A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged—Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)—demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

Autophagy Overactivity Regulate Terf1 and Terf2 in Positive and Negative-Telomerase Cancer Cell Lines

A recent suggestion for cancer therapy is targeting intracellular homeostatic signaling pathways like autophagy providing the balance between metabolism and cell cycling. Our study focused on investigating the relationship between autophagy activation by Beclin1 transfection and assessing Terf1 and Terf2 expression as shelterin proteins. The beclin1-containing plasmid was introduced to the U-2OS and Huh7 cell lines using Lipofectamine. The LC3-II as an intracellular autophagosomal marker was detected in transfected cells by flow cytometry. Also, the cells were treated with 3-methyladenine and metformin as autophagy inhibitors and inducers, respectively. Finally, the expression levels of Terf1 and Terf2 were analyzed by real-time PCR. Fluorescent images and flow cytometry results proved excellent GFP expression in the transfected cells. The results of real-time PCR demonstrated that autophagy induction by Beclin1 was increased Terf1 expression level in U-2OS cells up to 451%, while Huh7 cells suffered from the decreased expression of Terf1. Altogether, Terf2 expression was enhanced significantly in both cell lines after 48h treatment in comparison with 24h treatment. The obtained data provided that Beclin1-based activation of autophagy leads to overexpression of some protective shelterin proteins.

Energy metabolism adaptations and gene expression reprogramming in a cellular MAFLD model

Mitochondrial dysfunction plays a critical role in metabolic associated fatty liver disease (MAFLD). This study aims to characterize mitochondrial dysfunctions in a human MAFLD Huh7 cell model triggered by free fatty acid (FFA) (palmitate and oleate) overload for 24 hours. We investigate its impact on cellular energy metabolism and identify potential targets for MAFLD treatment. FFA-treated cells displayed an accumulation of lipid droplets and slightly decreased viability but no significant changes in mitochondrial superoxide levels. Bioenergetic analysis showed a shift to more respiration and less glycolytic fermentation. Comprehensive transcriptomics and proteomics analyses identified changes in the expression of genes prominently involved in fatty acid handling and metabolism. The expressions of seven genes were consistently and significantly (p<0.05) altered (4 upregulated and 3 downregulated genes) in both proteomics and transcriptomics. The FFA-treated Huh7 cell model is an appropriate in vitro model to study fatty acid metabolism and suitable to investigate the role of mitochondria, glycolysis, and multiple metabolic pathways in MAFLD. Our comprehensive analyses form a basis for drug discovery and screening using this model.

Ultrastructural Analysis of Apoptosis Induced By Zinc Oxide Nanosphere in Human Hepatoma Huh7 Through Mitochondria Dysfunction

The efficiently of hepatocellular carcinoma therapy predominantly depends on advancement of nanomedicine. Zinc oxide nanostructure is promising material in nanomdicine field due to their unique properties. Zinc oxide Nanosphere (ZnO Ns) was fabricated with a size 50 nm diameter by a developed Sol-gel approach, using non-toxic biotemplate yeast extract. The physicochemical properties of zinc oxide nanosphere were estimated by Fourier-transform infrared spectrum (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and transmission electron microscope (TEM). Liberated zinc ions released from the zinc oxide nanosphere suspended medium was determined by ICP-AS. The viability was tested by using HuH7 and Vero cells.ZnO nanosphere was more effectively on cell line than released Zn ions. The cell cycle arrested at G1/S. Also, the apoptosis assay by using Annexin-V/PI showed that apoptosis of HuH7 by ZnO nanosphere is concentration and time-dependent. The mechanism of apoptosis was evaluated via using techniques such as RT-PCR and flow cytometry. The results revealed significance up-regulated of Bax, P53, and Cytochrome C, while a Bcl2 results displayed significance down-regulated.Caspase 3 assay results showed that the apoptosis mechanism was intrinsic and extrinsic pathways. Also, ZnO nanosphere and free Zn+2 ions induced oxidative stress via increasing reactive oxygen species (ROS) and lipid peroxidation. The ultrastructural analysis of HuH7 cell was occurred by TEM.Transmission electron microscope analysis of HuH7 after treated with ZnO nanosphere at different times revealed a chromatin condensation of the nuclear periphery fragmentation appearance. Interestingly, the apoptosis of HuH7 cells induced by Zinc oxide nanosphere, showed the canonical ultrastructure features of apoptotic nuclei, and fragmented by budding. Furthermore, There were many vacuoles that filled in the cytoplasm, majority lipid droplets, which resembling those observed in foamy cells, and vesicles with intact membranes, which were recognized as swollen mitochondria.

Knockdown of MUC16 (CA125) Enhances the Migration and Invasion of Hepatocellular Carcinoma Cells

As an important global medical problem, hepatocellular carcinoma (HCC) has been recognized as the most frequent primary liver cancer and a leading cause of death among patients with cirrhosis. Surveillance of HCC using serum markers aims to reduce the disease-related mortality of HCC. MUC16 (mucin 16, also known as carbohydrate antigen 125, CA125) has been predicted as a tumor biomarker for many cancer types. Based on the high frequency mutation rate in a database from the Cancer Genome Atlas (TCGA), we investigated the effects of MUC16 knockdown and the regulatory profile of MUC16 in HepG2 and Huh7 cell lines. Knockdown of MUC16 was conducted via siRNA transfection, and the proliferation of cells was not affected by CCK8 assay results. Moreover, decreasing the expression of MUC16 enhanced the migration and invasion of cells, as shown by wound healing and transwell assays. Furthermore, RNA-seq was used to investigate the effect of MUC16 knockdown on the gene expression profile of HepG2 and Huh7 cells. Our study demonstrated the significant role of MUC16 in the inhibition of the migration and invasion of HepG2 and Huh7 cells.

Evaluation of pro-apoptotic potential of taxifolin against liver cancer

Liver cancer is the second most common cause of cancer-induced deaths worldwide. Liver cirrhosis and cancer are a consequence of the abnormal angio-architecture formation of liver and formation of new blood vessels. This angiogenesis is driven by overexpression of hypoxia-inducible factor 1-alpha (Hif1-α) and vascular endothelial growth factor (VEGF). Apart from this, protein kinase B (Akt) is also impaired in liver cancer. Despite the advancement in conventional treatments, liver cancer remains largely incurable. Nowadays, the use of naturally occurring anticancer agents particularly flavonoids is subject to more attention due to their enhanced physicochemical properties. Therefore, this study underlines the use of a natural anticancer agent taxifolin in the treatment of liver cancer using hepatocellular carcinoma cell line HepG2 and Huh7. The aim of our study is to devise a natural and efficient solution for the disease prevalent in Pakistan. The study involved the assessment of binding of ligand taxifolin using molecular docking. The binding of taxifolin with the proteins (Hif1-α, VEGF and Akt) was calculated by docking using Vina and Chimera. Further evaluation was performed by cell viability assay (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay), colony formation assay, cell migration assay, DNA ladder assay and flow cytometry. To see whether taxifolin directly affected expression levels, analysis of gene expression of Hif1-α, VEGF and Akt was performed using real-time polymerase chain reaction (qPCR) and western blotting. In silico docking experiments revealed that these proteins showed favorable docking scores with taxifolin. Treatment with taxifolin resulted in the inhibition of the liver cancer growth and migration, and induced apoptosis in HepG2 and Huh7 cell lines at an inhibitory concentration (IC50) value of 0.15 µM and 0.22 µM, respectively. The expression of HIF1-α, VEGF and Akt was significantly reduced in a dose- dependent manner. The inhibitory effect of taxifolin on hepatic cells suggested its chemopreventive and therapeutic potential. The studied compound taxifolin exhibited pronounced pro-apoptotic and hepatoprotective potential. Our study has confirmed the pro-apoptotic potential of taxifolin in liver cancer cell lines and will pave a way to the use of taxifolin as a chemotherapeutic agent after its further validation on the animal models and humans based epidemiological studies.

In Vitro Inhibitory Analysis of Rationally Designed siRNAs against MERS-CoV Replication in Huh7 Cells

MERS-CoV was identified for the first time in Jeddah, Saudi Arabia in 2012 in a hospitalized patient. This virus subsequently spread to 27 countries with a total of 939 deaths and 2586 confirmed cases and now has become a serious concern globally. Camels are well known for the transmission of the virus to the human population. In this report, we have discussed the prediction, designing, and evaluation of potential siRNA targeting the ORF1ab gene for the inhibition of MERS-CoV replication. The online software, siDirect 2.0 was used to predict and design the siRNAs, their secondary structure and their target accessibility. ORF1ab gene folding was performed by RNAxs and RNAfold software. A total of twenty-one siRNAs were selected from 462 siRNAs according to their scoring and specificity. siRNAs were evaluated in vitro for their cytotoxicity and antiviral efficacy in Huh7 cell line. No significant cytotoxicity was observed for all siRNAs in Huh7 cells. The in vitro study showed the inhibition of viral replication by three siRNAs. The data generated in this study provide preliminary and encouraging information to evaluate the siRNAs separately as well as in combination against MERS-CoV replication in other cell lines. The prediction of siRNAs using online software resulted in the filtration and selection of potential siRNAs with high accuracy and strength. This computational approach resulted in three effective siRNAs that can be taken further to in vivo animal studies and can be used to develop safe and effective antiviral therapies for other prevalent disease-causing viruses.

NaCT (SLC13A5) facilitates citrate import and metabolism under nutrient-limited conditions

Citrate lies at a critical node of metabolism linking tricarboxylic acid metabolism and fatty acid synthesis via acetyl-coenzyme A. Recent studies have linked the sodium citrate transporter (NaCT), encoded by SLC13A5, to dysregulated hepatic metabolism and pediatric epilepsy. To examine how NaCT-mediated citrate metabolism contributes to the pathophysiology of these diseases we applied 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cell lines and primary rat cortical neurons. Exogenous citrate contributed to intermediary metabolism at appreciable levels only under hypoxic conditions. In the absence of glutamine, citrate supplementation increased de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromised citrate uptake and catabolism. Citrate supplementation rescued Huh7 cell viability in response to glutamine deprivation and Zn2+ treatment, and these effects were mitigated by NaCT deficiency. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient limited conditions and may facilitate resistance to metal toxicity.

Antiplasmodial and cytotoxic activities of two Euphorbiaceae family plants, Euphorbia hirta and Euphorbia thymifolia

Background: Malaria is a major disease of developing countries and the main issue in its control of resistance against all available antimalarial drugs. Our aim is to find affordable therapy for this worldwide problem. The present study was conducted to evaluate the antiplasmodial and cytotoxic activities of Euphorbia hirta and Euphorbia thymifolia. Methods: The antiplasmodial activity was evaluated against chloroquine-sensitive (MRC-2) and chloroquine-resistant (RKL-9) strain of Plasmodium falciparum by schizont maturation inhibition and Plasmodium lactate dehydrogenase (PfLDH) activity assays. The cytotoxicity was performed by MTT assay on Huh7 liver carcinoma cell line. Results: IC50 for PfLDH activity of E. hirta and E. thymifolia methanol extract against P. faciparum MRC-2 strain was 9.6 and 12.6 µg/mL, respectively and against P. faciparum RKL-9 strain was 10.2 and 11.56 µg/mL, respectively. Methanol extract of E. thymifolia showed lower IC50 (19.3 µg/mL) than methanol extract of E. hirta (24.7 µg/mL) in MTT assay against Huh7 cell line. Conclusion: Both the plant methanol extracts showed a two-fold lower cytotoxic activity against hepatocyte derived carcinoma cell line (Huh7) compared to in vitro inhibitory activity against P. falciparum strains (MRC-2 and RKL-9).

The Effect of 2D and 3D Cell Cultures on E-Cadherin Profile and Drug Resistance in Huh7 Cell

Studies have validated three-dimensional (3D) culture of cells differs significantly from the two-dimensional (2D) model in terms of drug efficacy due to complex mechanisms, including up-regulation of drug efflux, acquisition of stem cell-like properties by the cancer cells, aberrant apoptotic, etc. This study aimed to delineate the change of expression profile of E-cadherin in hepatoma cells Huh7 under the 3D condition and 2D condition. Cells were culture in ultra-low attachment plates to form multicellular 3D spheroids. The expression profile of E-cadherin and drug resistance were compared between 2D monolayers and 3D spheroids. E-cadherin was located at cell boundaries and cell membrane in 3D spheroids of Huh7 cells, however, E-cadherin was expressed on the cytoplasm and nuclei in 2D monolayers. In addition, high expression of E-cadherin and hepatic stem cell markers were found in 3D spheroids, and 3D spheroids showed greater resistance to pharmacological compounds. It seems 3D culture promoted the membrane localization of E-cadherin, and the change of MDR-related markers enhanced resistance in Huh7 3D spheroids. This study increases the knowledge of the effect of 3D arrangement in cancer cells.