HL-7702 Cells Research Articles

Empagliflozin Inhibits Hepatic Gluconeogenesis and Increases Glycogen Synthesis by AMPK/CREB/GSK3β Signalling Pathway.

Increases in glucose production and decreases in hepatic glycogen storage induce glucose metabolic abnormalities in type 2 diabetes (T2DM). Empagliflozin, a sodium-dependent glucose transporter 2 (SGLT2) inhibitor, is an effective hypoglycemic drug; however, the effects of empagliflozin on hepatic gluconeogenesis and glycogenesis are still unclear. In this study, we investigated the effects and mechanisms of empagliflozin on hepatic gluconeogenesis and glycogenesis in vivo and in vitro. Empagliflozin was administered via gavage to db/db mice for 8 weeks, and human hepatocyte HL7702 cells were treated with empagliflozin after palmitic acid (PA) stimulation. Compared with the control db/db mice, empagliflozin-treated mice showed a significant reduction in urine glucose levels, blood glucose levels, body weight and intraperitoneal glucose tolerance test (IPGTT) blood glucose levels. Moreover, the expression levels and activities of key gluconeogenesis enzymes PEPCK and G6Pase were dramatically reduced in the empagliflozin-treated mice, and the protein expression levels of AMPK/CREB/GSK3β signalling pathway-related molecules were significantly changed. In HL7702 cells, empagliflozin ameliorated glucose production and PEPCK and G6Pase expression and activity. Empagliflozin could also prevent the decreases in glycogen content and regulate the protein expression levels of AMPK/CREB/GSK3β signalling pathway-related molecules. Then, we selected the AMPK agonist AICAR and inhibitor compound C to further verify the effects of the AMPK signalling pathway on hepatic gluconeogenesis and glycogen synthesis. The results of the 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AIACR) intervention in HL7702 cells were consistent with those of empagliflozin treatment, and the effects of empagliflozin were abolished by compound C. In summary, empagliflozin could maintain glucose homoeostasis by reducing gluconeogenesis and increasing glycogenesis through the AMPK/CREB/GSK3β signalling pathway.

Two-Pronged Anti-Tumor Therapy by a New Polymer-Paclitaxel Conjugate Micelle with an Anti-Multidrug Resistance Effect.

IntroductionCancerous tumors are still a major disease that threatens human life, with tumor multidrug resistance (MDR) being one of the main reasons for the failure of chemotherapy. Thus, reversing tumor MDR has become a research focus of medical scientists.MethodsHere, a reduction-sensitive polymer prodrug micelle, mPEG-DCA-SS-PTX (PDSP), was manufactured with a new polymer inhibitor of drug resistance as a carrier to overcome MDR and improve the anti-tumor effect of PTX.ResultsThe PDSP micelles display good stability, double-responsive drug release, and excellent biocompatibility. The PDSP micelles reduced the cytotoxicity of PTX to normal HL-7702 cells and enhanced that to SMMC-7721 and MCF-7 cells in vitro. Improved sensitivity of A549/ADR to PDSP was also observed in vitro. Furthermore, in vivo experiments show reduced systemic toxicity and enhanced therapeutic efficacy of PTX to H22 subcutaneous tumor-bearing mice.ConclusionThis work proves that the reduction-sensitive polymer prodrug micelles carried by the new polymer inhibitor can be used as an alternative delivery system to target tumors and reverse MDR for paclitaxel and other tumor-resistant drugs.

Four temporin-derived peptides exhibit antimicrobial and antibiofilm activities against methicillin-resistant.

Temporin-GHa (GHa) was cloned from , showing a weak antimicrobial activity. In order to improve its bactericidal efficacy, GHaR6R, GHaR7R, GHaR8R and GHaR9W were designed and synthesized. Compared to the parent peptide, the GHa-derived peptides show potent antimicrobial activities against methicillin-resistant (MRSA), which is the main pathogen with high morbidity and mortality that causes various infections in humans. These peptides exert bactericidal actions on MRSA by permeabilizing the cytoplasmic membranes and damaging membrane integrity. All of the four peptides exhibit excellent stability under harsh conditions, including extreme temperature and salts. Furthermore, they inhibit the formation of biofilm and eradicate mature biofilm of MRSA. The GHa-derived peptides decrease bacterial surface hydrophobicity, autoaggregation and polysaccharide intercellular adhesion synthesis in concentration-dependent manner. Real-time quantitative reverse transcription PCR analysis revealed that the peptides downregulate the expression of adhesion genes involved in biofilm formation. Except for GHaR7R, the other three peptides have low hemolytic toxicity against human erythrocytes. In the presence of human erythrocytes, GHaR7R, GHaR8R and GHaR9W interact with MRSA preferentially. GHaR6R, GHaR8R and GHaR9W show less toxicity toward normal cells HL-7702 and hFOB1.19. These results suggest that the GHa-derived peptides may be promising antimicrobial candidates against MRSA infections.

MicroRNA-22-3p and MicroRNA-149-5p Inhibit Human Hepatocellular Carcinoma Cell Growth and Metastasis Properties by Regulating Methylenetetrahydrofolate Reductase.

microRNAs are small endogenous noncoding RNAs that have emerged as key negative regulators that target gene expression through RISC. Our previous study showed that the methylenetetrahydrofolate reductase gene (MTHFR) plays a key role in one carbon metabolism, which is downregulated by miR-22-3p and miR-149-5p, and that it could exert a potential anti-cancer effect. Whether miR-22-3p/miR-149-5p can regulate MTHFR to exert anti-cancer effects has become the focus of our research. Normal (HL-7702 cells) and cancerous (QGY-7703/HepG2 cells) human hepatocellular cells were transfected with 100 nM hsa-miR-22-3p/hsa-miR-149-5p mimic or controls. After 24, 48, and 72 h, cell proliferation ability was tested using CCK-8. The changes in MTHFR expression at both the transcriptional and translational levels were determined by RT-qPCR and Western blotting, respectively. Cancerous cell invasion and migration ability were confirmed by means of a transwell assay. We found that ectopic miR-22-3p/miR-149-5p inhibits hepatocellular carcinoma cell proliferation but does not inhibit normal human hepatocyte proliferation. The transfection of ectopic miR-22-3p/miR-149-5p downregulated the MTHFR expression in QGY-7703 and HepG2 but not in HL-7702. QGY-7703 and HepG2 migration and invasion were inhibited by ectopic miR-22-3p/miR-149-5p. Additionally, we found that ectopic miR-22-3p/miR-149-5p significantly increased the expression of TP53INP1 and PDCD4 in QGY-7703. The results of the study suggest that miRNA-22-3p and miRNA-149-5p inhibit tumor growth and metastasis properties may be by regulating MTHFR and that they exert anticancer effects in hepatocellular carcinoma cells.

Protective effects of silibinin against ethanol- or acetaldehyde-caused damage in liver cell lines involve the repression of mitochondrial fission

Silibinin is a natural polyphenolic flavonoid, isolated from the seeds of the milk thistle of Silybum marianum (L.) Gaertn. Silibinin has been widely used clinically as a traditional medicine for liver diseases. This study investigated the protective role of silibinin in ethanol- or acetaldehyde-induced apoptosis in human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells, focusing on elucidation of the underlying mechanism in vitro. The toxicity of ethanol or acetaldehyde was evaluated by MTT assay. Apoptosis-related proteins, mitochondrial fission-associated proteins and mitochondrial fusion-associated proteins were analyzed by western blotting and immunofluorescence microscopy. Present experimental results demonstrated that silibinin improved cell viability, reduced the enzyme activities of AST/ALT and ALDH/ADH, inhibited apoptosis and recovered mitochondrial function in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. Silibinin reduced the expression of mitochondrial fission-associated proteins, dynamin-related protein 1 (DRP1), but increased mitochondrial fusion-associated proteins, optic atrophy 1 (OPA1) and mitofusin 1 (MFN1). Accordingly, inhibition of DRP1 activity with its pharmacological inhibitor or siDRP1 efficiently attenuated ethanol- or acetaldehyde-induced apoptosis, whereas activation of DRP1 by using staurosporine (STS) further increased apoptosis in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. The results show that silibinin protects cells against ethanol- or acetaldehyde-induced mitochondrial fission that results in apoptosis.

Quercetin protects human liver cells from o,p’-DDT-induced toxicity by suppressing Nrf2 and NADPH oxidase-regulated ROS production

Epidemiologic studies have revealed that Dichlorodiphenyltrichloroethane (DDT) and its metabolites are associated with liver diseases. However, there has been little emphasis on the mechanism underlying liver toxicity of o,p’-DDT and relevant effective inhibitors investigation. This study indicated o,p’-DDT exposure significantly decreased cell viability and promoted lactate dehydrogenase (LDH) release based on the investigation of cytotoxicity by trypan blue exclusion counts, MTT, and lactate dehydrogenase (LDH) assays. Comet, micronuclei, and DNA-protein crosslinks (DPC) assays demonstrated o,p’-DDT exposure increased the comet parameters, micronuclei frequency, and DPC coefficient. Meanwhile, we found o,p’-DDT induced mitochondria-dependent apoptosis, which is characterized by the loss of mitochondrial membrane potential (Δψm), decreased Bcl-2 expression, and increased protein levels of Bax, cytochrome c, activated-caspase-9, and activated-caspase-3. Furthermore, o,p’-DDT induced reactive oxygen species (ROS) overproduction, decreased the protein levels of nuclear factor erythroid-derived 2-like 2 (Nrf2) in the nuclear, and enhanced the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. However, quercetin treatment significantly antagonized o,p’-DDT-induced cytotoxicity, genotoxicity, and apoptosis as well as effects on ROS, Nrf2, and NADPH oxidase. Taken together, these findings suggested quercetin could alleviate o,p’-DDT-induced toxicity in HL-7702 cells via inhibiting ROS production, which is modulated by down-regulating nuclear Nrf2 levels and NADPH oxidase expression.

Ratiometric fluorescence resonance energy transfer for reliable and sensitive detection of intracellular telomerase RNA via strand displacement reaction amplification

Human telomerase RNA (hTR) is one essential component of telomerase and is overexpressed in tumor cells. Therefore, the reliable and sensitive detection of hTR is essential for the early cancer diagnosis. Herein, to avoid the false positive signals caused by co-existing components in the cell, a ratiometric fluorescence resonance energy transfer (FRET) strategy was developed to achieve reliable detection of intracellular hTR. Manganese dioxide nanosheets (MnO2NS) with good biocompatibility carry two fluorophore-labelled hairpin DNA probes into the cancer cell and then release the probes via decomposition of MnO2NS by intracellular L-glutathione reduced (GSH). Then, hTR triggered the cyclic strand displacement reaction (SDR) between two hairpin DNA probes to continuously form DNA duplexes, which made two fluorophores close to each other and led to an effective FRET. Fluorescence imaging demonstrated a higher expression level of hTR in HeLa cells than that in normal HL-7702 cells. The high specificity of hairpin DNA probes and SDR make it easy to discriminate the single-base mutation. Therefore, it provides a highly sensitive, simple and reliable method for the extracellular and intracellular detection of hTR.

Serum superoxide dismutase level is a potential biomarker of disease prognosis in patients with HEV-induced liver failure

BackgroundViral hepatitis E clinically ranges from self-limiting hepatitis to lethal liver failure. Oxidative stress has been shown to mediate hepatic inflammation during HBV-induced liver failure. We investigated whether a biomarker of oxidative stress may be helpful in assessing severity and disease outcomes of patients with HEV-induced liver failure.MethodsClinical data were obtained from patients with HEV-induced acute viral hepatitis (AVH, n = 30), acute liver failure (ALF, n = 17), and acute-on-chronic liver failure (ACLF, n = 36), as well as from healthy controls (HC, n = 30). The SOD and HMGB1 levels were measured in serum by ELISA. HL-7702 cells were cultured and stimulated by serum from HEV-infected patients or by HMGB1; oxidative status was investigated by CellROX and apoptosis was investigated by flow cytometry.ResultsPatients with HEV-induced liver failure (including ALF and ACLF) showed increased SOD levels compared with HEV-AVH patients and healthy controls. SOD levels > 400 U/mL were associated with a significantly higher risk of mortality in HEV-ALF and HEV-ACLF patients. Serum from HEV-infected patients led to ROS accumulation, HMGB1 secretion, and apoptosis in HL-7702 cells. Antioxidant treatment successfully inhibited HEV-induced HMGB1 secretion, and HMGB1 promoted apoptosis in HL-7702 cells.ConclusionHEV increased oxidative stress in the pathogenesis of HEV-induced hepatic diseases. Early testing of serum SOD may serve as a predictor of both HEV-ALF and HEV-ACLF outcomes. Moreover, development of strategies for modulating oxidative stress might be a potential target for treating HEV-induced liver failure patients.

Ginsenoside Rb1 Attenuates Triptolide-Induced Cytotoxicity in HL-7702 Cells via the Activation of Keap1/Nrf2/ARE Pathway

Triptolide (TP) is the major bioactive compound extracted from Tripterygium wilfordii Hook F. It exerts anti-inflammatory, antirheumatic, antineoplastic, and neuroprotective effects. However, the severe hepatotoxicity induced by TP limits its clinical application. Ginsenoside Rb1 has been reported to possess potential hepatoprotective effects, but its mechanism has not been fully investigated. This study was aimed at investigating the effect of ginsenoside Rb1 against TP-induced cytotoxicity in HL-7702 cells, as well as the underlying mechanism. The results revealed that ginsenoside Rb1 effectively reversed TP-induced cytotoxicity in HL-7702 cells. Apoptosis induced by TP was suppressed by ginsenoside Rb1 via inhibition of death receptor-mediated apoptotic pathway and mitochondrial-dependent apoptotic pathway. Pretreatment with ginsenoside Rb1 significantly reduced Bax/Bcl-2 ratio and down-regulated the expression of Fas, cleaved poly ADP-ribose polymerase (PARP), cleaved caspase-3, and -9. Furthermore, ginsenoside Rb1 reversed TP-induced cell cycle arrest in HL-7702 cells at S and G2/M phase, via upregulation of the expressions of cyclin-dependent kinase 2 (CDK2), cyclin E, cyclin A, and downregulation of the expressions of p53, p21, and p-p53. Ginsenoside Rb1 increased glutathione (GSH) and superoxide dismutase (SOD) levels, but decreased the reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Pretreatment with ginsenoside Rb1 enhanced the expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), total Nrf2, NAD(P)H: quinone oxidoreductases-1 (NQO-1), heme oxygenase-1 (HO-1), and Kelch-like ECH-associated protein 1 (Keap1)/Nrf2 complex. Therefore, ginsenoside Rb1 effectively alleviates TP-induced cytotoxicity in HL-7702 cells through activation of the Keap1/Nrf2/ARE antioxidant pathway.

Glutathione ameliorates Hypoxia/Reoxygenation (H/R) induced hepatocyte oxidative damage via regulating HO-1 signaling

In our study, CCK-8 assay and LDH release detection were performed to detect the optimal protective concentration of GSH on HL7702 cell viability during H/R injury. HL7702 cells were randomly divided into four groups: Control group, H/R group, H/R+GSH group, and H/R+GSH+HO-1-siRNA group. Then, reactive oxygen species (ROS) was evaluated by DHE staning, MDA, T-SOD measurements; Cell injury was detected by CCK-8, LDH release, and supernatant AST and ALT levels; Apoptosis was determined by Hoechst staining and caspase 3 level. Compared with controls, H/R caused significant HL7702 cell injury evidenced as reduced cell viability, increased LDH release and apoptotic cell death (P < 0.01), with concomitant increases in ROS and MDA production (P < 0.01), while treated with GSH in H/R group significantly attenuated oxidative injury, enhanced cell viability and downregulated cell apoptosis (P < 0.01) together with HO-1 upregulation (P < 0.01). Knockdown HO-1 by its siRNA cancelled the protective effects of GSH from H/R compared with GSH group (P < 0.01). HO-1 was induced in HL7702 exposed to H/R injury and its level was obviously overexpressed after H/R injury with GSH treatment, suggesting its protective potential in GSH against H/R injury. GSH increases the expression of HO-1, which enhanced the early antioxidative activity and played a protective role against HL7702 cells H/R injury.

Self-Assembled Tocopherol-Albumin Nanoparticles with Full Biocompatibility for Chemo-photothermal Therapy against Breast Cancer.

The combination of photothermal therapy (PTT) and chemotherapy has proven to be a promising strategy for cancer treatment. Various nanomaterials have shown great potential in combination therapy, including gold, graphene oxide, iron oxide, and other nanoparticles. However, their undefinable toxicity in vivo greatly slowed down their development for clinical applications. The present work aimed to develop a multifunctional nanoparticle for chemo-photothermal therapy composed of acknowledged biocompatible materials. A novel biocompatible nanoparticle (HIT-NPs) was self-assembled through the intrinsic interaction between D-α-tocopherol Succinate (TOS), human serum albumin (HSA) and indocyanine green (ICG). Doxorubicin (DOX) was then loaded due to the ion pairing between DOX and TOS. The feasibility of combined chemo-photothermal therapy induced by DOX-loaded HIT-NPs was carefully evaluated. In vitro, HIT-NPs showed no cytotoxicity on human normal liver cells (HL-7702 cells) but obvious killing effects on murine breast cancer cells (4T1 cells). The combined chemo-photothermal therapeutic effect on 4T1 cells was successfully obtained. DOX-loaded HIT-NPs could effectively accumulate in 4T1 subcutaneous tumors after intravenous injection, and the tumor temperature rapidly increased under laser exposure, indicating the feasibility of PTT in vivo. The self-assembled HIT-NPs could provide a promising platform for combined chemo- photothermal cancer therapy with full biocompatibility.

A primer extension activating 3D DNAzyme walker for in situ imaging and sensitive detection of telomerase activity.

Acquiring information on telomerase activity at multiple levels contributes to a better understanding of its role in various physiological and pathological processes. Herein, a primer extension activating 3D DNAzyme walker is developed for in situ imaging and sensitive detection of telomerase activity. This walker is constructed via co-modifying specially designed hairpin structured walking strands and track strands on a gold nanoparticle (AuNP). The walking strand contains a pre-blocked DNAzyme sequence and a telomerase primer hybridized to its root. The track strand embeds at an RNA cleavage site and is labeled with the FAM group. After this walker is taken up by cells, the telomerase primer is extended under the action of endogenous telomerase to liberate DNAzyme. The liberated DNAzyme cuts track strands in the presence of the cofactor Mn2+ to drive the walker's processive operation, resulting in an enhanced fluorescence recovery of the AuNP-quenched FAM fluorophore. In situ imaging of telomerase activity in three different cell lines (MCF-7 cells, HeLa cells and HL-7702 cells) was well implemented. The discrimination of cancer cells from normal cells and the screening of telomerase inhibitors have been achieved. The sensitive detection of telomerase activity in HeLa cell lysate has also been realized with a detection limit of 10 cells. This walker performed a new approach for monitoring telomerase activity from different levels, providing a potential tool for clinical diagnosis, prognostic evaluation and drug screening.

Dual-channel detection of viscosity and pH with a near-infrared fluorescent probe for cancer visualization.

Compared to ordinary cells, tumor cells have a unique microenvironment, characterized by high viscosity, low pH, high reactive oxygen species level and the overexpression of certain proteases. Therefore, viscosity and pH can be used as important parameters for visualizing cancer. We designed a spiro-oxazolidine compound (In-1) for the dual-channel detection of viscosity and pH, with the red channel for detecting viscosity and the blue channel for pH. Interestingly, In-1 can locate different organelles under different conditions. Under physiological conditions, In-1 efficiently targeted lysosomes and showed that the viscosity of lysosomes increases in cancer cells while the pH decreases, which can be used to distinguish and detect cancer cells and normal cells. When we treated HL-7702 cells with CCCP, the probe could effectively target the mitochondria, and the fluorescence intensity in the pH channel decreased. This indicates that In-1 can be used as a powerful tool to simultaneously monitor viscosity and pH in different organelles, and may have a guiding role in diseases caused by mitochondrial and lysosomal microenvironments.

A new class of nickel(II) oxyquinoline-bipyridine complexes as potent anticancer agents induces apoptosis and autophagy in A549/DDP tumor cells through mitophagy pathways.

A new class of nickel(II) oxyquinoline-bipyridine complexes, namely, [Ni(La1)2(Lb6)] (Ni1), [Ni(La1)2(Lb2)] ·CH3OH (Ni2), [Ni(La7)2(Lb11)]·2H2O (Ni3), [Ni(La1)2(Lb9)] (Ni4), [Ni(La1)2(Lb8)] (Ni5), [Ni(La2)2(Lb1)] (Ni6), [Ni(La2)2(Lb6)]·CH3OH (Ni7), [Ni(La2)2(Lb11)]·CH3OH (Ni8), [Ni(La2)2(Lb3)] (Ni9), [Ni(La2)2(Lb2)]·CH3OH (Ni10), [Ni(La2)2(Lb5)]·CH3OH (Ni11), [Ni(La2)2(Lb7)] (Ni12), [Ni(La3)2(Lb2)] (Ni13), [Ni(La4)2(Lb4)]·2CH3OH (Ni14), [Ni(La4)2(Lb8)]·2.5CH3OH (Ni15), [Ni(La4)2(Lb11)]·1.5CH3OH (Ni16), [Ni(La5)2(Lb7)] (Ni17), [Ni(La5)2(Lb10)]·CH3OH (Ni18), [Ni(La6)2(Lb11)]·3CH3OH (Ni19), [Ni(La7)2(Lb7)]·2CH3OH (Ni20), [Ni(La7)2(Lb8)]·2CH3OH (Ni21) and [Ni(La7)2(Lb1)]·2CH3OH (Ni22) bearing oxyquinoline (H-La1-H-La7) and bipyridine derivatives (Lb1-Lb11) were synthesized and characterized by elemental analysis, X-ray crystallography, infrared (IR) spectroscopy and electrospray mass spectrometry (ESI-MS). An MTT method suggested that the IC50 values of Ni1-Ni22 for A549/DDP tumor cells were 0.25-25.14 μM, but these complexes exhibited low cytotoxicity toward normal HL-7702 cells (>50 μM). Ni2 could induce A549/DDP tumor cell apoptosis, cause a decrease in the mitochondrial membrane potential (MMP, ΔΨm), and increase the intracellular [Ca2+] and reactive oxygen species (ROS) levels better than Ni10, Ni13, and Ni14. Autophagic and western blot assays showed that Ni2, Ni10, Ni13, and Ni14 could induce autophagy and regulate the expression of LC3 II/I, Beclin1, P62, PINK1, and Parkin proteins, and the inducibility activities were in the order of Ni2 > Ni14 > Ni13 > Ni10. Taken together, these results revealed that the nickel(II) oxyquinoline-bipyridine complex Ni2 inhibited cell growth in A549/DDP tumor cells via mitophagy pathways.

Effects of TMEM206 on the malignant behavior of HepG2 human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common cancer of the digestive system. Recently, transmembrane proteins (TMEMs) have been extensively studied in different types of malignant tumors. However, the influence of TMEM206 in hepatocellular carcinoma is unclear. The UALCAN database was used to investigate the expression of TMEM206 mRNA in liver cancer tissues and used the Human Protein Atlas (THPA) to study the expression of TMEM206 in HCC tissues. The expression of TMEM206 was measured in normal liver HL-7702 cells and HepG2, SMMC-7721, and Bel-7402 liver cancer cells. Next, a lentivirus was used to knockdown TMEM206 in HepG2 cells. Furthermore, after verifying knockdown, we studied the effect of TMEM206 downregulation on the malignant behavior of HepG2 and on the PI3K/AKT pathway. TMEM206 was highly expressed in liver cancer cells ( p &lt; 0.001). Downregulation of TMEM206 significantly inhibited the proliferation, migration and invasion of HepG2, significantly promoted the apoptosis of HepG2, and inhibited the expression of P-PI3K and P-AKT. TMEM206 can affect the malignant behavior of HCC. And the PI3K/AKT pathway was affected. This study provides new ideas for the treatment of HCC.

Discovery of novel β-carboline derivatives as selective AChE inhibitors with GSK-3β inhibitory property for the treatment of Alzheimer's disease

The natural product harmine, a representative β-carboline alkaloid from the seeds of Peganum harmala L. (Zygophyllaceae), possesses a broad spectrum of biological activities. In this study, a novel series of harmine derivatives containing N-benzylpiperidine moiety were identified for the treatment of Alzheimer's disease (AD). The results showed that all the derivatives possessed significant anti-acetylcholinesterase (AChE) activity and good selectivity over butyrylcholinesterase (BChE). In particular, compound ZLWH-23 exhibited potent anti-AChE activity (IC50 = 0.27 μM) and selective BChE inhibition (IC50 = 20.82 μM), as well as acceptable glycogen synthase kinase-3 (GSK-3β) inhibition (IC50 = 6.78 μM). Molecular docking studies and molecular dynamics simulations indicated that ZLWH-23 could form stable interaction with AChE and GSK-3β. Gratifyingly, ZLWH-23 exhibited good selectivity for GSK-3β over multi-kinases and very low cytotoxicity towards SH-SY5Y, HEK-293T, HL-7702, and HepG2 cell lines. Importantly, ZLWH-23 displayed efficient reduction against tau hyperphosphorylation on Ser-396 site in Tau (P301L) 293T cell model. Collectively, harmine-based derivatives could be considered as possible drug leads for the development of AD therapies.

Capsaicin inhibits cell proliferation by enhancing oxidative stress and apoptosis through SIRT1/NOX4 signaling pathways in HepG2 and HL-7702 cells.

Capsaicin could suppress the proliferation of cancer cells and inhibit many biochemical pathways associated with tumorigenesis and metastasis. This study investigates the effects of capsaicin in both hepatocellular carcinoma (HepG2) and normal hepatocytes (HL-7702) via the SIRT1/NOX4 signaling pathway. After determination of cytotoxic concentrations of capsaicin on HL-7702 and HepG2 cells, we measured total oxidant status (TOS), reduced glutathione (GSH), 8-hydroxydeoxyguanosine (8-OHdG), cytochrome c (CYC), caspase3 (CASP3), Bcl-2, Bax, sirtuin1 (SIRT1), and NADPH oxidases4 (NOX4) levels. Besides this, we analyzed the messenger RNA and protein levels of SIRT1 and NOX4. We found that capsaicin increased TOS, 8-OHdG, CASP3, CYC, Bax, and NOX4 levels, and decreased Bcl-2, GSH, and SIRT1 in a concentration-dependent manner in HepG2 cells. However, especially low capsaicin concentration (128.75 µM) enhanced GSH and SIRT levels and reduced TOS, CASP3, CYC, 8-OHdG, and NOX4 levels in HL-7702 cells (p < 0.05). Interestingly, 128.75 and 172.8 µM capsaicin treatment increased SIRT1 expression levels in HL-7702 cells, resulting in an increase in GSH levels and a decrease in TOS, CYC, CAPS3, and 8-OHdG levels through NOX4 inhibition. Furthermore, we demonstrated a significant decrease in SIRT1 protein levels and an increase in NOX4 protein levels and caspase-3/-7 activities in both HL-7702 and HepG2 cells treated with 261.5 µM capsaicin. Additionally, morphological changes in HL-7702 and HepG2 cells treated with capsaicin correlated with the enhancement in oxidative burden, DNA damage, and apoptosis. Our results show that capsaicin effectively might cause higher oxidative, apoptotic, and DNA damage in HepG2 cells than in HL-7702 cells through the SIRT1/NOX4 signaling pathway.

Dual-Channel Fluorescent Probe for the Simultaneous Monitoring of Peroxynitrite and Adenosine-5'-triphosphate in Cellular Applications.

Changes in adenosine triphosphate (ATP) and peroxynitrite (ONOO–) concentrations have been correlated in a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Herein, we report the development of a fluorescent probe ATP-LW, which enables the simultaneous detection of ONOO– and ATP. ONOO– selectively oxidizes the boronate pinacol ester of ATP-LW to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λex = 450 nm, λem = 562 nm or λex = 488 nm, λem = 568 nm). In contrast, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λex = 520 nm, λem = 587 nm). Due to the differences in emission between the ONOO– and ATP products, ATP-LW allows ONOO– levels to be monitored in the green channel (λex = 488 nm, λem = 500–575 nm) and ATP concentrations in the red channel (λex = 514 nm, λem = 575–650 nm). The use of ATP-LW as a combined ONOO– and ATP probe was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. Treatment of HL-7702 cells with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of signal intensity in the red channel and an increase in that of the green channel as expected for a reduction in ATP concentrations. Similar fluorescence changes were seen in the presence of SIN-1 (an exogenous ONOO– donor).

Cell membrane breakage and triggering T cell infiltration are involved in human telomerase reverse transcriptase (hTERT) promoter-driven novel peptide KK-64 for liver cancer gene therapy

ABSTRACT Liver cancer is an aggressive malignancy with exhibits both high mortality and morbidity. The current treatment options are associated with several limitations, novel specific anti-cancer drugs are urgently needed to improve liver cancer treatment. In this study, a new peptide KK-64 was designed, and it showed strong cytotoxicity against liver cancer cells. To obtain the tumor targeting property, a plasmid that contains KK-64 DNA fragment and driven by human telomerase reverse transcriptase (hTERT) promoter was constructed. pcTERT-kk-64 plasmid was found to specifically inhibit the viability of liver cancer cells HepG2, induce substantial apoptosis as well as damage to the cell membranes, but had minimal effects toward normal liver HL-7702 cells. Furthermore, pcTERT-kk-64 plasmids was also noted to significantly attenuate migration and invasion of HepG2 cells. The anti-tumor effect of pcTERT-kk-64 plasmid was also observed in H22 cell-bearing mice, and it appeared to cause significant tumor regression, trigger tumor cell apoptosis, and infiltrate cytotoxicity T cells to the tumor tissues after plasmids injection. Thus, pcTERT-kk-64 plasmids showed both strong cytotoxicity and tumor selectivity in vitro and in tumor-bearing mice in liver cancer models.

Quantification of active selenols in cells: a selenol-specific recognition europium-switched signal-amplification ICP-MS approach.

Selenium (Se) is a mysterious thus tempting element playing a dual bio-chemical function, mainly through selenol, during life processes. Quantification of the selenols is thus of great significance for understanding the biological roles of Se, but remains a big challenge. Herein we report a selenol-specific recognition-mediated and europium (Eu) signal-switched amplification inductively coupled plasma mass spectrometry (ICP-MS) approach for quantifying the free active selenols (act-SeH) in cells. A bifunctional molecule, 2,4-dinitrobenzenesulfonyl-piperidin-4-yl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic europium (DNBS-DOTA-Eu), was designed and synthesized for the specific recognition and highly sensitive quantification of act-SeH via switching Se to more sensitive Eu ICP-MS signals. The limit of detection (LOD, 3σ) of 3.41 pg/mL (22.43 pmol/L), corresponding to the absolute mass LOD of 6.82 ag act-SeH per cell, is almost 25 times lower than 83.76 pg/mL (1.06 nmol/L), 167.52 ag, when monitoring 80Se. The results indicate that act-SeH in the selenite-precultured cancerous HepG2 and paracancerous HL7702 cells are 0.090 ± 0.002 pg/cell (n = 7) and 0.021 ± 0.006 pg/cell (n = 7), more than 4.28 times higher in HepG2 than in HL7702. Preliminary application of this approach to the cells from real hepatic tissue samples suggested that act-SeH has a positive relationship with the degree of hepatic disease. act-SeH in cells appears to be a very promising relevant index for understanding the biochemical functions of Se, besides the total Se in cells and blood serum and/or plasma.