Human Hepatoma Cell Line HepG2 Research Articles

NDRG2 inhibits tumorigenesis of hepatocellular carcinoma by regulating metabolism of phospholipids and triglyceride: a metabonomic analysis

To explore the role of the tumor suppressor gene NDRG2 in regulating lipid metabolism in hepatoma cells. We analyzed the differential expression of NDRG2 gene between hepatocellular carcinoma tissues (n=809) and normal liver tissues (n=379) based on data from TNMplot database, and investigated the correlation between NDRG2 mRNA expression and the overall survival of the patients with hepatocellular carcinoma using THPA database, which was also used for analysis of NDRG2 expression levels in tumor cell lines for screening hepatoma cell lines. Human hepatoma cell line HepG2 was infected with a lentivirus containing NDRG2 cDNA, and the expression level of NDRG2 in the infected cells was detected using qPCR and Western blotting. Lipid metabolomics analysis was performed to analyze the regulatory effect of NDRG2 overexpression on lipid metabolism in HepG2 cells, and ELISA and Oil Red O staining were used to examine the changes in contents of phospholipids and triglyceride in NDRG2-overexpressing HepG2 cells. Analysis of the TNMplot database showed that NDRG2 expression level was significantly lower in hepatocellular carcinoma tissues than in normal liver tissues (P < 0.001). Analysis of THPA database showed that the patients with high NDRG2 mRNA levels had a longer survival time than those with low NDRG2 mRNA levels, and NDRG2 expression level was the highest in HepG2 cell line among the tumor cell lines. Metabolomics analysis showed that in HepG2 cells, NDRG2 overexpression led to changes in the contents of phospholipids, and among them lecithin PC, phosphatidyl glycerol PG, phosphatidyl ethanolamine PE, sphinophosphatidyl serine SM, and ceramide Cer exhibited significant changes. The results of ELISA and Oil Red O staining demonstrated that NDRG2 overexpression obviously reduced the contents of multiple phospholipids and significantly lowered the contents of triglyceride in HepG2 cells. NDRG2 regulates tumorigenesis of hepatocellular carcinoma by modulating the metabolism of phospholipids and triglyceride.

Effects of Raf kinase inhibitor protein on biological characteristics of hepatocellular carcinoma cells and its potential therapeutic effects

Raf kinase inhibitor protein (RKIP) is an important member of the phosphatidylethanolamine-binding protein family. This study investigated the inhibitory effect of RKIP on the malignant biological behavior of liver cancer cells in vivo and in vitro. An RKIP gene expression vector was constructed using the pcDNA3.1 vector and transfected into human hepatoma cell line HepG2 to overexpress the RKIP gene. Growth, proliferation, the cell cycle, apoptosis, migration, and invasion of the cell line were analyzed. For in vivo experiments, the vector was transfected into tumor tissue of hepatoma-bearing animals and then tumor growth was analyzed and compared with the control to assess its anti-tumor effect. Compared with the control group, cell growth in the RKIP gene transfection group (HEP-RKIP) was significantly slower. The average colony formation rate of the HEP-RKIP group was significantly lower than that of the other two groups (p < 0.05). The proportion of HEP-RKIP cells in G0/G1 phase was significantly higher than that of the control group (p < 0.05), while the proportion of cells in G2/M phase was significantly lower than that in the control group (p < 0.05). The apoptosis rate of HEP-RKIP cells was approximately 6.4%, which was significantly higher than that of the control group (p < 0.05). Migration of HEP-RKIP cells was significantly slower than that of the other two groups (p < 0.05). In an inhibition experiment of the liver tumor animal model, the tumor inhibition rate of the RKIP in vivo transfection group was significantly higher than that of each control group (p < 0.05). RKIP inhibits malignant biological behavior of liver cancer cells in vitro and has a tumor inhibitory effect in vivo, which may be a potential target for gene therapy of liver cancer.

Downregulated lncRNA DRAIC enhances the radiotherapy sensitivity of human HCC cell line HepG2 by targeting miR-223-3p

Abstract Objective This study aims to investigate the effects of the long noncoding RNA (lncRNA) DRAIC on the proliferation, apoptosis, and radiosensitivity of hepatocellular carcinoma (HCC) cells and the molecular mechanisms involved. Methods Cancer tissues and their corresponding adjacent tissues from 30 patients with HCC were collected, and the expression levels of DRAIC and miR-223-3p were detected via RT-qPCR. DRAIC interference and miR-223-3p overexpression vectors were transfected into HepG2 cells. In addition, DRAIC and miR-223-3p interference vectors were co-transfected into HepG2 cells. The constructed cells were irradiated at 4 Gy. Cell colony formation assay, MTT assay, and flow cytometry were performed to detect the radiosensitivity, proliferation inhibition rate, and apoptosis rate of HepG2 cells, respectively. Dual luciferase reporter gene assay was performed to detect the targeted regulation of DRAIC on miR-223-3p expression. Results The expression level of DRAIC in HCC tissues was higher than that in paracancer tissues, whereas the expression level of miR-223-3p was lower in HCC tissues than that in paracancer tissues (P &lt; 0.05). Inhibition of DRAIC expression or overexpression of miR-223-3p increased the proliferation inhibition and apoptosis rates of HepG2 cells (P &lt; 0.05). After irradiation, cell survival fraction decreased and cell proliferation inhibition and apoptosis rates increased (P &lt; 0.05). DRAIC targeted the regulation of miR-223-3p expression, and interference of miR-223-3p expression reversed the effects of inhibiting DRAIC expression on the proliferation, apoptosis, and radiosensitivity of HepG2 cells. Conclusion Inhibition of DRAIC expression can inhibit the proliferation of HepG2 cells, promote cell apoptosis, and enhance the radiosensitivity of cells via upregulation of miR-223-3p.

In vitro cytotoxicity activity of copper complexes of imine and amine ligands: A combined experimental and computational study

Two new Cu(II) complexes [CuL1 (µ1,3-NCS)]2, 2ClO4 (A) and [CuL2 (NCS)2] (B) with Schiff base ligand L1 and reduced Schiff base ligand L2 have been synthesized and characterized by various spectroscopic and electrochemical methods along with single crystal XRD techniques. X-ray diffraction analysis confirms the formation of binuclear (A) and mononuclear (B) complexes. Hirshfeld surfaces analysis of the two complexes and their associated two-dimensional fingerprint plots are plotted. The metal complexes were optimized by using the density functional theory (DFT) comprising Becke’s three parameters exchange potential and Lee-Yang-Parr correlation functional (B3LYP) method, with 6-31G(d,p) basis set for the ligand atoms and LANL2DZ for the central metal ions. The synthesized binuclear Complex A and mononuclear Complex B were screened for their in vitro anticancer activity against human colon adenocarcinoma cells (HT-29) and human hepatoma cell line (Hep-G2) and HeLa cell line. Activity results proved that Complex A has excellent antiproliferative activity against colon carcinoma whereas Complex B is active against hepatocellular carcinoma and cervical cancer.

Adaptive regulations of Nrf2 alleviates silver nanoparticles-induced oxidative stress-related liver cells injury

Silver nanoparticles (AgNPs) are widely used in various fields such as industry, agriculture, and medical care because of their excellent broad-spectrum antibacterial activity. However, their extensive use has raised concerns about their health risks. Liver is one of the main target organs for the accumulation and action of AgNPs. Therefore, evaluating the toxic effects of AgNPs on liver cells and its mechanisms of action is crucial for the safe application of AgNPs. In the study, polyvinylpyrrolidone (PVP)-coated AgNPs were characterized. The human hepatoma cell line (HepG2) and the normal hepatic cell line (L02) were exposed to different concentrations of AgNPs (20–160 μg/mL) and pretreated with the addition of N-acetylcysteine (NAC) or by Nrf2 siRNA transfection. NAC was able to inhibit the concentration-dependent increase in the level of apoptosis induced by AgNPs in HepG2 cells and L02 cells. Interestingly, HepG2 cells were more sensitive to AgNPs than L02 cells, and this may be related to the different ROS generation and responses to AgNPs by cancer cells and normal cells. In addition, NAC also alleviated the imbalance of antioxidant system and cell cycle arrest, which may be related to AgNPs-induced DNA damage and autophagy. The knockdown of nuclear factor erythroid-derived factor 2-related factor (Nrf2) found that AgNPs-induced ROS and apoptosis levels were further upregulated, but the cell cycle arrest was alleviated. On the whole, Nrf2 exerts a protective role in AgNPs-induced hepatotoxicity. This study complements the hepatotoxicity mechanisms of AgNPs and provides data for a future exploration of AgNPs-related anti-hepatocellular carcinoma drugs.

Novel ginsenoside derivatives induce apoptosis in HepG-2 cells via the MDM2-p53 signaling pathway

In this study, 6-aryl-5-cyanopyrimidines and quinazolinones were introduced into panaxadiol (PD) to synthesize 25 new panaxadiol derivatives. The cytotoxicity of these compounds against three cancer cells and one normal cell was examined by methylthiazoletetrazolium (MTT) cytotoxicity experiment. The findings demonstrated that PD cyanopyrimidine derivatives have superior anti-proliferative effects over quinazoline derivatives. Among them, PM14 had the strongest inhibitory activity on the proliferation of human hepatoma cell lines (HepG-2), the IC50 value was 2.13 ± 0.20 μM. 4′,6-diamidino-2-phenyl-indole (DAPI) staining showed that PM14 made HepG-2 nucleus shrink and had obvious apoptosis. Mechanistic studies confirmed that the derivative PM14 activates p53 signaling pathway by reducing the expression of MDM2 protein, and further induces an increase in the intracellular Bax/Bcl-2 ratio, down-regulated the expression of Caspase-3, up-regulated Cl-Caspase-3 expression, eventually leading to cell apoptosis. This lays the foundation for subsequent development of derivatives with stronger anti-proliferative activity.

Alizarin as a New Activator of the Aryl Hydrocarbon Receptor Signaling Pathway

Alizarin (1,2-dihydroxyanthraquinone) is a natural red dye extracted from the roots of Rubia cordifolia L. (family Rubiaceae). Alizarin has been used as a biological red stain for calcium. The aryl hydrocarbon receptor (AhR) has critical roles in multiple physiological pathways. This study aimed to determine whether alizarin is an unreported ligand of AhR. In the present study, we investigated the effects on cytochrome P450 (CYP) 1A1 mRNA, protein expression, AhR nuclear translocation, aryl hydrocarbon response element (AHRE) reporter activity, and AhR-specific antagonist following alizarin treatment of cells of the human hepatoma cell line, HepG2, and murine hepatoma cell line, Hepa-1c1c7. Alizarin induced CYP1A1 mRNA and protein expression in HepG2 and Hep-1c1c7 cells. Such induction was not present in C4 (B13NBii1) cells, which are AhR signal deficient, C12 (B15ECiii2) cells, which reduce AhR protein levels. The alizarin-induced responses were blocked by CH-223191, which is an AhR antagonist. Alizarin, the same as with the AhR ligand, induced the nuclear localization of AhR, as well as stimulated the transcriptional activity of AHRE. The results of this study suggest that alizarin is an AhR agonist.

The Biological Relevance of Papaverine in Cancer Cells.

Papaverine (PPV), a benzylisoquinoline alkaloid, extracted from the Papaverine somniferum plant, is currently in clinical use as a vasodilator. Research has shown that PPV inhibits phosphodiesterase 10A (PDE10A,) resulting in the accumulation of cyclic adenosine 3', 5'-monophosphate (cAMP) that affects multiple downstream pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), a mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF). The accumulation of cAMP can further affect mitochondrial metabolism through the activation of protein kinase A (PKA), which activates the mitochondrial complex I. Literature has shown that PPV exerts anti-proliferative affects in several tumorigenic cell lines including adenocarcinoma alveolar cancer (A549) and human hepatoma (HepG-2) cell lines. Cell cycle investigations have shown varying results with the effects dependent on concentration and cell type with data suggesting an increase in cells occupying the sub-G1 phase, which is indicative of cell death. These results suggest that PPV may be a beneficial compound to explore for the use in anticancer studies. More insight into the effects of the compound on cellular and molecular mechanisms is needed. Understanding the effects PPV may exert on tumorigenic cells may better researchers' understanding of phytomedicines and the effects of PPV and PPV-derived compounds in cancer.

Insulin Downregulates the Expression of ATP-binding Cassette Transporter A-I in Human Hepatoma Cell Line HepG2 in a FOXO1 and LXR Dependent Manner.

ATP-binding cassette transporter A-I (ABCA1) is an ubiquitously expressed protein whose main function is the transmembrane transport of cholesterol and phospholipids. Synthesis of ABCA1 protein in liver is necessary for high-density lipoprotein (HDL) formation in mammals. Thus, the mechanism of ABCA1 gene expression regulation in hepatocytes are of critical importance. Recently, we have found the insulin-dependent downregulation of other key player in the HDL formation-apolipoprotein A-I gene (J. Cell. Biochem., 2017, 118:382-396). Nothing is known about the role of insulin in the regulation of ABCA1 gene. Here we show for the first time that insulin decreases the mRNA and protein levels of ABCA1 in human hepatoma cell line HepG2. PI3K, p38, MEK1/2, JNK and mTORC1 signaling pathways are involved in the insulin-mediated downregulation of human ABCA1 gene. Transcription factors LXRα, LXRβ, FOXO1 and NF-κB are important contributors to this process, while FOXA2 does not regulate ABCA1 gene expression. Insulin causes the decrease in FOXO1, LXRα and LXRβ binding to ABCA1 promoter, which is likely the cause of the decrease in the gene expression. Interestingly, the murine ABCA1 gene seems to be not regulated by insulin in hepatocytes (in vitro and in vivo). We suggest that the reason for this discrepancy is the difference in the 5'-regulatory regions of human and murine ABCA1 genes.

Differential Inhibite Effect of Xanthohumol on HepG2 Cells and Primary Hepatocytes.

Xanthohumol (XN) is the major prenylated chalcone of the female inflorescences (cone) of the hop plant (Humulus lupulus). It is also a constituent of beer, the major dietary source of prenylated flavonoids. It has shown strong antitumorigenic activity towards various types of cancer cells. In the present study, we show the impact on human hepatocarcinoma cell line HepG2 cell and potential adverse effects on rat primary hepatocytes. Cell growth/viability assay (MTT) demonstrated that HepG2 cells are highly sensitive to XN at a concentration range of 25-100μM. The primary mode of tumor cell destruction was apoptosis as demonstrated by the binding of Annexin Ⅴ-FITC, we show that XN at a concentration of 25μM induced apoptosis in HepG2. Further evidence that XN kills HepG2 by inducing apoptosis was provided by the impact of XN on the cleavage of PARP-1 and caspases-3. In contrast, XN concentrations up to 100μM did not affect viability of primary rat hepatocytes in vitro, meanwhile, XN did not induce the apoptosis of primary rat hepatocytes in vitro. In summary, our data provide a rationale for clinical evaluation of XN for the treatment of liver cancer.

Kribbellichelins A and B, Two New Antibiotics from Kribbella sp. CA-293567 with Activity against Several Human Pathogens.

Current needs in finding new antibiotics against emerging multidrug-resistant superbugs are pushing the scientific community into coming back to Nature for the discovery of novel active structures. Recently, a survey of halophilic actinomyectes from saline substrates of El Saladar del Margen, in the Cúllar-Baza depression (Granada, Spain), led us to the isolation and identification of 108 strains from the rhizosphere of the endemic plant Limonium majus. Evaluation of the potential of these strains to produce new anti-infective agents against superbug pathogens was performed through fermentation in 10 different culture media using an OSMAC approach and assessment of the antibacterial and antifungal properties of their acetone extracts. The study allowed the isolation of two novel antibiotic compounds, kribbellichelin A (1) and B (2), along with the known metabolites sandramycin (3), coproporphyrin III (4), and kribelloside C (5) from a bioassay-guided fractionation of scaled-up active extracts of the Kribbella sp. CA-293567 strain. The structures of the new molecules were elucidated by ESI-qTOF-MS/MS, 1D and 2D NMR, and Marfey’s analysis for the determination of the absolute configuration of their amino acid residues. Compounds 1–3 and 5 were assayed against a panel of relevant antibiotic-resistant pathogenic strains and evaluated for cytotoxicity versus the human hepatoma cell line HepG2 (ATCC HB-8065). Kribbellichelins A (1) and B (2) showed antimicrobial activity versus Candida albicans ATCC-64124, weak potency against Acinetobacter baumannii MB-5973 and Pseudomonas aeruginosa MB-5919, and an atypical dose-dependent concentration profile against Aspergillus fumigatus ATCC-46645. Sandramycin (3) confirmed previously reported excellent growth inhibition activity against MRSA MB-5393 but also presented clear antifungal activity against C. albicans ATCC-64124 and A. fumigatus ATCC-46645 associated with lower cytotoxicity observed in HepG2, whereas Kribelloside C (5) displayed high antifungal activity only against A. fumigatus ATCC-46645. Herein, we describe the processes followed for the isolation, structure elucidation, and potency evaluation of these two new active compounds against a panel of human pathogens as well as, for the first time, the characterization of the antifungal activities of sandramycin (3).

PREPARATION AND CHARACTERIZATION OF ALGINATE CHITOSAN CROSSLINKED NANOPARTICLES BEARING DRUG FOR THE EFFECTIVE MANAGEMENT OF ULCERATIVE COLITIS

Objective: Delivery of anticancer molecule to the liver remains a “holy grail” in molecular medicine and nanobiotechnology with conventional therapy, as conventional cancer chemotherapy does not prove effective as drug molecule does not reach to the target site at therapeutic concentration. Tumor vasculature differs from the vasculature of normal tissue both in morphology and biochemistry. Most of these differences appear too related to angiogenesis (formation of new blood vessels from pre-existing ones). For the present study nanoparticles (NPs) were chosen as a delivery system, because they have many advantages, e. g. they can pass through the smallest capillary vessels because of their ultra-tiny volume, can penetrate cells and tissue gap to arrive at, pH, ion and/or temperature sensitivity of materials, can improve the utility of drugs and reduce toxic side effects. Methods: PLGA (poly lactide co glycolic acid) was used for the preparation of NPs because of its biodegradability and biocompatibility. It degrades by hydrolysis of ester linkages in the presence of water in to two monomers lactic acid and glycolic acid. There are a number of ligands available for hepatic delivery, among them lactobionic acid (containing galactose moiety) was selected for present work. Preparation of plain nanoparticles was carried out using emulsification–diffusion method. Optimization of the polymer concentration is the first step during the study and it was performed by varying the polymer concentration where as keeping other variables constant. The prepared formulation was optimized on the basis of particle size and polydispersity index. Amount of drug was optimized on the basis of particle size and percentage entrapment efficiency. Results: Particle size and zeta potential of the nanoparticle were determined by zetasizer showed that particles are in nano range (blow 200 nm) and have acceptable range of zeta potential. Shape and surface morphology were determined by TEM and SEM analysis. The conjugation of lactobionic acid with PLGA polymer was proved by FTIR. The in vitro release profiles of entrapped drug from formulations were determined using dialysis membrane. For stability studies, the LDNPs (conjugated NPs) are stored at the temperatures 4±1 °C and room temperature. Human hepatoma cell line HepG2 by SRB assay was selected and it clearly suggests a dose dependent cytotoxicity response i.e. decrease in cell survival fraction with increasing concentration of drug. The in vivo study are important in evaluating the targeting efficacy of designed dosage form and also helps in establishing the correlation between the results obtained from in vitro experimentation to that from in vivo studies. The formulations were administered by tail vein to mice of four groups Group I: PBS 7.4 (control); Group II: 5-FU solution; Group III: DNPs; Group IV: LDNPs. Conclusion: The proposed targeting strategy is expected to enhance the therapeutic index of conventional anticancer drug as well as reduce its cytotoxic effects to normal cells.

Biosynthesis of ZnO nanoparticles using Melia azedarach seed extract: Evaluation of the cytotoxic and antimicrobial potency

ZnO nanoparticles (NPs) synthesized by using an aqueous extract of Melia azedarach seed are reported in this paper. The structural, morphological, compositional, and optical properties of the NPs were evaluated. A hexagonal phase with spherical morphology and an average crystallite size of 57.74 nm was obtained. Optical studies indicated a maximum absorption peak around 296 nm and bandgap energy of 3.88 eV. Cytotoxicity assay of both the plant extract and the as-prepared ZnO nanoparticles against the human hepatoma (HepG2) cell line showed that the M. azedarach seed-mediated ZnO nanoparticles were less toxic compared to ZnO synthesized by the conventional method, while the plant itself showed dose-dependent low toxicity. ZnO nanoparticles by the green route also exhibited good antibacterial activity, with higher efficiency for Gram-negative bacteria than Gram-positive bacteria.

Riboflavin (Vitamin B2) Deficiency Induces Apoptosis Mediated by Endoplasmic Reticulum Stress and the CHOP Pathway in HepG2 Cells.

Riboflavin is an essential micronutrient and a precursor of flavin mononucleotide and flavin adenine dinucleotide for maintaining cell homeostasis. Riboflavin deficiency (RD) induces cell apoptosis. Endoplasmic reticulum (ER) stress is considered to induce apoptosis, and C/EBP homologous protein (CHOP) is a key pathway involved in this process. However, whether RD-induced apoptosis is mediated by ER stress and the CHOP pathway remains unclear and needs further investigation. Therefore, the current study presents the effect of RD on ER stress and apoptosis in the human hepatoma cell line (HepG2). Firstly, cells were cultured in a RD medium (4.55 nM riboflavin) and a control (CON) medium (1005 nM riboflavin). We conducted an observation of cell microstructure characterization and determining apoptosis. Subsequently, 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, was used in HepG2 cells to investigate the role of ER stress in RD-induced apoptosis. Finally, CHOP siRNA was transfected into HepG2 cells to validate whether RD triggered ER stress-mediated apoptosis by the CHOP pathway. The results show that RD inhibited cell proliferation and caused ER stress, as well as increased the expression of ER stress markers (CHOP, 78 kDa glucose-regulated protein, activating transcription factor 6) (p < 0.05). Furthermore, RD increased the cell apoptosis rate, enhanced the expression of proapoptotic markers (B-cell lymphoma 2-associated X, Caspase 3), and decreased the expression of the antiapoptotic marker (B-cell lymphoma 2) (p < 0.05). The 4-PBA treatment and CHOP knockdown markedly alleviated RD-induced cell apoptosis. These results demonstrate that RD induces cell apoptosis by triggering ER stress and the CHOP pathway.

Diaporine Potentiates the Anticancer Effects of Oxaliplatin and Doxorubicin on Liver Cancer Cells.

Recent studies have shown that diaporine, a novel fungal metabolic product, has a strong in vitro and in vivo anticancer effect on human non-small-cell lung and breast cancers. In this study, three human hepatocarcinoma cell lines (HepG2, Hep3B, and Huh7) were used to evaluate the efficacy of diaporine alone and in combination with the standard cytotoxic drugs oxaliplatin and doxorubicin for the treatment of liver cancer. We demonstrated that diaporine, oxaliplatin, and doxorubicin triggered a concentration- and time-dependent decrease in the number of HepG2 cells. Diaporine at a concentration of 2.5 μM showed almost 100% inhibition of cell counts at 72 h. Similar effects were observed only with much higher concentrations (100 μM) of oxaliplatin or doxorubicin. Decreases in cell numbers after 48 h treatment with diaporine, oxaliplatin, and doxorubicin were also demonstrated in two additional hepatoma cell lines, Hep3B and Huh7. The combination of these drugs at low concentration for 48 h in vitro noticeably showed that diaporine improved the inhibitory effect on the number of cancer cells induced by oxaliplatin or doxorubicin. Additionally, this combination effectively inhibited colony growth in vitro. We found that inhibition of phosphorylation of ERK1/2 significantly increased when diaporine was used in combination with other agents. In addition, we also found that when diaporine was used in combination with doxorubicin or oxaliplatin, their proapoptotic effect greatly increased. We further revealed that the induction of apoptosis in hepatoma cells after treatment is due, at least in part, to the inhibition of phosphorylation of AKT, leading to the activation of caspase-3, inactivation of poly (ADP-ribose) polymerase (PARP), and subsequently to DNA damage, as indicated by the increased level of H2AX. Based on these findings, we suggest that diaporine in combination with the standard cytotoxic drugs oxaliplatin and doxorubicin may play a role in the treatment of liver cancer.

Fractions of Hoslundia opposita Vahl and hoslundin induced apoptosis in human cancer cells via mitochondrial-dependent reactive oxygen species (ROS) generation

BackgroundCancer remains one of the leading causalities of several morbidity and mortality with negative impact on global economy due to low workforce and management/treatment cost. A number of conventional therapies have been explored in the management/treatment of cancer including chemotherapeutic intervention, radiotherapy, and surgery. Among these treatment modalities, chemotherapy remains the most popular first line of intervention in management/treatment of cancer, and natural products have been implicated as the main source of antineoplastic agents with phenomenal efficacy. However, current antineoplastic agents suffer from lack of selectivity and specificity necessitating the need for further research in the search for novel anticancer drug molecules. MethodsIn this present study, the anticancer activity of Hoslundia opposita leaves extracts were tested against a number of cell lines including human hepatoma cell line (HepG2), human breast cancer cell lines (MDA-MB-231), intestinal epithelial cell lines (Caco-2), and human keratinocyte HACAT cell lines. A bio-guided fractionation assay and the structural elucidation of the pure isolate (hoslundin) was conducted by 1D and 2D NMR spectroscopy. The cell viability, colony formation, and apoptotic activities were investigated using MTT assay, clonogenic assay, and caspase − 3 and − 7 kits respectively. Flow cytometry was employed in assessing the altered cell cycle expression. The production of the intracellular reactive oxygen species (ROS) levels and the reduction of the mitochondrial membrane potential (MMP) was determined at the cellular level using fluorescent probe dyes dihydro-fluorescin diacetate (DCFH-DA) and tetramethylrhodamin (TMRE), respectively. ResultsThe H. opposita fractions and its pure isolate (hoslundin) demonstrated a potent cytocidal activity against the tumorigenic cells (HepG2, MDA-MB-231, Caco-2) at concentration ranging from 25 to 100 µg/mL. The inhibition of the colony formation was significantly observed in HepG2 cell lines. More so, the cellular viability of the normal cells (HaCaT) was relatively unchanged in the presence of H. opposita fractions and its isolate proving the selectivity of the compounds towards tumourigenic cells. The H. opposita fractions and hoslundin exerted their anticancer activity via cell cycle arrest with the accumulation of the DNA content at the S-phase, activation of apoptosis in the caspase 3,7 activities and depolarized mitochondrial membrane potential mediated by mitochondrial-dependent ROS generation in the treated tumor cells. ConclusionThe anticancer activities of Hoslundia opposita Vahl and hoslundin exhibited significant efficacy against tumor cells and well tolerated in the presence of normal cells making them a potential antineoplastic agent.

Tamoxifen induced hepatic steatosis in high-fat feeding rats through SIRT1-Foxo1 suppression and LXR-SREBP1c activation.

Clinically, long-term use of tamoxifen (TAM) would lead to fatty liver disease in breast cancer patients, especially obese women. However, the exact mechanism of TAM-induced hepatic steatosis is still unclear. Meanwhile, there is no drug to prevent and treat it. In view of silent information regulator 1 (SIRT1) playing a key role in hepatic lipid metabolism regulation, this study was conducted to investigate whether SIRT1 is a potential therapeutic target for TAM-induced hepatic steatosis. In this study, obese female Wistar rats fed with high-fat diet (HFD) for 15weeks were given TAM (4, 8mg/kg, intragastric) for 14days. In vitro, human hepatocarcinoma cell line HepG2 was used to establish a high-fat model with 50μM oleic acid and TAM (10μM) was treated simultaneously for 72h. The results showed that TAM was more likely to upregulate the expression of lipid synthetase that caused the increase of lipid content in HepG2 cells and rat liver. The expression of SIRT1 was downregulated both in vitro and in vivo. SIRT1 agonist SRT1720 (15mg/kg, 30mg/kg, i.p.) could resist TAM-induced hepatic lipid synthetase overexpression to relieve TAM-induced hepatic steatosis. Meanwhile, the upregulation of p-forkhead box O1 and LXRα induced by TAM was reversed by SRT1720. These results indicated that TAM-induced hepatic steatosis was based on SIRT1-p-FoxO/LXRα-sterol regulatory element binding protein 1c pathway under HFD condition. SIRT1 agonist might be a potential therapeutic drug to relieve this side effect. Tamoxifen increased lipid synthesis and regulated lipid transport in HFD rat liver.p-FoxO1/LXRα-SREBP1c signaling was upregulated through the inhibition of SIRT1 in tamoxifen-induced hepatic steatosis under HFD condition.SIRT1 agonist SRT1720 could relieve tamoxifen-induced hepatic steatosis.

The Key Network of mRNAs and miRNAs Regulated by HIF1A in Hypoxic Hepatocellular Carcinoma Cells.

Purpose: Hypoxia plays an essential role in the progression of hepatocellular carcinoma (HCC), whereas hypoxia inducible factor-1 (HIF-1) is the key transcription factor allowing HCC to survive hypoxia. The aim of this study was to define the essential mRNAs and miRNAs regulated by HIF1A and dissect their functions, interactions, and tumor-infiltrating immune cells in HCC. Methods: A human HCC cell line HepG2 was used as a cell model of HCC. The CRISPR/Cas9 system was used to knock out HIF1A in HepG2 cells, and RNA sequencing was utilized to characterize differentially expressed mRNAs and miRNAs in the HIF1A-knockout HepG2 cells; the identified candidates were then analyzed by GO annotation and KEGG pathway enrichment to study their function and establish a PPI network. Quantitative (q) PCR was used to verify if there were significant differences in the expression of mRNAs, and the association of the selected mRNAs expression with immune cell infiltration levels was further analyzed using The Cancer Genome Atlas (TCGA) pan-cancer data. Results: Using RNA-sequencing, we discovered that there were 1535 mRNAs differentially expressed (adjusted p < 0.05, |fold change|>1.5) in the HIF1A-knockout HepG2 cells, among which there were 644 mRNAs upregulated and 891 mRNAs downregulated. GO annotation and KEGG pathway enrichment showed that these mRNAs were involved in glycolysis/gluconeogenesis, PI3K-Akt signaling pathways, and HIF-1 signaling pathways. In addition, we found that there were 309 miRNAs differentially expressed (adjusted p < 0.05, |fold change|>1.5) in the HIF1A-knockout HepG2 cells, of which there were 213 miRNAs upregulated and 96 miRNAs downregulated. Our further analyses uncovered that these miRNA putative targets were involved in the hippo signaling pathway, axon guidance, and tight junction. Moreover, the construction and analysis of the PPI network showed that OASL, IL6, and TAF1 were recognized as hub genes with the highest connectivity degrees. Importantly, in the HIF1A-knockout HepG2 cells, our qRT-PCR data confirmed the selected mRNA changes revealed by RNA-sequencing, and with TCGA pan-cancer data, we revealed that the expressional levels of these three genes, LUM, SCOC, and CCL2, were associated with immune cell infiltration levels. Conclusion: The identified potential key network of mRNAs and miRNAs regulated by HIF1A in the HCC cells suggests a key role of HIF1A in the tumorigenesis of HCC.

Blocking autophagy with chloroquine aggravates lipid accumulation and reduces intracellular energy synthesis in hepatocellular carcinoma cells, both contributing to its anti-proliferative effect

PurposeThe autophagy inhibitor chloroquine enhances the effect of targeted therapy using tyrosine kinase inhibitor in liver cancer. We would like to further understand the specific mechanism by which chloroquine inhibits the proliferation of tumor cells.MethodsWe used a human hepatocarcinoma cell line (HepG2) as cell culture model. In contrast to the control groups (treated only with complete medium), cells in experimental groups were treated either with complete medium + 40 ng/ml Hepatocyte growth factor (HGF), or with complete medium + 60 μM chloroquine or with complete medium + 40 ng/ml HGF + 60 μM chloroquine for 24 h. Cell number and ATP content were investigated using spectrophotometric assays. Cell proliferation and apoptosis were detected by immunohistochemistry. Cell morphological alterations were examined by Giemsa and H&E staining. Cellular lipid content was determined by Oil Red O staining and Triglyceride quantification assay. Autophagy-related proteins (LC3B and p62) and hepatocyte proliferation-related protein (S6K1) were examined using western blot. The autophagic flux of cells was assessed by mRFP-EGFP-LC3 transfection assay.ResultsWe found that chloroquine inhibited the proliferation of HepG2 cells, as evidenced by a decrease in cellular ATP content, Ki-67 and S6K1 protein expression and a reduction in cell number. This finding was associated with an increase in lipid content. As expected, chloroquine inhibited autophagy of HepG2 cells, as evidenced by the accumulation of LC3B-II and the significant upregulation of p62. mRFP-EGFP-LC3 transfection assay showed that indeed chloroquine blocked the autophagic flux in HepG2 cells.ConclusionChloroquine impaired proliferation of HepG2 cells might be due to intracellular accumulation of lipids and inhibition of energy synthesis.

The Chloroform Extracts of Vietnamese Sophora flavescens Ait. Inhibit the Proliferation of HepG2 Cells through Apoptosis Induction

The present study evaluated the effects of Sophora flavescens Ait. root extract on the proliferation of human hepatoma cell line HepG2. HPLC-UV analysis showed that the highest matrine and oxymatrine contents were obtained in the chloroform extract, compared to ethanol extract and ethyl acetate extract. The morphological analysis revealed that the chloroform extract of Sophora flavescens Ait. (SFA-CHCl3 extract) induced alterations of HepG2 cell morphology, resulting in the shrinkage of cells, the formation of debris, and cell detachment. The proliferation of HepG2 cells was inhibited by SFA-CHCl3 extract treatment. Cell cycle analysis exhibited that the cell proportion of the G0/G1 phase of HepG2 cells with SFA-CHCl3 extract treatment was decreased, while the cell proportion of the G2/M phase was increased. Flow cytometry analysis indicated a dramatic increase in the apoptotic percentage of HepG2 cells over the time of SFA-CHCl3 extract treatment. The SFA-CHCl3 extract also caused morphological changes in HepG2 nuclear, including chromatin condensation and DNA fragmentation. SFA-CHCl3 extract treatment induced the bax up-regulation and the bcl-2 down-regulation in HepG2 cells. These results revealed that SFA-CHCl3 extract could be a potential apoptosis inducer in HepG2 cells.