HepG2 Liver Research Articles

GW9662 ameliorates nonalcoholic steatohepatitis by inhibiting the PPARγ/CD36 pathway and altering the gut microbiota

Background & aimsPeroxisome proliferator-activated receptors (PPARs) are currently among the most focused-on therapeutic targets for non-alcoholic steatohepatitis (NASH), although no clinical transformation has been achieved to date. In this study, we aimed to evaluate the effects of GW9662 on choline-deficient, L-amino acid-defined high-fat diet (CDAA-HFD)-induced NASH mice and reveal the mechanism underlying this effect. MethodsGW9662 (1 mg/kg) was administered in CDAA-HFD mouse model of NASH. The effect of GW9662 on hepatic lipid metabolism was investigated using liver RNA-seq and HepG2 cells induced by oleic acid and palmitic acid. In addition, 16S rRNA gene sequencing was performed to analyze the effects of GW9662 on the composition and function of the fecal microbiota. ResultsGW9662 improved the CDAA-HFD caused elevation in the levels of ALT, AST, hepatic free fatty acids and triglycerides. The liver pathological analysis indicated that GW9662 alleviated the hepatic steatosis and fibrosis. The NAFLD activity score and RNA-Seq revealed that GW9662 mainly regulated the fatty acids transport and lipid synthesis by inhibiting PPARγ, CD36, FABP1, FASN, and SCD1, and through the up-regulation of PPARα. Moreover, GW9662 reduced the epididymal fat weight. GW9662 reversed the gut microbiota disorder by increasing the abundance of the beneficial bacteria Dubosiella and Lactobacillus and decreasing the abundance of harmful bacteria Lachnospiraceae_NK4A136_group, Helicobacteraceae, Desulfovibriaceae, and Rickenaceae. ConclusionsGW9662 ameliorated lipid metabolism by inhibiting the PPARγ/CD36 pathway and altering the composition of the gut microbiota in NASH mice. Therefore, the PPARγ antagonist GW9662 deserves more attention as a potential therapeutic agent for NASH.

Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model.

Ferroptosis is a non-conventional cellular death caused by lipid peroxide induced iron deposition. Intracellular lipid accumulation followed by generation of lipid peroxides is an Hallmark of Non Alcoholic Fatty Liver Disease (NAFLD). Melatonin (MLT) is an important pineal hormone with tremendous antioxidant and anti-inflammatory properties. Various studies targeted ferroptosis in different diseases using melatonin. However, none of them focused the intrinsic mechanism of MLT's action to counteract ferroptosis in NAFLD. Hence, the present study investigated the role of MLT in improvement of NAFLD induced ferroptosis. &#160;HepG2 cells were treated with Free Fatty Acids (FFAs) to induce in vitro NAFLD state and C57BL/6 mice were fed with HFD followed by MLT administration 1.</p> &#160;The results indicated that MLT administration caused the recovery from both FFA and HFD induced ferroptotic state via increasing GSH and SOD level, decreasing lipid ROS and MDA (Malondialdehyde) level, increasing Nrf2 and HO-1 level to defend cells against an oxidative environment. MLT also altered the expression of two key proteins GPX4 and SLC7A11 back to their normal levels, which would otherwise cause ferroptosis. MLT also protected against histopathological damage of both liver tissue and HepG2 cells as depicted by Oil Red O, HE staining and immunofluorescence microscopy. MLT also had control over pAMPKα as well as PPARγ and PPARα responsible for lipid homeostasis and lipogenesis.</p> &#160;In brief, MLT exerted its multifaceted effect in FFA and HFD induced NAFLD by retrieving cellular oxidative environment, reducing lipogenesis and lipid peroxidation and modulating Nrf2/HO-1 and GPX4/SLC7A11 axis to combat ferroptosis.</p>.

Effect of RPL27 knockdown on the proliferation and apoptosis of human liver cancer cells

RPL27 is linked to the development of various diseases including malignant tumors. RPL27 may play an oncogenic function in hepatocellular carcinoma (HCC), but this is unknown. So, the aim of this study was to investigate how the human liver cancer cell lines SNU449 and HepG2 responded to RPL27 knockdown in terms of proliferation and apoptosis. SNU449 and HepG2 were cultured and infected with shCon and shRPL27 lentiviral particles to induce RPL27 knockdown, and then RPL27 expression was detected using qPCR and Western blot. Cell proliferation was measured using CCK8, cell cloning, cell scraping, and transwell migration and invasion, while apoptosis was measured using flow cytometry (FCM). The qPCR revealed that mRNA expression of RPL27 decreased after knocking down RPL27 in cells. The CCK8 and cell cloning assay confirmed that knocking down RPL27 significantly reduced cell viability. The cell scratch assay and transwell assays showed that the proliferation rate decreased after knocking down RPL27. A substantial increase in apoptotic cells was discovered by FCM. According to WB, RPL27 knockdown increased the expression of Bax and Caspase-3 while decreasing the expression of bcl-2. The findings showed that RPL27 knockdown inhibited cell proliferation in SNU449 and HepG2 via inducing apoptosis, proving that RPL27 is a novel gene linked with HCC and is crucial for both proliferation and apoptosis. These outcomes imply that RPL27 may be a potential target for liver cancer diagnosis and therapy.

The Effects of Glypican-3 Deficiency on Radiosensitivity in Liver Cancer Cells

Glypican-3 (GPC-3), a heparan sulfate proteoglycan involved in cellular proliferation, modulates signaling of FGF/FGFR, IGF/IGFR, HGF/Met, Wnt/Frizzled, among others and correlates with survival. GPC-3 is overexpressed in the majority of hepatocellular carcinoma and hepatoblastoma, but not in normal hepatocytes. Accordingly, it is being investigated as a liver cancer-selective target for radiopharmaceutical imaging and therapy. However, the potential linkage between GPC-3 expression and radiosensitivity has not yet been defined. In this study, we investigated the effects of GPC-3 deficiency on radiosensitivity in liver cancer cell lines. CRISPR/Cas9 system was used to engineer GPC-3 knockout variants of liver cancer cell lines, HepG2 & Hep3B, both of which natively express GPC-3. Confirmation of knockout of GPC-3 was evaluated by RT-PCR, western blotting, flow cytometry, immunocytochemistry, and gDNA sequencing. Cell growth and migration were evaluated by BrdU insertion and wound-healing assays, respectively. In vitro radiosensitivity was examined by radiation-induced apoptosis/necrosis (Annexin V-APC and PI staining), cell cycle modification, γH2AX foci formation, and clonogenic assays (6 Gy). Wildtype and knockout lines were engrafted into athymic mice to assess tumor growth kinetics. RT-PCR, western blotting, flow cytometry, and immunocytochemistry all confirmed GPC-3 knockout in both HepG2 and Hep3B cell lines. Nucleotide deletion at exon 3 of the GPC-3 gene was confirmed by gDNA sequencing in HepG2ΔGPC3 and Hep3BΔGPC3. GPC-3 deficiency reduced liver cancer cell proliferation (HepG2ΔGPC3, p = 0.027, and Hep3BΔGPC3, p = 0.031) and migration (HepG2ΔGPC3: 1.5-fold, p<0.001, and Hep3BΔGPC3: 2.3-fold, p<0.001) significantly when compared with wild type. GPC-3 deficiency reduced cell survival and clonogenicity (HepG2ΔGPC3: DEF = 1.23, Hep3BΔGPC3: DEF = 1.23) in liver cancer cells exposed to irradiation (6 Gy). The delayed repair of double-stranded DNA damage was observed in irradiated GPC-3 deficient liver cancer cells. Tumor growth was dramatically delayed by GPC-3 deficiency. Tumor weight measured at 50 (Hep3B) and 60 (HepG2) days after liver cancer cell inoculation corroborated these effects. Knockout lines of HepG2 and Hep3B exhibited decreased cell proliferation, migration, and in vivo tumor growth compared to wildtype. GPC-3 deficiency was associated with increased sensitivity to radiation therapy. Studies identifying the pathways through which this radiosensitivity is mediated are ongoing.

A Novel Dual-Reporter System Reveals Distinct Characteristics of Exosome-Mediated Protein Secretion in Human Cells

BackgroundExosomes, a special subtype of extracellular vesicles derived from human cells, serve as vital mediators of intercellular communication by transporting diverse bioactive cargos, including proteins and enzymes. However, the underlying mechanisms governing exosome secretion and regulation remain poorly understood. In this study, we employed a dual-reporter system consisting of bioluminescent Gaussia luciferase and fluorescent proteins to investigate the dynamics and regulation of exosome secretion in cultured human cells.ResultsOur results demonstrated that the engineered dual-reporters effectively monitored both exosome-mediated and ER-Golgi-mediated secretory pathways in a specific and quantitative manner. Notably, we observed distinct characteristics of exosome-mediated protein secretion, including significantly lower capacity and different dynamics compared to the ER-Golgi pathway. This phenomenon was observed in human kidney 293T cells and liver HepG2 cells, emphasizing the conserved nature of exosome-mediated secretion across cell types. Furthermore, we investigated the impact of brefeldin A (BFA), an inhibitor of ER-to-Golgi membrane trafficking, on protein secretion. Interestingly, BFA inhibited protein secretion via the ER-Golgi pathway while stimulating exosome-mediated protein secretion under same experimental conditions.ConclusionsCollectively, our study highlights the utility of the dual-reporter system for real-time monitoring and quantitative analysis of protein secretion through conventional ER-Golgi and unconventional exosome pathways. Moreover, our findings unveil distinct features of exosome-mediated protein secretion, shedding light on its differential capacity, dynamics, and regulatory mechanisms compared to ER-Golgi-mediated proteins in human cells.

Cellular and bioenergetic effects of polystyrene microplastic in function of cell type, differentiation status and post-exposure time

The ubiquity of microplastics (MPs) in food sources and personal care products increasingly raises concerns on human health. However, little is known about the duration of the effects of MPs and whether effects depend on cellular differentiation status. Herein, cellular and bioenergetic effects of MPs in different exposure scenarios on four types of human cell lines derived from lung (A549 and BEAS-2B), colon (Caco-2) and liver (HepG2) were investigated. These cell lines are models for the major exposure routes in the body (inhalation, ingestion and physiological transport through the liver by the portal vein). To this aim, different scenarios were implemented by exposing undifferentiated and differentiated cells to single dosing of 2-μm polystyrene (PS) (102-105 particles/mL) for 48 h and 12 days. The undifferentiated Caco-2 cells with short exposure (48 h) showed the highest uptake rate of PS yet without significant cellular and mitochondrial responses. The biological effects, with the exception of ROS production, were not influenced by differentiation states of A549 and Caco-2 cells although differentiated cells showed much weaker ability to internalize PS. However, PS had significantly long-term impacts on cellular and mitochondrial functions even after the initial exposure period. In particular, Caco-2 cells that were post-exposed for 12 days after single PS dosing suffered higher oxidative stress and exhibited mitochondrial dysfunction than that for short exposure. Correspondingly, we observed that PS particles still remained in cell membrane and even in nuclei with high retention rate by 14-d post exposure during which metabolism and exchange of internalization and release occurred in cells. This indicates PS could induce chronic stress and even harmful effects on human cells after single intake that persists for a long time. This study paves the way for assessing the influence of PS on human health at low particle concentrations and with multiple exposure scenarios.

Chemo-/Regio-Selective Synthesis of Novel Functionalized Spiro[pyrrolidine-2,3'-oxindoles] under Microwave Irradiation and Their Anticancer Activity.

A novel series of nitrostyrene-based spirooxindoles were synthesized via the reaction of substituted isatins 1a-b, a number of α-amino acids 2a-e and (E)-2-aryl-1-nitroethenes 3a-e in a chemo/regio-selective manner using [3+2] cycloaddition (Huisgen) reaction under microwave irradiation conditions. The structure elucidation of all the synthesized spirooxindoles were done using 1H and 13C NMR and HRMS spectral analysis. The single crystal X-ray crystallographic study of compound 4l was used to assign the stereochemical arrangements of the groups around the pyrrolidine ring in spiro[pyrrolidine-2,3'-oxindoles] skeleton. The in vitro anticancer activity of spiro[pyrrolidine-2,3'-oxindoles] analogs 4a-w against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines shows promising results. Out of the 23 synthesized spiro[pyrrolidine-2,3'-oxindoles], while five compounds (4c, 4f, 4m, 4q, 4t) (IC50 = 34.99-47.92 µM; SI = 0.96-2.43) displayed significant in vitro anticancer activity against human lung (A549) cancer cell lines, six compounds (4c, 4f, 4k, 4m, 4q, 4t) (IC50 = 41.56-86.53 µM; SI = 0.49-0.99) displayed promising in vitro anticancer activity against human liver (HepG2) cancer cell lines. In the case of lung (A549) cancer cell lines, these compounds were recognized to be more efficient and selective than standard reference artemisinin (IC50 = 100 µM) and chloroquine (IC50 = 100 µM; SI: 0.03). However, none of them were found to be active as compared to artesunic acid [IC50 = 9.85 µM; SI = 0.76 against lung (A549) cancer cell line and IC50 = 4.09 µM; SI = 2.01 against liver (HepG2) cancer cell line].

Green-fabricated MgO nanoparticles: A potent antimicrobial and anticancer agent

MgO nanoparticles are gaining popularity because of their potential applications in several different industries, such as bioengineering, medicine, and environmental protection. The fabrication of MgO nanoparticles with enhanced biological properties remains difficult, despite the growing interest in this area. In this paper, we describe a method that minimizes environmental impact when manufacturing MgO nanoparticles using garlic (Allium sativum L.) extract. For ages, one of the most vital functions in human nutrition and medicine has been played by garlic. Garlic also contains phytochemicals that are effective against cancer and microbes. Our goal was to increase the medicinal relevance of MgO nanoparticles while decreasing our reliance on harmful chemicals. Various characterization techniques were employed to confirm the synthesis of MgO nanoparticles, including SEM, TEM, EDX, and XRD. The study findings indicate that the synthesis of MgO nanoparticles yielded a polycrystalline cubic configuration with a crystal size of 55–60 nm. The results of the EDX analysis suggest that only Mg and O elements are present, without any detectable impurities. The efficacy of MgO nanoparticles against gram-positive (Staphylococcus epidermidis), gram-negative bacteria (Escherichia coli) and a fungal pathogen (Candida albicans) was investigated. The results indicated that MgO nanoparticles exhibited high effectiveness against all three microorganisms. The study revealed that MgO nanoparticles exhibit potent anticancer properties against human liver (HepG2) and lung (A549) cancer cells. Reactive oxygen species production in cancer cells by MgO nanoparticles suggests oxidative stress pathway-mediated anticancer action. The present research highlights the significance of utilizing medicinal plants in the synthesis of nanoparticles for enhanced anticancer and microcidal properties.

Syzygium cumini (L.) Extract-Derived Green Titanium Dioxide Nanoparticles Induce Caspase-Dependent Apoptosis in Hepatic Cancer Cells.

An aqueous extract of Syzygium cumini seeds was utilized to green synthesize titanium dioxide nanoparticles (TiO2 NPs). UV-Visible, DLS, FTIR, XRD, FESEM, TEM, SAED, EDAX, and photoluminescence spectroscopy techniques were employed to characterize the prepared TiO2 nanoparticles. The rutile crystal structure of TiO2 NPs was revealed by XRD study. The TEM and FESEM images of the TiO2 NPs revealed an average particle size of 50-100 nm. We employed EDAX to investigate the elemental compositions of TiO2 NPs. The O-Ti-O stretching bands appeared in the FTIR spectrum of TiO2 NPs at wavenumbers of 495 cm-1. The absorption edge peaks of TiO2 NPs were found in the UV-vis spectra at 397 nm. The MTT study revealed that TiO2 NPs effectively inhibited the growth of liver cancer Hep3 and Hep-G2 cells. The results of the corresponding fluorescent staining assays showed that TiO2 NPs significantly increased ROS generation, decreased MMP, and induced apoptosis in both liver cancer Hep3 and Hep-G2 cells. TiO2 nanoparticles lessened SOD, CAT, and GSH levels while augmenting MDA contents in Hep3 and Hep-G2 cells. In both Hep3 and Hep-G2 cells treated with TiO2 NPs, the Bax, CytC, p53, caspase-3, -8, and -9 expressions were remarkably augmented, while Bcl-2 expression was reduced. Overall, these findings revealed that formulated TiO2 NPs treatment considerably inhibited growth and triggered apoptosis in Hep3 and HepG2 cells.

Facile phytosynthesis of gold nanoparticles using Nepeta bodeana Bunge: Evaluation of its therapeutics and potential catalytic activities

In the present study, the ethanol extract of leaves of Nepeta bodeana Bunge was utilized in environmentally friendly synthesis of gold nanoparticles (AuNPs) via an immediate, one-step, and cost-effective process. The biosynthesized AuNPs were characterized using a variety of techniques, including UV–visible spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), and laser Doppler electrophoresis (zeta potential), among others. Pink in hue, the manufactured AuNPs were practically spherical and had<20.5 nm in diameter. The peak of the localized surface plasmon resonance absorption band at 547 nm helped to explain the biogenic synthesis of AuNPs, which used the ethanolic extract of the Nepeta bodeana leaves. In addition to this, the biosynthesized AuNPs possessed the remarkable stability as a result of their zeta potential value of − 22.3 mV. The green synthesized AuNPs demonstrated the excellent catalytic activity because they were able to catalytically degrade methylene blue (MB) at a variety of concentrations. The results indicated that 94.2% of MB were degraded after 10 min. They also showed a remarkable antibacterial activity against both Gram-nrgative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Staphylococcus aureus and Bacillus cereus) bacteria. In addition, a proper antifungal activity with the minimum inhibitory concentration (MIC) values of 50 and 25 μg/mL against Candida albicans and Aspergillus niger, respectively, were determined. The phytosynthesized AuNPs indicated a considerable antioxidant activity of 83% and 74% in the applied DPPH and ABTS radical scavenging activity tests, respectively. The anti-inflammatory properties of the synthesized AuNPs were established to be dose-dependent and varied from 11% to 76%. The green synthesized AuNPs also exhibited a concentration-dependent cytotoxicity on human liver (HepG2) and breast (MCF-7) cancer cells, which were around 77.4 and 71.2% at 300 µg/mL, respectively. They showed as well a notable anti-diabetic activity with the 52% inhibition of α-amylase and the 62% glucose uptake activity by yeast cells at 300 µg/mL. All these findings suggested that the produced AuNPs could be explored for developing many therapeutic as well as catalytic useful products.

Investigation of In Vitro antiproliferative activity properties of Spartium junceum L. (Spanish broom) against MDA-MB-231 and HepG2 cancer cell lines

Cancer is among the top global public health burdens leading to millions of deaths each year. The study aims to investigate the antiproliferative effect of Spartium junceum L. flowers on different cancer cell lines. The ethanolic extract of the flowers was prepared in the present study. Phytochemical analysis of the plant extract revealed the presence of several phenolic compounds such as cinnamic acid and its derivatives (chlorogenic, p-coumaric, ferulic acids), protocatechuic acid, epicatechin and luteolin. This extract was tested against human breast (MDA-MB-231) and liver (HepG2) cancer cell lines to find out its antiproliferative activity. It was determined that the extract was effective against both cell lines with IC50 values of 2.37 ± 0.47 and 0.98 ± 0.01 µL/mL for MDA-MB-231 and HepG2, respectively. Particularly, the extract was found to be more effective in the liver cancer cell line than the breast cancer cell line. All these obtained findings led us to believe that this medicinal plant could be a promising antiproliferative agent candidate for the treatment of human liver and breast cancers.

Searching for relationships between the structure of selected simple coumarins, their antioxidant capacity and anticancer properties

The antioxidant capacity of 23 simple coumarins was studied by using different total antioxidant capacity assays, namely ABTS, CUPRAC, and FRAP. Simultaneously, the anticancer activity of the investigated derivatives was evaluated by MTT test on lung (A549), cervical (HeLa), prostate (PC3), and liver (HepG2) cancer cell lines. Furthermore, fifty percent inhibitory concentration (IC50) values were calculated, while for the derivatives with the strongest anticancer properties their impact on the viability of normal immortalized prostate cells (PNT2), compared to their cancer counterparts (prostate cancer cells PC3), was determined. The correlation between antioxidant capacity, anticancer potential and the structure of the studied compounds has been observed and discussed. Interestingly, it has been proved that selected derivatives (with two neighboring hydroxyl substituents attached to the aromatic ring) exhibit the strongest antioxidant capacity and anticancer properties. These findings increase our comprehension of the relationship between structure and radical scavenging activity of simple coumarins.

Flavonol Glycosides from Eugenia uniflora Leaves and Their In Vitro Cytotoxicity, Antioxidant and Anti-Inflammatory Activities

In view of the extensive use of Eugenia uniflora leaves for the management of tumours and other chronic inflammatory diseases in traditional medicine, an activity-guided fractionation of its leaf ethanolic extract led to the isolation of two flavonol glycosides. Cytotoxicity study was based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assay against the non-tumourigenic human embryonic kidney (HEK-293) cells, and the cancerous liver (Hep-G2) and cervical (HeLa) cell lines. Antioxidant tests were carried out using 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) and hydrogen peroxide (H2O2) radical scavenging assays, while an in vitro anti-inflammatory test was conducted using egg albumin denaturation (EAD) assay. Based on comprehensive spectroscopic and spectrometric evidence, the compounds were elucidated as myricitrin (1) and a newly described compound, 5,7-dihydroxy-3-(3,4,5-trihydroxy-6-methyltetrahydropyran-2-yloxy)-2-(2,4,5-trihydroxyphenyl)chromen-4-one, named “unifloratrin (2)”. The cytotoxicity of myricitrin (1) was comparable to 5-fluorouracil (standard drug), with a CC50 of 8.5 ± 2.2 µg/100 µL against HeLa cells. It also demonstrated better antioxidant activity, with an IC50 of 6.23 ± 1.09, 22.01 ± 2.59 and 30.46 ± 1.79 µM against DPPH, NO and H2O2 free radicals, respectively. At 20 µg/mL and an incubation time of 2 h, myricitrin was comparable to diclofenac (standard drug) in anti-inflammatory activity. This report may serve as a justification for the ethnomedicinal use of E. uniflora, while flavonol glycosides, such as myricitrin (1), could be further exploited as a candidate cytotoxic agent.

Development of a sample preparation method for micro-proteomics analysis of the formaldehyde-fixed paraffin-embedded liver tissue samples

Liver micro-proteomics based on the routinely used formaldehyde-fixed paraffin-embedded (FFPE) samples is valuable for innovative research, but the technical approach for sample preparation is often challenging. In this study, we aimed to develop a method for sample preparation for micro-proteomics on using the FFPE liver samples. We collected 2000 individual cells per batch from FFPE liver slices with laser capture microdissection and used them as test samples. We used the microscale fresh-frozen liver samples or HepG2 cells as control samples. For the FFPE samples, we first established a procedure for protein extraction. 2 h incubation at 95 °C in alkaline amine buffer supplemented with 4% sodium dodecyl sulfate allows improved production, efficiency, and quality of protein extraction. Then, we developed a dedicated protocol HDMSP for the micro-concentrated (<0.05 μg/μL) protein preparation for mass spectrometry (MS) based analysis, in which 2 μg/μL carboxyl magnetic beads and 70% acetonitrile are used to induce protein precipitation. For the 0.01 μg/μL protein control samples, protein recovery rate (PRR) by HDMSP is 72.1%, while the PRR is 5.9% if using a standard method solid phase-enhanced sample preparation. For the FFPE samples, the HDMSP PRR is 88.8%, and the subsequent MS analysis demonstrates increased depth, robustness, and quantitation accuracy for HDMSP relative to the control of in-gel digestion. Moreover, the physicochemical properties and subcellular location of the FFPE liver micro-proteome are comparable to those of the fresh-frozen control samples processed with filter-aided sample preparation (FASP). HDMSP is also comparable to FASP in terms of reproducibility and physicochemical properties in liver subcellular proteomes, and meanwhile reduces the sample preparation time by 15.9% and the experimental cost by 30.8%. Overall, the new method is simple and highly effective for preparing the microscale FFPE liver protein samples for MS analysis. This study provides a useful solution for FFPE liver micro-proteomics.

Novel pyridine-thiazolidinone-triazole hybrid glycosides targeting EGFR and CDK-2: Design, synthesis, anticancer evaluation, and molecular docking simulation

The observed toxicity of newly designed anticancer drugs is considered as an impediment challenging phenomenon of intense concern arising from diversity in drug targets and the latter concern is therefore still in need for further research. The current investigation reports the design and synthesis of a new series of pyridine-thiazole hybrids and their derived 1,2,3-triazole analogues incorporating with varied sugar moieties and their acyclic analogues 3–11 and 13–18. The newly synthesized compounds were investigated for their cytotoxic activities against human liver HepG-2 and breast MCF-7 cancer cell lines. The pyridine-thiazolidinone-triazole based targets 16–18 revealed the potent cytotoxic activities. Also, all synthesized derivatives afforded promising safety antiproliferative margins against the normal human diploid cell line WI-38. The promising analogues 16–18 were further studied for their inhibitory assessment against EGFR and CDK-2/cyclin A2 kinases. The pyridine-thiazolidinone-1,2,3-triazole based glycosides 17 and 18 displayed promising and relatively equipotent inhibitory activity against EGFR enzyme compared with the reference drug, erlotinib (IC50= 0.19, 0.12, and 0.15 μM, respectively). While, the three glycosides 16–18 showed superior inhibitory potency against CDK-2/cyclin A2 kinases compared to the standard roscovitine (IC50 = 0.26, 0.22, 0.18 and 0.42 μM, respectively). The impact of triazole glycoside 18 upon cell cycle and apoptosis induction was determined to detect its mechanism of action. Additionally, it upregulated the levels of caspase-3, Bax and Bcl-2 in MCF-7 cells. Finally, molecular docking study was simulated to achieve a better rationalization and gain an insight into the binding affinity of the potent derivatives with their targeted enzymes, which prompts their application as optimum leads for further modification in the anticancer field.

SDPS-13 CELL LINE STUDY OF NUCLEOSOME-BASED BIOMARKERS IN THE DIAGNOSIS AND DETECTION OF RELAPSES IN GLIOBLASTOMA

Abstract Currently, glioblastoma (GBM) diagnosis and monitoring rely on neuroimaging and histopathological confirmation. However, overall survival has not improved in last decades due to therapeutic failure and to a lack of biomarkers for relapses’ detection. Liquid biopsies (i.e. blood or cerebrospinal fluid) using nucleosomes-containing-histone post-translational modifications (PTMs) have the potential to become valuable biomarkers for diagnosis and monitoring GBM. Four glioblastoma cell lines (SF-126, U-87MG, U-118MG, and U-138MG) compared to a healthy microglia cell line (HMC3) and other solid cancer cell lines including pancreas (MIA PaCa-2), liver (HepG2), and cervix/uterus (HeLa) have been analyzed to identify their epigenetic profile. Nucleosomes were extracted and analyzed with the Nu.Q® Discover immunoassays platform (Belgian Volition) addressing 13 histone-PTMs. Quantitative results of PTMs expression were normalized on quantification of total nucleosomes (H3.1-nucleosomes). The immunoassay identified three candidate biomarkers compared to control cell lines (n=4): citrullinated-histone H3 (H3R8Cit) (control=4.68% ; GBM=13.88% ; p&amp;lt;0.05), phosphorylation of H3S10 (H3S10Ph) (control=2.31% ; GBM=5.57% ; p&amp;lt; 0.05), and a trend in H3K4Me2 elevation (control=0.43% ; GBM=1.07% ; p=0.066) in GBM cell lines(n=4). These results were confirmed by western blot. In addition, principal component analysis revealed a segregation between GBM and control cells, mainly driven by these three PTMs. Moreover, to demonstrate the capacity of the Nu.Q® assays to quantitatively monitor PTMs, GBM cell line SF-126 (n=3) was treated with an EZH2 inhibitor (responsible of H3K27 methylation) (iEZH2). After 24 hours and 48 hours of exposure, the level of expression of H3K27Me3 – expressed as ratio of total nucleosomes was decreased by 32% (control=14.67%±0.58% ; iEZH2=10.00%±1.73% ; p&amp;lt;0.05) and 41% (control=15.33%±1.53% ; iEZH2=9.00%±0.00% ; p&amp;lt;0.05), respectively. Those results were also confirmed by western blot analysis. In conclusion, we identified three specific in vitro epigenetic-based marks of GBM and demonstrated that the Nu.Q® technology is a valuable tool to monitor the degree of PTMs expression.

Anti-cancer effect of nano-encapsulated boswellic acids, curcumin and naringenin against HepG-2 cell line

Backgroundliver cancer is one of the most common cancers in the world. So far, there is no gold standard treatment for hepatocellular carcinoma. We conducted this in vitro study to assess the effect of three natural products: Boswellic acids, curcumin and naringin versus corresponding nanoparticles (NPs) on Hep G2 cells proliferation.MethodsBoswellic acid, curcumin, naringin-loaded NPs were prepared using nanoprecipitation method. Human liver (HepG2) cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM). The cell growth inhibition and cytotoxicity were evaluated by MTT assay.ResultsBoswellic acid, curcumin, naringin were able to inhibit HepG2 cells proliferation. IC50 at 24 h, 48 h showed significant lower values in NPs versus Free herbs. IC50 values of free Boswellic acids and NPs at 24 h were (24.60 ± 1.89 and 7.78 ± 0.54, P < 0.001), at 48 h were (22.45 ± 1.13 and 5.58 ± 0.27, P < 0.001) respectively. IC50 values of free curcumin and NPs at 24 h were (5.89 ± 0.8 and 3.46 ± 0.23, P < 0.05), at 48 h were (5.57 ± 0.94 and 2.51 ± 0.11, P < 0.05), respectively. For free and naringenin NPs, IC50 values at 24 h were (14.57 ± 1.78 and 7.25 ± 0.17, P < 0.01), at 48 h were (11.37 ± 1.45 and 5.21 ± 0.18, P < 0.01) respectively.ConclusionBoswellic acid, curcumin, naringin and their nanoprecipitation prepared nanoparticles suppressed Hep G2 cells proliferation.

Pepper Fruit Extracts Show Anti-Proliferative Activity against Tumor Cells Altering Their NADPH-Generating Dehydrogenase and Catalase Profiles.

Cancer is considered one of the main causes of human death worldwide, being characterized by an alteration of the oxidative metabolism. Many natural compounds from plant origin with anti-tumor attributes have been described. Among them, capsaicin, which is the molecule responsible for the pungency in hot pepper fruits, has been reported to show antioxidant, anti-inflammatory, and analgesic activities, as well as anti-proliferative properties against cancer. Thus, in this work, the potential anti-proliferative activity of pepper (Capsicum annuum L.) fruits from diverse varieties with different capsaicin contents (California < Piquillo < Padrón < Alegría riojana) against several tumor cell lines (lung, melanoma, hepatoma, colon, breast, pancreas, and prostate) has been investigated. The results showed that the capsaicin content in pepper fruits did not correspond with their anti-proliferative activity against tumor cell lines. By contrast, the greatest activity was promoted by the pepper tissues which contained the lowest capsaicin amount. This indicates that other compounds different from capsaicin have this anti-tumor potentiality in pepper fruits. Based on this, green fruits from the Alegría riojana variety, which has negligible capsaicin levels, was used to study the effect on the oxidative and redox metabolism of tumor cell lines from liver (Hep-G2) and pancreas (MIA PaCa-2). Different parameters from both lines treated with crude pepper fruit extracts were determined including protein nitration and protein S-nitrosation (two post-translational modifications (PTMs) promoted by nitric oxide), the antioxidant capacity, as well as the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), among others. In addition, the activity of the NADPH-generating enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and NADP-isocitrate dehydrogenase (NADP-ICDH) was followed. Our data revealed that the treatment of both cell lines with pepper fruit extracts altered their antioxidant capacity, enhanced their catalase activity, and considerably reduced the activity of the NADPH-generating enzymes. As a consequence, less H2O2 and NADPH seem to be available to cells, thus avoiding cell proliferation and possibly triggering cell death in both cell lines.

Solarplast®-An Enzymatically Treated Spinach Extract.

In the modern world we are constantly bombarded by environmental and natural stimuli that can result in oxidative stress. Antioxidant molecules and enzymes help the human body scavenge reactive oxygen species and prevent oxidative damage. Most organisms possess intrinsic antioxidant activity, but also benefit from the consumption of antioxidants from their diet. Leafy green vegetables such as spinach are a well-researched rich source of dietary antioxidant molecules. However, plant cell walls are difficult to digest for many individuals and the bio-accessibility of nutrients and antioxidants from these sources can be limited by the degree of digestion and assimilation. Through a specific enzymatic process, Solarplast® contains organic spinach protoplasts without the cell wall, which may facilitate higher yield and efficacy of beneficial antioxidant molecules. In this study, analytical techniques coupled to in vitro bioassays were used to determine the potential antioxidant activity of Solarplast® and determine its antioxidant enzymatic capabilities. Solarplast® demonstrated superior antioxidant activity when compared to frozen spinach leaves in TOC, FRAP and TEAC antioxidant assays. Several antioxidant enzymes were also increased in Solarplast®, when compared to frozen spinach. As a functional readout, Solarplast® attenuated hydrogen peroxide-, ethanol- and acetaminophen-induced increases in oxidative stress and cytotoxicity in both intestinal (HT-29) and liver (HepG2) cell lines. These findings suggest that Solarplast® may represent a non-GMO, plant-based food supplement to help reduce oxidative stress in the human body.

Photocatalytic Degradation of Methylene Blue and Anticancer Response of In2O3/RGO Nanocomposites Prepared by a Microwave-Assisted Hydrothermal Synthesis Process.

The incorporation of graphene with metal oxide has been widely explored in various fields, including energy storage devices, optical applications, biomedical applications, and water remediation. This research aimed to assess the impact of reduced graphene oxide (RGO) doping on the photocatalytic and anticancer properties of In2O3 nanoparticles. Pure and In2O3/RGO nanocomposites were effectively synthesized using the single-step microwave hydrothermal process. XRD, TEM, SEM, EDX, XPS, Raman, UV-Vis, and PL spectroscopy were carefully utilized to characterize the prepared samples. XRD data showed that synthesized In2O3 nanoparticles had high crystallinity with a decreased crystal size after RGO doping. TEM and SEM images revealed that the In2O3 NPs were spherical and uniformly embedded onto the surface of RGO sheets. Elemental analysis of In2O3/RGO NC confirmed the presence of In, O, and C without impurities. Raman analysis indicated the successful fabrication of In2O3 onto the RGO surface. Uv-Vis analysis showed that the band gap energy was changed with RGO addition. Raman spectra confirmed that In2O3 nanoparticles were successfully anchored onto the RGO sheet. PL results indicated that the prepared In2O3/RGO NCs can be applied to enhance photocatalytic activity and biomedical applications. In the degradation experiment, In2O3/RGO NCs exhibited superior photocatalytic activity compared to that of pure In2O3. The degradation efficiency of In2O3/RGO NCs for MB dye was up to 90%. Biological data revealed that the cytotoxicity effect of In2O3/RGO NCs was higher than In2O3 NPs in human colorectal (HCT116) and liver (HepG2) cancer cells. Importantly, the In2O3/RGO NCs exhibited better biocompatibility against human normal peripheral blood mononuclear cells (PBMCs). All the results suggest that RGO addition improves the photocatalytic and anticancer activity of In2O3 NPs. This study highlights the potential of In2O3/RGO NCs as an efficient photocatalyst and therapeutic material for water remediation and biomedicine.