WI-38 Human Fibroblasts Research Articles

Green Synthesis and Anticancer Activity of New Bis-Imidazole-Thiazole Hybrids Targeting Hepatocellular Carcinoma

Background: Given the inadequacies of current chemotherapy, there is a need for more effective anticancer agents. Imidazole and thiazole compounds have demonstrated significant biological activity, making them promising candidates. Aims and Objective: This study investigates the anticancer potential of imidazole and thiazole derivatives, focusing on liver cancer. The aim is to synthesize bis-imidazole-thiazole hybrids and evaluate their efficacy as anticancer agents against hepatocellular carcinoma. Methods: The hybrids were synthesized using (2,2'-((1,4-phenylenebis(2-mercapto-4-methyl1H-imidazole-1,5-diyl))bis(ethan-1-yl-1-ylidene))bis(hydrazine-1-carbothioamide), hydrazonoyl halides, and α-halo ketones, catalyzed by DABCO. This method is designed to be fast, yield high amounts of product, and be environmentally friendly. Structural confirmation was provided by FT IR, NMR, and MS spectroscopy. Results: The synthesized hybrids were tested in vitro against HepG-2 and WI-38 cell lines. Compounds 16b, 14a, 16a, and 7b showed significant inhibitory activity, with IC50 values indicating strong inhibition comparable to or better than the standard drug Sorafenib. Conclusion: The bis-imidazole-thiazole hybrids exhibit potent anticancer properties, particularly against hepatocellular carcinoma, making them potential candidates for future cancer therapies. Their selectivity and safety were further demonstrated by their effects on normal WI-38 human fibroblasts.

Elimination of radiation-induced senescent cancer cells and stromal cells invitro by near-infrared photoimmunotherapy.

Therapy-induced senescent cancer and stromal cells secrete cytokines and growth factors to promote tumor progression. Therefore, senescent cells may be novel targets for tumor treatment. Near-infrared photoimmunotherapy (NIR-PIT) is a highly tumor-selective therapy that employs conjugates of a molecular-targeting antibody and photoabsorber. Thus, NIR-PIT has the potential to be applied as anovel senolytic therapy. This study aims to investigate the efficacy of NIR-PIT treatment on senescent cancer and stromal cells. Two cancer cell lines (human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells) and two normal cell lines (mouse fibroblast transfected with human epidermal growth factor receptor 2 [HER2] cells and human fibroblast WI38 cells) were used. The cytotoxicity of NIR-PIT was evaluated using anti-epidermal growth factor receptor (EGFR) antibody panitumumab and anti-HER2 antibody transtuzumab. Cellular senescence was induced in A549 and MIA PaCa-2 cells by 10 Gy γ-irradiation. The up-regulation of cellular senescence markers and characteristic morphological changes in senescent cells, including enlargement, flattening, and multinucleation, were observed in cancer cells after 5 days of γ-irradiation. Then, NIR-PIT targeting EGFR was performed on these senescent cancer cells. The NIR-PIT induced morphological changes, including bleb formation, swelling, and the inflow of extracellular fluid, and induced a significant decrease in cellular viability. These results suggested that NIR-PIT may induce cytotoxicity using the same mechanism in senescent cancer cells. In addition, similar morphological changes were also induced in radiation-induced senescent 3T3-HER2 fibroblasts by NIR-PIT targeting human epidermal growth factor receptor 2. NIR-PIT eliminates both senescent cancer and stromal cells invitro suggesting it may be a novel strategy for tumor treatment.

Bio-beneficial spectrum of Tecoma stans flower extract in vitro for fighting prostate and ovarian cancers with its anti-diabetic and antioxidant activities

People have long used plants and plant-derived products to treat a wide range of illnesses. In the present work, Tecoma stans flower was extracted using 90% ethanol. Flavonoids and total phenolic constituents of T. stans flower extract were screened, and polyphenolic compounds were assessed using high-performance liquid chromatography (HPLC). Anti-diabetic via α-amylase and α-glucosidase assays, antioxidant via 2,2-diphenyl-1-picryl-hydrazyl-hydrate, ferric reducing antioxidant power, and total antioxidant capacity of T. stans flower extract were assessed. The cytotoxic action for T. stans flower extract was assessed versus WI-38 (human fetal lung fibroblast cells), PC3 (prostate cancer cell line), and SK-OV3 (ovarian cancer cell line). The T. stans extract showed promising in vitro anti-diabetic effect with IC50 = 12.08 ± 0.2 µg/mL and 22.83 ± 0.3 µg/mL for α-amylase and α-glucosidase, respectively. Moreover, T. stans showed good in vitro antioxidant action with IC50= 5.36 ± 0.2 µg/mL for DPPH testing, and the best antitumor impact versus PC3 cells with IC50 = 113.27 ± 1.59 µg/mL. Flow cytometric analysis confirmed the role of T. stans in acceleration in apoptosis of PC3 cells through regulation of oxidative enzymes. These results indicate that the derived materials from T. stans flower have multiple medicinal applications.

Development of a novel laboratory photodynamic therapy device: automated multi-mode LED system for optimum well-plate irradiation

HighlightsMulti-mode Automated Well-plate PDT LED Laboratory Irradiation System described.Automates and standardizes time-consuming experiments.LED wavelength and temperature stabilized for highly reproducible irradiations.Efficacy demonstrated in 5-aminolevulinic acid (5-ALA) treatments of HT-29 colon cancer cells and WI-38 human lung fibroblasts.

Factors Affecting the Evaluation of Collagen Deposition and Fibrosis In Vitro.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Design, synthesis and biological evaluation of rhein-piperazine-furanone hybrids as potential anticancer agents.

Novel rhein-piperazine-furanone hybrids, 5, were designed and synthesized efficiently from rhein. Cytotoxicity of all hybrids 5a-j against A549 human lung cancer cells was superior to the parent rhein and the reference cytarabine (CAR). Hybrid 5e (IC50 = 5.74 μM), the most potent compound, was 46- and 35-fold more toxic against A549 cells than rhein (IC50 = 265.59 μM) and CAR (IC50 = 202.57 μM), respectively. Moreover, hybrid 5e (IC50 = 69.28 μM) was less toxic to normal WI-38 human lung fibroblast cells with good selectivity (WI-38/A549, SI ≈ 12), being much higher than rhein (SI ≈ 1) and CAR (SI ≈ 2). Structure-activity relationship (SAR) analysis showed that cytotoxicity and selectivity against A549 lung cancer cells were greatly enhanced when methoxy-containing furanone was introduced to the hybrids (5e and 5h). Further, hybrid 5e showed better cytotoxicity against four types of human lung cancer cells (H460, A549, PC-9, and Calu-1; IC50 = 4.35-15.39 μM) than six other types of human cancer cells (SK-BR-3, SK-OV-3, 786-O, Huh-7, HCT116, and HeLa; IC50 = 13.77-60.45 μM), showing specificity. In particular, hybrid 5e showed the highest cytotoxicity (IC50 = 4.35 μM) and the highest selectivity (WI-38/H460, SI ≈ 16) against H460 human lung cancer cells. Flow cytometric analysis showed that hybrid 5e induced apoptosis in a concentration-dependent manner in H460 cells. The results show that the cytotoxicity and selectivity of rhein can be greatly enhanced by hybridization with furanone. Hybrid 5e is expected to be a leading candidate for anti-lung cancer drugs.

Topology and adenocarcinoma cell localization dataset on the labyrinthin diapeutic biomarker

ObjectiveThe discovery and characterization of tumor associated antigens is increasingly important to advance the field of immuno-oncology. In this regard, labyrinthin has been implicated as a neoantigen found on the cell surface of adenocarcinomas. Data on the (1) topology, (2) amino acid (a.a.) homology analyses and (3) cell surface localization of labyrinthin by fluorescent activated cell sorter (FACS) are studied in support of labyrinthin as a novel, pan-adenocarcinoma marker.ResultsBioinformatics analyses predict labyrinthin as a type II protein with calcium binding domain(s), N-myristoylation sites, and kinase II phosphorylation sites. Sequence homologies for labyrinthin (255 a.a.) were found vs. the intracellular aspartyl/asparaginyl beta-hydroxylase (ASPH; 758 a.a.) and the ASPH-gene related protein junctate (299 a.a.), which are both type II proteins. Labyrinthin was detected by FACS on only non-permeablized A549 human lung adenocarcinoma cells, but not on normal WI-38 human lung fibroblasts nor primary cultures of normal human glandular-related cells. Microscopic images of immunofluorescent labelled MCA 44-3A6 binding to A549 cells at random cell cycle stages complement the FACS results by further showing that labyrinthin persisted on the cell surfaces along with some cell internalization for greater than 20 min.

Abstract 1137: Identification of a high selective SMARCA2 degrader which effectively suppresses the SMARCA4-deficient tumors in vitro and in vivo

Abstract The SWI/SNF chromatin-remodeling complex plays essential roles in a variety of cellular processes including differentiation, proliferation and DNA repair. Mammalian SWI/SNF complexes always contain one of two mutually exclusive and structurally highly related ATPases: SMARCA2/BRM or SMARCA4/BRG1, as its catalytic subunit, which hydrolyze ATP to drive nucleosome movement. It is reported that absence of SMARCA4 are present in several cancers and these SMARCA4 deficient cells highly depend on its paralog SMARCA2 for survival. Furthermore, concurrent inhibition of SMARCA2 and SMARCA4 is fatal to normal cells. Therefore, it holds great therapeutic promise to treat SMARCA4 deficient cancers with selective SMARCA2 degraders while leaving SMARCA4 wild type cells unaffected. Here, we report that SCR-9140 is a high selective SMARCA2 degrader which demonstrates superior anti-tumor activity and low cytotoxicity against non-malignant cells. SCR-9140 strongly degrades SMARCA2 with DC50 of <1 nM and achieves the complete protein degradation (Dmax of >95%) and also shows 100-fold degradation selectivity over SMARCA4 with DC50 of around 100 nM and Dmax of <60% in HiBiT knock-in SW1573 cells. Across a panel of SMARCA4 deficient tumor cell lines, SCR-9140 potently inhibits proliferation with IC50 values ranging from 1 to 50 nM. Due to the high selectivity over SMARCA4, SCR-9140 neither inhibits proliferation of SW1573 (SMARCA2-WT SMARCA4-WT) cells nor NCI-H1581 (SMARCA2-DEL SMARCA4-DEL) cells with IC50s of over 2000 nM. The degraders with poor selectivity of SMARCA2/4 (DC50 ratio: < 10 folds, Dmax of SMARCA4 > 80%) strongly inhibit the SW1573 cells proliferation with IC50s < 100nM, while there are no significant effects on the NCI-H1581 cells ascribing to the absence of targets. Furthermore, SCR-9140 demonstrates weak cytotoxicity on non-malignant cells, such as WI-38 (human lung fibroblast cells), THLE-2 (human liver cells), and human PBMC, rendering it the greater therapeutic window. In a CDX model of NSCLC, SCR-9140 effectively inhibits tumor growth and TGI correlates well with SMARCA2 degradation in tumors. In conclusion, our potent and selective SMARCA2 degrader, SCR-9140 induces strong synthetic lethality in SMARCA4 deficient cells in vitro and in vivo and shows great safety margin in normal cells. Citation Format: Feng Zhou, Zhiyong Yu, Ruonan Chen, Feng Tang, Peng Gu, Wenqing Yang, Liting Xue, Ping Chen, Renhong Tang. Identification of a high selective SMARCA2 degrader which effectively suppresses the SMARCA4-deficient tumors in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1137.

In Vitro of Melaleuca viridiflora Sol. ex Gaertn Plant Investigation of Antimicrobial, Anticancer and Cytotoxic Activities

In this study; it was aimed to investigate the anticancer and cytotoxic activities on DU-145 prostate cancer and MCF-7 breast cancer cell lines and WI-38 human fibroblast cell line of essential oil (Nioli) obtained from Melaleuca viridiflora Sol. ex Gaertn. plant and its antimicrobial effects on various bacteria and yeast cells. Content analyzes of Nioli essential oil were made by Gas Chromatography-Mass Spectrometry (GK-KS). The antimicrobial effects of the essential oil were determined using Disk Diffusion and Minimum Inhibition Concentration (MIC) tests. Its cytotoxic effects were determined by the XTT (2,3-bis-(2-methoxy-4-) nitro-5)-sulfophenyl)-2H-tetrazolium-5-carboxanilide) test; were investigated at different concentrations in breast cancer (MCF-7), prostate cancer (DU-145) and healthy human fibroblast (WI-38) cell lines. In the disc diffusion method of essential oil; It was determined that it formed an inhibition zone against Klebsiella pneumoniae bacteria at 200, 100, 50, 25 and 12.5 µg/mL concentrations, and against Candida albicans yeast at 200 and 100 µg/mL concentrations. Nioli essential oil showed the highest antibacterial effect against Bacillus cereus. While it was moderately effective against Pseudomonas aeruginosa and Candida albicans, it reached MIC values effective against other bacteria. It was observed that the anticancer activity of Nioli essential oil was more effective in DU-145 prostate cancer cells compared to MCF-7 breast cancer cells, and it had no toxicity in WI-38 healthy human fibroblast cells.

Analysis of Anti-Cancer and Anti-Inflammatory Properties of 25 High-THC Cannabis Extracts.

Cannabis sativa is one of the oldest cultivated plants. Many of the medicinal properties of cannabis are known, although very few cannabis-based formulations became prescribed drugs. Previous research demonstrated that cannabis varieties are very different in their medicinal properties, likely due to the entourage effect—the synergistic or antagonistic effect of various cannabinoids and terpenes. In this work, we analyzed 25 cannabis extracts containing high levels of delta-9-tetrahydrocannabinol (THC). We used HCC1806 squamous cell carcinoma and demonstrated various degrees of efficiency of the tested extracts, from 66% to 92% of growth inhibition of cancer cells. Inflammation was tested by induction of inflammation with TNF-α/IFN-γ in WI38 human lung fibroblasts. The efficiency of the extracts was tested by analyzing the expression of COX2 and IL6; while some extracts aggravated inflammation by increasing the expression of COX2/IL6 by 2-fold, other extracts decreased inflammation, reducing expression of cytokines by over 5-fold. We next analyzed the level of THC, CBD, CBG and CBN and twenty major terpenes and performed clustering and association analysis between the chemical composition of the extracts and their efficiency in inhibiting cancer growth and curbing inflammation. A positive correlation was found between the presence of terpinene (pval = 0.002) and anti-cancer property; eucalyptol came second, with pval of 0.094. p-cymene and β-myrcene positively correlated with the inhibition of IL6 expression, while camphor correlated negatively. No significant correlation was found for COX2. We then performed a correlation analysis between cannabinoids and terpenes and found a positive correlation for the following pairs: α-pinene vs. CBD, p-cymene vs. CBGA, terpenolene vs. CBGA and isopulegol vs. CBGA. Our work, thus, showed that most of high-THC extracts demonstrate anti-cancer activity, while only certain selected extracts showed anti-inflammatory activity. Presence of certain terpenes, such as terpinene, eucalyptol, cymene, myrcene and camphor, appear to have modulating effects on the activity of cannabinoids.

Discovery and characterization of heterogeneous and multipotent fibroblast populations isolated from excised cleft lip tissue

BackgroundRegularly discarded lip tissue obtained from corrective surgeries to close the cleft lip represents an easily accessible and rich source for the isolation of primary fibroblasts. Primary fibroblasts have been described to show compelling similarities to mesenchymal stem cells (MSCs). Hence, cleft lip and palate (CLP) lip-derived fibroblasts could be thought as an intriguing cell source for personalized regenerative therapies in CLP-affected patients.MethodsInitially, we thoroughly characterized the fibroblastic nature of the lip-derived mesenchymal outgrowths by molecular and functional assays. Next, we compared their phenotype and genotype to that of bone marrow-mesenchymal stem cells (BM-MSCs) and of human lung-derived fibroblasts WI38, by assessing their morphology, surface marker expression, trilineage differentiation potential, colony-forming (CFU) capacity, and immunomodulation property. Finally, to better decipher the heterogeneity of our CLP cultures, we performed a single cell clonal analysis and tested expanded clones for surface marker expression, as well as osteogenic and CFU potential.ResultsWe identified intriguingly similar phenotypic and genotypic properties between CLP lip fibroblasts and BM-MSCs, which makes them distinct from WI38. Furthermore, our own data in combination with the complex anatomy of the lip tissue indicated heterogeneity in our CLP cultures. Using a clonal analysis, we discovered single cell-derived clones with increased levels of the MSC markers CD106 and CD146 and clones with variabilities in their commitment to differentiate into bone-forming cells and in their potential to form single cell-derived colonies. However, we were not able to gain clones possessing superior MSC-like capacities when compared to the heterogeneous parental CLP population. Additionally, all clones could still generate contractile forces and retained robust levels of the fibroblast specific marker FSP1, which was not detectable in BM-MSCs.ConclusionsOur results suggest that we isolate heterogeneous populations of fibroblasts from discarded CLP lip tissue, which show a prominently multipotent character in their entirety avoiding the need for elaborate subpopulation selections in vitro. These findings suggest that CLP lip fibroblasts might be a novel potential cell source for personalized regenerative medicine of clinical benefit for CLP patients.

Histamine activates an intracellular Ca2+ signal in normal human lung fibroblast WI-38 cells.

Histamine is an inflammatory mediator that can be released from mast cells to induce airway remodeling and cause persistent airflow limitation in asthma. In addition to stimulating airway smooth muscle cell constriction and hyperplasia, histamine promotes pulmonary remodeling by inducing fibroblast proliferation, contraction, and migration. It has long been known that histamine receptor 1 (H1R) mediates the effects of histamine on human pulmonary fibroblasts through an increase in intracellular Ca2+ concentration ([Ca2+]i), but the underlying signaling mechanisms are still unknown. Herein, we exploited single-cell Ca2+ imaging to assess the signal transduction pathways whereby histamine generates intracellular Ca2+ signals in the human fetal lung fibroblast cell line, WI-38. WI-38 fibroblasts were loaded with the Ca2+-sensitive fluorophore, FURA-2/AM, and challenged with histamine in the absence and presence of specific pharmacological inhibitors to dissect the Ca2+ release/entry pathways responsible for the onset of the Ca2+ response. Histamine elicited complex intracellular Ca2+ signatures in WI-38 fibroblasts throughout a concentration range spanning between 1 µM and 1 mM. In accord, the Ca2+ response to histamine adopted four main temporal patterns, which were, respectively, termed peak, peak-oscillations, peak-plateau-oscillations, and peak-plateau. Histamine-evoked intracellular Ca2+ signals were abolished by pyrilamine, which selectively blocks H1R, and significantly reduced by ranitidine, which selectively inhibits H2R. Conversely, the pharmacological blockade of H3R and H4R did not affect the complex increase in [Ca2+]i evoked by histamine in WI-38 fibroblasts. In agreement with these findings, histamine-induced intracellular Ca2+ signals were initiated by intracellular Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate (InsP3) receptors (InsP3R) and sustained by store-operated Ca2+ channels (SOCs). Conversely, L-type voltage-operated Ca2+ channels did not support histamine-induced extracellular Ca2+ entry. A preliminary transcriptomic analysis confirmed that WI-38 human lung fibroblasts express all the three InsP3R isoforms as well as STIM2 and Orai3, which represent the molecular components of SOCs. The pharmacological blockade of InsP3 and SOC, therefore, could represent an alternative strategy to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients.

Dolutegravir-containing HIV therapy reversibly alters mitochondrial health and morphology in cultured human fibroblasts and peripheral blood mononuclear cells.

Given the success of combination antiretroviral therapy (cART) in treating HIV viremia, drug toxicity remains an area of interest in HIV research. Despite newer integrase strand transfer inhibitors (InSTIs), such as dolutegravir (DTG) and raltegravir (RAL), having excellent clinical tolerance, there is emerging evidence of off-target effects and toxicities. Although limited in number, recent reports have highlighted the vulnerability of mitochondria to these toxicities. The aim of the present study is to quantify changes in cellular and mitochondrial health following exposure to current cART regimens at pharmacological concentrations. A secondary objective is to determine whether any cART-associated toxicities would be modulated by human telomerase reverse transcriptase (hTERT). We longitudinally evaluated markers of cellular (cell count, apoptosis), and mitochondrial health [mitochondrial reactive oxygen species (mtROS), membrane potential (MMP) and mass (mtMass)] by flow cytometry in WI-38 human fibroblast with differing hTERT expression/localization and peripheral blood mononuclear cells. This was done after 9 days of exposure to, and 6 days following the removal of, seven current cART regimens, including three that contained DTG. Mitochondrial morphology was assessed by florescence microscopy and quantified using a recently developed deep learning-based pipeline. Exposure to DTG-containing regimens increased apoptosis, mtROS, mtMass, induced fragmented mitochondrial networks compared with non-DTG-containing regimens, including a RAL-based combination. These effects were unmodulated by telomerase expression. All effects were fully reversible following removal of drug pressure. Taken together, our observations indicate that DTG-containing regimens negatively impact cellular and mitochondrial health and may be more toxic to mitochondria, even among the generally well tolerated InSTI-based cART.

In vivo and in vitro evidence for growth hormone-like bioactivity of Rhizoma Anemarrhenae extract

Certain herbs used in traditional Chinese medicine may produce a growth-enhancing effect by promoting the secretion of growth hormone (GH) by the pituitary gland or mimicking the function of GH. In this study, we aimed to identify herbs that could serve as GH alternatives. A reporter gene assay for GH was developed, and 100 different herbal extracts were assayed. We found that Rhizoma Anemarrhenae (RA) water extracts exhibited transactivation activities that stimulate the activation of signal transducer and activator of transcription 5 (STAT5). The growth-promoting effect of RA in NB2–11 cells was inhibited by co-treatment with GH receptor (GHR)-Fc fusion protein. Unlike GH, RA extracts did not enhance the growth of B16F10 melanoma cells. The activation of the Janus kinase 2-STAT5 signaling pathway was confirmed in both NB2–11 cells and WI-38 human normal lung fibroblasts; the activation was inhibited by co-treatment with GHR-Fc fusion protein. Docking analysis of the active ingredients of RA, including mangiferin, neomangiferin, isomangiferin, anemarsaponin E, 7-O-methylmangiferin, officinalisinin I, timosaponin BII, timosaponin AI, and timosaponin AIII, using SWISSDOCK indicated a direct interaction of these compounds with GHR. The growth-promoting effects and activation of STAT5 were also confirmed. Moreover, we found that RA extract significantly increased the height of the tibial growth plate and stimulated the production of insulin-like growth factor 1 in the serum, liver, and muscle tissues. Our findings provide evidence that herbal extracts, particularly, RA extracts, can promote growth by mimicking GH bioactivity.

P52 signaling promotes cellular senescence

BackgroundNuclear factor-κB is a multi-subunit transcription factor that plays a central role in cellular senescence. We previously reported that an increase in the p52 subunit is seen in senescent cells and aged tissue. In the current work, we examined the mechanism by which p52 is activated and whether the increase in p52 promotes senescence.ResultsUsing both primary mouse embryonic fibroblasts (MEFs) and WI-38 human lung fibroblasts, we examined cells after serial passage and following prolonged culture. An increase in p52 was found in the nucleus relative to pre-senescent cells. The increase in p52 protein was not reflected by an increase in NFKB2 mRNA or by an increase in the abundance of upstream activating kinases, IKKα and NIK. To examine whether p52 promotes senescence, we over-expressed mature p52 in primary MEFs. Significantly more senescence was seen compared to control, a finding not seen with p52 mutated at critical DNA binding residues. In addition, blocking p52 nuclear translocation with the peptide inhibitor, SN52, decreased β-galactosidase (β-gal) formation. Subsequent filtration studies demonstrated that proteins in conditioned media (CM) were necessary for the increase in p52 and mass spectrometry identified S100A4 and cyclophilin A (CYPA) as potential factors in CM necessary for induction of p52. The requirement of these proteins in CM for induction of p52 was confirmed using depletion and supplementation studies. In addition, we found that activation of STAT3 signaling was required for the increase in p52. Finally, genome wide ChIP-sequencing analysis confirmed that there is an increase in p52 chromatin enrichment with senescence and identified several downstream factors whose expression is regulated by increased p52 binding.ConclusionsThese results demonstrate that p52 nuclear translocation is increased in senescent cells by factors in conditioned media and that mature p52 induces cellular senescence. The data are consistent with the prior observation that p52 is elevated in aged tissue and support the hypothesis that p52 contributes to organismal aging.

Synthesis, Antiproliferative Activity, and Apoptotic Profile of New Derivatives from the Meta Stable Benzoxazinone Scaffold.

Cancer exerts a huge strain on the health system. The emerging resistance to the current chemotherapies demands the continuous development of new anticancer agents with lower cost, higher efficacy, and greater specificity. This study aimed at developing selective small molecules as targeted anticancer agents. The behavior of benzoxazinone 2 towards nitrogen nucleophiles, such as hydrazine hydrate, formamide, ethanolamine, aromatic amines, and thiosemicarbazide, was described. The behavior of the amino quinazolinone 3 towards carbon electrophiles and P2S5 was also investigated. The antiproliferative activity of 17 new benzoxazinone derivatives was examined against the growth of three human cancer cell lines; liver HepG2, breast MCF-7, and colon HCT-29, in addition to the normal human fibroblasts WI-38, and the selectivity index was calculated. The possible molecular pathways, such as the cell cycle and apoptosis, were investigated. Derivatives 3, 7, 8, 10, 13, and 15 had a significant (less than 10 μM) antiproliferative activity against the three cancer cell lines investigated. Derivative 7 showed the best antiproliferative profile comparable to that of doxorubicin. The selectivity index for all the effective derivatives ranged from ~5-12 folds, indicating high selectivity against the cancer cells. Derivative 15 caused ~ 7-fold and 8-fold inductions in the expression of p53 and caspase3, respectively. It also caused a ~ 60% reduction in the expression of both topoisomerase II (topoII) and cyclin-dependent kinase 1 (cdk1). Derivatives 3, 7, and 8 had a similar profile; ~ 6-8-fold increased in the expression of p53 and caspase3 but these compounds were devoid of any significant effect on the expression of topoII and cdk1. Derivatives 10 and 13 were also similar and resulted in a ~6-fold elevation in the expression ofcaspase3, and more than 60% downregulation in the expression of topoII. The results of the gene expression of topoII and caspase3 were confirmed by the measurement of the topoII concentration and caspase3 activity in the HepG2 cells. Six derivatives exerted their antiproliferative activity by arresting the cell cycle (decreasing cdk1), preventing the DNA duplication (downregulating topo II), and inducing apoptosis (inducing p53 and caspase3). One common feature in all the six active derivatives is the presence of a free amino group. These compounds have merit for further investigations.

Identification of gingerenone A as a novel senolytic compound.

Senescent cells accumulate with aging and have been shown to contribute to age-associated diseases and organ dysfunction. Eliminating senescent cells with senolytic drugs has been shown to improve age phenotypes in mouse models and there is some initial evidence that it may improve the health of persons with chronic diseases. In this study, we employed WI-38 human fibroblasts rendered senescent by exposure to ionizing radiation (IR) to screen several plant extracts for their potential senolytic and/or senomorphic activity. Of these, ginger extract (Zingiber officinale Rosc.) selectively caused the death of senescent cells without affecting proliferating cells. Among the major individual components of ginger extract, gingerenone A and 6-shogaol showed promising senolytic properties, with gingerenone A selectively eliminating senescent cells. Similar to the senolytic cocktail dasatinib and quercetin (D+Q), gingerenone A and 6-shogaol elicited an apoptotic program. Additionally, both D+Q and gingerenone A had a pronounced effect on suppressing the senescence-associated secretory phenotype (SASP). Gingerenone A selectively promotes the death of senescent cells with no effect on non-senescent cells and these characteristics strongly support the idea that this natural compound may have therapeutic benefit in diseases characterized by senescent cell accumulation.

Novel insights from a multiomics dissection of the Hayflick limit.

The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick's original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single-cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by t YAP1/TEAD1 and TGF-β2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.

Identification of a promising hit from a new series of pyrazolo[1,5-a]pyrimidine based compounds as a potential anticancer agent with potent CDK1 inhibitory and pro-apoptotic properties through a multistep in vitro assessment

A new series of sixteen new 2-arylamino-5,7-disubstituted-N-aryl-pyrazolo[1,5-a]pyrimidine-3-carboxamide derivatives was designed and synthesized. The antitumor activities of the new compounds were initially screened through the developmental therapeutics program at NCI-USA 60 cell line panel. 2-((2,4-dimethoxyphenyl)amino)-5,7-diphenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (7a) was identified as a potential hit with a mean percentage of growth inhibition of 48.5% over the 60-NCI cancer cell lines whereas the other fifteen compounds ranged from 0.5 to 10.72%. In MTT assay, compound 7a exhibited IC50 of 6.28 ± 0.26 µM and 17.7 ± 0.92 µM against HCT-116 colorectal cancer and WI-38 human lung fibroblast normal cell lines, respectively. In cell cycle analysis, compound 7a arrested cell cycle at G2/M phase. It was able to inhibit CDK1 (Cyclin-Dependent Kinase 1)/Cyc B (Cyclin B) complex at IC50 161.2 ± 2.7 nM. The apoptosis-inducing ability of compound 7a was assessed through apoptosis detection flow-cytometry and gene expression analysis of apoptosis markers and caspase cascade which revealed that compound 7a exerts pro-apoptotic effect and increased expression of p53, Bax, cytochrome c, caspases (-3,-8, and-9), and decreased expression of Bcl-2. This suggests that the pro-apoptotic effect is exerted through the intrinsic pathway. The molecular docking study revealed a unique binding mode at the ATP binding pocket of CDK1/Cyc B/Cks2 through its 2,4-dimethoxyphenyl-amino. These results suggest that compound 7a could be a promising hit as a targeted protein kinase inhibitor which exerts its antitumor effect through CDK1 inhibition and pro-apoptotic action.

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application.

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0–6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.