Human Liver HepG2 Research Articles

Investigating the Antioxidant and Anti-Cancer Properties of Milk Thistle Extract Against the HepG2 Cells

Background: Hepatocellular carcinoma (HCC) is the third among all cancer-related causes of death worldwide. The primary cause of HCC development is oxidative stress. Milk thistle (MT) contains numerous phytochemicals that contain antioxidant and hepatoprotective properties. The goal of this study was to investigate MT's chemical composition and antioxidant activity, as well as its bioactive compound's in vitro anti-cancer efficacy against HepG2 human liver cancer cells.Methods: We analyzed the bioactive components of MT using GC-MS and HPLC methods. The antioxidant activity was measured by the total phenolic, total flavonoid, DPPH, and radical ABTS scavenging. Furthermore, we evaluated the anti-hepatocellular carcinoma activity using the human cell line HepG2. The MTT test was used to calculate the IC50 values of MT. Cell cycle and apoptosis were measured by flow cytometry in the cell lines.Result: Our findings demonstrated that 11 bioactive compounds with antioxidant potential were identified via GC/MS screening. Eighteen bioactive compounds with anti-cancer and antioxidant capabilities were found through HPLC screening. Furthermore, HPLC analysis demonstrated both high quality and number of amino acids. MT stops the growth of HepG2 cells at an IC50 level of 21.727±0.89 μg/ml. Moreover, MT arrested HepG2 cells in the G1 phase and induced apoptosis.Conclusion: The findings of this study indicate that bioactive MT extract has potential therapeutic effects on cancer cells, highlighting the need for further research to explore its mechanisms of action and therapeutic applications in cancer treatment.Keywords: Milk Thistle; Free Radicals; Oxidative Stress; HCC; Anti-oxidants

Occurrence of Alternaria Toxins, Ochratoxin A and Aflatoxins B1 in Green Arabic Coffee (Coffea arabica) in Saudi Arabia: Risk Assessment on HepG-2 Cell Line and Male Albino Rats

Arabica coffee (Coffee arabica L.) is among the most popular and widely consumed drinks globally today. The current study aimed to detect and quantify of potential mycotoxins in coffee beans and to investigate their potential risk on HepG-2 human liver cancer cells and male albino rats. Eighteen fully matured coffee beans samples were collected from different geographical locations in Saudi Arabia. Mycotoxins detected and quantified utilizing high-performance liquid chromatography (HPLC). Cytotoxicity of the detected toxins was investigated utilizing the MTT assay method toward HepG-2 cells and male albino rats. Cytotoxic properties of the detected mycotoxins were investigated at doses of 31.25, 62.50, 125, 250 and 500 µg/ml following exposure durations of 24 and 48 h in comparison with control. HPLC analysis revealed the detection of four Alternaria toxins (ALT-Ts) namely Altenuene (ALT), Tenuazonic acid (TA), Alternariol (AOH), and Altenuisol being AOH reported as the frequently detected mycotoxin in 18 coffee beans samples representing 75 % with quantity ranged 0.98 to 8.97 μg/g. OTA was frequently detected (10 samples, 55.5%) in concentrations varied from 0.75 to 9.87 μg/g. In addition, Aflatoxin B1 was also detected with low quantity ranged 0.89 to 3.22 μg/g. Treatment of HepG-2 cell line with combined Alternaria toxins (ALT, TeA, AOH, and Altenuisol), Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) as well as their combination triggered a decline in cell viability that was proportional to the dose administered. The elevated doses (62.50, 125, 250 and 500 µg/ml) notably reduced cell viability and induced cellular damages when contrasted with the lowest dose (31.25 µg/ml) and control. The IC50 for the tested mycotoxins registered for ALT-Ts, AFB1 and OTA as well as their combination were 115.95, 56.68, 59.86 and 31.05 µg/ml after 48 h of exposure. Secreting inflammatory markers (Interleukin-6 (IL-6), interleukin-1β (IL-1β) and COX-2), and oxidative markers (total antioxidant capacity (TAC), glutathione (GSH) and nitrite (NO) were downregulated by all mycotoxins with the highest decrease induced by their mixture. Malonaldehydes (MDA) and TAC were significantly increased by all toxins in treated rats. Hematological results revealed that AFB1, OTA and ALT-Ts-treated rats exhibited a notable decline in hemoglobin (Hb), red blood cells (RBCs), white blood cells (WBCs), and packed cell volume % (PCV). However, single and double dose-treated rats with OTA, Mix1 and Mix2 exhibited a marked increase in WBCs. The results demonstrated a notable decline in liver and kidney functions in all mycotoxins-treated rats. The studied organs (liver, kidney and spleen) exhibited significant damage, with the most severe alterations observed in the Mix1 and Mix2-treated group in comparison with the groups receiving individual treatment. This indicates that the mixture of AFB1, ALT-Ts and OTA had an enhanced toxic consequence against treated HepG-2 cell line and rats. Our results indicated the synergistic effect of the combined different mycotoxins against HepG-2 human liver cancer cells and male albino rats. This work could help fill in data gaps and enhance risk assessment for human health by addressing some of the possible risks associated with Alternaria toxins

Protein corona formed on lipid nanoparticles compromises delivery efficiency of mRNA cargo.

Lipid nanoparticles (LNPs) are the most clinically advanced nonviral RNA-delivery vehicles, though challenges remain in fully understanding how LNPs interact with biological systems. In vivo, proteins form an associated corona on LNPs that redefines their physicochemical properties and influences delivery outcomes. Despite its importance, the LNP protein corona is challenging to study owing to the technical difficulty of selectively recovering soft nanoparticles from biological samples. Herein, we developed a quantitative, label-free mass spectrometry-based proteomics approach to characterize the protein corona on LNPs. Critically, this protein corona isolation workflow avoids artifacts introduced by the presence of endogenous nanoparticles in human biofluids. We applied continuous density gradient ultracentrifugation for protein-LNP complex isolation, with mass spectrometry for protein identification normalized to protein composition in the biofluid alone. With this approach, we quantify proteins consistently enriched in the LNP corona including vitronectin, C-reactive protein, and alpha-2-macroglobulin. We explore the impact of these corona proteins on cell uptake and mRNA expression in HepG2 human liver cells, and find that, surprisingly, increased levels of cell uptake do not correlate with increased mRNA expression in part likely due to protein corona-induced lysosomal trafficking of LNPs. Our results underscore the need to consider the protein corona in the design of LNP-based therapeutics.

A 3D Cell-Culture System That Uses Nano-Fibrillated Bacterial Cellulose to Prepare a Spherical Formulation of Culture Cells

A 3-dimensional (3D) cell culture is now being actively pursued to accomplish the in vivo-like cellular morphology and biological functions in cell culture. We recently obtained nano-fibrillated bacterial cellulose (NFBC). In this study, we developed a novel NFBC-based 3D cell-culture system, the OnGel method, and the Suspension method. HepG2 human liver cancer cells were cultured via these methods and formed spherical formulations in the optimized condition, 1.0% (w/v) of NFBC in the OnGel method, and 0.06-0.10% (w/v) of NFBC in the Suspension method. Non-cancerous cells such as human-induced pluripotent stem (iPS) cells and human mesenchymal stem cells (MSCs) also formed spherical formulations. It is noteworthy that both the size and cell viability of spheroids prepared via these methods were comparable to those cultured using commercially available 3D cell-culture systems. Both OnGel and Suspension methods are less complicated than the existing 3D cell-culture systems, which is an invaluable advantage for the preparation of cancer spheroids. The NFBC-based 3D cell-culture systems introduced here show great promise as a tool to prepare cultures for cell-derived spheroids for the progress of both in vitro and in vivo studies of the biological functioning of cells.

Exploring the protective role of caffeine against Taraxacum-Induced ribotoxic stress mediated through autophagy and mitochondrial depolarization

The ribotoxic stress response is a pathway that gets activated when ribosomes get impaired, leading to disruptions in protein synthesis, increased inflammatory signaling, and cell death if left unresolved. Taraxacum can induce apoptosis-associated ribosomal RNA (rRNA) cleavage, however, the exact working mechanism of Taraxacum-induced rRNA cleavage remains unclear. In this study, we used the RNA integrity (RIN) value and 28S/18S ratio to confirm the integrity of experiments. Our RNA sequencing data showed that Taraxacum formosanum (T. formosanum) upregulated 893 genes and downregulated 509 genes and triggered hallmark genes of spliceosomes, TNF-α signaling via NF-κB, inflammatory response, and IL6-JAK-STAT3 signaling. Additionally, T. formosanum imbalanced the levels of ribosomal proteins of the large and small subunits. We found that caffeine was the only screening agent that could rescue the cleavage of 28S and 18S rRNA induced by T. formosanum. However, caffeine failed to rescue T. formosanum-targeted mRNAs when the RIN values were relatively lower. T. formosanum induced the N-terminal clipping of histone H3, which was observed not only in human HeLa cervical cancer cells but also in human Huh6 and HepG2 liver cancer cells. Our study revealed that caffeine could reverse the effects of T. formosanum on the reduction of autophagy and the disruption of mitochondrial membrane potential. However, caffeine could only change the populations of necrotic and apoptotic cells but not T. formosanum-induced cell death. By providing detailed information on Taraxacum-induced rRNA cleavage and N-truncated histone H3’s mechanisms of gene regulation, we hope to understand their respective cellular death and survival stresses.

Pretargeted Multimodal Tumor Imaging by Enzymatic Self-Immobilization Labeling and Bioorthogonal Reaction.

Covalent modification of cell membranes has shown promise for tumor imaging and therapy. However, existing membrane labeling techniques face challenges such as slow kinetics and poor selectivity for cancer cells, leading to off-target effects and suboptimal in vivo efficacy. Here, we present an enzyme-triggered self-immobilization labeling strategy, termed E-SIM, which enables rapid and selective labeling of tumor cell membranes with bioorthogonal trans-cycloctene (TCO) handles in vivo. E-SIM utilizes P-TCO, an alkaline phosphatase (ALP) responsive quinone methide (QM) precursor with a TCO group, facilitating the rapid conjugation of high-density TCO handles onto tumor cell membranes via proximity labeling. These TCO groups then react efficiently with tetrazine (Tz)-bearing reporters via a fast bioorthogonal reaction, resulting in significant enrichment of reporters of various sizes and imaging modalities on tumor cell membranes. We demonstrate the efficacy of E-SIM labeling and bioorthogonal reaction for pretargeted multimodality imaging of tumors in vivo. Notably, we achieve selective and efficient installation of Tz-modified Renilla luciferase on tumor cells in vivo, thereby offering highly sensitive bioluminescence signals for detecting and guiding the surgical removal of small human HepG2 liver tumor peritoneal metastases. E-SIM represents a robust tool for precise tumor cell labeling in complex in vivo environments, feasible for pretargeted enrichment of various reporters in tumors for multimodal imaging applications.

Exploring the Anticancer Potential of MonoHER (7-Mono-O-(β-Hydroxyethyl)-Rutoside): Mitochondrial-Dependent Apoptosis in HepG2 Cells.

Flavonoids are a group of polyphenols, abundantly present in our diet. Although, based on their chemoprotective effects, intake of flavonoids is associated with a high anticancer potential as evidenced in in vitro and in vivo models, the molecular mechanism is still elusive. This study explores the antiproliferative and cytotoxic effects of the semi-synthetic flavonoid MonoHER (7-mono-O-(β-hydroxyethyl)-rutoside) in vitro on cancer cells. HepG2 liver, MCF7 breast, and H1299 lung cancer cells were grown under ambient conditions with or without MonoHER exposure. CCK8 assay was used to assess cell viability. Apoptosis, JC-1, and mitochondrial mass were determined using flow cytometry and confocal analysis. The effects of monoHER on apoptosis proteins were detected by confocal microscopy analysis and Western blot. It was found that MonoHER can reduce HepG2 cells' and MCF7 cells' viability, but not H1299 cells', and induced apoptosis only in HepG2 cells. MonoHER has the potential to enhance the expression of caspase-9 and caspase-3, to damage mitochondria, and to provoke the release of cytochrome C from the mitochondria. MonoHER can inhibit cell growth and induce apoptosis especially in HepG2 human liver cancer cells by triggering the mitochondrial signal transduction pathway, leading to the release of cytochrome C in the cytoplasm and the subsequent activation of caspase-9 and caspase-3. Future research should further explore MonoHER's mechanism of action, efficacy, and potential for clinical translation.

Exploration of the cytotoxic and microtubule disruption potential of novel imidazo[1,5-a]pyridine-based chalcones.

In continuation of our efforts to develop new anticancer compounds, a new series of imidazo[1,5-a]pyridine-chalcone derivatives was designed, synthesized, characterized, and evaluated for its cytotoxicity against five human cancer cell lines, i.e., breast (MDA-MB-231), colon (RKO), bone (Mg-63), prostate (PC-3), and liver (HepG2) cell lines, as well as a normal cell line (HEK). Among the synthesized compounds, two exhibited promising cytotoxicity against the MDA-MB-231 cell line with IC50 values of 4.23 ± 0.25 μM and 3.26 ± 0.56 μM. We also studied apoptotic induction of the compounds using annexin V-FITC/PI staining, and ROS-mediated mitochondrial damage was elucidated using DCFDA, followed by JC-1 staining. The potential activity of the compounds was further confirmed by immuno-fluorescence and molecular docking studies, which revealed the anticancer activity of active compounds through binding and microtubule disruption.

Dexlansoprazole is an aryl hydrocarbon receptor agonist.

Dexlansoprazole, a proton pump inhibitor, is commonly used to treat gastro-esophageal reflux disease and erosive esophagitis. The activated aryl hydrocarbon receptor (AhR) functions as a transcription factor by binding to the aryl hydrocarbon response element (AHRE) of its target genes, with cytochrome P450 (CYP) 1A1 being the most well-known target. In this study, we demonstrated that dexlansoprazole stimulates AhR activity, leading to increased CYP1A1 expression. Our findings indicate that treatment with 2μM dexlansoprazole is sufficient to induce CYP1A1 mRNA and protein expression, as well as AHRE-mediated transcriptional activity, in both human and mouse cells. Using AhR signal-deficient mutant cells and specific AhR antagonists-SR1, GNF351, and CH-223191-we confirmed that AhR is required for dexlansoprazole-induced CYP1A1 expression. Additionally, we showed that dexlansoprazole promotes AhR nuclear translocation, acting as an AhR agonist. However, due to its lower potency compared to FICZ and ITE in activating AhR, dexlansoprazole suppresses FICZ- and ITE-induced CYP1A1 expression in human liver HepG2 and ovarian granulosa HO23cell lines, suggesting that it functions as both an AhR agonist and a modulator. This study offers valuable insights into the potential clinical side effects of dexlansoprazole.

Exposure to a Titanium Dioxide Product Alters DNA Methylation in Human Cells.

The safety of titanium dioxide (TiO2), widely used in foods and personal care products, has been of ongoing concern. Significant toxicity of TiO2 has been reported, suggesting a risk to human health. To evaluate its potential epigenotoxicity, the effect of exposure to a TiO2 product to which humans could be exposed on DNA methylation, a primary epigenetic mechanism, was investigated using two human cell lines (Caco-2 (colorectal) and HepG2 (liver)) relevant to human exposure. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Gene promoter methylation was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNA methyltransferases, MBD2, and URHF1 was quantified by qRT-PCR. A decrease in global DNA methylation was observed in both cell lines. Across the cell lines, seven genes (BNIP3, DNAJC15, GADD45G, GDF15, INSIG1, SCARA3, and TP53) were identified in which promoters were methylated. Changes in promoter methylation were associated with gene expression. Results also revealed aberrant expression of regulatory genes, DNA methyltransferases, MBD2, and UHRF1. Findings from the study clearly demonstrate the impact of TiO2 exposure on DNA methylation in two cell types, supporting the potential involvement of this epigenetic mechanism in its biological responses. Hence, epigenetic studies are critical for complete assessment of potential risk from exposure.

Construction of Sorafenib Tosylate and Etoposide‐loaded Liposomes: A Path to Precision Liver Cancer Therapy and its Apoptosis Induction

AbstractNanotechnology is an effective tool in fighting against cancer, playing a crucial role in investigating and fabricating novel anticancer drugs. Recognizing the worldwide prevalence of cancer, we combined sorafenib tosylate (ST) and etoposide (ETP) within liposomes. We assessed their ability to kill human umbilical vein endothelial cells (HUVECs) and HepG2 liver cancer cells. The liposomes effectively contained ST and ETP, exhibiting a particle size distribution below 180 nm, a polydisperse index (PDI) below 0.2, a spherical shape, a strong negatively charged zeta potential, and encapsulation efficiencies of 59% for ST, 88% for ETP, and 57% for ST combined with 87% for ETP. The FTIR analysis indicates that the drugs were incorporated within liposomes. Encapsulation of the drugs in liposomes resulted in a more significant cytotoxic impact on HepG2 cells and a reduced cytotoxic impact on HUVECs. The morphological assessment of the HepG2 liver cancer cells was investigated using AO‐EB and Hoechst 33258 staining methods. Apoptosis mechanisms of HepG2 cells were examined by Annexin V and PI dual staining. Furthermore, the coadministration of ST and ETP, which were enclosed in liposomes, resulted in a synergistic impact on the drugs, leading to cell death by apoptosis.

Spontaneous reversal of small molecule-induced mitochondrial uncoupling: the case of anilinothiophenes.

Tissue specificity can render mitochondrial uncouplers more promising asleading compounds for creating drugs against serious diseases. In searchof tissue-specific uncouplers, we address anilinothiophenes as possible glutathione-S-transferase substrates (GST). Earlier, 'cyclic' uncoupling activity was reported for 5-bromo-N-(4-chlorophenyl)-3,4-dinitro-2-thiophenamine (BDCT) in isolated rat liver mitochondria (RLM). The mechanism by which BDCT induced two-phase changes in mitochondrial respiration (stimulation followed by deceleration) was unknown. To clarify this issue, we synthesized BDCT and its two analogues. Among these, 5-bromo-3,4-dinitro-N-(4-nitrophenyl)-2-thiophenamine (BDNT) appeared to be the most effective as a mitochondrial uncoupler, decreasing membrane potential and stimulating respiration at submicromolar concentrations. Importantly, BDNT exerted two-phase changes in both mitochondrial membrane potential and respiration rate of RLM, which were enhanced by the addition of glutathione (GSH) but inhibited by the compounds capable of GSH depleting, such as 1-chloro-2,4-dinitrobenzene (CDNB). By contrast, the phase of recoupling was not observed in rat heart mitochondria (RHM). Remarkably, BDNT elicited mitochondrial depolarization in primary human fibroblasts but not in cultured human liver (HepG2) cells. By detecting proton-selective electrical current through planar bilayer lipid membranes, we demonstrated the ability of BDCT and BDNT to transfer protons across membranes. BDNT proved to be an anionic protonophore with a pKa of 7.38. By using LC-MS and capillary electrophoresis, we directly showed the formation of BDNT conjugates with GSH upon incubation with RLM but not RHM. Therefore, we hypothesize that GST is involved in the disappearance of the anilinothiophene uncoupling activity in RLM, ensuring the tissue-specific behavior of the uncoupler.

Structural Elucidation and NMR Spectral Assignments of New Diphenyl Ether Derivatives From Liuweizhiji Gegen-Sangshen Oral Liquid.

Chemical investigation of the Liuweizhiji Gegen-Sangshen oral liquid afforded one new diphenyl ether derivative (1), together with one known compound (2). Their structures were established by 1D and 2D NMR, and HR-ESI-MS spectroscopic analysis and the absolute configuration of 1 was confirmed by ECD calculation. Compounds 1 and 2 were evaluated for the cytotoxic activities, and compounds 1 and 2 showed weak cytotoxic activities towards HepG2 human liver cancer cells, with IC50 values of 97.3 and 79.6 μM, respectively.

Synthesis of Sulfonamide-Based Schiff Bases as Potent Anticancer Agents: Spectral Analyses, Biological Activity, Molecular Docking, ADME, DFT, and Pharmacophore Modelling Studies.

The current study focuses on the synthesis and characterization of six benzenesulfonamide-based Schiff base derivatives (7-12) with various electron-withdrawing and electron-donating substituents (-F, -CI, -Br, -CH3, and -OCH3) and the assessment of their antiproliferative activities against human lung (A549) and liver (HepG2) cancer cell lines using in vitro and in silico approaches. The structures of the synthesized compounds (7-12) were elucidated by elemental analysis and FT-IR, 1D (1H, 13C, APT, and DEPT-135), and 2D (HMQC and HMBC) NMR spectroscopies. The cytotoxic activities of the targeted compounds were determined at various concentrations against these cancer cell lines for 72 h, using the MTT method. The targeted molecules (7-12) demonstrated remarkable antiproliferative activities, with IC50 values ranging from 6.032-9.533 μM against the A549 cell line and 5.244-9.629 μM against the HepG2 cell line. These compounds showed activities at lower or very similar concentrations to cisplatin against the A549 cell line and at much lower concentrations than cisplatin against the HepG2 cell line. Among them, compounds 10 and 12 were found to be more effective against A549 and HepG2 cells, respectively, than cisplatin. These compounds were analyzed by interacting with the 1BNA, 4HJO, and 4ASD crystal structures in molecular docking studies. The docking score of 4ASD-compound 12 interaction was calculated as -4.045 kcal/mol, 4HJO-compound 10 interaction was calculated as -5.179 kcal/mol and 1BNA-compound 10 interaction was calculated as -8.571 kcal/mol and it was determined that these compounds were theoretically better than Cisplatin. In the present study, ADME data were estimated using the web tool SwissADME. With ADME, it was calculated that the logP value of compounds 7-12 was less than 5, the HBD number was 1, the HBA number was 7 or 8, and the molecular weight was less than 500. Properties such as the electrophilic index and chemical hardness of the designed compounds were examined by density functional theory (DFT) using B3LYP/6-311G**. In conclusion, these compounds have emerged as promising new anti-cancer drug candidates.

Herbo-vitamin medicine Livogrit Vital ameliorates isoniazid induced liver injury (IILI) in human liver (HepG2) cells by decreasing isoniazid accumulation and oxidative stress driven hepatotoxicity

BackgroundTuberculosis (TB) is a leading cause of infection related mortality. Isoniazid is one of the frontline drugs for anti-TB therapy. Hepatotoxicity induced by isoniazid is a major cause of drug-discontinuation which may lead to development of resistant TB or increased mortality.PurposeTo characterize pharmacological properties of plant-based prescription medicine, Livogrit Vital (LVV) against isoniazid-induced liver injury (IILI) using HepG2 cells.MethodPhytometabolite characterization of LVV was performed by High-performance liquid chromatography (HPLC). The effects of LVV on cytosafety, IC50 shift, oxidative stress, ER stress, apoptosis, liver injury markers, and accumulation of isoniazid and hydrazine was performed on HepG2 cells induced with isoniazid. Silymarin was used as the positive control.ResultsHPLC based phytometabolite characterization of LVV revealed the presence of several anti-oxidant, anti-apoptotic, and hepatoprotective compounds. In isoniazid-induced HepG2 cells, LVV reduced cytotoxicity of isoniazid and shifted its IC50 value. Treatment with LVV reduced ROS generation and lipid peroxidation; enhanced GSH enzyme levels in isoniazid-induced HepG2 cells. As per the mechanistic evaluation, LVV modulated gene expression level of Caspase-3, FGF21, and IRE-1α. LVV treatment also normalized isoniazid-induced elevated Caspase-3 activity and cPARP1 protein levels, indicating its potentials to regulate liver cell apoptosis. Concomitantly, biomarkers of hepatotoxicity, ALT and GGT, also decreased by LVV treatment. Interestingly, LVV treatment reduced intracellular accumulation of isoniazid and its toxic metabolite hydrazine, in isoniazid-stimulated HepG2 cells.ConclusionTreatment of hepatic cells with the herbo-vitamin medicine, Livogrit Vital, regulates IILI by modulation of oxidative and ER stress, apoptosis, and bioaccumulation of isoniazid and hydrazine. Collectively, Livogrit Vital could well be explored as an adjuvant hepatoprotective agent alongwith anti-TB medicines.

Network pharmacology analysis revealed the mechanism and active compounds of jiao tai wan in the treatment of type 2 diabetes mellitus via SRC/PI3K/AKT signaling

Ethnopharmacological relevanceJiao-tai-wan (JTW) is a traditional Chinese herbal prescription, exerts its therapeutic effects on type 2 diabetes mellitus (T2DM). However, its mechanisms and active components remain unclear. Aim of the studyTo investigate the therapeutic mechanisms of JTW in treating type 2 diabetes mellitus (T2DM), focusing on identifying active components, their targets, and validating efficacy through SRC/PI3K/AKT signaling pathway modulation in vitro and in vivo. Materials and methodsActive ingredients were retrieved from the Traditional Chinese Medicine System Pharmacology (TCMSP) and Comprehensive Traditional Chinese Medicine Database (TCMID). Targets for these components were identified using the ChemMapper database based on 3D structural similarity. T2DM-related genes were sourced from the DisGeNET and Gene Expression Omnibus (GEO) databases. Protein-protein interaction (PPI) analysis and functional enrichment analysis were conducted to construct a pathway network of “herbs-active ingredients-candidate targets”, identifying core molecular mechanisms and key active ingredients. SwissDock was used for molecular docking to predict ligands for candidate targets. The diabetic models were established using C57BL/6 mice and human liver HepG2 cell lines. Their Effectiveness and key molecules were verified through biochemical detection and immunoblotting. ResultsTotal 30 active compounds, 597 active ingredient targets, 9631 T2DM-related genes, and 521 overlapping candidate targets were found for JTW on T2DM. Go enrichment indicated the core pathways enriched on insulin and glucose metabolism. The auto-docking demonstrated SRC has potential binds to ingredients of JTW. In vivo, JTW can reduce blood glucose, and blood lipid levels, and HOMA-IR, and increase HOMA-ISI levels in T2DM mice with reduced ALT, AST, MDA levels and increased SOD levels. Meanwhile, decreased phosphorylation of SRC, along with increased levels of phosphorylated PI3K, PI3K, and phosphorylated AKT, were observed. HE staining of liver tissues further confirmed that JTW administration improved liver morphology, reducing inflammation and necrosis. In vitro, JTW significantly ameliorates upstream dysregulation by reducing SRC phosphorylation while enhancing phosphorylated PI3K, PI3K, and AKT phosphorylation levels. ConclusionJTW may alleviate glucose, insulin resistance, and lipid metabolism disorders by the SRC/PI3K/AKT signaling pathway, that provide a novel view of potential active compounds and essential targets in treating T2DM.

Synthesis, Cytotoxicity, Apoptosis and Cell Cycle Arrest of a Ruthenium(II)-Substituted Keggin Polyoxotungstate

The ruthenium multi-substituted polyoxotungstate, K7[SiW9O37Ru4(H2O)3Cl3]·15H2O (S1), was synthesized by a conventional aqueous solution containing the trilacunary Keggin-anions β-Na9HSiW9O34·12H2O (S2) and RuCl3·nH2O (S3). Compound S1 was characterized by elemental analysis, energy-dispersive X-ray spectroscopy (EDS), thermogravimetric analysis (TG), infrared spectroscopy (IR), uliraviolet visible absorption spectroscopy (UV/Vis) and X-ray photoelectron spectroscopy (XPS). The cytotoxicitycy of S1 was tested in C33A (human cervical cancer), DLD-1 (human colon cancer), HepG2 (human liver cancer) and human normal embryonic lung fibroblasts cell (MRC-5). And the viability of these treated cells was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.To explore the mode of cell death induced by S1, morphological study of DNA damage and apoptosis assays were conducted. These analyses revealed that S1 exerted its cytotoxic effect in a dose-dependent manner, primarily triggering apoptotic cell death. Cell cycle analysis by flow cytometry indicated that compound S1 caused cell cycle arrest and accumulated cells in S phase.

The Anticancer Properties, Cell-cycle Cytotoxicity and Apoptosis of Cissus rotundifolia, Trema orientalis, and Buddleja polystachya with Ocular Applications

BackgroundBuddleja polystachya, Trema orientalis, and Cissus rotundifolia were applied locally for various ocular purposes, while receiving few scientific evaluations. PurposeThis study aimed to screen the anticancer properties and determine the cell-cycle cytotoxicity and apoptotic activity of the most promising plant extract. MethodsIn this study, MTT assays with MCF7 (human breast adenocarcinoma), HT29 (human colorectal adenocarcinoma) and HepG2 (human liver adenocarcinoma) were used. In addition to MRC5 (normal human foetal lung fibroblast) was carried out for preliminary activity screening and selectivity. The most promising extract was subjected to GC-MS analysis to determine the phytochemical composition. Additionally, a clonogenic assay was performed to measure tumor cell survival and subsequent proliferative capacity after drug exposure was conducted for the most active extract(s) and finally western blotting was used to determine the expression change of the two selected proteins (survivin and CCND1) in order to determine the exact mechanistic features of the most promising plant extract. ResultsThe six extracts showed variable IC50 values ranging from 1.77- 40.97 μg/mL. The most active extracts were C. rotundifolia coded as (stem; BEP-03A and leaves; BEP-03B) on HepG2 cells and showed ∼ 4 and 8 fold selectivity compared to normal MRC5 cells. Both extracts showed a dose-dependent clonogenic effect on HepG2 cells, which was comparable to the effect of doxorubicin. The extract (BEP-03B) caused a significant decrease in the expression of survivin and CCND1 compared to the control GAPDH at its highest dose (12 µg/mL). The GC-MS chromatogram of the leaf of C. rotundifolia extract (BEP-03B) revealed the presence of 17 compounds, as the main phytoconstituents representing 57.5% of the total compounds present in BEP-03B. Three steroidal components (12, 14 and 15) were the main components, while compound stigmast-5-en-3-ol (compound 15) was the main component. ConclusionsLeaves of Cissus rotundifolia (Forssk.) Vahl, possess a significant cytotoxic effect and it may produce this effect, through apoptosis induction, perturbation, and disruption of the cell cycle. The detected phytoconstituents in the plant extract might be involved in the tested cytotoxic activity and its molecular apoptotic mechanism. Future studies are required to isolate the active ingredient(s) and confirm the therapeutic application(s).

Determination of the interactions of a schiff base with different targets via molecular docking and cytotoxic activity studies

The pyrimidine nucleus is a vital pharmacophore that exhibits excellent pharmacological activity. A Schiff base containing a pyrimidine nucleus was obtained in two steps. This compound was assessed on the human prostate (PC3) and liver (HepG2) cancer cell lines using the MTT method. The prepared compound 3 was determined to have a high cytotoxic effect towards prostate cancer with an IC50 value of 9.32 μM. Since experimental prostate and liver cancer studies were examined, the molecular docking study's target structures were determined accordingly. In molecular docking studies, compound 3 interacted in silico with the crystal structure of the human HER2 kinase domain (PDB Id: 3PP0), the crystal structure of VEGFR-2 (PDB Id: 4ASD), and the crystal structure of EGFR tyrosine kinase (PDB Id: 4HJO), respectively. As a result of these interactions, binding energy values were calculated, and binding modes were determined. Additional in vitro and in vivo experiments targeting other cancer cell lines will provide deeper insight into the anticancer spectrum of this compound and new studies on this compound will be planned in the following years. Furthermore, optimizing and studying such pyrimidine-based Schiff base compounds for improved selectivity and potency against prostate cancer is also considered to be valuable as it may increase the potential to capture potential drug candidates.

Synthesis, Cytotoxicity, and Quantitative Structure–Activity Relationship Studies of Alkyl Triphenylphosphonium Pinostrobin Derivatives

AbstractPinostrobin, an isolated compound from Boesenbergia rotunda, has been shown to have potent anti‐proliferation and apoptosis effects in cancer stem‐like cells. However, its hydrophobic properties reduce its bioavailability. Eight new alkyl‐TPP+ pinostrobin derivatives were synthesized and assessed for their cytotoxicity against the human HepG2 liver cancer cell line using the alamarBlue assay. All derivatives exhibited moderate cytotoxicity, with the IC50 values ranging from 38.55–101.95 μM, and were more potent than pinostrobin and pinostrobin hydrazone (IC50>100 μM). Four alkyl‐TPP+ pinostrobin hydrazone amide derivatives showed more potent cytotoxicity than four alkyl‐TPP+ pinostrobin ester derivatives. QSAR analysis showed key 3D structural descriptors of TPP+‐based compounds responsible for cytotoxicity against HepG2 cells for the first time. The resultant 3D‐QSAR models using PLS and PCR methods were evaluated and found reliable in predicting the cytotoxicity of other TPP+‐based compounds.