various resources, affecting the properties of the resulting product and their application. Herein, carbon nanodots (CNDs) were synthesized with a green and simple hydrothermal method from sage leaves at 200 °C for 6 hours. The obtained CNDs are well dispersed in water with a negative surface charge (ζ-potential = -11 mV) and an average particle size of 3.6 nm. The synthesized CNDs showed concentration-dependent anticancer activity toward liver cancer (Hep3B) cell lines and decreased the viability of the cancer cells to 23% at the highest used concentration (250 μg ml-1 of CNDs). More interestingly, the cytotoxicity of the CNDs was tested in normal liver cell lines (LX2) revealed that the CNDs at all tested concentrations didn't affect their viability including at the highest concentration showing a viability of 86.7%. The cellular uptake mechanisms of CNDs were investigated and they are thought to be through energy-dependent endocytosis and also through passive diffusion. The main mechanisms of endocytosis were lipid and caveolae-mediated endocytosis. In addition, the CNDs have hindered the formation of 3D spheroids from the Hep3B hepatocellular carcinoma cell line. Hence, it would be concluded that the synthesized CNDs from sage are more highly selective to liver cancer cells than normal ones. The CNDs' cancer-killing ability would be referred to as the production of reactive oxygen species.

Transcriptome analysis revealed the role of mTOR and MAPK signaling pathways in the white strain of Hypsizygus marmoreus extracts-induced cell death of human hepatoma Hep3B cells.

The aim of this study was to investigate the anticancer mechanisms of white genius mushroom (WGM). WGM is a popular edible mushroom in Taiwan and has been demonstrated to mediate potent antiproliferation effects against human Hep3B liver cancer cells in our previous study. According to next generation sequencing technology and KEGG pathway enrichment analysis, mTOR and MAPK signaling pathways were markedly changed during treatment with WGM extracts in Hep3B cells. Therefore, this study examined the effects of WGM extracts on the expression of mTOR and MAPK signaling pathway-related proteins, such as PI3K, Akt, mTOR, Ras, Raf, MEK, ERK, p38 and JNK in Hep3B cells. According to the results of immunoblotting, we demonstrated that the protein expression of the members of PI3K/Akt/mTOR and MAPK signaling pathways were involved in WGM extracts-induced cell death. Furthermore, the inhibitors of PI3K/Akt/mTOR and MAPK signaling pathways such as rapamycin, MK2206, LY3214996 and SB202190, blocked the induction of cell death and vacuoles formation induced by WGM extracts. This study also demonstrated that WGM extracts is able to inhibit Hep3B cell migration and colony formation in a dose-dependent manner. In addition to being a very popular food, WGM should be a pharmacologically safe natural agent for cancer treatment. Therefore, WGM might be designed to develop into a dietary chemopreventive agent for the cancer treatment.

Design, Synthesis, and Biological Evaluation of 5-Formyl-pyrrolo[3,2-b]pyridine-3-carboxamides as New Selective, Potent, and Reversible-Covalent FGFR4 Inhibitors.

The FGF19-FGFR4 signaling pathway has been extensively studied as a promising target for the treatment of hepatocellular carcinoma (HCC). Several FGFR4-selective inhibitors have been developed, but none of them receives approval. Additionally, acquired resistance caused by FGFR4 gatekeeper mutations is emerging as a serious limitation for these targeted therapies. Herein, we report a novel series of 5-formyl-pyrrolo[3,2-b]pyridine derivatives as new reversible-covalent inhibitors targeting wild-type and gatekeeper mutant variants of FGFR4 kinase. The representative compound 10z exhibited single-digit nanomolar activity against wild-type FGFR4 and the FGFR4V550L/M mutant variants in biochemical and Ba/F3 cellular assays, while sparing FGFR1/2/3. Furthermore, 10z showed significant antiproliferative activity against Hep3B, JHH-7, and HuH-7 HCC cells with IC50 values of 37, 32, and 94 nM, respectively. MALDI-TOF-MS and X-ray protein crystallography studies were consistent with 10z acting as a reversible-covalent inhibitor of FGFR4, serving as a promising lead compound for further anticancer drug development.

Multi-Omics Analysis Revealed a Significant Alteration of Critical Metabolic Pathways Due to Sorafenib-Resistance in Hep3B Cell Lines.

Hepatocellular carcinoma (HCC) is the second prominent cause of cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, wherein sorafenib, a multiple target tyrosine kinase inhibitor, is used as the first line of treatment. Unfortunately, resistance to sorafenib is usually encountered within six months of treatment. Therefore, there is a critical need to identify the underlying reasons for drug resistance. In the present study, we investigated the proteomic and metabolomics alterations accompanying sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) library was used to identify the differentially abundant metabolites through MetaboScape 4.0 software (Bruker). For protein annotation and identification, the Uniprot proteome for Homo sapiens (Human) database was utilized through MaxQuant. The results revealed that 27 metabolites and 18 proteins were significantly dysregulated due to sorafenib resistance in Hep3B cells compared to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and calpain small subunit 1 were among the significantly altered proteins. The findings revealed that resistant Hep3B cells demonstrated significant alterations in amino acid and nucleotide metabolic pathways, energy production pathways and other pathways related to cancer aggressiveness, such as migration, proliferation and drug-resistance. Joint pathway enrichment analysis unveiled unique pathways, including the antifolate resistance pathway and other important pathways that maintain cancer cells’ survival, growth, and proliferation. Collectively, the results identified potential biomarkers for sorafenib-resistant HCC and gave insights into their role in chemotherapeutic drug resistance, cancer initiation, progression and aggressiveness, which may contribute to better prognosis and chemotherapeutic outcomes.

Lenvatinib Synergistically Promotes Radiation Therapy in Hepatocellular Carcinoma by Inhibiting Src/STAT3/NF-κB-Mediated Epithelial–Mesenchymal Transition and Metastasis

This study suggested that lenvatinib may incapacitate hepatocellular carcinoma (HCC) to radiation treatment by abrogating radiation-induced Src/signal transducer and the activator of transcription 3 signaling (STAT3)/nuclear factor-κB (NF-κB) to escalate radiation-induced extrinsic and intrinsic apoptosis. These findings uncover the role of targeting Src and its arbitrating epithelial-mesenchymal transition (EMT), which could increase the anti-HCC efficacy of radiation therapy (RT). Lenvatinib and sorafenib are multikinase inhibitors used to treat HCC. Lenvatinib is noninferior to sorafenib in the therapeutic response in HCC. However, whether lenvatinib intensifies the anti-HCC efficacy of RT is ambiguous. Several oncogenic kinases and transcription factors, such as Src, STAT3, and NF-κB, enhance the radiosensitivity of cancers. Therefore, we aimed to investigate the roles of the Src/STAT3/NF-κB axis in HCC after RT treatment and assessed whether targeting Src by lenvatinib may enhance the effectiveness of RT. Hep3B, Huh7, HepG2, and SK-Hep1 HCC cells and 2 types of animal models were used to identify the efficacy of RT combined with lenvatinib. Cellular toxicity, apoptosis, DNA damage, EMT/metastasis regulation, and treatment efficacy were validated by colony formation, flow cytometry, Western blotting, and in vivo experiments, respectively. Knockdown of Src by siRNA was also used to validate the role of Src in RT treatment. Silencing Src reduced STAT3/NF-κB signaling and sensitized HCC to radiation. Lenvatinib reversed radiation-elicited Src/STAT3/NF-κB signaling while enhancing the anti-HCC efficacy of radiation. Both lenvatinib and siSrc promoted the radiation effect of cell proliferation on suppression, inhibition of the invasion ability, and induction of apoptosis in HCC. Lenvatinib also alleviated radiation-triggered oncogenic and EMT-related protein expression. Our findings uncovered the role of the Src/STAT3/NF-κB regulatory axis in response to radiation-induced toxicity and confirmed Src as the key regulatory molecule for radiosensitization of HCC evoked by lenvatinib.

Combination Therapy Using Polyphenols: An Efficient Way to Improve Antitumoral Activity and Reduce Resistance.

Polyphenols represent a structural class of mainly natural organic chemicals that contain multiple phenol structural units. The beneficial properties of polyphenols have been extensively studied for their antitumor, anti-inflammatory, and antibacterial effects, but nowadays, their medical applications are starting to be extended to many other applications due to their prebiotic role and their impact on the microbiota. This review focused on the use of polyphenols in cancer treatment. Their antineoplastic effects have been demonstrated in various studies when they were tested on numerous cancer lines and some in in vivo models. A431 and SCC13 human skin cancer cell lines treated with EGCG presented a reduced cell viability and enhanced cell death due to the inactivation of β-catenin signaling. Additionally, resveratrol showed a great potential against breast cancer mainly due to its ability to exert both anti-estrogenic and estrogenic effects (based on the concentration) and because it has a high affinity for estrogen receptors ERα and Erβ. Polyphenols can be combined with different classical cytostatic agents to enhance their therapeutic effects on cancer cells and to also protect healthy cells from the aggressiveness of antitumor drugs due to their anti-inflammatory properties. For instance, curcumin has been reported to reduce the gastrointestinal toxicity associated with chemotherapy. In the case of 5-FU-induced, it reduced the gastrointestinal toxicity by increasing the intestinal permeability and inhibiting mucosal damage. Co-administration of EGCG and doxorubicin induced the death of liver cancer cells. EGCG has the ability to inhibit autophagic activity and stop hepatoma Hep3B cell proliferation This symbiotic approach is well-known in medical practice including in multiple chemotherapy.

Circular dorsal ruffles disturb the growth factor-induced PI3K-AKT pathway in hepatocellular carcinoma Hep3B cells

BackgroundCircular dorsal ruffles (CDRs) are rounded membrane ruffles induced on the dorsal surfaces of cells stimulated by growth factors (GF). They can serve as signal platforms to activate AKT protein kinase. After GF stimulation, phosphatidylinositol 3-kinase (PI3K) generates phosphatidylinositol (3,4,5)-triphosphate (PIP3) in the plasma membrane. PIP3 accumulates inside CDRs, recruits AKT into the structures, and phosphorylates them (pAKT). Given the importance of the PI3K-AKT pathway in GF signaling, CDRs are likely involved in cell growth. Interestingly, some cancer cell lines express CDRs. We hypothesized that CDRs contribute to carcinogenesis by modulating the AKT pathway. In the present study, we identified CDR-expressing cancer cell lines and investigated their cellular functions.MethodsCDR formation was examined in six cancer cell lines in response to epidermal growth factor (EGF) and insulin. The morphology of the CDRs was characterized, and the related signaling molecules were observed using confocal and scanning electron microscopy. The role of CDRs in the AKT pathway was studied using biochemical analysis. The actin inhibitor cytochalasin D (Cyto D) and the PI3K inhibitor TGX221 were used to block CDRs.ResultsGF treatment induced CDRs in the hepatocellular carcinoma (HCC) Hep3B cell line, but not in others, including HCC cell lines HepG2 and Huh7, and the LO2 hepatocyte cell line. Confocal microscopy and western blot analysis showed that the PI3K-PIP3-AKT pathway was activated at the CDRs and that receptor proteins were recruited to the structures. Cyto D and TGX221 completely blocked CDRs and partially attenuated GF-induced pAKT. These results indicate that CDRs regulate the receptor-mediated PI3K-AKT pathway in Hep3B cells and the existence of CDR-independent pAKT mechanisms.ConclusionsOur results showed that CDRs modulate the AKT pathway in Hep3B cells. Since CDRs were not observed in other HCC and hepatocyte cell lines, we propose that CDRs in Hep3B would determine the carcinoma characteristic of the cell by aberrantly triggering the AKT pathway. Signaling molecules involved in CDR formation are promising therapeutic targets for some types of HCC.9eb9aTWDk6mAQeLNgQGeJaVideo abstract

Design, synthesis, and biological evaluation of a new series of pyrazole derivatives: Discovery of potent and selective JNK3 kinase inhibitors

The design, synthesis, and biological activities of a new series of pyrazole derivatives are reported. The target compounds 1a-1w were initially investigated against NCI-60 cancer cell lines. Compounds 1f, 1h, 1k, and 1v exerted the highest anti-proliferative activity over the studied panel of cancer cell lines. Compound 1f showed the most potent activity, and it is more potent than sorafenib in 29 cancer cell lines of different types and more potent than SP600125 against almost all the tested cancer cell lines. It also exerted sub-micromolar IC50 values (0.54–0.98 µM) against nine cell lines. Moreover, the 23 target compounds were tested against Hep3B and HepG2 hepatocellular carcinoma cell lines, of which compounds 1b, 1c, and 1h showed the strongest anti-proliferative activity. The most potent anticancer compounds (1b, 1c, 1f, and 1h) demonstrated a high selectivity towards cancer cells vis-à-vis normal cells. Compounds1f and 1h induced apoptosis and mild necrosis upon testing against RPMI-8226 leukemia cells. Kinase profiling of this series led to the discovery of two potent and selective JNK3 inhibitors, compounds 1c and 1f with an IC50 values of 99.0 and 97.4 nM, respectively. Both compounds showed a good inhibitory effect against JNK3 kinase in the whole-cell NanoBRET assay. This finding was further supported through molecular modeling studies with the JNK3 binding site. Moreover, compounds 1c and 1f demonstrated a very weak activity against CYP 2D6, CYP 3A4, and hERG ion channels.

Identification of Key Pathways Involved in White Strain of Hypsizygus marmoreus Extracts-Induced Cell Death of Human Hepatoma Hep3B Cells by Next Generation Sequencing.

White strain of Hypsizygus marmoreus is named as white genius mushroom (WGM) and is a popular food in Taiwan. We have confirmed the cytotoxicity of WGM extracts on human Hep3B liver cancer cells. A total of 8711 significantly differential genes were identified through large-scale transcriptome sequencing. According to the KEGG pathway enrichment analysis, autophagy, mitophagy and apoptosis pathways were identified as significant in WGM extracts-treated cells. WGM extracts induced a dose-dependent generation of reactive oxygen species (ROS) and membrane-enclosed vacuoles in Hep3B cells. The inhibition of ROS by the ROS scavengers blocked the induction of cell death and vacuoles formation. We suggested that the cell death and membrane-enclosed vacuoles induced by WGM extracts are dependent on ROS production in Hep3B cells. (2E,6E)-3,7,11,15,19,23,27,31,35-Nonamethylhexatriaconta-2,6,34-triene-1,11,15,19,23,27,31-heptol and (18:2) lysophosphatidylcholine were identified in WGM extracts. In addition to being a very popular edible mushrooms, WGM may be developed into a dietary supplement or dietary chemopreventive agent for the cancer treatment.

P4HA2 promotes occurrence and progression of liver cancer by regulating the PI3K/Akt/mTOR signaling pathway

To investigate the role of proline 4-hydroxylase Ⅱ (P4HA2) in the occurrence and progression of liver cancer. GEPIA and Human Protein Atlas database were used to predict the expression of P4HA2 in hepatocellular carcinoma (HCC), and K-M plotter online database was used to analyze the relationship between P4HA2 expression and the prognosis of HCC. We also examined the expressions of P4HA2 in HCC cells and normal hepatocytes using qRT-PCR and Western blotting. With lentivirus-mediated RNA interference, P4HA2 expression was knocked down in hepatoma SNU-449 and Hep-3B cells, and the changes in cell proliferation, migration and invasion were assessed using cell counting kit-8 (CCK-8) assay, colony formation test, scratch test and Transwell assay. The changes in the expressions of epithelial-mesenchymal transition (EMT) and PI3K/Akt/mTOR signal pathway-related proteins were detected using Western blotting. Online database analysis showed that the expression of P4HA2 was significantly higher in HCC tissues than in normal liver tissues (P < 0.05). The expression levels of P4HA2 mRNA and protein were also significantly higher in HCC cell lines than in normal hepatocytes (P < 0.01). Lentivirus-mediated RNA interference of P4HA2 significantly lowered the expression levels of P4HA2 mRNA and protein in the hepatoma cells (P < 0.05) and caused obvious inhibition of cell proliferation, migration and invasion. P4HA2 knockdown significantly increased the expression of E-cadherin protein, lowered the expressions of N-cadherin and Snail, and obviously decreased the expressions of phosphorylated PI3K, AKT and mTOR (P < 0.05). P4HA2 enhances the proliferation, migration, invasion, and EMT of hepatoma cells by activating the PI3K/Akt/mTOR signaling pathway to promote the occurrence and progression of liver cancer.

Targeted inhibition of the expression of both MCM5 and MCM7 by miRNA-214 impedes DNA replication and tumorigenesis in hepatocellular carcinoma cells

MicroRNAs are noncoding RNAs with a typical length of 22 nucleotides that post-transcriptionally suppress gene expression by inducing target mRNA degradation and/or impairing translation in eukaryotes. Thousands of miRNA genes in the human genome are involved in various physiological and pathological processes. Each miRNA targets many different mRNAs, while each mRNA may be targeted by various miRNAs. Mini-chromosome maintenance (MCM2-7) protein complex functions as essential components of the pre-replicative complex (pre-RC) and forms a helicase together with other proteins to unwind the DNA duplex in S phase. MCM proteins are overexpressed in all cancer cells, while they are strictly regulated in normal cells, with no expression in non-proliferating normal cells. Here we report that miRNA-214–3p (miR-214) targets both MCM5 and MCM7. The level of miR-214 is lower in HepG2 and Hep3B hepatocellular carcinoma cells than the L-02 normal liver cells. Introduction of miRNA-214 mimic into HepG2 and Hep3B cells reduced the mRNA and protein levels of MCM5/7 and inhibited DNA replication, cell cycle progression, cell proliferation and colony formation. Comparatively, miRNA-214 mimic had little effect in L-02 cells. Importantly, miR-214 mimic can also inhibit the growth of HepG2 xenografts in nude mice. Our data suggest that miRNA-214 regulates DNA replication by targeting MCM5/7 and has the potential to be developed into a liver cancer drug. ImplicationsThis study supports the notion that DNA replication-initiation proteins (DRIPs), including MCM2-7 proteins, are attractive anticancer targets. Furthermore, the potential of miR-214 as an anticancer agent, with activity against liver cancer cells but not normal livre cells, may be of high significance.

IL-6 regulates induction of C-reactive protein gene expression by activating STAT3 isoforms

C-reactive protein (CRP) is synthesized in hepatocytes. The serum concentration of CRP increases dramatically during the acute phase response. In human hepatoma Hep3B cells, maximal CRP expression occurs in cells treated with the combination of IL-6 and IL-1β. IL-6 induces transcription of the CRP gene and IL-1β synergistically enhances the effects of IL-6. We investigated the role of IL-6-activated transcription factor STAT3, also known as STAT3α, in inducing CRP expression since we identified four consensus STAT3-binding sites centered at positions − 72, − 108, − 134 and − 164 on the CRP promoter. It has been shown previously that STAT3 binds to the site at − 108 and induces CRP expression. We found that STAT3 also bound to the other three sites, and several STAT3-containing complexes were formed at each site, suggesting the presence of STAT3 isoforms and additional transcription factors in the complexes. Mutation of the STAT3 sites at − 108, − 134 or − 164 resulted in decreased CRP expression in response to IL-6 and IL-1β treatment, although the synergy between IL-6 and IL-1β was not affected by the mutations. The STAT3 site at − 72 could not be investigated employing mutagenesis. We also found that IL-6 activated two isoforms of STAT3 in Hep3B cells: STAT3α which contains both a DNA-binding domain and a transactivation domain and STAT3β which contains only the DNA-binding domain. Taken together, these findings raise the possibility that IL-6 not only induces CRP expression but also regulates the induction of CRP expression by activating STAT3 isoforms and by utilizing all four STAT3 sites.