RPMI Medium Research Articles

Regulatory Effects of the Silymarin on Expression of OCT4, NANOG, and P53 in MCF7 Cell Lines

Background:: Breast cancer was known as the second most common cause of death in the world, natural sources compound derived from milk thistle called silymarin had already shown anticancer properties. Objective: In the present study, silymarin was used to treat MCF7 cells and inhibition of stem cell pluripotency genes, as well as cell proliferation. Methods: MCF7 cells were cultured in the presence of RPMI-1640 medium consisting of various silymarin extract concentrations (10, 100, 500, 1000, 2000, 3000, 4000, and 5000 µg/mL) for 24, 48, and 72 hours. The inhibitory effects of the compound on cellular proliferation were assessed via employing MTT assay techniques. Following confirming apoptosis, the fold changes of OCT4, NANOG and P53 expression were determined by quantitative Real-Time PCR. Results: There was a significant difference (p value<0.05) in cell viability when various concentrations of silymarin extract were used for 24, 48, and 72 h in comparison to the control. Real-Time- PCR analysis indicated that the expression of OCT4 and NANOG was downregulated while P53 upregulated in compare to untreated control cells (p value <0.05). Conclusion: According to these findings, the silymarin effects on MCF7 cell line and act via modulating OCT4, NANOG, and P53 pathway mediators. Silymarin may introduce this compound as a promising therapeutic compound against MCF7.

THE RESTORATION OF HYBRIDOMA CELLS LINES AFTER UNSUITABLE STORAGE

The ongoing war in Ukraine has disrupted the infrastructure of cryopreservation, compromising the viability of hybridoma cell lines that are critical to therapeutic antibody production. This study focuses on hybridoma cells that produce antibodies targeting protein C. They were damaged by wrong preservation conditions: in a freezer at -80 °C. The aim of the study was to identify optimal culture conditions for restoring the functionality and antibody production capacity of these compromised hybridoma cells. Materials and methods. The cryopreserved hybridoma clone, producing the IV-6A antibody, was used. After thawing, the cells were cultured in RPMI medium supplemented with 10% of three different commercially available Fetal Bovine Serum (FBS) variants: F7524 and F2442 (Sigma, USA) and One Shot format A5256701 (Gibco, USA). Cell vitality was monitored through daily observations with a light microscope. Quality assessment of antibody production was conducted through ELISA analysis. The binding capacity of the purified antibody was compared against a reference. Results. Hybridoma cells cultured in RPMI medium with F7524 or F2442 FBS exhibited a significant decrease in viability by day 4. Conversely, cells cultured with A5256701 FBS displayed high viability and an exponential growth pattern. ELISA confirmed that these cells retained the ability to produce the monoclonal antibody IV-6A. However, the purified antibody from the restored cell line showed a slightly less intensive binding capacity compared to the reference antibody. Conclusions. Despite the lasting improper storage conditions, the hybridoma cells were successfully restored retaining their ability to produce monoclonal antibodies specific to protein C.

Effect of hydroxyapatite nanoparticles coating of titanium surface on biofilm adhesion: An in vitro study

AimTo evaluate the adhesion of mono and duospecies biofilm on a commercially available dental implant surface coated with hydroxyapatite nanoparticles (nanoHA). Material and methodsTitanium discs were divided into two groups: double acid-etched (AE) and AE coated with nanoHA (NanoHA). Surface characteristics evaluated were morphology, topography, and wettability. Mono and duospecies biofilms of Streptococcus sanguinis (S. sanguinis) and Candida albicans (C. albicans) were formed. Discs were exposed to fetal bovine serum (FBS) to form the pellicle. Biofilm was growth in RPMI1640 medium with 10% FBS and 10% BHI medium for 6 h. Microbial viability was evaluated using colony-forming unit and metabolic activity by a colorimetric assay of the tetrazolium salt XTT. Biofilm architecture and organization were evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). ResultsAE surface had more pores, while NanoHA had even nanoHA crystals distribution. Roughness was similar (AE: 0.59 ± 0.07 µm, NanoHA: 0.69 ± 0.18 µm), but wettability was different (AE: Θw= 81.79 ± 8.55°, NanoHA: Θw= 53.26 ± 11.86°; P = 0.01). NanoHA had lower S. sanguinis viability in monospecies biofilm (P = 0.007). Metabolic activity was similar among all biofilms. In SEM both surfaces on C. albicans biofilm show a similar distribution of hyphae in mono and duospecies biofilms. AE surface has more S. sanguinis than the NanoHA surface in the duospecies biofilm. CLSM showed a large proportion of live cells in all groups. ConclusionsThe nanoHA surface reduced the adhesion of S. sanguinis biofilm but did not alter the adhesion of C. albicans or the biofilm formed by both species.

Fish arginase constrains excessive production of nitric oxide and limits mitochondrial damage during Aeromonas hydrophila infection

Bacteria-enhanced inducible nitric oxide synthase (iNOS) overproduces nitric oxide (NO) leading to mitochondrial and cellular damage. In mammals, arginase (ARG), the enzyme consuming the same substrate l-arginine with iNOS, was believed to inhibit iNOS activity by competing the substrate. But in fish, this conception has been widely challenged. In this study, the gene expression using real-time quantitative PCR (RT-qPCR) technology showed that when stimulated by Aeromonas hydrophila (A. hydrophila), grass carp (gc) iNOS was up-regulated in head kidney monocytes/macrophages (M0/MФ), and its changes were not detected in the whole tissue of liver or spleen, showing a high degree of cell-specific expression pattern. At the same time, gcARG2 had a high basal expression in tissues and was up-regulated by A. hydrophila stimulation. Next, phthalaldehyde-primaquine reaction was first used in the determination of intracellular urea in fish cells. It was found that the induced gcARG2 led to an increase in the intracellular urea content. Moreover, urea and NO production in M0/MФ were increased in a substrate dose-dependent manner from 30 to 100 μM of l-arginine and reached the highest yield at 300 and 3000 μM of l-arginine, respectively. Furthermore, head kidney M0/MФ was cultured in RPMI1640 medium containing physiological concentration (500 μM) of l-arginine to evaluate the effect of ARG. Under A. hydrophila stimulation, treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) showed that inhibition of arginase could further enhance the NO production stimulated by A. hydrophila. This in turn led to a cumulation in peroxynitrite (ONOO-) content and an injury of the mitochondrial membrane potential. Our study showed for the first time that fish ARG in head kidney M0/MФ can limit excessive production of NO and harmful products by iNOS to maintain mitochondrial and cellular homeostasis.

Effect of Celastrus Orbiculatus Extract on proliferation and apoptosis of human Burkitt lymphoma cells.

The lymphoma incidence rate is on the rise, with invasive forms particularly prone to relapse following conventional treatment, posing a significant threat to human life and wellbeing. Numerous studies have shown that traditional Chinese botanical drug medicine offers promising therapeutic benefits for various malignancies, with previous experimental findings indicating that Celastrus orbiculatus extract effectively combats digestive tract tumors. However, its impact on lymphoma remains unexplored. This study aims to investigate the impact and underlying mechanisms of COE on the proliferation and apoptosis of Burkitt lymphoma cells. We diluted COE in RPMI-1640 medium to create various working concentrations and introduced it to human Burkitt lymphoma Raji and Ramos cells. To evaluate cell viability, we used the CCK-8 assay, and we observed morphological changes using HE staining. We also conducted Annexin V-PI and JC-1 staining experiments to assess apoptosis. By combining the cell cycle experiment with the EDU assay, we gained insights into the effects of COE on DNA replication in lymphoma cells. Using Western blotting, we detected alterations in apoptosis-related proteins. In vivo experiments revealed that following COE intervention, tumor volume decreased, survival time was prolonged, spleen size reduced, and the expression of tumor apoptosis-related proteins changed. Our findings indicate that COE effectively inhibits lymphoma cell proliferation and promotes apoptosis by regulating these apoptosis-related proteins.

Spontaneous and LPS-induced cytokine production in supernatants of cell cultures isolated from a skin biopsy in a mouse model of acute psoriasis-like dermatitis

The purpose of the work performed was to obtain data on spontaneous and LPS-stimulated cytokine production in cell culture supernatants isolated from isolated from skin bioptates of C57BL/6 mice in an imiquimod-induced psoriasis model. Methods. Skin cells were isolated by spontaneous migration and enzymatic dissociation in a collagenase solution. Cells isolated from the skin were cultured for 24 hours in RPMI-1640 (spontaneous cytokine production) or RPMI-1640 medium with the addition of a solution (10 micrograms/ml) of E. coli LPS (induced). Cytokine levels were determined by flow laser cytofluorometry using the Mouse Th1/Th2/Th17 multiplex test system (Antigenix America, USA) on an FC-500 cytometer. Results. In the focus of imiquimod-induced inflammation, an increase in the spontaneous production of proinflammatory cytokines IL-1, IL-5, IL-17 was observed by 2.4-3.8 times. At the same time, a 1.9-fold inhibition of IFNy synthesis was detected when cultivating skin cells in the presence of bacterial LPS.

Optimal conditions for adenoviral transduction of immature dendritic cells without affecting the tolerogenic activity of DC-based immunotherapy

Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes. In the mouse heart transplant model, imDCs with unstable phenotype and function due to adenoviral transduction resulted in an increased proportion of Th1 and Th17 cells in recipients. However, these effects can be managed, and our proposed optimized transduction strategy significantly minimizes these adverse effects. Our study holds significant implications for the development and optimization of immunotherapy utilizing tolerogenic dendritic cells.

Dispersion and Dosimetric Challenges of Hydrophobic Carbon-Based Nanoparticles in In Vitro Cellular Studies.

Methodologies across the dispersion preparation, characterization, and cellular dosimetry of hydrophilic nanoparticles (NPs) have been developed and used extensively in the field of nanotoxicology. However, hydrophobic NPs pose a challenge for dispersion in aqueous culture media using conventional methods that include sonication followed by mixing in the culture medium of interest and cellular dosimetry. In this study, a robust methodology for the preparation of stable dispersions of hydrophobic NPs for cellular studies is developed by introducing continuous energy over time via stirring in the culture medium followed by dispersion characterization and cellular dosimetry. The stirring energy and the presence of proteins in the culture medium result in the formation of a protein corona around the NPs, stabilizing their dispersion, which can be used for in vitro cellular studies. The identification of the optimal stirring time is crucial for achieving dispersion and stability. This is assessed through a comprehensive stability testing protocol employing dynamic light scattering to evaluate the particle size distribution stability and polydispersity. Additionally, the effective density of the NPs is obtained for the stable NP dispersions using the volumetric centrifugation method, while cellular dosimetry calculations are done using available cellular computational modeling, mirroring approaches used for hydrophilic NPs. The robustness of the proposed dispersion approach is showcased using a highly hydrophobic NP model (black carbon NPs) and two culture media, RPMI medium and SABM, that are widely used in cellular studies. The proposed approach for the dispersion of hydrophobic NPs results in stable dispersions in both culture media used here. The NP effective density of 1.03-1.07 g/cm3 measured here for black carbon NPs is close to the culture media density, resulting in slow deposition on the cells over time. So, the present methodology for dispersion and dosimetry of hydrophobic NPs is essential for the design of dose-response studies and overcoming the challenges imposed by slow particle deposition.

2-aryloxazolines inhibit Candida clinical isolates growth and morphogenesis of Candida albicans and Candida tropicalis

New antifungal molecules are being researched addressing new targets and mechanisms of action. Therefore, the present study aimed to evaluate the antifungal activity of 2-aryloxazoline derivatives (4i and 9i) on Candida clinical isolates and on morphogenesis of competent-filament species. Both compounds inhibited Candida spp. growth at concentrations ≤0.03–2 μg/mL, including less susceptible and resistant isolates to standard antifungals. However, neither fungicidal effect of compounds nor synergistic/antagonistic interactions in combination with antifungals were observed. On the other hand, C. albicans treated with 2-aryloxazoline exhibited some ultrastructural changes such as the integrity loss of the plasma membrane and cell wall. In C. albicans, the proteinase activity was reduced after treatment with high concentrations of 2-aryloxazoline, but this inhibition was not observed in C. tropicalis. The compounds inhibited up to 50% of the total biomass of C. albicans and C. tropicalis biofilms but did not inhibit the metabolic activity. Additionally, 2-aryloxazolines caused a dose-dependent reduction in the pseudohyphae/hyphae formation of both Candida species (C. albicans and C. tropicalis) in RPMI medium and other filament-inducing media. Notably, the compounds caused a total inhibition of pseudohyphae/hyphae formation in Spider broth, a partial inhibition in Lee medium, and no inhibition in SM supplemented with N-acetylglucosamine. This effect on the yeast-hyphae transition was associated with blockage of cAMP and MAPK pathways due to the gene downregulation of the transcription factors TEC1, EFG1, and CEK1 in 4i-treated C. albicans cells. In this regard, the adhesins (HWP1 and ALS3) and candidalysin (ECE1) genes were also downregulated as a result of the interference of compounds on the filamentation signaling pathways. Therefore, 2-aryloxazolines showed promising results inhibiting Candida growth and morphogenesis; however, other approaches should be carried out to better elucidate other possible mechanisms of action.

Abstract 2976: The potential use of butyrate and propionate to inhibit oncogenic phenotypes in oral squamous cell carcinoma and triple negative breast cancer

Abstract Background Oral squamous cell carcinoma (OSCC) and triple-negative breast cancer (TNBC) are aggressive cancers with low survival rates, particularly in Hispanics. Since the underpinning mechanisms of oncogenesis for both OSCC and TNBC are multifactorial, the development of novel targeted therapies is limited. The tumor microbiome has been shown to modulate the microenvironment. The microbial product short-chain fatty acids (SCFA) may represent a putative target for treatment as they can potentially inhibit cell growth, proliferation, migration, and induce morphologic changes in cancer cells. We evaluated the impact of the SCFAs butyrate, acetate, and propionate on the oncogenic phenotypes of OECM-1 and MDA-231 cell line models for OSCC and TNBC respectively. Methods OECM-1 and MDA-231 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS. Cytotoxicity was determined using an alamarBlue assay by treating the cells with SCFA in a series of 7 concentrations starting at 125 mM (1:2 dilution factor) for 24, 48, and 72 hours (h). Proliferation quantification was performed using trypan blue dye after treatment with SCFA for 24h, 48h and 72h. Migration and invasion were measured after treatment for 24h with SCFAs. Comparative analysis of our results was performed using GraphPad Prism v10. Results Higher concentrations of all SCFA decreased cell viability in OSCC and TNBC at all time points. In OECM-1 cells, the average inhibitory concentration of 50% (IC50) was 3mM for butyrate, 382mM for acetate, and 64mM for propionate while in MDA-MB-231 cells, the average IC50 was 4.3mM for butyrate, 462mM for acetate, and 20mM for propionate. Both OECM-1 and MDA-MB-231 cells treated with butyrate and propionate at 5mM resulted in a slower migration after 24h of treatment. Additionally, there was a significant reduction of cell number and proliferation rate after 48h and 72h of butyrate treatment at the concentrations 1mM (p=0.0425) and 5mM (p=0.0007) in OSCC. Conclusions Our results show that butyrate and propionate can reduce oncogenic phenotypes in OSCC and TNBC and may represent a potential target for the development of alternative treatment strategies for both cancers. Future work includes measuring the impact of these SCFA in epithelial-mesenchymal transition, loss of adhesion, and cell death. Citation Format: Angel J. Amaral Maisonet. The potential use of butyrate and propionate to inhibit oncogenic phenotypes in oral squamous cell carcinoma and triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2976.

In Vitro Antischistosomal Activity of Bridelia ferruginea, Clausena anisata, Khaya senegalensis, and Vernonia amygdalina.

Schistosomiasis is caused by parasitic flatworms and the disease is endemic to most countries in sub-Saharan Africa including Ghana. The current therapeutic agent for managing this disease solely relies on praziquantel. The continual dependence on this single available drug could lead to possible drug resistance. This study seeks to evaluate the antischistosomal activity of the following Ghanaian medicinal plants: Khaya senegalensis, Vernonia amygdalina, Clausena anisata, and Bridelia ferruginea. Methodology. Two concentrations (100 μg/mL and 50 μg/mL) of each extract were tested in a 96-well plate containing 30 newly transformed schistosomula (NTS). Moreover, six worms of both sexes of adult Schistosoma mansoni were exposed to the extracts diluted in the RPMI medium. The assay was performed in a 24-well plate. The parasitic worms were examined using an inverted optical microscope. At 100 μg/mL and 50 μg/mL, all extracts performed better and showed strong activity (p < 0.001) against NTS; thus, 98.08%, 100%, 80.77%, and 100% for Clausena, Vernonia, Bridelia, and Khaya, respectively, when compared to praziquantel. Strong activity was recorded when the extracts underwent testing against Schistosoma mansoni adults at 100 μg/mL; 96.35%, 100%, and 94.55% for Vernonia, Bridelia, and Khaya, respectively, except for Clausena which exhibited weak activity, i.e., 56.02%. There was no significant difference between Vernonia, Bridelia, and Khaya when compared to praziquantel. At 100 μg/mL, Khaya senegalensis, Vernonia amygdalina, and Bridelia ferruginea extracts demonstrated strong activity against both schistosomula and adult Schistosoma mansoni. These data can serve as baseline information in the quest to find alternative therapeutic agents to treat schistosomiasis.

Pulmonary surfactant impacts in vitro activity of selected antifungal drugs against Candida krusei and Candida albicans

PurposeThis study investigates how surfactants affect the in-vitro anti-infective efficacy of micafungin, caspofungin, anidulafungin, and amphotericin B in treating pulmonary mycoses.MethodsMIC values for antifungal agents were determined against Candida krusei (now Pichia kudriavzevii) ATCC 6258, Candida albicans ATCC 90028, and 18 clinical isolates using the broth microdilution method in RPMI medium, following EUCAST recommendations. MIC assays included testing with and without Curosurf® surfactant at 1 mg/mL for C. krusei ATCC 6258 and all C. krusei isolates. Subsequent Time-kill studies in Sabouraud broth involved testing both C. albicans ATCC 90028 and C. krusei ATCC 6258 strains at concentrations equal their respective MIC values, with and without surfactant, using all four antifungals. CFU/mL were assessed at multiple time points up to 24 h. TKCs with different surfactant concentrations for C. krusei ATCC 6258 and mini-TKCs at various concentrations relative to the MIC of C. krusei isolates and the reference strain were conducted with micafungin, anidulafungin, and caspofungin.ResultsMIC results showed that 1 µg/mL surfactant reduced killing of micafungin and anidulafungin against C. krusei, while caspofungin was unaffected. Amphotericin B's MIC decreased by half. TKCs demonstrated significant effects of surfactant on micafungin and anidulafungin against C. krusei, with complete abolition of anidulafungin's activity against C. albicans.ConclusionThis in-vitro study highlights the concentration-dependent inhibitory effect of surfactant on antifungal activity against C. krusei and, to some extent, C. albicans, necessitating further clinical validation for invasive lung mycoses treatment.

Host defense peptides mitigate the spread of antibiotic resistance in physiologically relevant condition.

Antibiotic resistance represents a significant challenge to public health and human safety. The primary driver behind the dissemination of antibiotic resistance is the horizontal transfer of plasmids. Current conjugative transfer assay is generally performed in a standardized manner, ignoring the effect of the host environment. Host defense peptides (HDPs) possess a wide range of biological targets and play an essential role in the innate immune system. Herein, we reveal that sub-minimum inhibitory concentrations of HDPs facilitate the conjugative transfer of RP4-7 plasmid in the Luria Broth medium, and this observation is reversed in the RPMI medium, designed to simulate the host environment. Out of these HDPs, indolicidin (Ind), a cationic tridecapeptide from bovine neutrophils, significantly inhibits the conjugation of multidrug resistance plasmids in a dose-dependent manner, including blaNDM- and tet(X4)-bearing plasmids. We demonstrate that the addition of Ind to RPMI medium as the incubation substrate downregulates the expression of conjugation-related genes. In addition, Ind weakens the tricarboxylic acid cycle, impedes the electron transport chain, and disrupts the proton motive force, consequently diminishing the synthesis of adenosine triphosphate and limiting the energy supply. Our findings highlight the importance of the host-like environments for the development of horizontal transfer inhibitors and demonstrate the potential of HDPs in preventing the spread of resistance plasmids.

First insights on the susceptibility of native coccidicidal fungi Mucor circinelloides and Mucor lusitanicus to different avian antiparasitic drugs

BackgroundThe combined application of predatory fungi and antiparasitic drugs is a sustainable approach for the integrated control of animal gastrointestinal (GI) parasites. However, literature addressing the possible interference of antiparasitic drugs on the performance of these fungi is still scarce. This research aimed to assess the in vitro susceptibility of six native coccidicidal fungi isolates of the species Mucor circinelloides and one Mucor lusitanicus isolate to several antiparasitic drugs commonly used to treat GI parasites’ infections in birds, namely anthelminthics such as Albendazole, Fenbendazole, Levamisole and Ivermectin, and anticoccidials such as Lasalocid, Amprolium and Toltrazuril (drug concentrations of 0.0078–4 µg/mL), using 96-well microplates filled with RPMI 1640 medium, and also on Sabouraud Agar (SA).ResultsThis research revealed that the exposition of all Mucor isolates to the tested anthelminthic and anticoccidial drug concentrations did not inhibit their growth. Fungal growth was recorded in RPMI medium, after 48 h of drug exposure, as well as on SA medium after exposure to the maximum drug concentration.ConclusionsPreliminary findings from this research suggest the potential compatibility of these Mucor isolates with antiparasitic drugs for the integrated control of avian intestinal parasites. However, further in vitro and in vivo studies are needed to confirm this hypothesis.

Abstract B039: Butyrate and propionate-reduced oncogenic phenotypes in triple-negative breast cancer

Abstract Objectives Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer affecting 5% of all patients with no known treatment. Hispanic women show a higher risk of developing this type of cancer at an early age. Heterogeneity of TNBC between patients makes it challenging to devolop a effective treatment. The compostion of microbiome amongst the patients has a high variablity that could affect the production of the microbial products. The changes in microbial products have not been previously evaluated and could possibly contribute to chronic inflammation in the patients. Short-chain fatty acids (SCFA), i.e. butyrate, acetate, and propionate which are metabolical products of the microbiome that can potentially inhibit cell growth, proliferation and migration of cancer cells, may represent an alternative treatment for TNBC. Here, we measured the impact of SCFAs treament on viability and migration of MDA-231 cell lines. Methods MDA-231 cell line is an aggressive triple-negative breast cancer (TNBC) cell line common for evaluating new therapeutic strategies. MDA-231 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and we evaluated oncogenic phenotypes (cell viability and migration) after SCFA treatment. Cells were treated with butyrate, acetate, and propionate in cell culture media at seven concentrations starting at 125mM (1:2 dilution factor) for 24, 48 and 72 hours (h). We determined the cell viability using Alamar blue and determined the half-maximal inhibitory concentration (IC50) after SCFA treatment at all time points. Furthermore, we performed a wound-healing assay for 24h to evaluate the impact of SCFA on cell migration. MDA-231 cells were cultured to a 100% confluency and a scratch was performed in the cell monolayer to create an artificial gap, simulating the wound. Immediately after performing the wound, the cells were treated with SCFA at concentrations of 1mM and 5mM. Results Higher concentrations of butyrate and propionate reduced cell viability for MDA-231 was reduced. Particularly, butyrate showed an IC50 at 24h, 48h and 72h of 4.7mM, 4.34 mM and 3.6mM, respectively. However, propionate showed an IC50 only at 48h and 72h of 21.09 mM and 19.5 mM, respectively. There was no impact in cell viability after acetate treatment. In relation to migration, MDA-231 cell lines treated with butyrate and propionate at 5mM show a slower migration rate at 24h when compared to the untreated cells. Conclusions Our results suggest that butyrate and propionate can reduce oncogenic phenotypes in MDA-231. Future work will measure the impact of SCFA on cell proliferation, motility, and epithelial-mesenchymal transition. SCFAs may represent a promising target for novel treatment strategies for TNCB. Citation Format: Liah M Román-Calderón, Ariana S García-López, Jeannette L Salgado-Montilla, Gabriel Borges-Vélez, Esther A Peterson-Peguero, Josué Pérez-Santiago. Butyrate and propionate-reduced oncogenic phenotypes in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B039.

Therapeutic Potential of Bipolar Androgen Therapy for Castration-Resistant Prostate Cancer: In Vitro and In Vivo Studies.

Androgen deprivation therapy (ADT) is a primary treatment for advanced prostate cancer (PCa), but resistance often leads to castration-resistant PCa (CRPC). CRPC remains androgen receptor (AR)-dependent, and AR overexpression causes vulnerability to high doses of androgen in CRPC. Bipolar androgen therapy (BAT) refers to the periodic administration of testosterone, resulting in oscillation between supraphysiologic and near-castrate serum testosterone levels. In this study, we evaluated the efficacy of BAT against CRPC in a preclinical setting. To emulate CRPC characteristics, PCa cell lines (LNCaP, VCaP, and 22Rv1) were cultured in phenol red-free RPMI-1640 medium supplemented with 10% dextran-coated charcoal treated FBS (A- cell line). Cell viability, AR, and AR-V7 expression were evaluated using the Cell Counting Kit-8 and Western blotting. In vivo studies involved 12 castrated NOG mice injected with LNCaP/A- cells, treated with testosterone pellets or controls in 2-week cycles. Tumor sizes were measured post a 6-week treatment cycle. Bicalutamide inhibited PCa cell viability but not in the adapted cell lines. Supraphysiologic androgen levels suppressed AR-expressing PCa cell growth in vitro. In vivo, high AR-expressing LNCaP cells proliferated under castrate conditions, while BAT-treated xenografts exhibited significant growth inhibition with low Ki-67 and mitotic indexes and a high cell death index. This study provides preliminary evidence that BAT is effective for the treatment of CRPC through rapid cycling between supraphysiologic and near-castrate serum testosterone levels, inducing an anti-tumor effect.

In Vitro Study of the Antiparasitic Activity of Vitellaria paradoxa Gaertn (Sapotaceae) on the Parasite Onchocerca ochengi

Onchocerciasis is a parasitic disease that affects millions of animals and people around the world. A recipe based on Vitellaria paradoxa is used in traditional medicine against this disease in Chad. The objective of this study was to evaluate the antiparasitic activities of extracts from the roots, fruits, leaves and bark of Vitellaria paradoxa on the parasitic nematode Onchocerca ochengi, a model similar to Onchocerca volvulus used in the research of control drugs. against onchocerciasis. The identification of parasites and the hydroethanolic extracts of the different planar organs were carried out at the Laboratory of Research, Diagnostics and Scientific Expertise (LaboReDES) of the Faculty of Human Sciences, University of N'Djamena using the methods of Ndjonka et al., 2012. The extracts of this plant were tested at different concentrations (0.1; 0.2; 0.3; 0.4 mg/mL) in the RPMI-1640 culture medium. The antiparasitic activity was noted according to the mortality rate after 72 hours of incubation of Onchocerca ochengi at 37°C. The LC50 values were then graphically determined at the end of this incubation. They were 0.05 mg/mL for the roots; 0.06 mg/mL for fruits; 0.05 mg/mL for the leaves and 0.06 mg/mL for the bark. These results confirm the effectiveness of Vitellaria paradoxa used in traditional medicine to treat parasitosis. This plant also has the same effects as ivermectin. The V. paradoxa root test on microfilariae shows an LC50 value of 0.05 mg/mL in 24 hours of incubation. This study showed that the hydroethanolic extracts of the bark of Vitellaria paradoxa had antiparasitic activities, allowing the development of a new molecule active against these parasitic infections and also the extracts of this plant could be used as alternatives to treat infections in case of parasite resistance to ivermectin.

Repurposing Anthracycline Drugs as Potential Antibiotic Candidates and Potentiators to Tackle Multidrug-Resistant Pathogens.

The escalating mortality rate resulting from multidrug-resistant (MDR) bacteria has intensified the urgency for innovative antimicrobial agents. Currently, the antimicrobial activity of compounds is usually assessed by testing the minimum inhibitory concentration (MIC) on a standardized laboratory medium. However, such screening conditions differ from the in vivo environment, making it easy to overlook some antibacterial agents that are active in vivo but less active in vitro. Herein, by using tissue medium RPMI, we uncover that anthracyclines, especially mitoxantrone (MX), exhibit improved bacteriostatic and bactericidal effects against various MDR bacteria in host-like media. Transcriptome results reveal that LPS modification-related genes of bacterial membrane surfaces and metabolic genes are significantly down-regulated in RPMI media. Mechanistic studies demonstrate that MX leads to more substantial membrane damage, increased ROS production, and DNA damage in host-mimicking conditions. Furthermore, we demonstrate that MX and colistin exhibit strong synergistic effects against mcr-positive strains in host-mimicking media by disrupting iron homeostasis. In an experimental murine infection model, MX monotreatment demonstrates therapeutic efficacy in reducing bacterial burdens. Overall, our work suggests that mimicking the host condition is an effective strategy to identify new antimicrobial agents and highlights the therapeutic potential of anthracycline drugs in combating MDR pathogens.

Anti-leishmanial effects of Eryngium planum and Ecbilliun elaterum methanolic extract against Leishmania major

Leishmaniasis is a vector-borne disease, one of the most important neglected tropical diseases. Existing anti-leishmanial treatments are not effective for a long time and associated with toxic side effects so searching for a new, effective and safe alternative treatments against infectious diseases is greatly needed. This study is aimed to assess the leishmaniacidal effects of methanolic extracts of Eryngium planum (E. planum) and Ecbilliun elaterum (E. elaterum) on Leishmania major (L. major), In vitro. The selected plants were collected from northern areas of Iran. The methanolic extract from the aerial parts of plants were prepared using maceration methods. GC- Mass analysis was used to determine the compounds of the plants. Promastigotes of L. major was cultured in RPMI-1640 medium and the anti-leishmanial and cytotoxicity effects of extracts at concentrations of 100, 200, 400 and 800 µg/ml were assessed using MTT assay. The data obtained from gas chromatography revealed that α-Pinene, Caryophyllene oxide, β‐Caryophyllene, Bicyclogermacrene and α‐Bisabolol are the main compounds extracted from E. planum and α‐Pinene, Germacrene D, Caryophyllene oxide, γ‐Eudesmol and α‐Bisabolol are the main components of E. elaterum. The results of MTT Assay revealed that E. planum at concentrations of 800 µg/ml after 24 h at 400 µg/ml after 48 h and the E. elaterium at concentrations of 800 µg/ml after 48 h at 400 µg/ml after 72 h had similar anti-leishmanial effects to the positive control. These results indicated that E. planum and E. elaterum are the potential sources for the discovery of novel anti-leishmanial treatments.

EFFECT OF EXTRACELLULAR VESICLES DERIVED FROM HUMAN MACROPHAGES ON OSTEOSARCOMA CELLS

Macrophages (Mφ) are immune cells that play a crucial role in both innate and adaptive immunity as they are involved in a wide range of physiological and pathological processes. Depending on the microenvironment and signals present, Mφ can polarize into either M1 or M2 phenotypes, with M1 macrophages exhibiting pro-inflammatory and cytotoxic effects, while M2 macrophages having immunosuppressive and tissue repair properties. Macrophages have been shown to play key roles in the development and progression or inhibition of various diseases, including cancer. For example, macrophages can stimulate tumor progression by promoting immunosuppression, angiogenesis, invasion, and metastasis. This work aimed to investigate the effect of extracellular vesicles (EVs)-derived from polarized macrophages on an osteosarcoma cell line. Monocytes were extracted from buffy coats and cultured in RPMI medium with platelet lysate or M-CSF. After 6 days of seeding, Mφ were differentiated into M1 and M2 with INF-γ/LPS and IL-4/IL-13, respectively. The medium with M1 or M2 derived EVs was collected and EVs were isolated by differential centrifugation and size exclusion chromatography and its morphology and size were characterized with SEM and NTA, respectively. The presence of typical EVs markers (CD9, CD63) was assessed by Western Blot. Finally, EVs from M1 or M2-polarized Mφ were added onto osteosarcoma cell cultures and their effect on cell viability and cell cycle, proliferation, and gene expression was assessed. The EVs showed the typical shape, size and surface markers of EVs. Overall, we observed that osteosarcoma cells responded differentially to EVs isolated from the M1 and M2-polarized Mφ. In summary, the use of Mφ-derived EVs for the treatment of osteosarcoma and other cancers deserves further study as it could benefit from interesting traits of EVs such as low immunogenicity, nontoxicity, and ability to pass through tissue barriers.Acknowledgements: Carlos III Health Institute and the European Social Fund for contract CP21/00136 and project PI22/01686.