Seedless black Vitis vinifera polyphenols suppress hepatocellular carcinoma in vitro and in vivo by targeting apoptosis, cancer stem cells, and proliferation.

Hepatocellular carcinoma (HCC) is an aggressive tumor and one of the most challenging cancers to treat. Here, we evaluated the in vitro and in vivo ameliorating impacts of seedless black Vitis vinifera (VV) polyphenols on HCC. Following the preparation of the VV crude extract (VVCE) from seedless VV (pulp and skin), three fractions (VVF1, VVF2, and VVF3) were prepared. The anticancer potencies of the prepared fractions, compared to 5-FU, were assessed against HepG2 and Huh7 cells. In addition, the effects of these fractions on p-dimethylaminoazobenzene-induced HCC in mice were evaluated. The predicted impacts of selected phenolic constituents of VV fractions on the activity of essential HCC-associated enzymes (NADPH oxidase "NADPH-NOX2", histone deacetylase 1 "HDAC1", and sepiapterin reductase "SepR") were analyzed using molecular docking. The results showed that VVCE and its fractions induced apoptosis and collapsed CD133+ stem cells in the studied cancer cell lines with an efficiency greater than 5-FU. VVF1 and VVF2 exhibited the most effective anticancer fractions in vitro; therefore, we evaluated their influences in mice. VVF1 and VVF2 improved liver morphology and function, induced apoptosis, and lowered the fold expression of various crucial genes that regulate cancer stem cells and other vital pathways for HCC progression. For most of the examined parameters, VVF1 and VVF2 had higher potency than 5-FU, and VVF1 showed more efficiency than VVF2. The selected phenolic compounds displayed competitive inhibitory action on NADPH-NOX2, HDAC1, and SepR. In conclusion, these findings declare that VV polyphenolic fractions, particularly VVF1, could be promising safe anti-HCC agents.

Investigation of prunetrin induced G2/M cell cycle arrest and apoptosis via Akt/mTOR/MAPK pathways in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) stands as a leading cause of mortality, and despite recent advancements in the overall survival rates, the prognosis remains dismal. Prunetin 4-O-glucoside (Prunetrin or PUR), an active compound derived from Prunus sp., was explored for its impact on HepG2 and Huh7 cells. The cytotoxicity assessment revealed a notable reduction in cell viability in both cell lines, while exhibiting non-toxicity towards HaCaT cells. Colony formation studies underscored PUR's inhibitory effect on cell proliferation, dose-dependently. Mechanistically, PUR downregulated cell cycle proteins (CDC25c, Cdk1/CDC2, and Cyclin B1), inducing G2/M phase arrest, corroborated by flow cytometry. Western blot analyses exhibited dose-dependent cleavages of PARP and caspase 3, indicative of apoptosis. Treatment with the apoptotic inhibitor z-vmd-fmk provided evidence of PUR-induced apoptosis. Annexin V and PI flow cytometry further affirmed apoptotic induction. Enhanced expression of cleaved-caspase 9 and the pro-apoptotic protein Bak, coupled with reduced anti-apoptotic Bcl-xL, and affirmed PUR's induction of intrinsic apoptosis. Additionally, PUR activated the MAPK pathway, evidenced by elevated phospho p38 and phospho ERK expressions in both cell lines. Notably, a concentration-dependent decrease in mTOR and Akt expressions indicated PUR's inhibition of the Akt/mTOR pathway in HepG2 and Huh7 cells. These findings illuminate PUR's multifaceted impact, revealing its potential as a promising therapeutic agent against HepG2 and Huh7 cells through modulation of cell cycle, apoptosis, and key signaling pathways.

Hepatocellular carcinoma patients serum modulates the regenerative capacities of adipose mesenchymal stromal cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers causing the highest mortality rate worldwide. Treatment options of surgery, radiation, cytotoxic drugs and liver transplantation suffer significant side effects and a high frequency of relapse. Stem cell therapy has been proposed as a new effective therapy, however, controversial reports are emerging on the role of mesenchymal stem cells in cancer. In this work, we aimed to assess the regenerative capacities of adipose mesenchymal stem cells when exposed to serum from HCC patients, by assessing the effect of the sera on modulating the regenerative capacities of h-AMSCs and the cancer properties in HCC cells. This will pave the way for maximizing the efficacy of MSCs in cancer therapy. Our data show that HCC serum-treated hA-MSCs suffered oncogene-induced senescence as shown by their altered morphology and ameliorated proliferation and differentiation. The cells were enlarged with small irregular nuclei, swollen rough endoplasmic reticulum cisternae, and aging lysosomes typified by dark residual bodies. HCC serum-treated Huh-7 cancer cells on the other hand displayed higher tumor aggressiveness as depicted by altered morphology, increased cellular proliferation and migration, and decreased percentage of early and late apoptotic cells. Our findings provide evidence that exposure of hA-MSCs to the serum of HCC patients decreases their regenerative capacities and should be considered when employed as a potential therapy in HCC patients.

Transcriptome analysis of Huh7 cells upon Chikungunya virus infection and capsid transfection reveals regulation of distinct cellular and metabolic pathways.

Chikungunya virus (CHIKV) causes persistent arthritis and neurological problems imposing a huge burden globally. The present study aims to understand the interaction mechanism of Chikungunya virus and CHIKV-capsid in Huh7 cells. The RNA-sequencing and qRT-PCR method was used for the transcript and gene profiles of CHIKV virus and CHIKV capsid alone. Transcriptional analysis showed capsid induced 1114 and 956 differentially expressed genes (DEGs) to be upregulated and downregulated respectively, while in virus, 933 genes were upregulated and 956 were downregulated. Total 202 DEGs were common in both capsid and virus; and nine were validated using qRT-PCR. Identified DEGs were found to be associated with metabolic pathways such as Diabetes, cardiac disease, and visual impairment. Further, knock-down study on one of the DEGs (MafA) responsible for insulin regulation showed low viral proteins expression suggesting a reduction in virus-infection. Thus, the study provides insight into the interplay of the virus-host factors assisting virus replication.

Comparison of Alternative Splicing Landscapes Revealed by Long-Read Sequencing in Hepatocyte-Derived HepG2 and Huh7 Cultured Cells and Human Liver Tissue.

The long-read RNA sequencing developed by Oxford Nanopore Technologies provides a direct quantification of transcript isoforms, thereby making it possible to present alternative splicing (AS) profiles as arrays of single splice variants with different abundances. Additionally, AS profiles can be presented as arrays of genes characterized by the degree of alternative splicing (the DAS-the number of detected splice variants per gene). Here, we successfully utilized the DAS to reveal biological pathways influenced by the alterations in AS in human liver tissue and the hepatocyte-derived malignant cell lines HepG2 and Huh7, thus employing the mathematical algorithm of gene set enrichment analysis. Furthermore, analysis of the AS profiles as abundances of single splice variants by using the graded tissue specificity index τ provided the selection of the groups of genes expressing particular splice variants specifically in liver tissue, HepG2 cells, and Huh7 cells. The majority of these splice variants were translated into proteins products and appeal to be in focus regarding further insights into the mechanisms underlying cell malignization. The used metrics are intrinsically suitable for transcriptome-wide AS profiling using long-read sequencing.

Replication and Expression of the Consensus Genome of Hepatitis B Virus Genotype C from the Chinese Population.

Hepatitis B virus (HBV) genotype C is a prevalent HBV genotype in the Chinese population. Although genotype C shows higher sequence heterogeneity and more severe liver disease than other genotypes, its pathogenesis and immunological traits are not yet fully elucidated. In this study, we first established and chemically synthesized the consensus sequence based on representative 138 full-length HBV genotype C genomes from the Chinese population. The pHBV1.3C plasmid system, containing a 1.3-fold full-length HBV genotype C consensus sequence, was constructed for subsequent validation. Next, we performed functional assays to investigate the replicative competence of pHBV1.3C in vitro through the transient transfection of HepG2 and Huh7 cells and validated the in vivo function via a hydrodynamic injection to BALB/c recipient mice. The in vitro investigation revealed that the extracellular HBV DNA and intracellular replicative intermediate (i.e., pregenomic RNA, pgRNA) were apparently measurable at 48 h, and the HBsAg and HBcAg were still positive in hepatoma cells at 96 h. We also found that HBsAg and HBeAg accumulated at the extracellular and intracellular levels in a time-dependent manner. The in vivo validation demonstrated that pHBV1.3C plasmids induced HBV viremia, triggered morphological changes and HBsAg- or HBcAg- positivity of hepatocytes, and ultimately caused inflammatory infiltration and focal or piecemeal necrosis in the livers of the murine recipients. HBV protein (HBsAg) colocalized with CD8+ T cells or CD4+ T cells in the liver. F4/80+ Kupffer cells were abundantly recruited around the altered murine hepatocytes. Taken together, our results indicate that the synthetic consensus sequence of HBV genotype C is replication-competent in vitro and in vivo. This genotype C consensus genome supports the full HBV life cycle, which is conducive to studying its pathogenesis and immune response, screening novel antiviral agents, and further optimizing testing and therapeutics.

Excessive N-acetylcysteine exaggerates glutathione redox homeostasis and apoptosis during acetaminophen exposure in Huh-7 human hepatoma cells.

Acetaminophen (APAP) hepatotoxicity is one of the biggest drawbacks of this relatively safe and widely used drug. In addition to its hepatotoxicity, APAP also cause comparable levels of toxicity on human hepatoma cells. Here we show activation of the intrinsic caspase-9/3 pathway of apoptosis followed by gasdermin E (GSDME) cleavage and subsequent ballooning in APAP (10 mM, 72 h)-treated Huh-7 human hepatocarcinoma cells. N-acetylcysteine (NAC), an antioxidant currently used as an antidote for APAP overdose, does not alleviate APAP toxicity in Huh-7 cells; NAC overdose (10 mM) rather aggravates APAP toxicity. NAC overdose not only aggravates cell death, but also decreases the cellular GSH/GSSG ratio, an indicator of redox homeostasis of glutathione. These results show for the first time that APAP-induced apoptosis in hepatoma cells is followed by secondary necrosis via the caspase-3/GSDME pathway. NAC overdose (10 mM) not only worsens the glutathione redox status, but also accelerates this pathway.

Effect of mTOR inhibitors on sodium taurocholate cotransporting polypeptide (NTCP) function in vitro.

The sodium taurocholate cotransporting polypeptide (NTCP; gene name SLC10A1) is the primary hepatic basolateral uptake transporter for conjugated bile acids and the entry receptor for the hepatitis B and D virus (HBV/HDV). Regulation of human NTCP remains a knowledge gap due to significant species differences in substrate and inhibitor selectivity and plasma membrane expression. In the present study, various kinase inhibitors were screened for inhibition of NTCP function and taurocholate (TCA) uptake using NTCP-transfected HuH-7 cells. This study identified everolimus, an mTOR inhibitor and macrocyclic immunosuppressive drug, as an NTCP inhibitor with modest potency (IC50 = 6.7-8.0 µM). Further investigation in differentiated HuH-7 cells expressing NTCP and NTCP-overexpressing Flp-In T-REx 293 cells revealed that the mechanism of action of everolimus on NTCP is direct inhibition and mTOR-independent. Structural analogs of everolimus inhibited NTCP-mediated TCA uptake, however, functional analogs did not affect NTCP-mediated TCA transport, providing further evidence for direct inhibition. This work contributes to the growing body of literature suggesting that NTCP-mediated bile acid uptake may be inhibited by macrocyclic peptides, which may be further exploited to develop novel medications against HBV/HDV.

Stability of Dengue 2 Nonstructural Glycoprotein 1 (NS1) Is Affected by the Nature of Basic Residue at Position NS1-324.

Dengue is the most prevalent mosquito-borne viral disease. It is caused by the infection of any of the four dengue virus (DENV) serotypes DENV-1 to DENV-4. The DENV non-structural glycoprotein 1 (NS1) plays an important role in virus replication and the immunopathogenesis of virus infection. The NS1 protein has been identified as both a cell-associated homodimer and a soluble secreted lipoprotein nanoparticle. The nature of the residues at positions NS1-272 and NS1-324 in the ß-ladder domain may have an effect on the biological behaviors of DENV-2 NS1 protein in human hepatoma Huh7 cells. The stability of the NS1 protein from the Reunion 2018 DENV-2 strain was affected by the presence of lysine residues at positions 272 and 324. In the present study, we evaluated the impact of mutations into lysine at positions 272 and 324 on recombinant NS1 protein from the DES-14 DENV-2 strain bearing arginine residue on these two positions. The DES-14 NS1 protein mutant bearing a lysine at position 324 was deficient in protein stability and secretion compared to wild-type protein. The defect in the DES-14 NS1 protein mutant was associated to oxidative stress and pro-inflammatory cytokine activation in Huh7 cells. The ubiquitin-proteasome proteolytic pathway might play a key role in the stability of DENV-2 protein bearing a lysine residue at position 324.

Lipid-induced alteration in retinoic acid signaling leads to mitochondrial dysfunction in HepG2 and Huh7 cells.

A surfeit of mitochondrial reactive oxygen species (ROS) and inflammation serve as obligatory mediators of lipid-associated hepatocellular maladies. While retinoid homeostasis is essential in restoring systemic energy balance, its role in hepatic mitochondrial function remains elusive. The role of lecithin-retinol acyltransferase (LRAT) in maintenance of retinoid homeostasis is appreciated earlier; however, its role in modulating retinoic acid (RA) bioavailability upon lipid-imposition is unexplored. We identified LRAT overexpression in high-fat diet (HFD)-fed rats and palmitate-treated hepatoma cells. Elevation in LRAT expression depletes RA production and deregulates RA signaling. This altered RA metabolism enhances fat accumulation, accompanied by inflammation that leads to impaired mitochondrial function through enhanced ROS generation. Hence, LRAT inhibition could be a novel approach preventing lipid-induced mitochondrial dysfunction in hepatoma cells.

Induction of apoptosis by emestrin from the plant endophytic fungus Emericella nidulans ATCC 38163 in Huh-7 human hepatocellular carcinoma cells.

This research aimed to investigate the anticancer properties of emestrin, a major constituent of Emericella nidulans ATCC 38163 through the induction of apoptosis in Huh-7 human hepatocellular carcinoma cells. In this study, this fungus was isolated from the fresh leaves of Ruprechtia salicifolia (Cham. & Schltdl.) C.A. Mey, and identified by morphology and 18S rDNA followed by large-scale fermentation in liquid biomalt broth medium. Epidithiodioxopiperazine derivative emestrin along with ten known metabolites were isolated and identified from the fungal extract. The cytotoxic assay revealed that emestrin had the strongest cytotoxicity against Huh-7 and A-549 cells with IC50 values of 4.89 and 6.3 µM, respectively. Using annexin V-FITC assay, treatment of Huh-7 cells with 4.89 µM for 24 h resulted in a significant increase in the percentage of early and late apoptosis (3.16% and 22.84%, respectively) compared to untreated cells. Additionally, Bax and bcl-2 protein levels were regulated, which induced apoptosis in treated cells. These results indicate that emestrin induces mitochondrial pathway to stimulate apoptosis and inhibits cell proliferation in hepatocellular carcinoma.

Computational Design and Experimental Evaluation of MERS-CoV siRNAs in Selected Cell Lines.

Middle East respiratory syndrome coronavirus (MERS-CoV) is caused by a well-known coronavirus first identified in a hospitalized patient in the Kingdom of Saudi Arabia. MERS-CoV is a serious pathogen affecting both human and camel health globally, with camels being known carriers of viruses that spread to humans. In this work, MERS-CoV genomic sequences were retrieved and analyzed by multiple sequence alignment to design and predict siRNAs with online software. The siRNAs were designed from the orf1ab region of the virus genome because of its high sequence conservation and vital role in virus replication. The designed siRNAs were used for experimental evaluation in selected cell lines: Vero cells, HEK-293-T, and Huh-7. Virus inhibition was assessed according to the cycle threshold value during a quantitative real-time polymerase chain reaction. Out of 462 potential siRNAs, we filtered out 21 based on specific selection criteria without off-target effect. The selected siRNAs did not show any cellular toxicity in the tested cell lines at various concentrations. Based on our results, it was obvious that the combined use of siRNAs exhibited a reduction in MERS-CoV replication in the Vero, HEK-293-T, and Huh-7 cell lines, with the highest efficacy displayed in the Vero cells.

Cell-cycle and apoptosis related and proteomics-based signaling pathways of human hepatoma Huh-7 cells treated by three currently used multi-RTK inhibitors.

Sorafenib, lenvatinib and regorafenib, the multi-RTK inhibitors with potent anti-angiogenesis effects, are currently therapeutic drugs generally recommended for the patients with advanced hepatocellular carcinoma (HCC). To date, however, there have been no published studies on the mechanism underling differential effects of the three drugs on HCC cell proliferation, and the proteomic analysis in HCC cell lines treated by regorafenib or lenvatinib. The present study for the first time performed a direct comparison of the cell cycle arrest and apoptosis induction in the Huh-7 cells caused by sorafenib, regorafenib and lenvatinib at respective IC50 using flow cytometry technique, as well as their pharmacological interventions for influencing whole cell proteomics using tandem mass tag-based peptide-labeling coupled with the nLC-HRMS technique. Sorafenib, regorafenib and lenvatinib at respective IC50 drove the remaining surviving Huh-7 cells into a G0/G1 arrest, but lenvatinib and regorafenib were much more effective than sorafenib. Lenvatinib produced a much stronger induction of Huh-7 cells into early apoptosis than sorafenib and regorafenib, while necrotic cell proportion induced by regorafenib was 2.4 times as large as that by lenvatinib. The proteomic study revealed 419 proteins downregulated commonly by the three drugs at respective IC50. KEGG pathway analysis of the downregulated proteins indicated the ranking of top six signaling pathways including the spliceosome, DNA replication, cell cycle, mRNA surveillance, P53 and nucleotide excision repair involved in 33 proteins, all of which were directly related to their pharmacological effects on cell cycle and cell apoptosis. Notably, lenvatinib and regorafenib downregulated the proteins of PCNA, Cyclin B1, BCL-xL, TSP1, BUD31, SF3A1 and Mad2 much more strongly than sorafenib. Moreover, most of the proteins in the P53 signaling pathway were downregulated with lenvatinib and regorafenib by more than 36% at least. In conclusion, lenvatinib and regorafenib have much stronger potency against Huh-7 cell proliferation than sorafenib because of their more potent effects on cell cycle arrest and apoptosis induction. The underling mechanism may be at least due to the 33 downregulated proteins centralizing the signal pathways of cell cycle, p53 and DNA synthesis based on the present proteomics study.

Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein.

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.

A novel nitidine chloride nanoparticle overcomes the stemness of CD133+EPCAM+ Huh7 hepatocellular carcinoma cells for liver cancer therapy.

BACKGROUND: Stemness of CD133+EPCAM+ hepatocellular carcinoma cells ensures cancer resistance to apoptosis,which is a challenge to current liver cancer treatments. In this study, we evaluated the tumorcidal activity of a novel nanoparticle of nitidine chloride (TPGS-FA/NC, TPGS-FA: folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate, NC: nitidine chloride), against human hepatocellular carcinoma (HCC) cell line Huh7 growth in vitro and in vivo. METHODS: Huh7 cells were treated with TPGS-FA/NC. Cell proliferation was assessed using MTT and colony assays. The expression of cell markers and signaling proteins was detected using western blot analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Huh7 cells. TPGS-FA/NC (7.5, 15, 30, 60, 120 µg/mL) dose-dependently inhibited the proliferation of HCC cells, which associated with a reduction in AQP3 and STAT3 expression. Importantly,TPGS-FA/NC (10, 20, and 40 µg/mL) significantly reduced the EpCAM+/CD133+cell numbers, suppressed the sphere formation. The in vivo antitumor efficacy of TPGS-FA/NC was proved in Huh7 cell xenograft model in BALB/c nude mice, which were administered TPGS-FA/NC(4 mg· kg - 1· d - 1, ig) for 2 weeks. RESULTS: TPGS-FA/NC dose-dependently suppressed the AQP3/STAT3/CD133 axis in Huh7 cells. In Huh7 xenograft bearing nude mice, TPGS-FA/NC administration markedly inhibited Huh7 xenograft tumor growth . CONCLUSIONS: TPGS-FA/NC inhibit HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the clinical therapy of hepatocellular carcinoma.

Oncolytic Vaccinia Virus Harboring Aphrocallistes vastus Lectin Inhibits the Growth of Hepatocellular Carcinoma Cells.

Oncolytic vaccinia virus has been developed as a novel cancer therapeutic drug in recent years. Our previous studies demonstrated that the antitumor effect of oncolytic vaccina virus harboring Aphrocallistes vastus lectin (oncoVV-AVL) was significantly enhanced in several cancer cells. In the present study, we investigated the underlying mechanisms of AVL that affect virus replication and promote the antitumor efficacy of oncolytic virus in hepatocellular carcinoma (HCC). Our results showed that oncoVV-AVL markedly exhibited antitumor effects in both hepatocellular carcinoma cell lines and a xenograft mouse model. Further investigation illustrated that oncoVV-AVL could activate tumor immunity by upregulating the expression of type I interferons and enhance virus replication by inhibiting ISRE mediated viral defense response. In addition, we inferred that AVL promoted the ability of virus replication by regulating the PI3K/Akt, MAPK/ERK, and Hippo/MST pathways through cross-talk Raf-1, as well as metabolism-related pathways. These findings provide a novel perspective for the exploitation of marine lectins in oncolytic therapy.

The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes.

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.

Design and synthesis of ludartin derivatives as potential anticancer agents against hepatocellular carcinoma.

Our previous study demonstrated that guaiane-type sesquiterpenoid ludartin showed potent antihepatoma activity against two human hepatocellular carcinoma cell lines, HepG2 and Huh7, with IC50 values of 32.7 and 34.3 µM, respectively. In this study, 34 ludartin derivatives were designed, synthesized and evaluated for their cytotoxic activities against HepG2 and Huh7 cell lines using an MTT assay in vitro. As a result, 17 compounds increased the activity against HepG2 cells, and 20 compounds enhanced the activity against Huh7 cells; 14 derivatives 2, 4-7, 9, 11, 17, 24, 28-30 and 32-33 were superior to ludartin on both HepG2 and Huh7 cells. In particular, dimeric derivative 33 as the most active compound showed 20-fold and 17-fold enhancement of cytotoxicity against HepG2 and Huh7 cells compared to that of ludartin. These results suggested that compound 33 could serve as a promising lead compound against liver cancer. Graphical abstract.

Discovery of 5,7-Dimethoxy-2-(3,4,5-trimethoxyphenoxy)-chromen-4-one with Lipid Lowering Effects in Hepatocytes.

The population with nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is increasing. However, no medicine is indicated for treating these diseases clinically nowadays. Therefore, there is an urgent need to develop a new drug to overcome NAFLD and NASH. Capillarisin, a 2-phenoxychromone originating from Artemisia capillaris Thunb., is well-known for its liver-protective effects. As a result, a series of 2-phenoxychromones was prepared and evaluated for its protective activity against lipid droplet formation in oleic acid (OA)-treated Huh7 cells by means of high-content screening. In the light of the results, the compounds with trimethoxy groups on the phenyl ring possessed better inhibitory properties against lipid accumulation in Huh7 cells, compared to other functional groups on the same ring. Nonetheless, the compounds with a hydroxy group at the C-5 position of the chromone exhibited apparent cytotoxicity. Finally, the active compound, 5,7-dimethoxy-2-(3,4,5-trimethoxyphenoxy)-chromen-4-one (7e), with an IC50 value of 32.2 ± 2.1 µM against lipid accumulation and no significant cytotoxicity, reduced the accumulation of lipid droplets by up-regulating peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) to facilitate the catabolism of fat, which shows promise for further optimization to manage NAFLD and NASH.

Matrine Combined with Mammalian Target of Rapamycin Inhibitor Enhances Anti-Tumor Efficacy of dendritic cell Vaccines in hepatocellular carcinoma.

Dendritic cells (DCs), as the most important antigen-presenting cells, play a crucial role in T cell activation. The latest research showed that inhibition of the mammalian target of rapamycin (mTOR) could enhance DCs maturation, promoting antigen presentation. Matrine has been identified as one of the key alkaloids isolated from the roots of Sophora flavescens. In present study, we combined matrine and mTOR inhibitor KU0063794 to observe the DCs functions, especially the antigen presentation ability. DCs were activated by phosphate-buffered saline (PBS), lipopolysaccharide (LPS), LPS+KU0063794, LPS+Matrine, and LPS+KU0063794+Matrine. The surface markers in DCs, proliferation of T cells and cytokines were detected by flow cytometry, cell counting kit-8 (CCK-8) and enzyme-linked immunosorbent assay (ELISA), respectively. The lactate dehydrogenase (LDH) release test was used to detect the antitumor efficacy. The tumor growth curves were plotted by calculating tumor volume. The apoptosis was detected by Terminal-deoxynucleoitidyl Transferase-Mediated Nick End Labeling (TUNEL) method. Matrine combined with KU0063794 could enhance the maturity of DCs, T cells proliferation and cytokines secretion (P < 0.05). The cytotoxic T lymphocytes (CTL) killing efficacy of LPS+KU0063794+Matrine group was higher than other groups (P < 0.05). In vivo, the tumor weights and volumes in LPS+KU0063794+Matrine group were lower than other groups. The detections of tumor apoptosis were increased in LPS+KU0063794+Matrine group (P < 0.05). DC vaccine with mTOR inhibitor and matrine could significantly improve the efficacy of antitumor immunity in vitro and vivo. These findings illustrated that mTOR inhibitor and matrine, as two immunomodulators, could enhance DC activation and differentiation.