Cancer Cell Lines Huh7 Research Articles

An iron chelation-based combinatorial anticancer therapy comprising deferoxamine and a lactate excretion inhibitor inhibits the proliferation of cancer cells

BackgroundAlthough iron chelation has garnered attention as a novel therapeutic strategy for cancer, higher levels of efficacy need to be achieved. In the present study, we examined the combinatorial effect of deferoxamine (DFO), an iron chelator, and α-cyano-4-hydroxy cinnamate (CHC), a suppressor of lactate excretion, on the proliferation of cancer cell lines.MethodsWe established a deferoxamine (DFO)-resistant cell line by culturing HeLa cells in media containing increasing concentrations of DFO. Metabolome and gene expression analyses were performed on these cells. Synergistic effect of the drugs on the cells was determined using an in vitro proliferation assay, and the combination index was estimated.ResultsDFO-resistant HeLa cells exhibited enhanced glycolysis, salvage cycle, and de novo nucleic acid synthesis and reduced mitochondrial metabolism. As DFO triggered a metabolic shift toward glycolysis and increased lactate production in cells, we treated the cancer cell lines with a combination of CHC and DFO. A synergistic effect of DFO and CHC was observed in HeLa cells; however, the same was not observed in the human liver cancer cell line Huh7. We hypothesized that the efficacy of the combination therapy in cancer cells depends on the degree of increase in lactate concentration upon DFO treatment.ConclusionCombination therapy involving administration of DFO and CHC is effective in cancer cells wherein DFO treatment results in an elevation in lactate levels. Our findings illustrate that the DFO-induced enhanced glycolysis provides specific targets for developing an efficient anticancer combinatorial therapy involving DFO. These findings will be beneficial for the development of novel cancer chemotherapeutics.

Chimeric HDAC and the cytoskeleton inhibitor broxbam as a novel therapeutic strategy for liver cancer

Broxbam, also known as N-hydroxy-4-{1-methoxy-4-[4′-(3′-bromo-4′,5′-dimethoxyphenyl)-oxazol-5′-yl]-2-phenoxy} butanamide, is a novel chimeric inhibitor that contains two distinct pharmacophores in its molecular structure. It has been previously demonstrated to inhibit the activity of histone deacetylases (HDAC) and tubulin polymerisation, two critical components required for cancer growth and survival. In the present study, the potential suitability of broxbam for the treatment of liver cancer was investigated. The effects of broxbam on cell proliferation and apoptosis, in addition to the under-lying molecular mechanism of action, were first investigated in primary liver cancer cell lines Huh7, HepG2, TFK1 and EGI1. Real-time proliferation measurements made using the iCEL-Ligence system and viable cell number counting following crystal violet staining) revealed that broxbam time- and dose-dependently reduced the proliferation of liver cancer cell lines with IC50 values <1 µM. In addition, a significant inhibition of the growth of hepatoblastoma microtumours on the chorioallantoic membranes (CAM) of fertilised chicken eggs by broxbam was observed according to results from the CAM assay, suggesting antineoplastic potency in vivo. Broxbam also exerted apoptotic effects through p53- and mitochondria-driven caspase-3 activation in Huh7 and HepG2 cells according to data from western blotting (p53 and phosphorylated p53), mitochondrial membrane potential measurements (JC-1 assay) and fluorometric capsase-3 measurements. Notably, no contribution of unspecific cytotoxic effects mediated by broxbam were observed from LDH-release measurements. HDAC1, -2, -4 and -6 expression was measured by western blotting and the HDAC inhibitory potency of broxbam was next evaluated using subtype-specific HDAC enzymatic assays, which revealed a largely pan-HDAC inhibitory activity with the most potent inhibition observed on HDAC6. Silencing HDAC6 expression in Huh7 cells led to a drop in the expression of the proliferation markers Ki-67 and E2F3, suggesting that HDAC6 inhibition by broxbam may serve a predomi-nant role in their antiproliferative effects on liver cancer cells. Immunofluorescence staining of cytoskeletal proteins (α-tubulin & actin) of broxbam-treated HepG2 cells revealed a pronounced inhibition of tubulin polymerisation, which was accompanied by reduced cell migration as determined by wound healing scratch assays. Finally, data from zebrafish angiogenesis assays revealed marked antiangiogenic effects of broxbam in vivo, as shown by the suppression of subintestinal vein growth in zebrafish embryos. To conclude, the pleiotropic anticancer activities of this novel chimeric HDAC- and tubulin inhibitor broxbam suggest that this compound is a promising candidate for liver cancer treatment, which warrants further pre-clinical and clinical evaluation.

Cheminformatics Application in the Phytochemical and Biological Study of Eucalyptus globulus L. Bark as a Potential Hepatoprotective Drug

Natural products are considered as a good source of antifibrotic agents, but identifying and isolating bioactive molecule(s) is still challenging. Fortunately, numerous computational techniques have evolved to save time and efforts in this field. The aim of the current study was to utilize several cheminformatics software to study the chemical and biological features of the bark of Eucalyptus globulus cultivated in Egypt. Sirius software, with the aid of online databases, was used to process liquid chromatography–mass spectrometry (LC–MS) chemical profiling and predict precise molecular formulae, chemical classes, and structures. Accordingly, 37 compounds were tentatively identified, including 15 reported here for the first time from this species. Also, the BioTransformer tool was successfully applied for in silico virtual study of the human metabolism of these compounds, and 1960 different products were obtained through various metabolic pathways. Finally, an electronic library of the identified compounds and their metabolites were developed and docked in silico against eight different protein targets that are involved in the liver fibrosis process. The results revealed that the extract may have a potential hepatoprotective effect through several mechanisms and that the metabolites have the highest binding affinities to the relevant enzymes than their parent compounds. The extract was found to show potent cytotoxic activity against the liver cancer cell lines HEPG2 and HUH-7, and its absorption was enhanced through nanoformulation, as proved using the ex vivo everted gut sac method.

Biological Activity and Stability of Aeruginosamides from Cyanobacteria.

Aeruginosamides (AEGs) are classified as cyanobactins, ribosomally synthesized peptides with post-translational modifications. They have been identified in cyanobacteria of genera Microcystis, Oscillatoria, and Limnoraphis. In this work, the new data on the in vitro activities of three AEG variants, AEG A, AEG625 and AEG657, and their interactions with metabolic enzymes are reported. Two aeruginosamides, AEG625 and AEG657, decreased the viability of human breast cancer cell line T47D, but neither of the peptides was active against human liver cancer cell line Huh7. AEGs also did not change the expression of MIR92b-3p, but for AEG625, the induction of oxidative stress was observed. In the presence of a liver S9 fraction containing microsomal and cytosolic enzymes, AEG625 and AEG657 showed high stability. In the same assays, quick removal of AEG A was recorded. The peptides had mild activity against three cytochrome P450 enzymes, CYP2C9, CYP2D6 and CYP3A4, but only at the highest concentration used in the study (60 µM). The properties of AEGs, i.e., cytotoxic activity and in vitro interactions with important metabolic enzymes, form a good basis for further studies on their pharmacological potential.

Antitumoral Properties of a Pincer-Type Isonicotinohydrazone-Hg(II) Complex

Objective: The aim of this study was to evaluate the antitumoral properties of the novel Hg(SCN)2 complex of a pincer-type isonicotinohyrazone ligand (PTIH). Materials and Methods: Hydrazine ligands were prepared through a Schiff base condensation between the 2-Acetylpyridine and isonicotinoyl acid hydrazide. PTIH was synthetized by a branch tube method using Hg(SCN)2. In vitro cell killing activity, the induction of apoptosis, the influence on motility, and the effects on mRNA expression to a motility-related gene (E-cadherin) of PTIH were evaluated in A549, HepG2, HUH7 cancer cell lines, and BEAS2B non-cancer cell line. Results: PTIH was found to be cytotoxic to cancer cells, compared to non-cancers, and induced apoptosis. Additionally, it suppressed cell migration in A549 cells, leading to an increase in the levels of E-cadherin mRNA expression, and decreased colony formation in HepG2 and HUH7 cells. Through UV titration studies, it was determined that PTIH showed albumin binding and interacted with DNA by electrostatically and/or groove binding. Conclusion: PTIH exerted more cytotoxic effects in some cancer cells than in normal cells. This feature and other anticancer properties make it a promising agent.

The Mechanism of miR-141 Regulating the Proliferation and Metastasis of Liver Cancer Cells by Targeting STAT4.

Background In recent years, it has been reported that miRNA can be used as one of the markers of tumor diagnosis, treatment, and prognosis (including liver cancer), and it plays an important role in tumorigenesis. However, there are still very few studies on the mechanism and role of miR-141 in liver cancer. Methods qRT-PCR was used to test the expressions of miR-141 and STAT4 in collected liver cancer tissues and adjacent tissues, cultured liver cancer cell lines MHCC97H, Hep3B, and Huh7, and normal human liver cells HL7702. After processing the results of the qRT-PCR experiment, liver cancer cell MHCC97H which has the lowest expression level was decided to be taken as the research object. miR-NC, miR-141 mimics, si-NC, si-STAT4, miR-141 mimics and pcDNA-NC, and miR-141 mimics and pcDNA-STAT4 were transfected into MHCC97H cells, respectively. The MTT assay was used to detect the proliferation of each group of cells, and the Transwell test was used to detect the effect of miR-141 on cell proliferation, migration, and invasion. The interaction between miR-141 and STAT4 was verified by the dual-luciferase reporter experiment, and the expression level of Cyclin D1 and MMP2 was detected by the western blot. Results Compared with normal cell HL7702, the expression level of miR-141 in liver cancer cell lines was relatively low (P < 0.05) and the expression level of STAT4 in liver cancer cell lines was relatively high (P < 0.05) after testing the expression level of STAT4; transfecting miR-141 mimics or Si-SLBP can inhibit cell proliferation, migration, and invasion; dual-luciferase reporter experiments confirmed that miR-141 can specifically bind to the 3′UTR of STAT4; cotransfection of miR-141 mimics and pcDNA-STAT4 can antagonize the effects of miR-141 mimics on cell proliferation, migration, and invasion. Conclusion miR-141 can target the STAT4 gene expression to inhibit the proliferation, migration, and invasion of liver cancer cells.

MicroRNA-506-3p targets SIRT1 and suppresses AMPK pathway activation to promote hepatic steatosis.

Nonalcoholic fatty liver disease (NAFLD) is a complex type of liver disease that represents an important global health threat. The mechanistic basis of this disease remains incompletely understood. The present study sought to explore whether microRNA (miR)-506-3p served a functional role in the onset and/or progression of NAFLD. To that end, high levels of glucose were used to treat liver cancer cell lines (HepG2 and Huh7) to model hepatic steatosis, and the expression levels of miR-506-3p and its downstream target genes were assessed. The cells of this hepatic steatosis model were transfected with miR-506-3p mimic molecules to explore the effect of miR-506-3p overexpression on cell viability, target gene expression and AMP-activated protein kinase (AMPK) phosphorylation. Via bioinformatics approaches, sirtuin 1 (SIRT1) was identified as a potential miR-506-3p target gene with relevance in NAFLD, and this interaction was confirmed via luciferase reporter assay. In the hepatic steatosis model of the present study, miR-506-3p expression level was significantly increased, whereas SIRT1 mRNA/protein levels and AMPK phosphorylation levels were markedly decreased. Transfection of the cells with miR-506-3p mimics led to significant SIRT1 downregulation, while miR-506-3p inhibitor molecules exhibited the opposite effect, with similar trends observed in the phosphorylation status of AMPK. These results suggested that miR-506-3p can inhibit SIRT1 expression and associated AMPK phosphorylation in HepG2 and Huh7 cells in an in vitro hepatic steatosis model system. These data indicated that the miR-506-3p/SIRT1/AMPK axis may be valuable as a therapeutic target in patients affected by NAFLD.

If It Works, Don't Touch It? A Cell-Based Approach to Studying 2-[18F]FDG Metabolism.

The glucose derivative 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) is still the most used radiotracer for positron emission tomography, as it visualizes glucose utilization and energy demand. In general, 2-[18F]FDG is said to be trapped intracellularly as 2-[18F]FDG-6-phosphate, which cannot be further metabolized. However, increasingly, this dogma is being questioned because of publications showing metabolism beyond 2-[18F]FDG-6-phosphate and even postulating 2-[18F]FDG imaging to depend on the enzyme hexose-6-phosphate dehydrogenase in the endoplasmic reticulum. Therefore, we aimed to study 2-[18F]FDG metabolism in the human cancer cell lines HT1080, HT29 and Huh7 applying HPLC. We then compared 2-[18F]FDG metabolism with intracellular tracer accumulation, efflux and the cells’ metabolic state and used a graphical Gaussian model to visualize metabolic patterns. The extent of 2-[18F]FDG metabolism varied considerably, dependent on the cell line, and was significantly enhanced by glucose withdrawal. However, the metabolic pattern was quite conserved. The most important radiometabolites beyond 2-[18F]FDG-6-phosphate were 2-[18F]FDMannose-6-phosphate, 2-[18F]FDG-1,6-bisphosphate and 2-[18F]FD-phosphogluconolactone. Enhanced radiometabolite formation under glucose reduction was accompanied by reduced efflux and mirrored the cells’ metabolic switch as assessed via extracellular lactate levels. We conclude that there can be considerable metabolism beyond 2-[18F]FDG-6-phosphate in cancer cell lines and a comprehensive understanding of 2-[18F]FDG metabolism might help to improve cancer research and tumor diagnosis.

Chemical and biological characterization of lipid profile from Hydroclathrus clathraus

This research addresses studying the lipid content of Hydroclathrus clathraus brown algae from the Red Sea, Egypt Region and their assess acute oral LD50 of sterol fraction in albino mice and anticancer activity against different cancer cell lines including; colon cancer cell line (Caco-2) and liver cancer cell line (Huh-7) using MMT, apoptosis analysis, EGFR and genes’ expression. Total lipids, glycolipid, sulfolipid, and phospholipid of Hydroclathrus clathraus content were 1mg/g, 0.08 mg/g, 0.25±0.01 mg/g and 0.57±0.04 mg/g respectively. The LD50 of Hydroclathrus clathraus sterols was greater than 3g/kg in the mice. The major fatty acid is palmitic acid (67.04%). Polyunsaturated fatty acids content was arachidonic (8.05%), α-Linolenic acid (4.36%) and Linoleic acid (1.69%). The major sterols fractions were Hexacosane (18.54%), Octacosane (14.96%) and Nonadecane (13.31%). Cholesterol (8.32%), β-Sitosterol (2.08%) and Campesterol (1.36%) were presented as minor sterols. However, algal sterol showed the best potential antitumor activity with IC50 of 0.22 µg/ml and 3.09µg/ml against CaCo2 and HuH7. These sterols fraction reduced tumor genesis by three mechanisms that firstly, the cells' apoptosis which may reduce the regulatory suppressive activity of the epidermal growth factor receptor (EGFR) enzyme, secondly, gene expression of bcl2 was downregulated and thirdly, it induced cell cycle arrest at the G1/S. Thus, algal sterols can be used as medicine to treat disorders liver and colon cancers

Cooperative Role of Thrombopoietin and Vascular Endothelial Growth Factor-A in the Progression of Liver Cirrhosis to Hepatocellular Carcinoma.

Primary thrombopoietic mediator thrombopoietin (THPO) is mainly produced by the liver; it may act as a growth factor for hepatic progenitors. Principal angiogenesis inducer vascular endothelial growth factor-A (VEGF-A) is critical for the complex vascular network within the liver architecture. As a cross-regulatory loop between THPO and VEGF-A has been demonstrated in the hematopoietic system, the two growth factors were hypothesized to cooperatively contribute to the progression from liver cirrhosis (LC) to hepatocellular carcinoma (HCC). The mRNA and protein expression levels of THPO, VEGF-A, and their receptors were examined, compared, and correlated in paired cancerous and LC tissues from 26 cirrhosis-related HCC patients, using qRT-PCR and immunohistochemistry. THPO and VEGF-A were alternatively silenced by small interfering RNA (siRNA) in human liver cancer cell lines Huh7 and HepG2. THPO and VEGF-A expressions significantly increased in tumor versus LC tissues. HCC and paired LC cells expressed similar levels of THPO receptor (R), whereas vascular endothelial growth factor receptor (VEGFR) -1 and VEGFR-2 levels were higher in HCC than in corresponding LC tissue samples. A significant linear correlation emerged between THPO and VEGF-A transcripts in HCC and, at the protein level, THPO and THPOR were significantly correlated with VEGF-A in tumor tissues. Both HCC and LC expressed similar levels of gene and protein hypoxia inducible factor (HIF)-1α. Positive cross-regulation occurred with the alternative administration of siRNAs targeting THPO and those targeting VEGF-A in hypoxic liver cancer cell lines. These results suggest THPO and VEGF-A might act as interdependently regulated autocrine and/or paracrine systems for cellular growth in HCC. This might be clinically interesting, since new classes of THPOR agonistic/antagonistic drugs may provide novel therapeutic options to correct the frequent hemostatic abnormality seen in HCC patients.

Study on synergistic anti-tumor effect of Shuangdan Capsules combined with 5-FU on hepatocellular carcinoma cells Huh-7 and xenograft mice

This paper discussed the synergistic anti-tumor effect of Shuangdan Capsules combined with 5-fluorouracil(5-FU) on human liver cancer cell line Huh-7 and tumor bearing mice. The effects of Shuangdan Capsules combined with 5-FU on the activity and vascular endothelial growth factor(VEGF) receptor protein expression of Huh-7 cells were investigated, and the effects of drug combination on tube formation of HUVEC cell were also verified. In addition, the mice model of Huh-7 was established to observe the anti-tumor effect of drug combination and the distribution of tumor blood flow in tumor bearing mice by using molecular imaging. HPLC analysis showed that Shuangdan Capsules mainly consisted of danshensusodium, protocatechuic aldehyde, paeoniflorin, rosmarinic acid, alkannic acid, salvianolic acid B, and paeonol. In MTT experiment, the inhibition rate of Shuangdan Capsules(20 mg·L~(-1)) and 5-FU(1 μmol·L~(-1)) on Huh-7 cells was 60%, and the CI value was 0.59, suggesting that these two drugs had synergistic anti-hepatoma cells effect. The expression of VEGF receptor in Huh-7 cells was inhibited by the combination of these two drugs. In addition, the process of HUVEC was slow, and the number, length and area of the lumen branches decreased significantly. In vivo, Shuangdan Capsules combined with 5-FU inhibited the growth and prolongation of survival of Huh-7 cells in subcutaneous transplanted tumor nude mice; serum expression of CD31 and VEGF in nude mice were decreased, while caspase-3 was increased. Meanwhile, the drug combination significantly inhibited the expressions of MMP2 and VEGF in tumor tissues. Ultrasound showed that Shuangdan Capsules combined with 5-FU also inhibited tumor angiogenesis and reduced blood flow of tumor tissue. The results showed that Shuangdan Capsules combined with 5-FU may inhibit tumor angiogenesis by inhibiting VEGF and MMP2 expressions, thereby blocking tumor growth.

New Tailored RNA-Targeted Organometallic Drug Candidates against Huh7 (Liver) and Du145 (Prostate) Cancer Cell Lines.

New organometallic drug candidates [Ph2Sn(HL)], 1, and [Ru(η6--p-cymene)(HL)Cl], 2, were designed and synthesized by in situ reaction of a Schiff base ligand (HL) and diphenyltin dichloride and [RuCl2(p-cymene)]2, respectively. The drug candidates 1 and 2 have been characterized by spectroscopic methods (Fourier-transform infrared spectroscopy, UV–vis, and 1H/13C NMR), elemental analysis, and single X-ray crystallographic studies (in case of 1). The ground-state geometry optimization of 1 and 2 was performed by density functional theory calculations. The interaction of 1 and 2 with tRNA was assessed by absorption spectroscopy, cyclic voltammetry, circular dichroism, and ethidium bromide displacement assay using fluorescence emission spectroscopy to determine their potential to act as antitumor agents. The cytotoxicity of 1 and 2 was screened against human liver carcinoma (Huh7), prostate cancer (Du145), and the normal prostate cell line (PNT 2). The results implicated a dose-dependent growth inhibition of the two cancer cells at concentrations (2.5–15 μM) of 1 and 2 with the treatment after 48 h. Interestingly, 1 revealed good selective activity toward the liver cancer cell line (Huh7). Furthermore, both the drug candidates 1 and 2 were found to be nontoxic toward the PNT 2 normal cell line. These studies lay a paradigm for rational efficacious drug design for chemotherapeutic intervention in cancers using new tailored organometallic drug entities; organotin(IV) and organoruthenium(II) have been demonstrated to be viable for the safe administration and specific targeted drug uptake by the resistant cancerous cell lines at low intracellular concentrations.

MiR-328-3p overexpression attenuates the malignant proliferation and invasion of liver cancer via targeting Endoplasmic Reticulum Metallo Protease 1 to inhibit AKT phosphorylation.

BackgroundLiver cancer is one of the most common cancers worldwide. microRNAs (miRNAs) have been recognized as minimally invasive prognostic markers for distinct types of cancer. This study evaluates the mitigation role of miR-328-3p on liver cancer in vitro and in vivo.MethodsLiver cancer cell line Huh-7 and HepG2 were used for in vitro experiments. Compared with the control group, miR-328-3p overexpression inhibited the proliferation, invasion, and promoted apoptosis of Huh-7 cells. miR-328-3p and endoplasmic reticulum metalloprotease 1 (ERMP1) had an excellent targeting relationship. Compared with the pcDNA-ERMP1 transfection group, the ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in miR-328-3p mimic and pcDNA-ERMP1 co-transfection group were significantly decreased. Animal models were set up using four-week-old immunodeficient BABL/c female nude mice. Huh-7 cells transfected with lentivirus holding miR-328-3p or empty vector were injected into the right dorsal side of BABL/c nude mice, respectively. Tumor volume was measured every five days. After one month, animals were sacrificed, xenograft tumors were dissected and weighed for RT-PCR and immunohistochemical assays.ResultsCompared with control group, miR-328-3p overexpression significantly inhibited tumor weight (0.46±0.07 vs. 0.11±0.05 g, P<0.05) and tumor volume (1876±321 vs. 543±168 mm3, P<0.05) after thirty days. miR-328-3p overexpression significantly downregulated the percentage of Ki67 positive cells, N-cadherin positive cells and vimentin positive cells.ConclusionsThese findings suggested that miR-328-3p could be a new treatment or a novel marker for liver cancer prognosis.

Phytogenic Synthesis of Nickel Oxide Nanoparticles (NiO) Using Fresh Leaves Extract of Rhamnus triquetra (Wall.) and Investigation of Its Multiple In Vitro Biological Potentials.

Chemically nickel oxide nanoparticles (NiONPs) involve the synthesis of toxic products, which restrict their biological applications. Hence, we developed a simple, eco-friendly, and cost-efficient green chemistry method for the fabrication of NiONPs using fresh leaf broth of Rhamnus triquetra (RT). The RT leaves broth was used as a strong reducing, capping, and stabilizing agent in the formation of RT-NiONPs. The color change in solution from brown to greenish black suggests the fabrication of RT-NiONPs which was further confirmed by absorption band at 333 nm. The synthesis and different physicochemical properties of RT-NiONPs were investigated using different analytical techniques such as UV-Vis (ultraviolet−visible spectroscopy), XRD (X-ray powder diffraction), FT-IR (Fourier-transform infrared spectroscopy), SEM (scanning electron microscopy), TEM (transmission electron microscopy), EDS (energy-dispersive X-ray spectroscopy), DLS (dynamic light scattering) and Raman. Further, RT-NiONPs were subjected to different in vitro biological activities and revealed distinctive biosafe and biocompatibility potentials using erythrocytes and macrophages. RT-NiONPs exhibited potential anticancer activity against liver cancer cell lines HUH7 (IC50: 11.3 µg/mL) and HepG2 (IC50: 20.73 µg/mL). Cytotoxicity potential was confirmed using Leishmanial parasites promastigotes (IC50: 27.32 µg/mL) and amastigotes (IC50: 37.4 µg/mL). RT-NiONPs are capable of rendering significant antimicrobial efficacy using various bacterial and fungal strains. NiONPs determined potent radical scavenging and moderate enzyme inhibition potencies. Overall, this study suggested that RT-NiONPs can be an attractive and eco-friendly candidate. In conclusion, current study showed potential in vitro biological activities and further necessitate different in vivo studies in various animal models to develop leads for new drugs to treat several chronic diseases.

Silence of cytoskeleton-associated protein 2 represses cell proliferation and migration and promotes apoptosis in liver cancer cell lines.

To investigate the roles of cytoskeleton-associated protein 2 (CKAP2) in proliferation, apoptosis, and migration in liver cancer cells and the potential mechanisms. Human normal hepatocyte L02 and liver cancer cell lines HepG2, Huh7, and SMMC-7721 were cultured. The CKAP2 expression was detected by real-time PCR and Western blotting. HepG2 cells were randomly divided into a control group, a negative control (NC) group, and a CKAP2 silencing (siCKAP2) group. CCK-8 and BrdU assays were used to evaluate cell viability and proliferation, respectively. Transwell assay was employed to determine cell migration and invasion. The protein levels of cleaved-caspase 3, Bax, E-cadherin, N-cadherin, Vimentin, phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) were determined by Western blotting. Compared with normal hepatocyte L02, CKAP2 was highly expressed in liver cancer cell lines HepG2, Huh7, and SMMC-7721 (all P<0.05). Compared with the NC group, cell viability and proliferation rate of the siCKAP2 group were decreased (both P<0.05). The apoptotic rate, protein expression of cleaved-caspase 3 and Bax in the siCKAP2 group were significantly higher than those in the NC group (all P<0.05). Compared with the NC group, cell migration and invasion rates of the siCKAP2 group were significantly attenuated (both P<0.05). Compared with the NC group, E-cadherin protein expression in siCKAP2 group was increased, while protein expression levels of Vimentin, N-cadherin, p-JAK2, and p-STAT3 were decreased (all P<0.05). CKAP2 gene silence inhibits proliferation, migration, and invasion, and promotes apoptosis in liver cancer cells, while JAK2/STAT3 signaling pathway may be involved in these processes.

Synthesis, anticancer, and computational studies of 1, 3, 4‐oxadiazole‐purine derivatives

AbstractTheophylline‐7‐acetic acid (acefylline) (3) and its derivatives are pharmacologically active compounds and generally recognized as bronchodilators for the treatment of respiratory diseases like acute asthma for over 70 years. In this article, synthesis of 2‐((5‐((1,3‐dimethyl‐2,6‐dioxo‐2,3‐dihydro‐1H‐purin‐7(6H)‐yl)methyl)‐1,3,4‐oxadiazol‐2‐yl)thio)‐N‐arylacetamides (10a‐j) has been reported. All the synthesized derivatives (10a‐j) were structurally verified by FT‐IR, 1H NMR, 13C NMR and evaluated for their anti‐cancer (using MTT assay), hemolytic and thrombolytic potential. N‐(4‐Chlorophenyl)‐2‐(5‐((1,3‐dimethyl‐2,6‐dioxo‐2,3‐dihydro‐1H‐purin‐7(6H)‐yl)methyl)‐1,3,4‐oxadiazol‐2‐ylthio)acetamide (10g) was found to be the most active against human liver cancer cell lines (Huh7) having cell viability 53.58 ± 1.28 using 100 μg/mL concentration of compound which was further in‐silico modelled to describe the possible mechanistic insights for its anti‐proliferative activity. The results of hemolytic and thrombolytic activities indicated that these derivatives were less toxic and hold considerable potential as a drug candidate. 2‐(5‐((1,3‐Dimethyl‐2,6‐dioxo‐2,3‐dihydro‐1H‐purin‐7(6H)‐yl)methyl)‐1,3,4‐oxadiazol‐2‐ylthio)‐N‐(2‐fluorophenyl)acetamide (10c) of the series was found to be least toxic with 0.1% hemolysis relative to ABTS (95.5%) as positive control. 2‐(5‐((1,3‐Dimethyl‐2,6‐dioxo‐2,3‐dihydro‐1H‐purin‐7(6H)‐yl)methyl)‐1,3,4‐oxadiazol‐2‐ylthio)‐N‐(tetrahydro‐2H‐pyran‐4‐yl)acetamide (10j) exhibited potent clot lysis activity (90%) as compared to negative control DMSO (0.57%).

Facile green synthesis approach for the production of chromium oxide nanoparticles and their different in vitro biological activities.

Green synthesis of nanoparticles using plants has become a promising substitute for the conventional chemical synthesis methods. In the present study, our aim was to synthesize chromium oxide nanoparticles (Cr2 O3 NPs) through a facile, low-cost, eco-friendly route using leaf extract of Rhamnus virgata (RV). The formation of Cr2 O3 NPs was confirmed and characterized by spectroscopic profile of UV-Vis, EDX, FTIR, and XRD analyses. The UV-visible spectroscopy has confirmed the formation of Cr2 O3 NPs by the change of color owing to surface plasmon resonance. The bioactive functional groups present in the leaf extract of RV involved in reduction and stabilization of Cr2 O3 NPs were determined by FTIR analysis. Based on XRD analysis, crystalline nature of Cr2 O3 NPs was determined. The morphological shape and elemental composition of Cr2 O3 NPs were investigated using SEM and EDX analyses, respectively. With growing applications of Cr2 O3 NPs in biological perspectives, Cr2 O3 NPs were evaluated for diverse biopotentials. Cr2 O3 NPs were further investigated for its cytotoxicity potentials against HepG2 and HUH-7 cancer cell lines (IC50 : 39.66 and 45.87 μg/ml), respectively. Cytotoxicity potential of Cr2 O3 NPs was confirmed against promastigotes (IC50 : 33.24 μg/ml) and amastigotes (IC50 : 44.31 μg/ml) using Leishmania tropica (KMH23 ). The Cr2 O3 NPs were further evaluated for antioxidants, biostatic, alpha-amylase, and protein kinase inhibition properties. Biocompatibility assay was investigated against human macrophages which confirmed the nontoxic nature of Cr2 O3 NPs. Overall, the synthesized Cr2 O3 NPs are biocompatible and nontoxic and proved to possess significant biopotentials. In future, different in vivo studies are needed to fully investigate the cytotoxicity and mechanism of action associated with these Cr2 O3 NPs.

SMAC mimetic birinapant inhibits hepatocellular carcinoma growth by activating the cIAP1/TRAF3 signaling pathway.

The present study investigated the effects and molecular mechanism of the second mitochondria-derived activator of caspase (SMAC) mimetic birinapant on the proliferation and apoptotic rate of liver cancer cells. Western blotting and reverse transcription-quantitative PCR were used to detect the protein and mRNA expression levels of cellular inhibitor of apoptosis 1 (cIAP1) and tumor necrosis factor receptor-associated factor 3 (TRAF3) in the liver cancer cell lines Huh7, H22 and HepG2, and the hepatocyte line AML12. Annexin V-FITC and Transwell assays were used to assess the effect of birinapant pretreatment on the apoptotic rate and invasive ability of liver cancer cells. Lentivirus-mediated silencing of TRAF3 was performed in liver cancer cells. Western blotting was used to detect the lentivirus silencing efficiency. A subcutaneous hepatocellular carcinoma model was established in nude mice and 15 days after tumor induction the subcutaneous tumors were measured in each group. Immunohistochemistry assays were used to detect the protein expression levels of proliferating cell nuclear antigen and caspase-3. The results suggested that the expression levels of cIAP1 and TRAF3 were lower in Huh7, H22 and HepG2 cells compared with AML12 cells. Pretreatment with birinapant promoted apoptosis and inhibited invasion of liver cancer cells by activating the cIAP1/TRAF3 axis. Birinapant also promoted apoptosis and inhibited the growth of subcutaneous hepatocellular carcinoma tumors in nude mice. The present results suggested that the SMAC mimetic birinapant may promote apoptosis, and inhibit the proliferation and invasion of liver cancer cells. The molecular mechanism responsible for the effects of birinapant may be related to activation of the cIAP1/TRAF3 signaling pathway by birinapant in liver cancer cells.

Anti-Proliferative Effects of an Extra-Virgin Olive Oil Extract Enriched in Ligstroside Aglycone and Oleocanthal on Human Liver Cancer Cell Lines.

Oleocanthal and ligstroside aglycone are olive oil-derived polyphenols. The former interferes with tumor growth with minor or no cytotoxicity on non-tumorigenic primary cell lines. The information about the bioactivity of ligstroside aglycone are scanty, with the exception of a known antioxidant power. Hepatocellular carcinoma is a malignant tumor with high mortality rates. Systemic chemotherapy is only marginally effective and is frequently complicated by toxicity. Previous observations have shown that hepatocellular carcinoma cell lines become more sensitive to taxol when it is combined with Tumor Necrosis Factor α (TNFα). The present work aimed to assess the effects of a polyphenolic extract containing both oleocanthal and ligstroside aglycone on proliferation and/or death in three liver cancer cell lines (HepG2, Huh7 and Hep3B). The possibility to enhance such effect by the addition of TNFα was also investigated. Both cell proliferation and death were enhanced by the exposure to the polyphenolic extract. Such effect was associated with induction of autophagy and could be potentiated by TNFα. The presence of ligstroside aglycone in the extract lowered the oleocanthal concentration required for cytotoxicity. These results show for the first time that the effects of a polyphenol extract can be potentiated by TNFα and that modulation of autophagy likely account for these effects.

Hispidulones A and B, two new phenalenone analogs from desert plant endophytic fungus Chaetosphaeronema hispidulum.

Two new phenalenone analogs hispidulones A (1) and B (2) were isolated from the specially bioenvironmental desert plant endophytic fungus Chaetosphaeronema hispidulum. The structure of these two compounds were elucidated by extensive spectra analysis including HR-ESI-MS, NMR (1H, 13C, 1H-1H COSY, HSQC, and HMBC), CD, and electronic circular dichroism (ECD) combined with quantum-chemical calculations adopting time-dependent density functional theory (TDDFT) approaches. The W long-ranged 1H-1H COSY and HMBC correlations are very important in the structural elucidation of these two compounds. Hispidulone A (1) possesses a cyclohexa-2,5-dien-1-one moiety, whereas hispidulone B (2) contains a hemiacetal OCH3 group, which are very rare in the structures of phenalenone analogs. According to structural features of these two compounds together considering the literature, the possible biosynthetic pathway of 1 and 2 was postulated. Hispidulone B (2) displayed cytotoxic activities against three cancer cell lines A549, Huh7, and HeLa with IC50 values of 2.71 ± 0.08, 22.93 ± 1.61, and 23.94 ± 0.33 μM.