Inhibition of biofilm formation, quorum sensing and virulence factor production in Pseudomonas aeruginosa PAO1 by selected LasR inhibitors / Inhibición de la formación de biopelículas, detección de quórum y producción de factores de virulencia en Pseudomonas aeruginosa PAO1 mediante inhibidores de LasR seleccionados

The quorum sensing network of Pseudomonas aeruginosa mediates the regulation of genes controlling biofilm formation and virulence factors. The rise of drug resistance to Pseudomonas aeruginosa infections has made quorum sensing–regulated biofilm formation in clinical settings a major issue. In the present study, LasR inhibitors identified in our previous study were evaluated for their antibiofilm and antiquorum sensing activities against P. aeruginosa PAO1. The compounds selected were (3-[2-(3,4-dimethoxyphenyl)-2-(1H-indol-3-yl)ethyl]-1-(2-fluorophenyl)urea) (C1), (3-(4-fluorophenyl)-2-[(3-methylquinoxalin-2-yl)methylsulfanyl]quinazolin-4-one) (C2) and (2-({4-[4-(2-methoxyphenyl)piperazin-1-yl]pyrimidin-2-yl}sulfanyl)-N-(2,4,6-trimethylphenyl)acetamide) (C3). The minimum inhibitory concentrations of C1 and C2 were 1000 μM, whereas that of C3 was 500 μM. At sub-MICs, the compounds showed potent antibiofilm activity without affecting the growth of P. aeruginosa PAO1. Electron microscopy confirmed the disruption of biofilm by the selected compounds. The antiquorum sensing activity of the compounds was revealed by the inhibition of violacein in Chromobacterium violaceum and the inhibition of swimming and swarming motilities in P. aeruginosa PAO1. Furthermore, the compounds also attenuated the production of quorum sensing–mediated virulence factors. The qRT-PCR revealed the downregulation of quorum sensing regulatory genes, namely lasI, lasR, rhlI, rhlR, lasB, pqsA and pqsR. The selected compounds also exhibited lower cytotoxicity against peripheral blood lymphocytes. Thus, this study could pave a way to explore these compounds for the development of therapeutic agent against Pseudomonas aeruginosa biofilm–related infections.(AU)

Inhibition of biofilm formation, quorum sensing and virulence factor production in Pseudomonas aeruginosa PAO1 by selected LasR inhibitors.

The quorum sensing network of Pseudomonas aeruginosa mediates the regulation of genes controlling biofilm formation and virulence factors. The rise of drug resistance to Pseudomonas aeruginosa infections has made quorum sensing-regulated biofilm formation in clinical settings a major issue. In the present study, LasR inhibitors identified in our previous study were evaluated for their antibiofilm and antiquorum sensing activities against P. aeruginosa PAO1. The compounds selected were (3-[2-(3,4-dimethoxyphenyl)-2-(1H-indol-3-yl)ethyl]-1-(2-fluorophenyl)urea) (C1), (3-(4-fluorophenyl)-2-[(3-methylquinoxalin-2-yl)methylsulfanyl]quinazolin-4-one) (C2) and (2-({4-[4-(2-methoxyphenyl)piperazin-1-yl]pyrimidin-2-yl}sulfanyl)-N-(2,4,6-trimethylphenyl)acetamide) (C3). The minimum inhibitory concentrations of C1 and C2 were 1000 µM, whereas that of C3 was 500 µM. At sub-MICs, the compounds showed potent antibiofilm activity without affecting the growth of P. aeruginosa PAO1. Electron microscopy confirmed the disruption of biofilm by the selected compounds. The antiquorum sensing activity of the compounds was revealed by the inhibition of violacein in Chromobacterium violaceum and the inhibition of swimming and swarming motilities in P. aeruginosa PAO1. Furthermore, the compounds also attenuated the production of quorum sensing-mediated virulence factors. The qRT-PCR revealed the downregulation of quorum sensing regulatory genes, namely lasI, lasR, rhlI, rhlR, lasB, pqsA and pqsR. The selected compounds also exhibited lower cytotoxicity against peripheral blood lymphocytes. Thus, this study could pave a way to explore these compounds for the development of therapeutic agent against Pseudomonas aeruginosa biofilm-related infections.

Cancer Resistance to Immunotherapy: Molecular Mechanisms and Tackling Strategies.

Cancer immunotherapy has fundamentally altered cancer treatment; however, its efficacy is limited to a subset of patients in most clinical settings. The immune system plays a key role in cancer progression from tumor initiation to the metastatic state. Throughout the treatment course, communications between the immune cells in the tumor microenvironment and the immune macroenvironment, as well as interactions between the immune system and cancer cells, are dynamic and constantly evolving. To improve the clinical benefit for patients who do not respond completely to immunotherapy, the molecular mechanisms of resistance to immunotherapy must be elucidated in order to develop effective strategies to overcome resistance. In an attempt to improve and update the current understanding of the molecular mechanisms that hinder immunotherapy, we discuss the molecular mechanisms of cancer resistance to immunotherapy and the available treatment strategies.

Apigetrin Promotes TNFα-Induced Apoptosis, Necroptosis, G2/M Phase Cell Cycle Arrest, and ROS Generation through Inhibition of NF-κB Pathway in Hep3B Liver Cancer Cells.

Apigetrin (7-(ß-D-glucopyranosyloxy)-4',5-dihydroxyflavone), a glycoside bioactive dietary flavonoid derived from Taraxacum officinale and Teucrium gnaphalodes, is known to possess anticancer, antioxidant, and anti-inflammatory effects on numerous cancers. In the present study, we examined the effect of apigetrin in Hep3B hepatocellular cancer cell line (HCC). Apigetrin inhibited cell growth and proliferation of Hep3B cells, as confirmed by MTT and colony formation assay. We used apigetrin at concentrations of 0, 50, and 100 µM for later experiments. Of these concentrations, 100 µM of apigetrin showed a significant effect on cell inhibition. In apigetrin-treated Hep3B cells, cell cycle arrest occurred at the G2/M phase. Apoptosis and necroptosis of Hep3B cells treated with apigetrin were confirmed by Annexin V/propidium iodide (PI) staining and flow cytometry results. Morphological observation through 4',6-diamidino-2-phenylindole (DAPI) staining showed intense blue fluorescence representing chromatin condensation. Hematoxylin staining showed necroptotic features such as formation of vacuoles and swelling of organelles. Apigetrin increased reactive oxygen species (ROS) levels in cells, based on fluorescence imaging. Furthermore, the underlying mechanism involved in the apoptosis and necroptosis was elucidated through western blotting. Apigetrin up-regulated TNFα, but down-regulated phosphorylation of p-p65, and IκB. Apigetrin inhibited the expression of Bcl-xl but increased Bax levels. Up-regulation of cleaved PARP and cleaved caspase 3 confirmed the induction of apoptosis in apigetrin-treated Hep3B cells. Additionally, necroptosis markers RIP3, p-RIP3, and p-MLKL were significantly elevated by apigetrin dose-dependently, suggesting necroptotic cell death. Taken together, our findings strongly imply that apigetrin can induce apoptosis and necroptosis of Hep3B hepatocellular cancer cells. Thus, apigetrin as a natural compound might have potential for treating liver cancer.

Investigation on the cellular mechanism of Prunetin evidenced through next generation sequencing and bioinformatic approaches against gastric cancer.

Gastric cancer is the common type of malignancy positioned at second in mortality rate causing burden worldwide with increasing treatment options. More accurate and reliable diagnostic methods/biomarkers are urgently needed. The application of transcriptomics technologies possesses the high efficiency of identifying key metabolic pathways and functional genes in cancer research. In this study, we performed a transcriptome analysis on Prunetin treated AGS cells. A total of 1,118 differentially expressed (DE) genes on Prunetin treated AGS cancer cells, among which 463 were up-regulated and 655 were down-regulated. Notably, around 40 genes were found to be related with necroptosis, among which 16 genes were found to be in close association with Receptor Interacting Protein Kinase (RIPK) family. Validation of the RIPK genes through GEPIA identified 8 genes (NRP1, MNX1, SSRP1, PRDX2, PLRG1, LGALS4, SNX5 and FXYD3) which are highly expressed in stomach cancer were significantly down-regulated in PRU treated samples. In conclusion, the sequencing data explores the expression of RIPK mediated genes through necroptosis signaling network in treating gastric cancer. The futuristic validations on the 8 genes as candidate biomarkers will offer a treatment approach against gastric cancer using PRU.

Structural and Functional Properties of Activator Protein-1 in Cancer and Inflammation.

The transcriptional machinery is composed of numerous factors that help to regulate gene expression in cells. The function and the fundamental role of transcription factors in different human diseases and cancer have been extensively researched. Activator protein-1 (AP-1) is an inducible transcription factor that consists of a diverse group of members including Jun, Fos, Maf, and ATF. AP-1 involves a number of processes such as proliferation, migration, and survival in cells. Dysfunctional AP-1 activity is seen in several diseases, especially cancer and inflammatory disorders. The AP-1 proteins are controlled by mitogen-activated protein kinases (MAPKs) and the NF-κB pathway. AP-1 inhibitors can be actively pursued as drug discovery targets in cancer therapy when used as a treatment to halt tumor progression. The consumption of phytochemicals in the diet is related to decreasing the incidence of cancer and proves to exhibit anticancer properties. Natural product targets AP-1 are effective cancer prevention and treatment options for various cancer types. Targeting AP-1 with natural products is an effective cancer treatment option for different cancer types. This review summarizes AP-1 subunit proteins, their structures, AP-1-related signaling, and its modulation by natural bioactive compounds.

Prunetinoside Inhibits Lipopolysaccharide-Provoked Inflammatory Response via Suppressing NF-κB and Activating the JNK-Mediated Signaling Pathway in RAW264.7 Macrophage Cells.

Inflammation is a multifaceted response of the immune system at the site of injury or infection caused by pathogens or stress via immune cells. Due to the adverse effects of chemical drugs, plant-based compounds are gaining interest in current research. Prunetinoside or prunetin-5-O-glucoside (PUG) is a plant-based active compound, which possesses anti-inflammatory effects on immune cells. In this study, we investigate the effect of PUG on mouse macrophage RAW264.7 cells with or without stimulation of lipopolysaccharide (LPS). Cytotoxicity results showed that PUG is non-cytotoxic to the cells and it reversed the cytotoxicity in LPS-stimulated cells. The levels of nitric oxide (NO) and interleukin-6 (IL-6) were determined using a NO detection kit and IL-6 ELISA kit, respectively, and showed a significant decrease in NO and IL-6 in PUG-treated cells. Western blot and qRT-PCR were performed for the expression of two important pro-inflammatory cytokines, COX2 and iNOS, and found that their expression was downregulated in a dose-dependent manner. Other pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNFα, had reduced mRNA expression after PUG treatment. Furthermore, a Western blot was performed to calculate the expression of NF-κB and MAPK pathway proteins. The results show that PUG administration dramatically reduced the phosphorylation of p-Iκbα, p-NF-κB 65, and p-JNK. Remarkably, after PUG treatment, p-P38 and p-ERK remain unchanged. Furthermore, docking studies revealed that PUG is covalently linked to NF-κB and suppresses inflammation. In conclusion, PUG exerted the anti-inflammatory mechanism by barring the NF-κB pathway and activating JNK. Thus, prunetinoside could be adopted as a therapeutic compound for inflammatory-related conditions.

Inhibitory effect of membrane­free stem cell components derived from adipose tissues on skin inflammation in keratinocytes.

Inflammatory disorders of the skin are major public health concerns due to constant exposure to external stimuli. Skin cells are associated with prominent immune mechanisms to defend against adverse reactions. In the present study, the anti­inflammatory properties of membrane­free stem cell components (MFSCC) from adipose tissue­derived stem cells (ADSCs) and their basic preventive effects on skin wrinkle formation using human keratinocytes (HaCaT) and fibroblast (Detroit 551) cells, were investigated. Initially, a human inflammation antibody array was used on tumor necrosis factor­α (TNF­α)/interferon­Î³ (IFN­Î³)­induced and MFSCC­treated HaCaT cells. Array spots revealed three differential proteins, interleukin (IL)­1 F1 (IL­1α), IL­6, and TIMP2. Of these three proteins, IL­6 was significantly downregulated by MFSCC treatment. Western blot analysis revealed that IL­6 and its key downstream proteins JAK2 and STAT3 were suppressed in MFSCC­treated HaCaT cells. Further analysis revealed that MFSCC decreased the expression of TNF­α/IFN­Î³­induced phosphorylated (p)­IκB­α, p­p65, p­JNK, p­ERK, and p­p38 by inhibiting the activation of MAPK and NF­κB pathways. Treatment of Detroit 551 cells with MFSCC increased COL1A1 and elastin but suppressed matrix metalloproteinase (MMP)­1 and MMP­8 protein expression levels. Collectively, these data indicated that MFSCC exhibited a primary inhibitory effect on inflammation and wrinkle formation in skin. These results provide a basis for further extensive studies and application of MFSCC in treating skin inflammatory disorders.

Differences of Key Proteins between Apoptosis and Necroptosis.

Many different types of programmed cell death (PCD) have been identified, including apoptosis and necroptosis. Apoptosis is a type of cell death that is controlled by various genes. It is in charge of eliminating aberrant cells such as cancer cells, replenishing normal cells, and molding the body as it develops. Necroptosis is a type of programmed cell death that combines necrosis and apoptosis. In other words, it takes on a necrotic appearance, although cells die in a controlled manner. Various investigations of these two pathways have revealed that caspase-8, receptor-interacting serine/threonine-protein kinase 1 (RIPK1), and RIPK3 are crucial proteins in charge of the switching between these two pathways, resulting in the activation or inhibition of necroptosis. In this review, we have summarized the key proteins between apoptosis and necroptosis.

Potential Antioxidant and Anti-Inflammatory Function of Gynura procumbens Polyphenols Ligand.

The antioxidant and anti-inflammatory potentials of polyphenols contained in Gynura procumbens (GP) extract were systematically analyzed. Polyphenols in GP were analyzed for nine peaks using high-performance liquid chromatography (HPLC) combined with mass spectrometry (MS), and quantitatively determined through each standard. A total of nine polyphenolic compounds were identified in the samples and their MS data were tabulated. To determine the potential of bioactive ingredients targeting DPPH and COX-2, we analyzed them by ultrafiltration combined with LC. The results identified the major compounds exhibiting binding affinity for DPPH and COX-2. Caffeic acid, kynurenic acid, and chlorogenic acid showed excellent binding affinity to DPPH and COX-2, suggesting that they can be considered as major active compounds. Additionally, the anti-inflammatory effect of GP was confirmed in vitro. This study will not only be used to provide basic data for the application of GP to the food and pharmaceutical industries, but will also provide information on effective screening methods for other medicinal plants.

Inhibition of Cell Proliferation and Metastasis by Scutellarein Regulating PI3K/Akt/NF-κB Signaling through PTEN Activation in Hepatocellular Carcinoma.

Scutellarein (SCU) is a well-known flavone with a broad range of biological activities against several cancers. Human hepatocellular carcinoma (HCC) is major cancer type due to its poor prognosis even after treatment with chemotherapeutic drugs, which causes a variety of side effects in patients. Therefore, efforts have been made to develop effective biomarkers in the treatment of HCC in order to improve therapeutic outcomes using natural based agents. The current study used SCU as a treatment approach against HCC using the HepG2 cell line. Based on the cell viability assessment up to a 200 µM concentration of SCU, three low-toxic concentrations of (25, 50, and 100) µM were adopted for further investigation. SCU induced cell cycle arrest at the G2/M phase and inhibited cell migration and proliferation in HepG2 cells in a dose-dependent manner. Furthermore, increased PTEN expression by SCU led to the subsequent downregulation of PI3K/Akt/NF-κB signaling pathway related proteins. In addition, SCU regulated the metastasis with EMT and migration-related proteins in HepG2 cells. In summary, SCU inhibits cell proliferation and metastasis in HepG2 cells through PI3K/Akt/NF-κB signaling by upregulation of PTEN, suggesting that SCU might be used as a potential agent for HCC therapy.

Iridin Induces G2/M Phase Cell Cycle Arrest and Extrinsic Apoptotic Cell Death through PI3K/AKT Signaling Pathway in AGS Gastric Cancer Cells.

Iridin is a natural flavonoid found in Belamcanda chinensis documented for its broad spectrum of biological activities like antioxidant, antitumor, and antiproliferative effects. In the present study, we have investigated the antitumor potential of iridin in AGS gastric cancer cells. Iridin treatment decreases AGS cell growth and promotes G2/M phase cell cycle arrest by attenuating the expression of Cdc25C, CDK1, and Cyclin B1 proteins. Iridin-treatment also triggered apoptotic cell death in AGS cells, which was verified by cleaved Caspase-3 (Cl- Caspase-3) and poly ADP-ribose polymerase (PARP) protein expression. Further apoptotic cell death was confirmed by increased apoptotic cell death fraction shown in allophycocyanin (APC)/Annexin V and propidium iodide staining. Iridin also increased the expression of extrinsic apoptotic pathway proteins like Fas, FasL, and cleaved Caspase-8 in AGS cells. On the contrary, iridin-treated AGS cells did not show variations in proteins related to an intrinsic apoptotic pathway such as Bax and Bcl-xL. Besides, Iridin showed inhibition of PI3K/AKT signaling pathways by downregulation of (p-PI3K, p-AKT) proteins in AGS cells. In conclusion, these data suggest that iridin has anticancer potential by inhibiting PI3K/AKT pathway. It could be a basis for further drug design in gastric cancer treatment.

A Network Pharmacological Approach to Reveal the Pharmacological Targets and Its Associated Biological Mechanisms of Prunetin-5-O-Glucoside against Gastric Cancer.

Gastric cancer (GC) is an aggressive malignancy with increased mortality rate and low treatment options. Increasing evidence suggests that network pharmacology will be a novel method for identifying the systemic mechanism of therapeutic compounds in diseases like cancer. The current study aimed to use a network pharmacology approach to establish the predictive targets of prunetin-5-O-glucoside (PG) against gastric cancer and elucidate its biological mechanisms. Primarily, genes associated with the pathogenesis of GC was identified from the DiGeNET database and targets of PG was obtained from the Swiss target prediction database. In total, 65 correlative hits were identified as anti-gastric cancer targets of PG. Functional enrichment and pathway analysis revealed significant biological mechanisms of the targets. Interaction of protein network and cluster analysis using STRING resulted in three crucial interacting hub targets namely, HSP90AA1, CDK2, and MMP1. Additionally, the in vitro cytotoxic potential of PG was assessed on three gastric cancer cells (AGS, MKN-28, and SNU-484). Furthermore, the crucial targets were validated using molecular docking, followed by their expressions being evaluated by western blot and Human Protein Atlas. The findings indicate that the pharmacological action of PG against GC might be associated with the regulation of three core targets: HSP90AA1, CDK2, and MMP1. Thus, the network pharmacology undertaken in the current study established the core active targets of PG, which may be extensively applied with further validations for treatment in GC.

Transcriptome analysis of sinensetin-treated liver cancer cells guided by biological network analysis.

Hepatocellular carcinoma is recognized as one of the most frequently occurring malignant types of liver cancer globally, making the identification of biomarkers critically important. The aim of the present study was to identify the genes involved in the anticancer effects of flavonoid compounds so that they may be used as targets for cancer treatment. Sinensetin (SIN), an isolated polymethoxyflavone monomer compound, possesses broad antitumor activities in vitro. Therefore, the identification of a transcriptome profile on the condition of cells treated with SIN may aid to better understand the genes involved and its mechanism of action. Genomic profiling studies of cancer are increasing rapidly in order to provide gene expression data that can reveal prognostic biomarkers to combat liver cancer. In the present study, high-throughput RNA sequencing (RNA-seq) was performed to reveal differential gene expression patterns between SIN-treated and SIN-untreated human liver cancer HepG2 cells. A total of 43 genes were identified to be differentially expressed (39 downregulated and 4 upregulated in the SIN-treated group compared with the SIN-untreated group). An extensive network analysis for these 43 genes resulted in the identification of 10 upregulated highly interconnected hub genes that contributed to the progression of cancer. Functional enrichment analysis of these 10 hub genes revealed their involvement in the regulation of apoptotic processes, immune response and tumor necrosis factor production. Additionally, the mRNA expression levels of these 10 genes were evaluated using reverse transcription-quantitative PCR, and the results were consistent with the RNA-seq data. Overall, the results of the present study revealed differentially expressed genes involved in cancer after SIN treatment in HepG2 cells and may help to develop strategies targeting these genes for treating liver cancer.

Quantitative Proteomics Analysis for the Identification of Differential Protein Expression in Calf Muscles between Young and Old SD Rats Using Mass Spectrometry.

Aging is associated with loss of muscle mass and strength that leads to a condition termed sarcopenia. Impaired conditions, morbidity, and malnutrition are the factors of devaluation of muscle fibers in aged animals. Satellite cells play an important role in maintaining muscle homeostasis during tissue regeneration and repair. Proteomic profiling on the skeletal muscle tissues of different age group rats helps to determine the differentially expressed (DE) proteins, which may eventually lead to the development of biomarkers in treating the conditions of sarcopenia. In this study, nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) analysis was implemented in the calf tissues of young and old groups of rats. The mass spectrometry (MS) analysis revealed the presence of 335 differentially expressed proteins between the two different age conditions, among which those based on log-fold change 25 proteins were upregulated and 77 were downregulated. The protein-protein interaction network analysis revealed 18 upregulated proteins with three distinct interconnected networks and 57 downregulated proteins with two networks. Further, gene ontology (GO) enrichment analysis showed the biological process, cellular component, and molecular function of the differential proteins. Pathway enrichment analysis of the DE proteins identified nine significantly enriched pathways with a list of eight significant genes (Cryab, Hspb2, Acat1, Ak1, Adssl1, Anxa5, Gys1, Ogdh, Gc, and Adssl1). Quantification of significant genes by quantitative real-time polymerase chain reaction (qRT-PCR) confirmed the downregulation at the mRNA level. Western blot analysis of their protein expression showed concordant results on two candidate proteins (Ogdh and annexin 5) confirming their differential regulation between the two age groups of rats. Thus, these proteomic approaches on young and aged rats provide insights into the development of protein targets in the treatment of sarcopenia (muscle loss).

Cellular Function of Annexin A1 Protein Mimetic Peptide Ac2-26 in Human Skin Keratinocytes HaCaT and Fibroblast Detroit 551 Cells.

Inflammation of the skin is the most common dermatological problem in human. The anti-inflammatory mediated responses of the skin cells provide a mechanism for combating these conditions. Annexin A1 (AnxA1) is one of the proteins that has been shown to have a potent anti-inflammatory effect. However, the effects and mechanisms of AnxA1 in skin keratinocyte and fibroblast have not been reported yet. In the current study, we hypothesized that Ac2-26, AnxA1 mimetic peptide, ameliorates inflammation and wrinkle formation in human skin cells. Therefore, we aimed to identify whether Ac2-26 has anti-inflammatory and anti-wrinkle effects in human keratinocyte (HaCaT) and fibroblast (Detroit 551) cells, respectively. Human HaCaT cells were stimulated by TNF-α/IFN-γ with or without Ac2-26, to identify the anti-inflammatory effect. Human Detroit 551 cells were treated with Ac2-26 to verify the anti-wrinkle effect. Initially, cell cytotoxicity was carried out in each cell line treated using Ac2-26 by MTT assay. Human MDA, IL-8, and procollagen secretion were detected by ELISA assay. The inflammatory chemokines were measured by qRT-PCR analysis. To demonstrate the mechanism, MAPK, NF-κB, JAK/STAT, and MMPs were analyzed by Western blotting. As a result, we identified that Ac2-26 significantly decreased the expression of TNF-α/IFN-γ-stimulated pro-inflammatory chemokines, including IL-1ß, IL-6, IL-8, MDC, TARC, and TNF-α, by inhibiting the activation of MAPK, NF-κB, and JAK/STAT pathway in TNF-α/IFN-γ-stimulated HaCaT human keratinocytes. In addition, we also identified that Ac2-26 significantly induced collagen synthesis by generating pro-collagen, and suppressed collagen degradation by inhibiting the collagenase MMP-1 and MMP-8 expression. Collectively, these results suggest that Ac2-26 shows anti-inflammatory and anti-wrinkling effect. These effects may lead to the development of preventive and therapeutic application for inflammation-related skin disease and wrinkle formation.

Sinensetin Induces Autophagic Cell Death through p53-Related AMPK/mTOR Signaling in Hepatocellular Carcinoma HepG2 Cells.

Sinensetin (SIN) has been reported to exhibit anti-inflammatory and anti-cancer activity. However, the cellular and molecular mechanism by which SIN promotes hepatocellular carcinoma (HCC) cell death remains unclear. In the present study, we investigated the induction of cell death by SIN and its underlying mechanism in HepG2 cells, an HCC cell line. We found that SIN significantly induced cell death in HepG2 cells, whereas the proliferation rate of Thle2, human liver epithelial cells, was unaffected by SIN. SIN-treated HepG2 cells were not affected by apoptotic cell death; instead, autophagic cell death was induced through the p53-mediated AMPK/mTOR signaling pathway. Inhibition of p53 degradation led to both autophagy and apoptosis in HepG2 cells. p53 translocation led to SIN-induced autophagy, whereas p53 translocation inhibited SIN-induced apoptosis. However, SIN showed apoptosis in the p53-mutant Hep3B cell line. Molecular docking simulation of the p53 core domain showed effective binding with SIN, which was found significant compared with the known p53 activator, RITA. Collectively, these data suggest that SIN may be a potential anti-cancer agent targeting autophagic cell death in human liver cancer.

Compound Prunetin Induces Cell Death in Gastric Cancer Cell with Potent Anti-Proliferative Properties: In Vitro Assay, Molecular Docking, Dynamics, and ADMET Studies.

Gastric cancer is the common type of malignancy positioned at second in mortality rate causing burden worldwide with increasing treatment options. Prunetin (PRU) is an O-methylated flavonoid that belongs to the group of isoflavone executing beneficial activities. In the present study, we investigated the anti-proliferative and cell death effect of the compound PRU in AGS gastric cancer cell line. The in vitro cytotoxic potential of PRU was evaluated and significant proliferation was observed. We identified that the mechanism of cell death was due to necroptosis through double staining and was confirmed by co-treatment with inhibitor necrostatin (Nec-1). We further elucidated the mechanism of action of necroptosis via receptor interacting protein kinase 3 (RIPK3) protein expression and it has been attributed by ROS generation through JNK activation. Furthermore, through computational analysis by molecular docking and dynamics simulation, the efficiency of compound prunetin against RIPK3 binding was validated. In addition, we also briefed the pharmacokinetic properties of the compound by in silico ADMET analysis.

Comparative proteomic analysis uncovers potential biomarkers involved in the anticancer effect of Scutellarein in human gastric cancer cells.

Scutellarein (SCU), a flavone that belongs to the flavonoid family and abundantly present in Scutellaria baicalensis a flowering plant in the family Lamiaceae, has been reported to exhibit anticancer effects in several cancer cell lines including gastric cancer (GC). Although our previous study documented the mechanisms of Scutellarein­induced cytotoxic effects, the literature shows that the proteomic changes that are associated with the cellular response to SCU have been poorly understood. To avoid adverse side­effects and significant toxicity of chemotherapy in patients who react poorly, biomarkers anticipating therapeutic responses are imperative. In the present study, we utilized a comparative proteomic analysis to identify proteins associated with Scutellarein (SCU)­induced cell death in GC cells (AGS and SNU484), by integrating two­dimensional gel electrophoresis (2­DE), mass spectrometry (MS), and bioinformatics to analyze the proteins. Proteomic analysis between SCU­treated and DMSO (control) samples successfully identified 41 (AGS) and 31 (SNU484) proteins by MALDI­TOF/MS analysis and protein database search. Comparative proteomics analysis between AGS and SNU484 cells treated with SCU revealed a total of 7 protein identities commonly expressed and western blot analysis validated a subset of identified critical proteins, which were consistent with those of the 2­DE outcome. Molecular docking studies also confirmed the binding affinity of SCU towards these critical proteins. Phosphatidylinositol 4,5­bisphosphate 3­kinase catalytic subunit ß isoform (PIK3CB) protein expression was accompanied by a distinct group of cellular functions, including cell growth, and proliferation. Cancerous inhibitor of protein phosphatase 2A (CIP2A), is one of the oncogenic molecules that have been shown to promote tumor growth and resistance to apoptosis and senescence­inducing therapies. In the present study, both PIK3CB and CIP2A proteins were downregulated in SCU­treated cells, which boosts our previous results of SCU to induce apoptosis and inhibits GC cell growth by regulating these critical proteins. The comparative proteomic analysis has yielded candidate biomarkers of response to SCU treatment in GC cell models and further validation of these biomarkers will help the future clinical development of SCU as a novel therapeutic drug.

Apigetrin induces extrinsic apoptosis, autophagy and G2/M phase cell cycle arrest through PI3K/AKT/mTOR pathway in AGS human gastric cancer cell.

Apigetrin is a flavonoid glycoside phytonutrient derived from fruits and vegetables that is well known for a variety of biological activities such as antioxidant and anti-inflammatory activities. In the current study, we determined the effect of apigetrin on AGS gastric cancer cell. Apigetrin reduced cancer cell proliferation and induced G2/M phase cell cycle arrest by regulating cyclin B1, cdc25c and cdk1 protein expression in AGS cell. Apigetrin treatment caused apoptotic cell death in AGS cells, characterized by the accumulation of apoptosis portion, cleavage of caspase-3 and poly ADP-ribose polymerase (PARP). Apigetrin-treated cells increased the expression of extrinsic apoptosis pathway proteins and mRNA. However, intrinsic apoptosis pathway related proteins were not altered. In addition, AGS cells treated with apigetrin increased autophagic cell death, featured by the formation of autophagic vacuole and acidic vesicular organelles. Autophagy marker proteins, such as LC3B-II and beclin-1, were increased, and p62, an autophagy flux marker protein, was also increased by endoplasmic reticulum stress. Also, the phosphorylation of PI3K/AKT/mTOR pathway proteins and its downstream targets in apigetrin-treated AGS cells was identified to be decreased. Taken together, these data suggest that apigetrin-treated AGS cells induced G2/M phase cell cycle arrest, extrinsic apoptosis and autophagic cell death through PI3K/AKT/mTOR pathway, which can lead to the inhibition of gastric cancer development. Thus, our findings strongly indicate that apigetrin is a basic natural derived compound that could be used as a nutrient source with potential anticancer activities against gastric cancer.