24-h Treatment Research Articles

In vitro assessment of antioxidant and anti-aging properties of ethanol extracts from Amaranthus dubius, Perilla frutescens (L.) Britton, and Phyllanthus emblica: Promising natural agents for age-related disease prevention

Age-related diseases (ARDs) and cellular senescence are significant concerns as the aging population increases in Thailand and the ASEAN region. Oxidative stress, induced by free radicals that result in the formation of senescent cells (SCs), is a primary contributor to ARDs. Antioxidant-rich plants have potential in mitigating free radical-related diseases, but their safety for normal cells requires careful investigation. This study aimed to evaluate the anti-oxidative and anti-senescence properties of Amaranthus dubius leaves (AD), Perilla frutescens (L.) Britton seeds (PF), and Phyllanthus emblica fruits (PE), commonly used in culinary and traditional medicine in northern Thailand. The study assessed the phytochemical composition of the plant extracts by GC-MS, their total phenolic content, ability to scavenge free radicals and H2O2, and their potential for DNA damage protection, cellular senescence prevention, and restoration. The results showed that all plant extracts possessed antioxidant, H2O2 scavenging, and DNA damage protection properties, with PE displaying the highest efficacy. AD and PF were potentially non-toxic, whereas PE was observed to be harmful to HDF-a after 24-h treatment, with all extracts exhibiting potential harm after 72 h. Additionally, all extracts showed preventive properties against oxidative stress and cellular senescence but were not restorative, with PE exhibiting the greatest performance. In conclusion, PE displayed the most significant preventive properties against oxidative stress and cellular senescence, making it a potential candidate for supplement development to potentially mitigate the risk of ARDs occurrence.

Discovery of a heat-generated compound DHD derived from Patrinia villosa water extract with inhibitory effects on colon cancer cells viability and migration.

Introduction: The plant Patrinia villosa Juss. (PV) has long been used as a medicinal herb for treating intestinal disorders. Pharmacological activities such as anti-oxidation, anti-inflammation, and anti-cancer effects of compounds isolated from PV have been reported, but these bioactive compounds were not derived from PV water extract (PVW). Therefore, in the present study, we aimed to identify the active component(s) of PVW which exhibit inhibitory activities in colon cancer cells viability and migration. Methods: Human colon cancer HCT116 cells were treated with the isolated compounds of PVW and then subjected to MTT and transwell migration assays. Results: Our results showed that an active compound in PVW, 8,9-didehydro-7-hydroxydolichodial (DHD) inhibited cell viability of HCT116 cells, with IC50 value at 6.1 ± 2.2μM. Interestingly, DHD was not detected in the herbal material of PV. Further investigation revealed that DHD is in fact a heat-generated compound derived from a natural compound present in PV, namely valerosidate. Valerosidate also reduced cell viability in HCT116 cells, with IC50 value at 22.2 ± 1.1μM. Moreover, both DHD (2.75μM) and valerosidate (10.81μM) suppressed cell migration in HCT116 cells, with inhibitory rates at 74.8% and 74.6%, respectively. In addition, western blot results showed that DHD (5.5μM) could significantly increase p53 expression by 34.8% and PTEN expression by 13.9%, while valerosidate (21.6μM) could increase expressions of p53 and PTEN by 26.1% and 34.6%, respectively in HCT116 cells after 48h treatment. Discussion: Taken together, this is the first report that a naturally-occurring valerosidate present in PV could actually transform to DHD by thermal hydrolysis, and both compounds exhibited inhibitory effects on cell viability and migration in HCT116 cells via increasing the expressions of tumor suppressors (p53 and PTEN). Our findings demonstrated that valerosidate is present in raw herb PV but not in PVW, while DHD is present in PVW rather than in raw herb PV. This difference in chemical profiles of raw herb and boiled water extract of PV may affect the anti-cancer activity, and hence further investigations are warranted.

Pretreatment of prostate cancer cells with salinomycin and Wnt inhibitor increases the efficacy of cabazitaxel by inducing apoptosis and decreasing cancer stem cells.

Cancer stem cells (CSCs) are associated with metastasis and recurrence in prostate cancer as well as other cancers. We aimed to enhance the sensitivity of cabazitaxel in prostate cancer cell therapy by targeting CSCs with a Wnt inhibitor and salinomycin pretreatment. PC3, DU-145, and LNCaP human prostate cancer cells were exposed to Wnt/β-catenin pathway inhibitor CCT036477 (iWnt) with salinomycin for 48h, followed by cabazitaxel treatment for 48h. Cell viability, mRNA, and protein expression changes were evaluated by MTT, RT-qPCR, and Western blot assays, respectively. Apoptosis was determined by image-based cytometry, and cell migration was assessed by wound healing assay. Three-dimensional culture was established to assess the malignant phenotype and stemness potential of transformed or cancer cells. CD44 + CSCs were isolated using magnetic-activated cell sorting system. Pretreatment of PC3, DU-145, and LNCaP cells with salinomycin iWnt significantly sensitized the cells to cabazitaxel therapy. Spheroid culture confirmed that the treatment modality was more effective than a single administration of chemotherapy. The pretreatment of PC3 cells increased the rate of apoptosis compared to single administration of cabazitaxel, which downregulated Bcl-2 and upregulated caspase 3, caspase 8 expressions. The pretreatment suppressed cell migration, downregulated the expression of Sox2 and Nanog, and significantly reduced CD44 + CSC numbers. Notably, the treatment modality reduced pAKT, p-P38 MAPK, and pERK1/2. The data suggest that pretreatment of prostate cancer cells with salinomycin and Wnt inhibitor may increase the efficacy of cabazitaxel therapy by inhibiting cell proliferation and migration, and eliminating cancer stem cells.

Curcumol, a major terpenoid from Curcumae Rhizoma, attenuates human uterine leiomyoma cell development via the p38MAPK/NF-κB pathway.

Uterine fibroids (UFs) are the most common benign tumors in women of reproductive age. Curcumae Rhizoma, the main essential oil component of which is curcumol, is widely used for the treatment of phymatosis in China due to its antitumor, anti-inflammatory, antithrombin, anti-tissue fibrosis and anti-oxygen pharmacological activities, but its potential for the treatment of UFs has not been evaluated. This study aimed to investigate the effects and mechanisms of curcumol intervention in human uterine leiomyoma cells (UMCs). Putative targets of curcumol intervention in UFs were identified using network pharmacology strategies. Molecular docking was performed to assess the binding affinity of curcumol to core targets. A concentration gradient of curcumol (0, 50, 100, 200, 300, 400 and 500μM) or RU-486 (mifepristone, 0, 10, 20, 40, 50, and 100μM) was applied to UMCs, and cell viability was detected by the CCK-8 assay. Cell apoptosis and cell cycle were examined by flow cytometry, and cell migration was assessed by a wound-healing assay. Additionally, the mRNA and protein expression levels of critical pathway components were evaluated by RT‒PCR and western blotting. Finally, the actions of curcumol on different tumor cell lines were summarized. Network pharmacology predicted 62 genes with roles in the treatment of UFs with curcumol, and MAPK14 (p38MAPK) displayed a higher interaction degree. GO enrichment and KEGG analyses revealed that the core genes were abundantly enriched in the MAPK signaling pathway. The molecular binding of curcumol to core targets was relatively stable. In UMCs, 200, 300 and 400μM curcumol treatment for 24h decreased cell viability compared with that in the control group, and the greatest effect was detected at 48h and maintained until 72h. Curcumol arrested cells in the G0/G1 phase and subsequently suppressed mitosis, promoted early apoptosis and reduced the degree of wound healing in a concentration-dependent manner in UMCs. Furthermore, 200μM curcumol decreased the mRNA and protein expression of p38MAPK, the mRNA expression of NF-κB, and the protein expression of Ki-67 and increased the mRNA and protein expression of Caspase 9. Curcumol (300 and 400μM) decreased the mRNA and protein expression of p38MAPK, NF-κB, and Ki-67 and increased the protein expression of Caspase 9 in UMCs. Curcumol was demonstrated to treat tumor cell lines, including breast cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer and nasopharyngeal carcinoma, but its effects on benign tumors have not yet been reported. Curcumol suppresses cell proliferation and cell migration while arresting the cell cycle in the G0/G1 phase and inducing cell apoptosis in UMCs via a mechanism related to p38MAPK/NF-κB pathway regulation. Curcumol may be a potential therapeutic and preventive agent in the treatment of benign tumors such as UFs.

The degradation of chicken feathers by Ochrobactrum intermedium results in antioxidant and metal chelating hydrolysates and proteolytic enzymes for staphylococcal biofilm dispersion.

The increase in the generation of chicken feathers, due to the large production of the poultry industry, has created the need to search for ecologically safer ways to manage these residues. As a sustainable alternative for recycling keratin waste, we investigated the ability of the bacterium Ochrobactrum intermedium to hydrolyze chicken feathers and the valorization of the resulting enzymes and protein hydrolysate. In submerged fermentation with three different inoculum sizes (2.5, 5.0, and 10.0mg of bacterial cells per 50mL of medium), the fastest degradation of feathers was achieved with 5.0mg cells, in which a complete decomposition of the substrate (96h) and earlier peaks of keratinolytic and caseinolytic activities were detected. In the resulting protein hydrolysate, we noticed antioxidant and Fe2+ and Cu2+ chelating activities. ABTS scavenging, Fe3+-reducing ability and metal chelating activities of the fermentative samples followed the same trend of feather degradation; as feather mass decreased in the media, these activities increased. Furthermore, we noticed about 47% and 60% dispersion of established 7-day biofilms formed by S. aureus after enzymatic treatment for 5h and 24h, respectively. These findings highlight the potential use of this bacterium as an environmentally friendly alternative to treat this poultry waste and offer valuable products.

Proteomic responses in the human dopaminergic LUHMES cell line to imidacloprid and its metabolites imidacloprid-olefin and desnitro-imidacloprid

Neonicotinoids (neonics) are amongst the most commonly used class of pesticides globally. In the United States, imidacloprid (IMI) is extensively used for agriculture and in other common applications such as house-hold pest control. Regular exposure to IMI, and several of its known metabolites including IMI-olefin and desnitro-imidacloprid (DN-IMI), has been shown to be harmful to many organisms including mammals, birds, and fish. Studies show that neonics bind human nicotinicacetylcholine receptors (nAChRs) and cause cellular toxicity. In the dopaminergic Lund human mesencephalic (LUHMES) cell line, IMI and other neonics (10–100 μM) have been recently shown to activate intracellular calcium signaling through nAChRs. Thus, we examined proteomic responses of LUHMES cells to a 48-h treatment with 50 μM IMI, IMI-olefin, or DN-IMI. Our findings show differential effects of these neonics on cellular protein expression. Bioinformatic analysis of significantly altered proteins indicates an effect of IMI, IMI-olefin, and DN-IMI on protein synthesis and ribosomal function. These findings suggest a role for protein synthesis and transcriptional regulation in neonic-mediated dopaminergic neurotoxicity.

Essential oils of the ginger plants Meistera caudata and Conamomum vietnamense: chemical compositions, antimicrobial, and mosquito larvicidal activities.

The current study describes the chemical identification, antimicrobial, and mosquito larvicidal activities of essential oils from Meistera caudata and Conamomum vietnamense, growing in Vietnam. Essential oils were extracted from the leaves and rhizomes, and characterized by the GC-FID/MS (gas chromatography-flame ionization detection/mass spectrometry) analysis. Monoterpenes (33.1-89.2 %) were the main chemical class found in these oils. β-Pinene (30.8 %) and α-pinene (23.8 %) were two major compounds in M.caudata leaf oil. C.vietnamense leaf and rhizome essential oils were dominated by 1,8-cineole (47.9-62.0 %) and limonene (10.3-16.2 %). With the same MIC (minimum inhibitory concentration) value of 25 μg/mL, C.vietnamense leaf and rhizome essential oils strongly inhibited the growth of Gram-positive bacteria Staphylococcus aureus ATCC 29213 and Bacillus subtilis ATCC 6501, respectively. For 24 and 48-h treatments, C.vietnamense leaf essential oil strongly controlled the growth of mosquito Aedes aegypti with the respective LC50 values of 7.67 and 6.73 μg/mL, and the respective LC90 values of 13.37 and 10.83 μg/mL. In the same manner, C.vietnamense rhizome essential oil also showed strong mosquito larvicidal activity against Aedes albopictus with the LC50 values of 12.37 and 12.00 μg/mL, and the LC90 values of 20.56 and 18.58 μg/mL, respectively. C.vietnamense essential essential oils containing a high amount of 1,8-cineole are generally better than M.caudata essential essential oils in both two biological assays.

Antigenotoxic effect of hyperoside against Mitomycin C and hydrogen peroxide-induced genotoxic damage on human lymphocytes

Hyperoside is a flavonol glycoside isolated from various plant genera such as Hypericum and Crataegus. It has an important place in the human diet and is used medically to relieve pain and ameliorate cardiovascular functions. However, a comprehensive profile of the genotoxic and antigenotoxic effects of hyperoside is not known. The current study aimed to investigate the genotoxic and antigenotoxic effects of hyperoside against genetic damages induced by two genotoxins (MMC and H2O2) using chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) assays in human peripheral blood lymphocytes in vitro. Blood lymphocytes were incubated with 7.8–62.5 μg/mL concentrations of hyperoside alone and simultaneously with 0.20 μg/mL Mitomycin C (MMC) or 100 μM Hydrogen peroxide (H2O2). Hyperoside did not exhibit genotoxic potential in the CA, SCE, and MN assays. Moreover, it did not cause a decrease in mitotic index (MI) which is an indicator of cytotoxicity. On the other hand, hyperoside significantly decreased CA, SCE, and MN (except for MMC treatment) frequencies induced by MMC and H2O2. Hyperoside, increased mitotic index against both mutagenic agents at 24-h treatment when compared to positive control. Our results demonstrate that hyperoside exhibited antigenotoxic effects rather than genotoxic in vitro human lymphocytes. Therefore, hyperoside may be a potential preventive agent in inhibiting chromosomal and oxidative damage induced by genotoxic chemicals.

Single therapeutic dose of an antiviral UL29 siRNA swarm diminishes symptoms and viral load of mice infected intranasally with HSV-1.

Herpes simplex virus type 1 (HSV-1) is a human pathogen that causes recurrent infections. Acyclovir-resistant strains exist and can cause severe complications, which are potentially untreatable with current therapies. We have developed siRNA swarms that target a 653 base pair long region of the essential HSV gene UL29. As per our previous results, the anti-UL29 siRNA swarm effectively inhibits the replication of circulating HSV strains and acyclovir-resistant HSV strains in vitro, while displaying a good safety profile. We investigated a single intranasal therapeutic dose of a siRNA swarm in mice, which were first inoculated intranasally with HSV-1 and given treatment 4h later. We utilized a luciferase-expressing HSV-1 strain, which enabled daily follow-up of infection with in vivo imaging. Our results show that a single dose of a UL29-targeted siRNA swarm can inhibit the replication of HSV-1 in orofacial tissue, which was reflected in ex vivo HSV titers and HSV DNA copy numbers as well as by a decrease in a luciferase-derived signal. Furthermore, the treatment had a tendency to protect mice from severe clinical symptoms and delay the onset of the symptoms. These results support the development of antiviral siRNA swarms as a novel treatment for HSV-1 infections.

Chemical chaperone 4-PBA mitigates TNF alpha mediated ER stress in hASM cells

Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα selectively activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) ER stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyrate (4-PBA) may ameliorate ER stress activation. In the present study, we hypothesize that 4-PBA mitigates TNFα induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from 5 donors with no history of current smoking or respiratory diseases. hASM cells were serum-deprived for 48 h, and phenotype confirmed by expression of smooth muscle actin and fusiform morphology. hASM cells from the same donor were then separated into 4 12-h treatment groups: 1) TNFα (20 ng/mL), 2) TNFα + 4-PBA (30 min pretreatment), and 3) untreated control. Protein expression and phosphorylation of IRE1α and XBP1s were determined by Western blot. Gene expression of XBP1s was analyzed by PCR. We found that TNFα induced IRE1α phosphorylation and XBP1 splicing, which were significantly reduced by pretreatment with 4-PBA. These results support our hypothesis and suggest that, in the presence of chemical chaperone 4-PBA, TNFα induced ER stress is significantly decreased. R01HL 157984 (GCS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Defective Induction of IL-27-Mediated Immunoregulation by Myeloid DCs in Multiple Sclerosis.

The purpose of this study was to examine whether myeloid dendritic cells (mDCs) from patients with multiple sclerosis (MS) and healthy controls (HCs) become similarly tolerogenic when exposed to IL-27 as this may represent a potential mechanism of autoimmune dysregulation. Our study focused on natural mDCs that were isolated from HCs and MS patient peripheral blood mononuclear cells (PBMCs). After a 24-h treatment with IL-27 ± lipopolysaccharide (LPS), the mDCs were either harvested to identify IL-27-regulated gene expression or co-cultured with naive T-cells to measure how the treated DC affected T-cell proliferation and cytokine secretion. mDCs isolated from HCs but not untreated MS patients became functionally tolerogenic after IL-27 treatment. Although IL-27 induced both HC and untreated MS mDCs to produce similar amounts of IL-10, the tolerogenic HC mDCs expressed PD-L2, IDO1, and SOCS1, while the non-tolerogenic untreated MS mDCs expressed IDO1 and IL-6R. Cytokine and RNA analyses identified two signature blocks: the first identified genes associated with mDC tolerizing responses to IL-27, while the second was associated with the presence of MS. In contrast to mDCs from untreated MS patients, mDCs from HCs and IFNb-treated MS patients became tolerogenic in response to IL-27. The genes differentially expressed in the different donor IL-27-treated mDCs may contain targets that regulate mDC tolerogenic responses.

Investigation on the Biological Safety and Activity of a Gluconolactone-Based Lotion for Dermocosmetic Application.

(1) Background: Cosmeceuticals are formulas enriched with active ingredients that exert efficacy on different skin molecular targets. (2) Methods: Cell viability and the absence of potential irritant risk were evaluated on keratinocytes (HaCaT), fibroblasts (NHDF), adipocytes (3T3-L1), sebocytes (PCi-SEB_CAU) and reconstructed human epidermis (RHE), respectively. Several treatments were performed to evaluate the ability of the lotion to stimulate the production of collagen and elastin, stimulate the differentiation of keratinocytes and reduce the number of senescent cells following UVB stimulation. In addition, the modulation of genes involved in the production, storage and accumulation of sebum were investigated. (3) Results: The results obtained demonstrated the biosafety of the formula in all cell lines tested. The 24-h treatment with non-cytotoxic concentrations determined an increase in the expression of the collagen (COL1A1), elastin (ELN) and involucrin (IVL) genes, while a reduction of peroxisome proliferator-activated receptor-gamma (PPARγ) gene expression and a reduction of SA-βgal-positive cells were found. Moreover, the treatment did not interfere with normal steroid 5-alpha reductase (5RDA3) gene expression levels. (4) Conclusions: Data collected demonstrated the biosafety of the lotion, the non-comedogenic property and a multi targets anti-aging effect. In particular, data collected on the booster lotion make it a valid way to counteract the pore dilatation aging related.

Deletion of Cyclic GMP-AMP Synthase Aggravates Concanavalin A-Induced Acute Hepatic Injury by Facilitating Leukocyte Chemotaxis

Growing evidence demonstrates that cyclic GMP-AMP synthase (cGAS), as a cytosolic DNA sensor, is essential for activating innate immunity and regulating inflammatory response against cellular damage. However, its role in immune-mediated hepatitis remains unclear. Here by challenging the cGAS knockout (KO) and their littermate wide-type (WT) mice with intravenous ConA injection to induce acute immune-mediated liver injury, we found that lack of cGAS drastically aggravated liver damage post ConA treatment for 24h, reflected by increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and amplified hepatic necrosis. The number of apoptotic hepatocytes was also significantly increased in the KO mice. RNA-sequencing analysis revealed that leukocyte chemotaxis and migration-related genes were remarkably upregulated in the KO livers. Consistently, immunofluorescence assays illustrated that the infiltrating F4/80-positive macrophages, Ly6G-positive neutrophils, and CD3-positive T cells were all significantly increased in the KO liver sections. The hepatic expression of the pro-inflammatory genes was elevated as well. Supporting the in vivo findings, the knockdown of cGAS in cultured macrophages showed promoted migration potential and enhanced pro-inflammatory gene expression. These results collectively demonstrated that deletion of cGAS could aggravate ConA-induced acute liver injury, at least at the 24-h time point, and its mechanism might be related to facilitating leukocyte chemotaxis and promoting liver inflammatory response.

Preparation of sludge-based micro-electrolysis filler and its application in pharmaceutical wastewater treatment by an up-flow aerated filter

Sewage sludge (SS) and raw pharmaceutical wastewater (RPW) are both toxic and harmful wastes, which are a menace to human and animal health and the ecosystem. A sludge-base micro-electrolysis filler (SMEF) was gained by SS and Fe powder as the primary raw materials. The preparation process of the SMEF was achieved based on the tetracycline hydrochloride (TCH) removal efficiency. The physicochemical characteristics (e.g., surface area, morphology features, function groups, and valence state of Fe) of the obtained SMEF were decided. With an Fe/SS ratio of 1/2, a sintering temperature of 1050°C, a sintering time of 30min, an initial pH of 3, and a filler dosage of 100g/L, the SMEF demonstrated a high degradation ability for TCH with a removal rate reached 95.62% in 24h. Kinetic analysis showed that the adsorption process of TCH was consistent with the pseudo-first-order Lagergren kinetic model. Moreover, degradation mechanism analysis showed that TCH was gradually degraded through dehydroxylation, demethylation, ring opening, oxidation, and reduction in solution. The SMEF had a good continuous removal performance for contaminants in RPW in an up-flow aerated filter. The removal efficiency of TOC and TN reached 46.60 and 42.27% within 24h, respectively. The treated pharmaceutical wastewater was considered non-biotoxic after 24-h treatment with the SMEF. This study presents a innovative Fe-C micro-electrolysis filler based on SS and is important to the environmental-friendly recycling of SS and sustainable treatment of RPW, achieving the purpose of waste disposal.

Glucose-conjugated glutenin nanoparticles for selective targeting and delivery of camptothecin into breast cancer cells.

Receptor-mediated drug delivery systems are a promising tool for targeting malignant cells to suppress/inhibit the malignancy without disturbing healthy cells. Protein-based nanocarrier systems possess numerous advantages for the delivery of variety of chemotherapeutics, including therapeutic peptides and genes. In the present work, glucose-conjugated camptothecin-loaded glutenin nanoparticles (Glu-CPT-glutenin NPs) were fabricated to deliver camptothecin to MCF-7 cells via GLUT-1 transporter protein. Initially, Glu-conjugated glutenin polymer was successfully synthesized through reductive amination reaction, and this was confirmed by FTIR and 13C-NMR. Then, camptothecin (CPT) was loaded into Glu-conjugated glutenin polymer forming Glu-CPT-glutenin NPs. The nanoparticles were studied for their drug releasing capacity, morphological shape, size, physical nature, and zeta potential. The fabricated Glu-CPT-glutenin NPs were found to be spherical in shape and amorphous in nature with 200-nm size range and a zeta potential of - 30mV. Furthermore, MTT assay using Glu-CPT-glutenin NPs confirmed concentration-dependent cytotoxicity against MCF-7 cells after 24-h treatment, and IC50 was found to be 18.23μgmL-1. In vitro cellular uptake study demonstrated that the Glu-CPT-glutenin NPs had enhanced endocytosis and delivered CPT in MCF-7 cells. A typical apoptotic morphological change of condensed nuclei and distorted membrane bodies was found after treatment with IC50 concentration of NPs. The released CPT from NPs also targeted mitochondria of MCF-7 cells, significantly increasing the level of reactive oxygen species and causing the damage of mitochondrial membrane integrity. These outcomes confirmed that the wheat glutenin can positively serve as a significant delivery vehicle and enhance the anticancer potential of this drug.

Abstract 5729: Imiqualines for pancreatic cancer: first-in-class potent and synergistic inhibitors of microtubule polymerisation

Abstract Background: The survival rate for patients with Pancreatic Ductal Adenocarcinoma (PDAC) is dramatically poor with a five-year survival rate less than 10%. The research of new treatments, which could complement the current therapeutic arsenal constituted by Gemcitabine, FOLFIRINOX (fluorouracile, leucovorin, irinotecan, oxaliplatin) and nab-paclitaxel, is a major challenge. Imiqualines are new original small heterocyclic chemical molecules based on the quinoxalinic moiety. Among these first in class compounds, the lead EAPB02303 (1) displays outstanding nanomolar activities comparable to those of the current best anticancer agents on a panel of human cancer cell lines, notably on poorly sensitive cancer like PDAC and melanoma. We tested here if EAPB02303 could be an attractive first in class molecule in PDAC and we conducted in-deep molecular characterization and bioinformatics studies to decipher its mechanism of action. Methods: We characterized EAPB02303 effect on tumor growth in-vitro by conducting sulforhodamine B assay on a panel of PDAC cells including cells derived from PDX (Patient Derived Xenograft) and 3D models with Cancer Associated Fibroblasts. We assessed in-vivo activity on subcutaneous PDAC xenografts mouse models. We then studied EAPB02303 effect on cell cycle, apoptosis and microtubule polymerisation by flow cytometry and immunofluorescence. We analyzed mRNAseq and Reverse Phase Protein Assay (RPPA) data of PDAC cell lines treated with EAPB02303 at multiple time points and concentrations to identify signaling pathways and key proteins implicated in EAPB02303 effect. We performed differential gene expression and gene set enrichment analysis by using EdgeR, Deseq2 and fgsea packages. We also used PharmacoGx package to seek for similar transcriptomic profiles among the CMAP perturbational database. Results: We showed that EAPB02303 exerts activity at low nanomolar concentrations in-vitro in PDAC cell lines and 3D models, and is able to reduce tumor growth in our xenografts in-vivo mouse models. We also found a potent synergy with Paclitaxel at lower concentrations of both compounds. Furthermore, we found that EAPB02303 induces mitosis arrest and impairment of spindle assembly after 24h treatment. Cells also underwent apoptosis after 48h treatment. mRNAseq and RPPA data showed activation of several signaling pathways including MAPK kinases. CMAP database mining revealed a high connectivity score of transcriptomic signatures between EAPB02303 and inhibitors of microtubule polymerization. All these data suggest that EAPB02303 is a new microtubule-disrupting agent with in-vivo activity in PDAC and in-vitro synergy with Paclitaxel, showing potential for future clinical investigations. (1) Imidazo[1,2a]quinoxalines and derivatives thereof for treating cancers. WO 2009 043934A1. Deleuze-Masqeufa C. et al. Citation Format: Kevin Bigot, Véronique Garambois, Nadia Vie, Marine Bruciamacchie, Pierre-Emmanuel Colombo, Diego Tosi, Cindy Patinote, Yann Maggipinto, Pierre-Antoine Bonnet, Céline Gongora, Carine Deleuze-Masquefa, Christel Larbouret. Imiqualines for pancreatic cancer: first-in-class potent and synergistic inhibitors of microtubule polymerisation. [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 5729.

Abstract 5282: Mitotane induces GDF-15 expression in vitro and in vivo: A potential therapy-induced mechanism of immune escape in adrenocortical carcinoma

Abstract Context: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with poor prognosis and limited treatment options. The only approved therapy is mitotane, an adrenal toxicant, alone or in combination with cytotoxic chemotherapy. Response rate is only ~20%. Results of later line immune checkpoint inhibition (ICI) in ACC are disappointing, most likely due to an immunologically cold, immunosuppressive microenvironment. Among the factors induced in mitotane-treated ACC cell lines, we previously found growth/differentiation factor 15 (GDF-15), an anti-inflammatory cytokine physiologically expressed during pregnancy. Aim: As GDF-15 is an emerging mediator of tumor immune escape, we investigated changes in GDF-15 secretion upon mitotane treatment in ACC in vitro and in vivo, and explored a potential correlation with response to therapy and survival. Methods: Four different human ACC cell lines (NCI-H295R, CU-ACC1, CU-ACC2 and JIL-2266) were treated with mitotane. GDF-15 secretion and expression were determined by ELISA and immunoblot. GDF-15 levels in sera of 142 ACC patients (94 prior and 48 during mitotane) were analyzed by ELISA and correlated with survival from the time point of blood collection. GDF-15 serum levels were quantified in three responders and seven non-responders to ICI treatment. Results: In vitro, 24h treatment with 25 µM mitotane-induced GDF-15 secretion and expression in all cell lines by up to one order of magnitude. In 32 patient samples, GDF-15 serum concentrations increased from 0.6±1 ng/l prior mitotane to 2.1 ± 2.7 ng/l after mitotane initiation, p<0.0001.Importantly, patients with low serum GDF-15 levels before and during mitotane treatment had a significantly longer overall survival compared to patients with higher GDF-15 serum levels. This association retained statistical significance after adjustment for the known prognostic factors Ki67 and ENSAT tumour stage in the mitotane treated group (95% CI 1.15-.1.73, HR1.41, p=0.001). In an exploratory cohort of three responders and seven non-responders to ICI we found a trend for higher GDF-15 serum concentrations in the non-responders. Conclusion: Mitotane induces GDF-15 both in vivo and in vitro and potentially contributes to the poor response rates to ICI in ACC. Citation Format: Isabel Weigand, Tanja Anderlik, Jochen Schreiner, Hanna Remde, Otilia Kimpel, Laura-Sophie Landwehr, Eva Hoster, Florian Wedekink, Alexandra S. Triebig, Tanja Maier, Jörg Wischhusen, Martin Reincke, Martin Fassnacht, Matthias Kroiss. Mitotane induces GDF-15 expression in vitro and in vivo: A potential therapy-induced mechanism of immune escape in adrenocortical carcinoma. [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 5282.

Greenhouse test of spraying dsRNA to control the western flower thrips, Frankliniella occidentalis, infesting hot peppers

BackgroundThe western flower thrips Frankliniella occidentalis is an insect pest that damages various crops, including hot peppers. It is a vector of a plant pathogen, tomato spotted wilt virus. To control this pest, chemical insecticides have been used in the past, but the control efficacy is unsatisfactory owing to rapid resistance development by F. occidentalis.Methodology: This study reports a novel control technology against this insect pest using RNA interference (RNAi) of the vacuolar-type ATPase (vATPase) expression. Eight subunit genes (vATPase-A ∼ vATPase-H) of vATPase were obtained from the F. occidentalis genome and confirmed for their expressions at all developmental stages.ResultsDouble-stranded RNAs (dsRNAs) specific to the eight subunit genes were fed to larvae and adults, which significantly suppressed the corresponding gene expressions after 24-h feeding treatment. These RNAi treatments resulted in significant mortalities, in which the dsRNA treatments at ∼2,000 ppm specific to vATPase-A or vATPase-B allowed complete control efficacy near 100% mortality in 7 days after treatment. To prevent dsRNA degradation by the digestive proteases during oral feeding, dsRNAs were formulated in a liposome and led to an enhanced mortality of the larvae and adults of F. occidentalis. The dsRNAs were then sprayed at 2,000 ppm on F. occidentalis infesting hot peppers in a greenhouse, which resulted in 53.5–55.9% control efficacy in 7 days after treatment. Even though the vATPases are conserved in different organisms, the dsRNA treatment was relatively safe for non-target insects owing to the presence of mismatch sequences compared to the dsRNA region of F. occidentalis.ConclusionThese results demonstrate the practical feasibility of spraying dsRNA to control F. occidentalis infesting crops.

Abstract 4749: Effects of miR-34a-5p and miR-181b-5p silencing and induction on their potential targets in uterine leiomyosarcoma cells

Abstract Background: Leiomyosarcoma (LMS) is a malignant tumor of the uterine body that represents 40-60% of all gynecological sarcomas. Up to now, there is no specific treatment or diagnostic biomarkers for this tumor. The identification and characterization of miRNAs signature could help to its management. In addition, miRNAs represent targets for therapies due to their ability to modulate various signaling pathways simultaneously. Thus, the present study aimed to evaluate the effects of genetic manipulation of miR-34a and miR-181b on their predicted target genes in LMS cells. Methods: SK-UT-1 cells from uterine LMS were transfected with synthetic miRNA mimics and inhibitors (siRNA) of miR-34a and miR-181b. The efficacy of transfection was assessed by miRNA expression (qRT-PCR) assays. In order to identify potential genetic interactions network of miR-34a and 181b, predicted target genes were selected by in silico analysis (miRTargetLink 2.0 and miRDB). After SK-UT-1 cells transfection, the expression of target genes was evaluated by qRT-PCR. Results: SK-UT-1 cells showed greater efficiency of mimics and siRNA transfections after 48h treatment for both miR-34a and 181b. MDM4, TP53, BCL2, KMT2D, NOTCH2, and CCND1 were identified as predicted target genes of miR-34a; and the TIMP3, FGFR1, BCL2, NOTCH2, ATM, and IRS1 target genes for miR-181b. Expression profile of selected target genes was assessed after each treatment. The miR-34a mimic significantly increased the expression of MDM4 after 96h of treatment, while the BCL2 and TP53 showed an increase in expression with siRNA after 48h of treatment. CCND1 showed a greater expression in both treatments at 48h. An increase in KMT2D and NOTCH2 expression was also observed after treatment, even though without statistically significant. Treatment with miR-181b mimic led to increased FGFR1 expression within 72h. Although some expression alterations were observed in TIMP3, BCL2, NOTCH2, ATM, and IRS1 genes after mimic and siRNA treatments, there was no statistical significance. Conclusion: The effect of miR-34a upregulation leads to induction MDM4 and CCND1 expression, the first being a TP53 inhibitor and the second, responsible for cell cycle maintenance. The miR-34a downregulation had an inductor effect on expression of MDM4, BCL2 (apoptosis regulator), TP53 (tumor suppressor), and CCND1. The miR-181b upregulation induced the expression of fibroblast growth factor receptor, FGFR1. In uterine LMS cells, miR-34a seems to exert an important role in regulating the expression of cell cycle and apoptosis-related genes. Citation Format: Bruna C. de Almeida, Laura G. dos Anjos, Katia C. Carvalho. Effects of miR-34a-5p and miR-181b-5p silencing and induction on their potential targets in uterine leiomyosarcoma cells. [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 4749.

Gut Microbiota Metabolites Differentially Release Gliotransmitters from the Cultured Human Astrocytes: A Preliminary Report

Butyrate and indole-3-propionic acid represent the CNS-available gut microbiota metabolites exhibiting potentially beneficial effects on human brain function and being tested as antidepressants. Astrocytes represent one of the putative targets for the gut metabolites; however, the mechanism of action of butyrate and indole-3-propionic acid is not well understood. In order to test this mechanism, a human astrocyte cell-line culture was treated with the compounds or without them, and the supernatants were collected for the analysis of ATP and glutamate gliotransmitter release with the use of luminescent and fluorescent methods, respectively. A 10-min incubation of astrocytes with 1-5 mM butyrate increased the ATP gliotransmitter release by 78% (95%CI: 45-119%), p < 0.001. The effect was found to be mediated by the cytosolic Ca2+ mobilization. Both 10-min and 24-h treatments with indole-3-propionic acid produced no significant effects on the release of gliotransmitters. The results for glutamate release were inconclusive due to a specific glutamate release pattern discovered in the tested model. This preliminary report of butyrate-induced ATP gliotransmitter release appears to provide a novel mechanistic explanation for the beneficial effect of this gut microbiota metabolite on brain function; however, the results require further evaluation in more composed models.