Human Embryonic Liver Research Articles

Synthesis, biological evaluation, and molecular docking of novel 1,3,4-substituted-thiadiazole derivatives as potential anticancer agent

In an attempt to develop potent anti-cancer agents, a new 1,3,4-substituted-thiadiazole derivatives (8b-g), starting from 4-substituted-thiazol-2-chloroacetamides (4b-g), were synthesized and evaluated for their cytotoxic effects on multiple human cancer cell lines, including the hepatocellular carcinoma (HEPG-2), human lung carcinoma (A549), human breast carcinoma (MCF-7) and pseudo-normal human embryonic liver (L02) cancer cell lines by an MTT assay. Among all synthesized compounds, compound 8d showed the potent anti-cancer activities with GI50 values of 2.98, 2.85 and 2.53 μM against MCF-7, A549 and HepG-2 cell lines respectively as compared to standard drug Doxorubicin. Furthermore, molecular modelling studies have spotlighted the anchoring role of 1,3,4-substituted-thiadiazole moiety in bonding and hydrophobic interaction with the key amino acid residues. Therefore, these results can provide promising starting points for further development of best anti-cancer agents.Graphical

Protective Effect of Selenium-enriched Peptide from Cardamine violifolia on Ethanol-induced L-02 Hepatocyte Injury.

In this study, we investigated the protective effect of selenium (Se)-enriched peptide isolated from Cardamine violifolia (SPE) against ethanol-induced liver injury. Cell proliferation assays show that different concentrations of SPE protect human embryonic liver L-02 cells against ethanol-induced injury in a dose-dependent manner. Treatment with 12 μmol/L Se increases the cell survival rate (82.44%) and reduces the release of alanine aminotransferase, aspartate transaminase, lactate dehydrogenase, and apoptosis rate. SPE treatment with 12 μmol/L Se effectively reduces the concentration of intracellular reactive oxygen species and increases the contents of intracellular superoxide dismutase (51.64 U/mg), catalase (4.41 U/mg), glutathione peroxidase (1205.28 nmol/g), and glutathione (66.67 μmol/g), thereby inhibiting the effect of ethanol-induced oxidative damage. The results of the transcriptomic analysis show that the glutathione metabolism and apoptotic pathway play significant roles in the protection of L-02 hepatocytes by SPE. Real-time qPCR analysis shows that SPE increases the mRNA expression of GPX1 and NGFR. The results of this study highlight the protective effects of SPE against ethanol-induced liver injury.

Abstract 4188: Kyinno PBMC humanized mouse platform: Pioneering service in biomedical research

Abstract Tumor immunotherapy is the use of the human immune system to kill tumors. Compared with traditional chemotherapy and radiotherapy, immunotherapy has significant advantages such as significant efficacy, long-lasting effects, and low toxicity. Wild-type mice are widely used as preclinical models for immunotherapy due to their normal immune system, but they have many shortcomings, such as only being able to inoculate mouse tumor cells, differences between mouse and human immune systems, etc., which cannot accurately simulate the interaction between drugs and tumor cells in the human body. Humanized mouse models include PBMCs humanized mouse models, HSC humanized mouse models, and human embryonic bone, liver, and thymus tissue (BLT) transplantation models. Among them, the PBMCs humanized mouse model is widely used due to its simple preparation, short cycle, and high levels of hCD3+ T cells in the peripheral blood of mice. To assist in the development of immunotherapy, KYINNO has established a PBMC humanized mouse platform that can perform in vivo drug efficacy verification of various immunotherapeutic drugs such as CD3 bispecific antibodies or multi-specific antibodies, immune checkpoint inhibitors, etc. We screen many PBMC donors and select PBMC donors with light GvHD and good hCD3+ T cell reconstruction effects. The drug administration window of the PBMC humanized model has been extended to 6 weeks. Platform verification has been performed on subcutaneous and intravenous injection models of various tumors. Overall, we successfully established the PBMC humanized mouse platform, which is a powerful tool for immunotherapy. Citation Format: Tongtong Liu, Guojin Wu, Feng He, Longtan Luo, Zhengcheng Guo, Shuliang Li, Jinying Ning, Feng Hao. Kyinno PBMC humanized mouse platform: Pioneering service in biomedical research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4188.

Design, synthesis of new 4,5-dibenzylidene-9,10-diphenyl-1,2,7,8,9,10 hexahydroacridine-3,6-dione derivatives using extract of Vitexnegundo: Cytotoxic activity & molecular docking study

Among phytochemical and pharmaceutical investigations, the green production of silver nanoparticles has emerged as the crown jewel of this investigation. The production of acridinedione substances is greatly aided by the high catalytic activity of these nanoparticles. Structures and morphologies were determined by using a wide range of analytical techniques, including nuclear magnetic resonance (1H &13C), Fourier transform infrared spectroscopy, mass spectrometry, elemental investigation, scanning electron microscopy, also transmission electron microscopy. Acridinedione compounds' cytotoxicity was measured using the MTT assay with doxorubicin as the reference drug in a number of cancerous and healthy cell lines, comprising Human embryonic kidney cell (HEK293), liver cell (LO2), and lung cell (MRC5). The cytotoxicity compound 1h was shown to be especially active (HepG2, LC50-0.5 µM; MCF-7, LC50-0.64 µM; HeLa, LC50-0.52 µM). By eliciting IC50 values larger than 100 µg/M in normal cell lines (HEK-293), (LO2), and (MRC5), the synthesised compounds proved to be safe. In addition, we present the results from an in-silico analysis of the Methoxsalen protein (1Z11). The binding affinity of compound 1h for the 1Z11 protein is greater than that of other compounds (-11.7 kcal/mol), but it is less than that of Doxorubicin (-10.2 kcal/mol). Hence, the chemical 1h may be used to create powerful cancer drugs.

Toxicity of polystyrene nanoplastics to human embryonic kidney cells and human normal liver cells: Effect of particle size and Pb2+ enrichment

Nanoplastics pollution in drinking water has aroused wide concern, but their effects on human health are still poorly understood. Herein we explore the responses of human embryonic kidney 293T cells and human normal liver LO2 cells to polystyrene nanoplastics, mainly focusing on the effects of particle sizes and enrichment of Pb2+. When the exposed particle size is higher than 100 nm, there is no obvious death for these two different cell lines. As the particle size decreases from 100 nm, cell mortality goes up. Although the internalization of polystyrene nanoplastics in LO2 cells is at least 5 times higher than that in 293T cells, the mortality of LO2 cells is lower than that of 293T cells, illustrating that LO2 cells are more resistant to polystyrene nanoplastics than 293T cells. Additionally, the Pb2+ enrichment on polystyrene nanoplastics in water can further enhance their toxicity, which should be taken seriously. The cytotoxicity of polystyrene nanoplastics to cell lines works through a molecular mechanism involving oxidative stress-induced damage of mitochondria and cell membranes, resulting in a decrease in ATP production and an increase in membrane permeability. Referenced to nanoplastics pollution in drinking water, there is no necessary to panic about the adverse effects of plastic itself on human health, but the enrichment of contaminants should get more attention. This work provides a reference for the risk assessment of nanoplastics in drinking water to human health.

Developmental toxicity assessments for TBBPA and its commonly used analogs with a human embryonic stem cell liver differentiation model

Tetrabromobisphenol A (TBBPA) is widely used in industrial production as a halogenated flame retardant (HFR). Its substitutes and derivatives are also commonly employed as HFRs. Consequently, they can be frequently detected in environmental and human samples. The potential developmental toxicity of TBBPA and its analogs, particularly to the human liver, is still controversial or not thoroughly assessed. Therefore, in this study, we focused on the early stages of human liver development to explore the toxic effects of those HFRs, by using a human embryonic stem cell liver differentiation model. We concluded that nanomolar treatments (1, 10, and 100 nM) of those pollutants may not exert significant interference to liver development and functions. However, at 5 μM doses, TBBPA and its analogs severely affected liver functions, such as glycogen storage, and caused lipid accumulation. Furthermore, TBBPA-bis(allyl ether) showed the most drastic effects among the six compounds tested. Taken together, our findings support the view that TBBPA can be used safely, provided its amounts are strictly controlled. Nonetheless, TBBPA alternatives or derivatives may exhibit stronger adverse effects than TBBPA itself, and may not be safer choices for manufacturing applications when utilized in a large and unrestricted way.

Combined effect of polystyrene microplastics and bisphenol A on the human embryonic stem cells-derived liver organoids: The hepatotoxicity and lipid accumulation

Human are exposed to microplastics (MP) via inhalation or ingestion daily and inevitably. The liver is an important target organ of MP. Bisphenol A (BPA) is one of commonly used plasticizers. It is added in plastics, but also generally detected in the biological samples of human beings. However, the combined toxic effect of MP and BPA on human liver is unclear. In this study, a novel 3D in vitro model, the liver organoid (LO) derived from human-pluripotent stem cells, has been utilized to explore the 1 μm polystyrene (PS)-induced hepatotoxicity with BPA individually and jointly. Conclusively, all the changes in the cytotoxicity, cellular and molecular makers regarding the energy supplement, hepatic injury, oxidative stress, inflammatory response, disruption in the lipid accumulation, as well as epigenetics regulation induced by BPA or PS on the LOs individually were comparable to previous study. The BPA levels in the culture medium were declined by the added PS. The combined adverse effect of PS and BPA on the LOs was identified to be synergistic upon deteriorated hepatotoxicity and interfered the gene panels related to multiple processes of lipid metabolism, together with the proteins of HNF4A, CD36, ACC1, CPT1A, CYP2E1, ERα and ERβ. Specifically, PS didn't change the ERα or ERβ individually, but when the LOs were co-exposed to PS and BPA, the ERα further elevated significantly and synergistically. Our findings highlight the metabolic-related health risk due to co-exposure to MP and BPA, even at low-doses equivalent to human internal exposure level. Based on these findings, the potential adverse outcome pathway related to PS and BPA singly and jointly were proposed, predicting two possible outcomes to be hepatic steatosis. Moreover, the ERα and HNF4A were proposed to be potential candidate markers to investigate the “vector-like effect” of PS in the present of BPA.

Synchrotron radiation-based Fourier transform infrared microspectroscopy investigation of WRL68 cells treated with doxorubicin

Doxorubicin is an effective chemotherapeutic agent applied in a wide variety of cancers. Despite its potent anticancer activity towards cancer cells, doxorubicin is also toxic to noncancerous cells. Therefore, doxorubicin can cause serious side effects in various organs, especially when dose escalation is required for patients with advanced disease. The liver is the major detoxification organ that metabolizes drugs, and hepatotoxicity is one of the most common adverse effects of doxorubicin administration. However, the exact mechanisms of doxorubicin-induced hepatotoxicity have not been clearly identified, and how doxorubicin treatment affects the biomolecular contents of normal human hepatocytes has rarely been studied. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy is a state-of-the-art analytical technique for characterizing the biomolecules present in cells. In this research, the biomolecular alterations of doxorubicin-treated normal human hepatocytes compared to untreated control cells were investigated at the single-cell level by combining SR-FTIR microspectroscopy with the Cell Counting Kit-8 (CCK-8) assay and flow cytometry. WRL68 human normal embryonic liver cells, which have been shown to be very promising for assessing the cytotoxicity of toxic compounds and investigating hepato-toxicology, were used in this research. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to further analyse the biomolecular contents of WRL68 cells. The order of lipid acyl chains and protein α-helix structures in doxorubicin-treated WRL68 cells was found to be distinctly changed, while the nucleic acids were altered relatively less. No alteration in the carbohydrate content was distinguishable after doxorubicin treatment. These results provide more comprehensive information about the biomolecular changes in hepatocytes induced by doxorubicin treatment and help to elucidate the mechanism of doxorubicin-induced hepatotoxicity. This research also proves that SR-FTIR microspectroscopy, combined with PCA and OPLS-DA, is a promising approach for investigating drug-cell interaction systems.

Identification of Selective CYP3A7 and CYP3A4 Substrates and Inhibitors Using a High-Throughput Screening Platform.

Cytochrome P450 (CYP) 3A7 is one of the major xenobiotic metabolizing enzymes in human embryonic, fetal, and newborn liver. CYP3A7 expression has also been observed in a subset of the adult population, including pregnant women, as well as in various cancer patients. The characterization of CYP3A7 is not as extensive as other CYPs, and health authorities have yet to provide guidance towards DDI assessment. To identify potential CYP3A7-specific molecules, we used a P450-Glo CYP3A7 enzyme assay to screen a library of ∼5,000 compounds, including FDA-approved drugs and drug-like molecules, and compared these screening data with that from a P450-Glo CYP3A4 assay. Additionally, a subset of 1,000 randomly selected compounds were tested in a metabolic stability assay. By combining the data from the qHTS P450-Glo and metabolic stability assays, we identified several chemical features important for CYP3A7 selectivity. Halometasone was chosen for further evaluation as a potential CYP3A7-selective inhibitor using molecular docking. From the metabolic stability assay, we identified twenty-two CYP3A7-selective substrates over CYP3A4 in supersome setting. Our data shows that CYP3A7 has ligand promiscuity, much like CYP3A4. Furthermore, we have established a large, high-quality dataset that can be used in predictive modeling for future drug metabolism and interaction studies.

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor

BackgroundhPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages.MethodsTwo hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other’s (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations.ResultsA hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors.ConclusionThe present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering.

Pharmacognostic Profile and Screening of Anti-Proliferative Potential of Methanolic Extract of Tripterygium wilfordii Plant on WRL-68 Cell Line and Function of Polycystin-1.

Tripterygium wilfordii is a medicinal plant that plays a crucial role in health care programs, especially in developing countries, and had anti-tumor, anti-inflammatory, anti-fertility, anti-bacterial, and other therapeutic effects. This study was designed to determine the anti-proliferative effects of methanolic extract of T. wilfordii on the WRL-68 cell line and the function of polycystin-1 (PC-1). The half-maximal inhibitory concentration (IC50) values were recorded in WRL-68 and AsPC-1 cell lines as 193 µg/ml and 149.2 µg/ml, respectively, at 2-2.55 and 2-2.2 µg/ml methanolic plant concentrations. The maximum cytotoxic activities of the extract on the growth inhibition of WRL-68 and AsPC-1 were generally observed at 97.64% and 95.94% at extract concentrations of 50 µg/ml and 25 µg/ml, respectively. The pharmacognostic profile of T. wilfordii extract was found to be alkaloids, tannins, terpenoides, flavonoids, glycosides, and phenols. The extracts of T. wilfordii were tested through gas chromatography-mass spectrometry showing four peaks representing mostly of 3-Oxobutanol; ethyl acetate; acetic acid ethyl ester; chlorbromuron; 1-(methylthio)-, (E)-; n-Hexadecanoic acid; tetradecanoic acid; and 9-Octadecenoic acid. Therefore, the results of this study revealed that the methanolic extract of T. wilfordii was more potential in inducing anti-proliferative activity of WRL-68 and AsPC-1 human cell lines than the control. In addition, the current study was the first study that reported the anti-proliferative potential of T. wilfordii in the treatment of human embryonic liver WRL-68 cancer cells.

The Effect of Conjugation with Octaarginine, a Cell-Penetrating Peptide on Antifungal Activity of Imidazoacridinone Derivative.

Acridine cell-penetrating peptide conjugates are an extremely important family of compounds in antitumor chemotherapy. These conjugates are not so widely analysed in antimicrobial therapy, although bioactive peptides could be used as nanocarriers to smuggle antimicrobial compounds. An octaarginine conjugate of an imidazoacridinone derivative (Compound 1-R8) synthetized by us exhibited high antifungal activity against reference and fluconazole-resistant clinical strains (MICs ≤ 4 μg mL−1). Our results clearly demonstrate the qualitative difference in accumulation of the mother compound and Compound 1-R8 conjugate into fungal cells. Only the latter was transported and accumulated effectively. Microscopic and flow cytometry analysis provide some evidence that the killing activity of Compound 1-R8 may be associated with a change in the permeability of the fungal cell membrane. The conjugate exhibited low cytotoxicity against human embryonic kidney (HEK-293) and human liver (HEPG2) cancer cell lines. Nevertheless, the selectivity index value of the conjugate for human pathogenic strains remained favourable and no hemolytic activity was observed. The inhibitory effect of the analysed compound on yeast topoisomerase II activity suggested its molecular target. In summary, conjugation with R8 effectively increased imidazoacridinone derivative ability to enter the fungal cell and achieve a concentration inside the cell that resulted in a high antifungal effect.

A Human IPS Model Implicates Embryonic B-Myeloid Fate Restriction As a Developmental Susceptibility to ETV6-RUNX1

Childhood acute lymphoblastic leukemia (cALL) is clinically distinct from that in adults with higher incidence, better prognosis, a distinct mutational spectrum and that the pre-leukemic initiating events frequently occur in utero . One hypothesis for this difference is that childhood cancers arise in transient cell compartments unique to early human development. We have explored this in ETV6-RUNX1-associated cALL, where evidence from twin studies and neonatal heel prick testing has shown that this translocation occurs in utero and is an initiating event . Consistent with this, epidemiological evidence and changes in placental structure suggest that leukemic initiation occurs in the first trimester when both extra-embryonically and intra-embryonically-derived waves of hematopoiesis coexist. We therefore characterized B-lymphoid development in first trimester human embryos aiming to identify a candidate compartment vulnerable to the pre-leukemic initiating effect of ETV6-RUNX1. Using CD19 as a marker associated with definitive B lineage restriction in adult hematopoiesis, we established that the first CD19+ B cell progenitors emerged in the human fetal liver (FL) at Carnegie Stage (CS) 17 and were distinct from their cord blood or adult bone marrow counterparts in that the majority expressed surface IL7 receptor. Analysis of IL7R expression also uncovered a CD19-IL7R+ candidate B progenitor compartment. CD19-IL7R+ cells produced CD19+ B cells in vitro, possessed IGH DJ recombination, but also exhibited myeloid (but not megakaryocyte-erythroid) potential. Single cell analysis revealed co-expression of lymphoid and myeloid lineage programs in CD19-IL7R+ progenitors at CS20, whereas at CS17 their transcriptional programs appeared markedly more myeloid, indicating that lymphoid potential is first acquired in CD19-IL7R+ progenitors in this developmental window. Myeloid cells derived from CS20 IL7R progenitors harbored IGH DJ rearrangements, confirming their origins from a RAG-expressing lymphoid progenitor. We tested whether these distinct aspects of embryonic lymphoid development could be modeled using human pluripotent stem cells (hPSCs). In vitro B cell differentiation of hPSCs produced IL7R expressing CD19+ B cells as well as a CD19-IL7R+ progenitor that also “switched” from myeloid to B-myeloid programming during culture. At the global transcriptional level the hPSC-derived lymphoid hierarchy maps closely with that from the embryo, with both separating from the adult, suggesting that hPSCs provide a developmentally relevant model of early fetal B lymphopoiesis. We next used CRIPSR-directed homologous recombination to knock in the cDNA of RUNX1 into intron V of ETV6 resulting in expression of ETV6-RUNX1 under the endogenous ETV6 promoter. ETV6-RUNX1 hPSCs displayed a partial block in B lymphoid differentiation at the CD19-IL7R+ progenitor to proB cell transition and competitive co-culture demonstrated a selective advantage to the ETV6-RUNX1+ CD19-IL7R+ progenitors over their wild type counterparts. Single cell transcriptional analysis showed that the ETV6-RUNX1+ CD19+ B cells that do emerge express an abnormal B-myeloid gene expression signature akin to that seen in the CD19-IL7R+ progenitor. Consistent with this programming, when grown in myeloid cytokines, ETV6-RUNX1+ CD19+ B cells exhibited both a survival advantage and an aberrant ability to form IGH DJ rearranged macrophage colonies. Both the transcriptional and functional phenotypes were dependent on ETV6-RUNX1, as demonstrated by their reversion upon cre-mediated excision of the knock-in cassette. Our data support a model whereby ETV6-RUNX1 inhibits effective resolution of a lympho-myeloid program present in early fetal CD19-IL7R+ progenitors, impeding B lineage differentiation and resulting in the production of B cells with aberrant myeloid gene expression and potential. Such programming may explain the relatively high levels of myeloid antigen expression and lineage promiscuity seen in ETV6-RUNX1 and other cALLs. This hPSC model of ETV6-RUNX1 pre-leukemia will afford systematic evaluation of the contribution of second hit mutations seen in cALL and offers a tractable platform for drug screening. In conclusion we propose that a novel IL7R+ lympho-myeloid progenitor in the human embryonic fetal liver is a candidate target cell for in utero pre-leukemic initiation in childhood B-ALL. DisclosuresNo relevant conflicts of interest to declare.

FIRST REPORT OF THE ANTICANCER EFFICIENCY OF AGARICUS BRASILIENSIS MUSHROOM ON HUMAN EMBRYONIC LIVER WRL68 AND HUMAN PANCREATIC ASPC-1 CANCER CELLS THERAPY

Mushrooms are food traditionally consumed in Asia, Europe and America. They are being studied for medicinal benefits. Extensive studies have shown that Agaricus brasiliensis mushroom used as a medical productto combat cancers. Our data reveal that the determined inhibitory concentration fifty(IC50) values were observed maximum dose responses (IC50) of WRL68 andAsPC-1 cancer cells reported of 172.6 µg/ml and 158.2 µg/ml respectively at 2.23 and 2.1 µg/ml ethanolic mushroom concentrations. The highly cytotoxic activity of the extract on growth inhibition AsPC-1 and WRL68were generally observed 97.9% and 95% at extract concentrations of 25 µg/ml and 50µg/ml respectively. Finally, Phytochemical profile of Agaricus brasiliensis mushroom extract found to beflavonoids, glycosides, saponins, phenols,alkaloids, tannins.The extracts of Agaricus brasiliensiswas tested throughGas Chromatography-Mass(GC-MS). There were five different compounds analyzed from the extracts of Agaricus brasiliensis. The compounds in the ethanolic extract of A. brasiliensis mushroom were comprised mostly of Acetic acid ethyl ester(38.39%),followed by(3-Methyl-oxiran-2-yl)-methanol (34.71%), Chlorbromuron (22.86%), n-Hexadecanoic acid (3.85%) and Heptane, 1-(1-butenyloxy) (0.19%). However, no studies were done using Agaricus brasiliensismushroom extract against WRL68 and AsPC-1 human cell lines. Therefore, the anticancer efficiency of Agaricus brasiliensis mushroom on human embryonic liver WRL68 andhuman pancreatic AsPC-1 cancer cellstherapy is being reported for the first time in the current study.

Synthesis and Cytotoxic Activity of Novel Indole Derivatives and Their in silico Screening on Spike Glycoprotein of SARS-CoV-2.

This work investigated the interaction of indole with SARS-CoV-2. Indole is widely used as a medical material owing to its astounding biological activities. Indole and its derivatives belong to a significant category of heterocyclic compounds that have been used as a crucial component for several syntheses of medicine. A straightforward one-pot three-component synthesis of indole, coupled with Mannich base derivatives 1a–1j, was synthesized without a catalyst. The products were confirmed by IR, 1H-NMR, 13C-NMR, mass spectra, and elemental analysis. The indole derivatives were tested for cytotoxic activity, using three cancer cell lines and normal cell lines of Human embryonic kidney cell (HEK293), liver cell (LO2), and lung cell (MRC5) by MTT assay using doxorubicin as the standard drug. The result of cytotoxicity indole compound 1c (HepG2, LC50−0.9 μm, MCF−7, LC50−0.55 μm, HeLa, LC50−0.50 μm) was found to have high activity compared with other compounds used for the same purpose. The synthesized derivatives have revealed their safety by exhibiting significantly less cytotoxicity against the normal cell line (HEK-293), (LO2), and (MRC5) with IC50 > 100 μg/ml. Besides, we report an in silico study with spike glycoprotein (SARS-CoV-2-S). The selective molecules of compound 1c exhibited the highest docking score −2.808 (kcal/mol) compared to other compounds. This research work was successful in synthesizing a few compounds with potential as anticancer agents. Furthermore, we have tried to emphasize the anticipated role of indole scaffolds in designing and discovering the much-awaited anti-SARS CoV-2 therapy by exploring the research articles depicting indole moieties as targeting SARS CoV-2 coronavirus.

The Novel Target of Liver Cancer: MicroRNA-4324 Regulates Cell Proliferation and Migration via Targeting Neuraminidase 3

Background: MicroRNA-4324 has been reported to regulate various biological malignant cancer. Nonetheless, the expression and molecular mechanism of miR-4324 in liver cancer remain rarely known. This study aimed to investigate the effect miR-4324 on the proliferation, invasion and migration of hepatoma cells. Methods: The mRNA level of miR-4324 was assessed in four hepatoma cell lines (HepG2, Huh7, MHCC97, HB611) and human embryonic liver cell, HHL5. MiR-4324 was over-expressed in hepatoma cells. Subsequently, the effects of miR-4324 on cell proliferation, migration and invasion and the underlying molecular mechanisms were detected. Results: Our data indicated that miR-4324 was down-regulation in hepatoma cell lines compared with HHL5. Overexpression of miR-4324 inhibits cellular proliferation, colony-formation, migration and invasion abilities of hepatoma cells. However, the biological effects of miR-4324 overexpression on hepatoma cells were reversed after overexpressing NEU3. Conclusions: Our findings concluded that miR-4324 inhibits biological functions of hepatoma cells by targeting NEU3 and it might be a potential target for the treatment of liver cancer.

In vitro cytotoxicity of Reishi mushroom extract against two human cell lines

Reishi mushroom Ganoderma lucidum is commonly used in conventional medicine due to its high medicinal properties. Phytochemical screening of G. lucidum mushroom extract found to be flavonoids, glycosides, terpenoids, phenols, Carbohydrates. The current study was applied to demonstrate the In vitro cytotoxicity of ethanolic extract of G. lucidum mushroom against two human cell lines breast cancer cell line MCF-7 and human embryonic liver cell line WRL-68. The measured inhibitory concentration fifty(IC50) values were observed maximum dose responses (IC50) of WRL68 and MCF-7 at 2.0 µg/ml ethanolic mushroom concentration reported of 210.2% and 185.1% compared to the dose-response (IC50) of antidrug doxorubicin (control) at 2.0µg/ml ethanolic mushroom concentration counted to be 18.68. The highly cytotoxic activity of the extract on cell viability MCF-7 and WRL-68 were generally observed 100% and 97% at extract concentrations of 12.5 µg/ml and 25µg/ml respectively compared to antidrug was determined to be 94% cell viability at 6.25 µg/ml concentration.

Recapitulation of Human Embryonic Heartbeat to Promote Differentiation of Hepatic Endoderm to Hepatoblasts.

Hepatic development requires multiple sequential physicochemical environmental changes in an embryo, and human pluripotent stem cells (hPSCs) allow for the elucidation of this embryonic developmental process. However, the current in vitro methods for hPSC-hepatic differentiation, which employ various biochemical substances, produce hPSC-derived hepatocytes with less functionality than primary hepatocytes, due to a lack of physical stimuli, such as heart beating. Here, we developed a microfluidic platform that recapitulates the beating of a human embryonic heart to improve the functionality of hepatoblasts derived from hepatic endoderm (HE) in vitro. This microfluidic platform facilitates the application of multiple mechanical stretching forces, to mimic heart beating, to cultured hepatic endoderm cells to identify the optimal stimuli. Results show that stimulated HE-derived hepatoblasts increased cytochrome P450 3A (CYP3A) metabolic activity, as well as the expression of hepatoblast functional markers (albumin, cytokeratin 19 and CYP3A7), compared to unstimulated hepatoblasts. This approach of hepatic differentiation from hPSCs with the application of mechanical stimuli will facilitate improved methods for studying human embryonic liver development, as well as accurate pharmacological testing with functional liver cells.

Thunbergia laurifolia Leaf Extract Increased Levels of Antioxidant Enzymes and Protected Human Cell-Lines In Vitro Against Cadmium.

Thunbergia laurifolia or Rang Jued has been used as an herbal tea and in folk medicine as a detoxifying agent. Cd contamination is globally widespread and a serious public health problem. The aim of this study was to determine the endogenous antioxidant enzyme activities and malondialdehyde (MDA) production of the crude dried extract (CDE) of T. laurifolia leaves, using human embryonic kidney (HEK293) and human liver (HepG2) cells as in vitro models. Moreover, the cytotoxicity including anti-cadmium (Cd) toxicity in both cells were measured. The experimental design had 3 treatment groups with combined, pre-, and post-treatments for investigating the anti-Cd toxicity, and cell viability was determined with MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The CDE showed low cytotoxicity and increased catalase (CAT) and glutathione peroxidase (GPx) activities with decreased malondialdehyde (MDA) levels in both cell types. It was found that the CDE protected against Cd-induced toxicity in both cell types, and a synergistic combination therapy effect was seen when CaNa2EDTA, a chelating agent, was applied. Therefore, CDE can protect against Cd-induced oxidative stress in cells, possibly due to its antioxidant properties. Moreover, using the extract or drinking the herbal tea together with chelating agent should have an efficacy advantage over using the CDE or the chelating agent singly.

The phosphatidylethanolamine biosynthesis pathway provides a new target for cancer chemotherapy

Since human induced pluripotent stem cells (iPSCs) develop into hepatic organoids through stages thatresemble human embryonic liver development, they can be used to study developmental processes and disease pathology. Therefore, we examined the early stages of hepatic organoid formation to identify key pathways affecting early liver development. Single-cell RNA-sequencing and metabolomic analysis was performed on developing organoid cultures at the iPSC, hepatoblast (day 9) and mature organoid stage. The importance of the phosphatidylethanolamine biosynthesis pathway to early liver development was examined in developing organoid cultures using iPSC with a CRISPR-mediated gene knockout and an over the counter medication (meclizine) that inhibits the rate-limiting enzyme in this pathway. Meclizine's effect on the growth of a human hepatocarcinoma cell line in a xenotransplantation model and on the growth of acute myeloid leukemia cells invitro was also examined. Transcriptomic and metabolomic analysis of organoid development indicated that the phosphatidylethanolamine biosynthesis pathway is essential for early liver development. Unexpectedly, early hepatoblasts were selectively sensitive to the cytotoxic effect of meclizine. We demonstrate that meclizine could be repurposed for use in a new synergistic combination therapy for primary liver cancer: a glycolysis inhibitor reprograms cancer cell metabolism to make it susceptible to the cytotoxic effect of meclizine. This combination inhibited the growth of a human liver carcinoma cell line invitro and in a xenotransplantation model, without causing significant side effects. This drug combination was also highly active against acute myeloid leukemia cells. Our data indicate that phosphatidylethanolamine biosynthesis is a targetable pathway for cancer; meclizine may have clinical efficacy as a repurposed anti-cancer drug when used as part of a new combination therapy. The early stages of human liver development were modeled using human hepatic organoids. We identified a pathway that was essential for early liver development. Based upon this finding, a novel combination drug therapy was identified that could be used to treat primary liver cancer and possibly other types of cancer.