Reduction In Cellular Viability Research Articles

Pan-inhibition of the three H2S synthesizing enzymes restrains tumor progression and immunosuppression in breast cancer

BackgroundHydrogen sulfide (H2S) is a significant endogenous mediator that has been implicated in the progression of various forms of cancer including breast cancer (BC). Cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are the three principal mammalian enzymes responsible for H2S production. Overexpression of CBS, CSE and 3MST was found to be associated with poor prognosis of BC patients. Moreover, H2S was linked to an immune-suppressive tumor microenvironment in BC. Recently it was observed that BC cells, in response to single or dual inhibition of H2S synthesizing enzymes, develop an escape mechanism by overexpressing alternative sources of H2S generation. Thus, the aim of this work is to escape the H2S compensatory mechanism by pan repressing the three enzymes using microRNAs (miRNAs) and to investigate their impact on the oncogenic and immunogenic profile of BC cells.MethodsBC female patients (n = 25) were recruited. In-silico analysis was used to identify miRNAs targeting CBS, CSE, and 3MST. MDA-MB-231 cells were cultured and transfected using oligonucleotides. Total RNA was extracted using Biazol, reverse transcribed and quantified using qRT-PCR. H2S levels were measured using AzMc assay. BC hallmarks were assessed using trans-well migration, wound healing, MTT, and colony forming assays.ResultsmiR-193a and miR-548c were validated by eight different bioinformatics software to simultaneously target CBS, CSE and 3MST. MiR-193a and miR-548c were significantly downregulated in BC tissues compared to their non-cancerous counterparts. Ectopic expression of miR-193a and miR-548c in MDA-MB-231 TNBC cells resulted in a marked repression of CBS, CSE, and 3MST transcript and protein levels, a significant decrease in H2S levels, reduction in cellular viability, inhibition of migration and colony forming ability, repression of immune-suppressor proteins GAL3 GAL9, and CD155 and upregulation of the immunostimulatory MICA and MICB proteins.ConclusionThis study sheds the light onto miR-193a and miR-548c as potential pan-repressors of the H2S synthesizing enzymes. and identifies them as novel tumor suppressor and immunomodulatory miRNAs in TNBC.

Polystyrene microplastics impair the functions of cultured mouse Leydig (TM3) and Sertoli (TM4) cells by inducing mitochondrial-endoplasmic reticulum damage

Many laboratory studies demonstrated that the exposure to microplastics causes testosterone deficiency and spermatogenic impairment in mammals; however, the mechanism underlying this process remains still unclear. In this study, we investigated the effects of polystyrene microplastics (PS-MP) on the proliferation and functionality of cultured Leydig (TM3) and Sertoli (TM4) cells, focusing on the mitochondrial compartment and its association with the endoplasmic reticulum (ER). The in vitro exposure to PS-MP caused a substantial reduction in cellular viability in TM3 and TM4 cells. In TM3 cells PS-MP inhibited the protein levels of StAR and of steroidogenic enzymes 3β-HSD and 17β-HSD, and in TM4 cells PS-MP inhibited the protein levels of the androgen receptors other than the activity of lactate dehydrogenase (LDH). PS-MP inhibited the functions of TM3 and TM4, as evidenced by the decrease of the phosphorylation of ERK1/2 and Akt in both cell lines. The oxidative stress caused by PS-MP decreased antioxidant defense in TM3 and TM4 cells, promoting autophagic and apoptotic processes. Furthermore, we found mitochondrial dysfunction and activation of ER stress. It is known that mitochondria are closely associated with ER to form the Mitochondrial-Associated Endoplasmic Reticulum Membranes (MAM), the site of calcium ions transfer as well as of lipid biosynthesis-involved enzymes and cholesterol transport from ER to the mitochondria. For the first time, we studied this aspect in PS-MP-treated TM3 and TM4 cells and MAMs dysregulation was observed. This study is the first to elucidate the intracellular mechanism underlying the effects of PS-MPs in somatic testicular cells, corroborating that PS-MP might be one of the causes of an increase in male infertility through the impairment of steroidogenesis in Leydig cells and of the nurse function of Sertoli cells. Thus, our findings contributed with new information to the mechanism underlying the effects of PS-MP on the male reproductive system.

Statins affect human iPSC-derived cardiomyocytes by interfering with mitochondrial function and intracellular acidification.

Statins are effective drugs in reducing cardiovascular morbidity and mortality by inhibiting cholesterol synthesis. These effects are primarily beneficial for the patient's vascular system. A significant number of statin users suffer from muscle complaints probably due to mitochondrial dysfunction, a mechanism that has recently been elucidated. This has raised our interest in exploring the effects of statins on cardiac muscle cells in an era where the elderly and patients with poorer functioning hearts and less metabolic spare capacity start dominating our patient population. Here, we investigated the effects of statins on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-derived CMs). hiPSC-derived CMs were exposed to simvastatin, atorvastatin, rosuvastatin, and cerivastatin at increasing concentrations. Metabolic assays and fluorescent microscopy were employed to evaluate cellular viability, metabolic capacity, respiration, intracellular acidity, and mitochondrial membrane potential and morphology. Over a concentration range of 0.3-100µM, simvastatin lactone and atorvastatin acid showed a significant reduction in cellular viability by 42-64%. Simvastatin lactone was the most potent inhibitor of basal and maximal respiration by 56% and 73%, respectively, whereas simvastatin acid and cerivastatin acid only reduced maximal respiration by 50% and 42%, respectively. Simvastatin acid and lactone and atorvastatin acid significantly decreased mitochondrial membrane potential by 20%, 6% and 3%, respectively. The more hydrophilic atorvastatin acid did not seem to affect cardiomyocyte metabolism. This calls for further research on the translatability to the clinical setting, in which a more conscientious approach to statin prescribing might be considered, especially regarding the current shift in population toward older patients with poor cardiac function.

Enhancing tumor-specific recognition of programmable synthetic bacterial consortium for precision therapy of colorectal cancer

Probiotics hold promise as a potential therapy for colorectal cancer (CRC), but encounter obstacles related to tumor specificity, drug penetration, and dosage adjustability. In this study, genetic circuits based on the E. coli Nissle 1917 (EcN) chassis were developed to sense indicators of tumor microenvironment and control the expression of therapeutic payloads. Integration of XOR gate amplify gene switch into EcN biosensors resulted in a 1.8-2.3-fold increase in signal output, as confirmed by mathematical model fitting. Co-culturing programmable EcNs with CRC cells demonstrated a significant reduction in cellular viability ranging from 30% to 50%. This approach was further validated in a mouse subcutaneous tumor model, revealing 47%-52% inhibition of tumor growth upon administration of therapeutic strains. Additionally, in a mouse tumorigenesis model induced by AOM and DSS, the use of synthetic bacterial consortium (SynCon) equipped with multiple sensing modules led to approximately 1.2-fold increased colon length and 2.4-fold decreased polyp count. Gut microbiota analysis suggested that SynCon maintained the abundance of butyrate-producing bacteria Lactobacillaceae NK4A136, whereas reducing the level of gut inflammation-related bacteria Bacteroides. Taken together, engineered EcNs confer the advantage of specific recognition of CRC, while SynCon serves to augment the synergistic effect of this approach.

In Vitro Evaluation of the Effect of Oleanolic Acid as a Potential Root Canal Medicament on Viability and Proliferation of Dental Pulp Stem Cells

Abstract Objective In light of the potential drawbacks associated with certain intracanal medicaments, such as triple antibiotic paste (TAP) and calcium hydroxide (Ca(OH2)), the introduction of herbal agents has ushered in a new era in the field of dentistry. Consequently, this study aimed to explore the impact of oleanolic acid (OA) on the viability and proliferation of dental pulp stem cells (DPSCs), comparing its effects to those of conventional intracanal medicaments, TAP and Ca(OH2). Materials and Methods DPSCs were derived from the third molars of an adult donor. Flow cytometry was utilized to do a phenotypic study on DPSCs. The methyl-thiazol tetrazolium (MTT) test was used to evaluate cellular viability. The cells were subjected to various concentrations of TAP and Ca(OH)2 (5, 2.5, 1, 0.5, and 0.25 mg/mL), in addition to OA (40, 20, 10, 5, and 2.5 µM). A cell proliferation experiment assessed the cell growth precisely at 3, 5, and 7 days. Results DPSCs were characterized by flow cytometry. The mesenchymal markers (CD73, CD90, and CD105) had a positive expression. However, the hematological markers (CD14, CD34, and CD45) showed negligible expression. A notable reduction in cellular viability was seen in cells subjected to concentrations exceeding 0.5 mg/mL of TAP and Ca(OH)2 compared to the cells that were not treated (p < 0.05). The cells treated with different concentrations of OA 2.5, 5, 10, and 20 µM did not exhibit any significant variance in cell viability compared to untreated cells (p > 0.05). Moreover, the concentrations of OA (20, 10, and 5 µM) showed high proliferation level compared to TAP and Ca(OH2) especially 5µM of OA after 7 days (p < 0.05). Conclusion Our results revealed that OA exerted significant effect on the viability and proliferation of DPSCs compared to TAP and Ca(OH2).

Evaluation of Cytotoxicity, Cell Attachment, and Elemental Characterization of Three Calcium Silicate-Based Sealers.

We investigated three calcium silicate-based sealers with respect to their chemical characterization, cytotoxicity, and attachment to RAW264.7 cells. BioRoot RCS (BR), Bio-C Sealer (BC), and Sealer Plus BC (SPBC) were assessed using Fourier transform infrared spectroscopy (FTIR), X-ray fluorescence spectroscopy (XRF), and energy-dispersive X-ray spectroscopy (EDX) (n = 4) for elemental characterization, and using scanning electron microscopy (SEM) to evaluate cell morphology and adhesion. Cytotoxicity was determined at different dilutions (1:1, 1:2, and 1:5) using the succinate dehydrogenase activity (MTT assay). Statistical analysis was performed for normal distribution using the Shapiro-Wilk test and for homoscedasticity using Levene's test, and one-way ANOVA, Tukey's/Dunnett's post hoc tests for cell viability and XRF (α = 0.05). Calcium silicate hydrate and calcium hydroxide were detected by FTIR in all groups. EDX detected a higher calcium content for BR and SPBC and aluminum only in the premixed sealers. XRF detected the highest calcium release in BR (p < 0.05). The surface morphology showed irregular precipitates for all the sealers. SPBC at a 1:2 dilution resulted in the lowest cell viability compared to BR (p < 0.05) and BC (p < 0.05). The calcium silicate-based sealers produced a statistically significant reduction in cellular viability at a 1:1 dilution compared to the control group (p < 0.0001). All the sealers maintained viability above 70%.

Conjugated polymer nanoparticles as sonosensitizers in sono-inactivation of a broad spectrum of pathogens

Sonodynamic inactivation (SDI) of pathogens has an important advantage when compared to optical excitation-based protocols due to the deeper penetration of ultrasound (US) excitation in biological media or animal tissue. Sonosensitizers (SS) are compounds or systems that upon US stimulation in the therapeutic window (frequency = 0.8–3 MHz and intensity < 3 W/cm2) can induce damage to vital components of pathogenic microorganisms. Herein, we report the synthesis and application of conjugated polymer nanoparticles (CPNs) as an efficient SS in SDI of methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae and Candida tropicalis. A frequent problem in the design and testing of new SS for SDI is the lack of proper sonoreactor characterization which leads to reproducibility concerns. To address this issue, we performed dosimetry experiments in our setup. This enables the validation of our results by other researchers and facilitates meaningful comparisons with different SDI systems in future studies. On a different note, it is generally accepted that the mechanisms of action underlying SS-mediated SDI involve the production of reactive oxygen species (ROS). In an attempt to establish the nature of the cytotoxic species involved in our CPNs-based SDI protocol, we demonstrated that singlet oxygen (1O2) does not play a major role in the observed sonoinduced killing effect. SDI experiments in planktonic cultures of optimally growing pathogens using CPNs result in a germicide effect on the studied pathogenic microorganisms. The implementation of SDI protocols using CPNs was further tested in mature biofilms of a MRSA resulting in ∼40 % reduction of biomass and ∼70 % reduction of cellular viability. Overall, these results highlight the unique and unexplored capacity of CPNs to act as sonosensitizers opening new possibilities in the design and application of novel inactivation protocols against morbific microbes.

Dopamine receptors D1 and D2 show prognostic significance and potential therapeutic applications for endometrial cancer patients

ObjectiveCatecholaminergic signaling has been a target for therapy in different type of cancers. In this work, we characterized the ADRβ2, DRD1 and DRD2 expression in healthy tissue and endometrial tumors to evaluate their prognostic significance in endometrial cancer (EC), unraveling their possible application as an antitumor therapy. Methods109 EC patients were included. The expression of the ADRβ2, DRD1 and DRD2 proteins was evaluated by immunohistochemistry and univariate and multivariate analysis to assess their association with clinic-pathological and outcome variables. Finally, HEC1A and AN3CA EC cell lines were exposed to different concentrations of selective dopaminergic agents alone or in combination to study their effects on cellular viability. ResultsADRβ2 protein expression was not associated with clinico-pathological parameters or prognosis. DRD1 protein expression was reduced in tumors samples but showed a significant inverse association with tumor size and stage. DRD2 protein expression was significantly associated with non-endometrioid EC, high grade tumors, tumor size, worse disease-free survival (HR = 3.47 (95%CI:1.35–8.88)) and overall survival (HR = 2.98 (95%CI:1.40–6.34)). The DRD1 agonist fenoldopam showed a reduction of cellular viability in HEC1A and AN3CA cells. The exposure to domperidone, a DRD2 antagonist, significantly reduced cell viability compared to the control. Finally, DRD1 agonism and DRD2 antagonism combination induced a significant reduction in cell viability of the AN3CA cells compared to monotherapy, close to being an additive response than a synergistic effect (CI of 1.1 at 0.5% Fa). ConclusionDRD1 and DRD2 expression levels showed a significant association with clinico-pathological parameters. Both the combined activation of DRD1 and blockage of DRD2 may form an innovative strategy to inhibit tumor growth in EC.

Incorporation of Targeting Biomolecule Improves Interpolymer Complex-Superparamagnetic Iron Oxide Nanoparticles Attachment to and Activation of T2 MR Signals in M2 Macrophages.

Inflammatory diseases are the leading cause of death in the world, accounting for 3 out of 5 deaths. Despite the abundance of diagnostic tools for detection, most screening and diagnostic methods are indirect and insufficient as they are unable to reliably discriminate between high-risk or low-risk stages of inflammatory diseases. Previously, we showed that the selective activation of interpolymer complexed superparamagnetic iron oxide nanoparticles (IPC-SPIOs) under oxidative conditions can be detected by a change in T2 magnetic resonance (MR) contrast. In this work, IPC-SPIOs were further modified by incorporating mannose as a targeting biomolecule to enhance nanoparticle delivery to M2 macrophages at inflammatory sites. Uncoated SPIOs were synthesized via coprecipitation from a mixture of FeCl2 and FeCl3, PEGylated by adsorbing PEG 300 kDa (40 mg/mL in water) to SPIOs (3 mg/mL in water) over 24 hours, and complexed by mixing 0.25 mg/mL aqueous poly(gallol) with 2 mg/mL PEG-SPIOs and adding 1 M of phosphate buffer in a 9:9:2 ratio. Mannose-PEG attachment was accomplished conducting a second complexation of mannose-PEG to IPC-SPIOs. M2 macrophages were treated with 150, 100, and 75 µg/mL of IPC-SPIOs and mannose-IPC-SPIOs to investigate activation of T2 MRI signals. Surface modification resulted in a slight reduction in ROS scavenging capacity; however, nanoparticle uptake by M2 macrophages increased by over 50%. The higher uptake did not cause a reduction in cellular viability. In fact, mannose-IPC-SPIOs induced significant T2 MR contrast in M2 macrophages compared to IPC-SPIOs and nanoparticles exposed to M1 macrophages. M2 macrophages activated over 30% of mannose-IPC-SPIOs after 6 hours of exposure compared to M1 macrophages and untargeted M2 macrophages. These findings demonstrated that mannose-IPC-SPIOs specifically targeted M2 macrophages and scavenged cellular ROS to activate T2 MR signal, which can be used to detect inflammation.

Evaluation of ciprofloxacin used as an intranasal antibiotic.

The objective of this research was to examine the effects of topical ciprofloxacin on cultured nasal epithelial cells of human origin. Human nasal epithelial cells were collected from patients who voluntarily donated tissue left over following septorhinoplasty. The samples were from individuals without any indication of rhinosinusitis. An assay that may be employed to investigate toxic effects at the cellular level is MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). This test reveals where DNA becomes fragmented, the nuclei condense, the outer cell membrane is altered, or the cytoskeleton appears disrupted. The present study employed this technique. Nasal epithelium in cell culture was exposed to ciprofloxacin for 24 hours at a temperature of 37°C, following which the MTT assay was undertaken before examining the cells by confocal microscopy to look for alterations indicating cytotoxicity. Another test of toxicity, the artificial scratch technique, was also used. Cells treated in this way were assessed using a light microscope. The nasal epithelial cells in culture that were exposed to topical ciprofloxacin for 24 hours were less viable than controls and this result was statistically significant. For this length of exposure, the IC50 was calculated as 1.565 mg/mL. The peak reductions in cellular viability occurred with exposure at a concentration of 1.25 mg/mL and 0.625 mg/mL. These was only a mild decrease in viability at other concentrations, but these results were of statistical significance. The MTT assay and confocal microscopy confirmed this result. Cultured nasal epithelium not exposed to ciprofloxacin (i.e., controls) exhibited a compact morphological appearance when examined with confocal microscopy. The epithelial cells had a regular fusiform boundary, and the nuclei were intact. By contrast, the cultures with exposure to the antibiotic were of decreased size and their outline changed from fusiform to round. The epithelial cells cultures where scratch injury was induced were examined by light microscopy, the extent of closure of the denuded area being assessed after 24 hours. It was noted that the area opened up by the experimental scratch was not closed completely by 24 hours later. This result shows that ciprofloxacin decreases the viability of nasal epithelial cells. Topical application of ciprofloxacin to the nasal lining is not recommended, since this resulted in decreased cellular viability, cellular shrinking and alteration in outline from fusiform to round in cultured nasal epithelial cells. These changes indicate that topically applied ciprofloxacin is toxic to nasal epithelial cells. The outcomes of this study should be studied and correlated in vivo models.

The superiority of Dexpanthenol or Vaseline as excipient in nasal formulations.

Dexpanthenol is an ingredient in multiple topical pharmaceutical preparations thanks to its high penetration and localized concentration. It is included in many ointments or lotions for dermatological use, assisting in healing and reducing pruritus. Vaseline is a synthetic product obtained by distilling crude oil. It is commercially available in several grades. The study presented here examined how topically applied agents (dexpanthenol or vaseline) affect nasal epithelial cells in culture. In particular, the study aimed to identify any alterations to epithelial cells which might indicate toxicity. The nasal epithelial cells used were sourced from mucosal tissue fragments left over the following septorhinoplasty on five patients not suffering from rhinosinusitis. The first step was to dissect the mucosal fragments into smaller pieces on a sterilized Petri dish. These fragments were then placed into the DMEM-F12 cell culture medium, which had been freshly prepared. The dexpanthenol and vaseline were diluted in dimethylsulfoxide (DMSO) to a concentration of 5 mg/mL. The cells in the wells were exposed to varying concentrations of dexpanthenol or vaseline. The actual concentration of the test reagent to which the epithelial cells were exposed ranged from 0.15 mg/mL to 5 mg/mL. The exposure period was 24 hours. The cells were finally examined using a Leica SP5II confocal microscope. The features sought were DNA fragmentation, condensation of the nuclei, changes in the outer membrane, or cytoskeletal abnormality. These features, if present, indicate cytotoxicity. The viability of the cultured nasal epithelial cells was unaltered by a 24-hour exposure to dexpanthenol, nor was the cellular proliferation rate affected at the level of statistical significance. There was evidence of a cytotoxic effect from exposing nasal epithelial cells to vaseline in liquid form for 24 hours. There was a reduction in cellular viability in the plates where the highest dose of vaseline (5 mg/mL) was used. Cellular viability was not affected significantly at any of the doses below 5 mg/mL. The absence of cytotoxic effects from the application of dexpanthenol to the nasal mucosa indicates that this agent may be safely used within the nose. The cytotoxic effects of liquid vaseline observed in this trial (condensed nuclear chromatin, loss of cellular volume) indicate that this agent may be harmful when used intranasally. For patients who require nasal packing due to nose bleeds or following endoscopic sinus surgical procedures, dexpanthenol should be preferred to vaseline from the point of view of maximizing healing of a nasal injury.

Formulation, Characterisation and Evaluation of the Antihypertensive Peptides, Isoleucine-Proline-Proline and Leucine-Lysine-Proline in Chitosan Nanoparticles Coated with Zein for Oral Drug Delivery.

Isoleucine-Proline-Proline (IPP) and Leucine-Lysine-Proline (LKP) are food-derived tripeptides whose antihypertensive functions have been demonstrated in hypertensive rat models. However, peptides display low oral bioavailability due to poor intestinal epithelial permeability and instability. IPP and LKP were formulated into nanoparticles (NP) using chitosan (CL113) via ionotropic gelation and then coated with zein. Following addition of zein, a high encapsulation efficiency (EE) (>80%) was obtained for the NP. In simulated gastric fluid (SGF), 20% cumulative release of the peptides was achieved after 2 h, whereas in simulated intestinal fluid (SIF), ~90% cumulative release was observed after 6 h. Higher colloidal stability (39–41 mV) was observed for the coated NP compared to uncoated ones (30–35 mV). In vitro cytotoxicity studies showed no reduction in cellular viability of human intestinal epithelial Caco-2 and HepG2 liver cells upon exposure to NP and NP components. Administration of NP encapsulating IPP and LKP by oral gavage to spontaneously hypertensive rats (SHR) attenuated systolic blood pressure (SBP) for 8 h. This suggests that the NP provide appropriate release to achieve prolonged hypotensive effects in vivo. In conclusion, chitosan-zein nanoparticles (CZ NP) have potential as oral delivery system for the encapsulation of IPP and LKP.

Nano-pesticidal potential of Cassia fistula (L.) leaf synthesized silver nanoparticles (Ag@CfL-NPs): Deciphering the phytopathogenic inhibition and growth augmentation in Solanum lycopersicum (L.).

Plant-based synthesis of silver nanoparticles (Ag-NPs) has emerged as a potential alternative to traditional chemical synthesis methods. In this context, the aim of the present study was to synthesize Ag-NPs from Cassia fistula (L.) leaf extract and to evaluate their nano-pesticidal potential against major phyto-pathogens of tomato. From the data, it was found that particle size of spherical C. fistula leaf synthesized (Ag@CfL-NPs) varied from 10 to 20 nm, with the average diameter of 16 nm. Ag@CfL-NPs were validated and characterized by UV-visible spectroscopy (surface resonance peak λmax = 430 nm), energy dispersive spectrophotometer (EDX), Fourier transform infrared (FTIR), and X-ray diffraction pattern (XRD), and electron microscopy; scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The FTIR spectra verified the participation of various living molecules (aromatic/aliphatic moieties and proteins) in synthesized Ag@CfL-NPs. The anti-phytopathogenic potential of Ag@CfL-NPs was assessed under in vitro conditions. Increasing doses of Ag@CfL-NPs exhibited an inhibitory effect against bacterial pathogen Pseudomonas syringae and 400 μg Ag@CfL-NPs ml–1 caused a reduction in cellular viability, altered bacterial morphology, and caused cellular death Furthermore, Ag@CfL-NPs reduced exopolysaccharides (EPS) production and biofilm formation by P. syringae Additionally, Ag@CfL-NPs showed pronounced antifungal activity against major fungal pathogens. At 400 μg Ag@CfL-NPs ml–1, sensitivity of tested fungi followed the order: Fusarium oxysporum (76%) > R. solani (65%) > Sarocladium (39%). Furthermore, 400 μg Ag@CfL-NPs ml–1 inhibited the egg-hatching and increased larval mortality of Meloidogyne incognita by 82 and 65%, respectively, over control. Moreover, pot studies were performed to assess the efficacy of Ag@CfL-NPs to phyto-pathogens using tomato (Solanum lycopersicum L.) as a model crop. The applied phyto-pathogens suppressed the biological, physiological, and oxidative-stress responsiveness of tomatoes. However, 100 mg Ag@CfL-NPs kg–1 improved overall performance and dramatically increased the root length, dry biomass, total chlorophyll, carotenoid, peroxidase (POD), and phenylalanine ammonia lyase (PAL) activity over pathogens-challenged tomatoes. This study is anticipated to serve as an essential indication for synthesis of efficient nano-control agents, which would aid in the management of fatal phyto-pathogens causing significant losses to agricultural productivity. Overall, our findings imply that Ag@CfL-NPs as nano-pesticides might be used in green agriculture to manage the diseases and promote plant health in a sustainable way.

Abstract 5360: Temozolomide-resistant glioblastoma cells are collaterally sensitive to ferroptosis through NRF2 high expression

Abstract Glioblastoma patients have a poor prognosis with a low median survival rate mainly due to temozolomide (TMZ) resistance. NRF2 is an important transcript factor involved in chemotherapy resistance due to its ability to regulate genes related to the antioxidant response and to prevent cell death processes, such as ferroptosis. However, the relation between NRF2 and iron-dependent cell death is contradictory and poorly understood. This study aimed to analyze the role of NRF2 in ferroptosis modulation in glioblastoma cells. To this end, it was analyzed two human glioblastoma cell lines (U251MG and T98G) after treatment with TMZ, ferroptosis inducers (Erastin, RSL3, and Sorafenib), and ferroptosis inhibitor (Ferrostatin-1). Also, we performed gene expression analysis of glioma patients. Our results demonstrated that T98G compared to the U251MG was more resistant to chemotherapy and showed elevated levels of NRF2 expression and its targets xCT, HMOX1, and ABCC1. Interestingly, T98G cells revealed higher sensitivity and lipoperoxidation levels after ferroptotic treatment. Next, we established T98G NRF2 silenced cells and we observed a significant reduction in cellular viability after TMZ treatment when compared to wild-type cells. On the other hand, T98G-shNRF2 was more resistant to ferroptosis induction, indicating that NRF2 plays a key role in the modulation of chemoresistance and ferroptosis. After, we showed that NRF2 controls levels of ABCC1/MRP1 in glioblastoma cells, and ABCC1 silencing promotes sensitivity to TMZ and resistance to Erastin. These results support a possible mechanism of ferroptosis modulation by NRF2 on TMZ-resistant gliomas through ABCC1, which has been recently associated with ferroptosis induction by promoting efflux of glutathione out of the cell. Furthermore, we confirmed that NRF2 has a positive correlation with ABCC1 in glioma patients, and higher ABCC1 expression was associated with tumor aggressiveness. Also, we validated ABCC1 as an independent prognostic factor for poor overall survival on glioma. Finally, we observed that T98G cells have sensitivity to the ferroptosis inducer FDA-approved, sorafenib. Altogether our data suggest that high levels of NRF2 may result in ferroptosis sensitivity on glioblastoma through the high levels of expression of its pro-ferroptotic target ABCC1, once the xCT system is blocked by Erastin. Thus, glioblastoma cell vulnerability to ferroptosis by NRF2 and ABCC1 high expression can be an Achilles’ heel to reverse drug resistance on glioblastoma through the treatment with ferroptosis inducers. Citation Format: Izadora de Souza, Linda Karolynne Monteiro, Camila Banca Guedes, Marcela Latancia, Marina Tomaz Andrade, Matheus Molina Silva, Bruna Contieri, Bruna Felício Milazzotto Ribeiro, Mariana Lazarini, Luciana Gomes, Clarissa Rocha. Temozolomide-resistant glioblastoma cells are collaterally sensitive to ferroptosis through NRF2 high expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5360.

Preclinical Efficacy and Toxicity Analysis of the Pan-Histone Deacetylase Inhibitor Gossypol for the Therapy of Colorectal Cancer or Hepatocellular Carcinoma.

Gossypol, a sesquiterpenoid found in cotton seeds, exerts anticancer effects on several tumor entities due to inhibition of DNA synthesis and other mechanisms. In clinical oncology, histone deacetylase inhibitors (HDACi) are applied as anticancer compounds. In this study, we examined whether gossypol harbors HDAC inhibiting activity. In vitro analyses showed that gossypol inhibited class I, II, and IV HDAC, displaying the capability to laterally interact with the respective catalytic center and is, therefore, classified as a pan-HDAC inhibitor. Next, we studied the effects of gossypol on human-derived hepatoma (HepG2) and colon carcinoma (HCT-116) cell lines and found that gossypol induced hyperacetylation of histone protein H3 and/or tubulin within 6 h. Furthermore, incubation with different concentrations of gossypol (5–50 µM) over a time period of 96 h led to a prominent reduction in cellular viability and proliferation of hepatoma (HepG2, Hep3B) and colon carcinoma (HCT-116, HT-29) cells. In-depth analysis of underlying mechanisms showed that gossypol induced apoptosis via caspase activation. For pre-clinical evaluation, toxicity analyses showed toxic effects of gossypol in vitro toward non-malignant primary hepatocytes (PHH), the colon-derived fibroblast cell line CCD-18Co, and the intestinal epithelial cell line CCD 841 CoN at concentrations of ≥5 µM, and embryotoxicity in chicken embryos at ≥2.5 µM. In conclusion, the pronounced inhibitory capacity of gossypol on cancer cells was characterized, and pan-HDACi activity was detected in silico, in vitro, by inhibiting individual HDAC isoenzymes, and on protein level by determining histone acetylation. However, for clinical application, further chemical optimization is required to decrease cellular toxicity.

Induction of Synthetic Lethality by Activation of Mitochondrial ClpP and Inhibition of HDAC1/2 in Glioblastoma.

Novel therapeutic targets are critical to unravel for the most common primary brain tumor in adults, glioblastoma (GBM). We have identified a novel synthetic lethal interaction between ClpP activation and HDAC1/2 inhibition that converges on GBM energy metabolism. Transcriptome, metabolite, and U-13C-glucose tracing analyses were utilized in patient-derived xenograft (PDX) models of GBM. Orthotopic GBM models were used for in vivo studies. We showed that activation of the mitochondrial ClpP protease by mutant ClpP (Y118A) or through utilization of second-generation imipridone compounds (ONC206 and ONC212) in combination with genetic interference of HDAC1 and HDAC2 as well as with global (panobinostat) or selective (romidepsin) HDAC inhibitors caused synergistic reduction of viability in GBM model systems, which was mediated by interference with tricarboxylic acid cycle activity and GBM cell respiration. This effect was partially mediated by activation of apoptosis along with activation of caspases regulated chiefly by Bcl-xL and Mcl-1. Knockdown of the ClpP protease or ectopic expression of a ClpP D190A mutant substantially rescued from the inhibition of oxidative energy metabolism as well as from the reduction of cellular viability by ClpP activators and the combination treatment, respectively. Finally, utilizing GBM PDX models, we demonstrated that the combination treatment of HDAC inhibitors and imipridones prolonged host survival more potently than single treatments or vehicle in vivo. Collectively, these observations suggest that the efficacy of HDAC inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

The Effect of Irradiated Riboflavin in Human Tenon’s Fibroblast – A Study on Cellular Viability

ABSTRACT Purpose/ Aim The main purpose of this work is to study the cellular viability effect of irradiated riboflavin in cultured human tenon fibroblasts. Materials and Methods The tenon tissue was harvested from a patient undergoing strabismus surgery. The human tenon fibroblast cell culture and isolation were performed according to the standard laboratory cell culturing protocol. The cells were divided into three groups: control, treatment with irradiated and non-irradiated riboflavin. There were five different concentrations (0.00156%, 0.003125%, 0.00625%, 0.0125%, 0.025%) in each group of riboflavin. The fibroblasts were treated with riboflavin and the cellular viability was assessed at 24-hour and 48-hour post treatment with MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide colorimetric assay. The absorbance values were analysed using Magellan microplate reader data analysis. A triplicate of readings was taken. The data were presented as mean ± standard deviation of the triplicates. Statistical analysis was performed with Statistical Package for Social Sciences (SPSS) analysis version 23. Results Irradiated riboflavin caused a concentration-dependent cell death in human tenon fibroblast cell culture (p < .05). The antiproliferative difference between irradiated and non-irradiated riboflavin was significant up to 48 hours (p < .05). Post hoc multiple comparisons showed higher concentrations of irradiated riboflavin (0.0125% and 0.025%) caused more reduction in cellular viability in human tenon fibroblast cells (p < .05). The duration of treatment is not a causative factor in this study. Conclusions This pilot experiment demonstrated that irradiated riboflavin induced cell death in human tenon fibroblast culture in a concentration-dependent manner, but is not time-dependent. Further exploratory investigations should be performed to determine the mechanism of cell death. We postulate that apoptosis occurred in these irradiated riboflavin-treated cells.

TAMI-17. INDUCTION OF SYNTHETIC LETHALITY BY ACTIVATION OF MITOCHONDRIAL CLPP AND INHIBITION OF HDAC1/2 IN GLIOBLASTOMA

Abstract Activation of the mitochondrial ClpP protease is an innovative therapeutic concept and the identification of synthetic lethal interactions may foster the development of novel therapies for glioblastoma (GBM). By integration of a transcriptome, metabolite and U-13C-glucose tracing analyses, we showed that activation of the mitochondrial ClpP protease through constitutively active ClpP (Y118A) or utilization of second-generation imipridone compounds (ONC206 and ONC212) in combination with genetic interference of HDAC1 and HDAC2 as well as with global (Panobinostat) and selective (Romidepsin) HDAC inhibitors caused synergistic reduction of viability in established, neuro-sphere and patient-derived xenograft (PDX) cultures of human GBM, which was mediated by interference with tricarboxylic acid cycle activity and GBM cell respiration. Notably, human astrocytes were significantly less susceptible to the combination treatment of HDAC-inhibitors and ClpP activators. The reduction of GBM viability occurred independent of TP53 status and was accompanied by activation of cell death with apoptotic features along with cleavage of caspases regulated chiefly by Bcl-xL and Mcl-1. Importantly, knockdown of the ClpP protease or ectopic expression of a ClpP D190A mutant almost completely rescued from the inhibition of oxidative energy metabolism as well as from the reduction of cellular viability by ClpP activators and the combination treatment, suggesting critical involvement of this protein. Finally, utilizing GBM PDX models, we demonstrated that the combination treatment of HDAC-inhibitors and imipridones reduced tumor growth and prolonged host survival more potently than single treatments or vehicle in vivo. Collectively, these observations suggest that the efficacy of HDAC inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

Anticancer Impact of Nitric Oxide (NO) and NO Combination with SMYD-3 Inhibitor on Breast Carcinomas.

Despite enormous advances in the detection and treatment of breast cancer, it still remains the leading cancer diagnosis and has the second highest mortality rate. Thus, breast cancer research is a high priority for academics and clinicians alike. Based on previous research indicating the potential of nitric oxide (NO) and SMYD-3 inhibition, this work sought to expand upon these concepts and combine the two approaches. Both NO (from S-Nitrosoglutathione (GSNO)), termed Group 1, and a combination therapeutic, inhibitor-4 (SMYD-3 inhibitor) plus NO (from GSNO), termed Group 2, were evaluated for their efficacy on breast carcinoma cell lines MCF7 and MDA-MB-231, and the normal MCF10A breast cell line, using cellular viability, colony formation capacity, cytotoxicity, and cellular apoptosis analysis. These results indicated that, in Group 1, breast carcinoma lines MCF7 and MDA-MB-231, cells experienced a moderate reduction in cellular viability (~20–25%), a large reduction in colony formation capacity (~80–90%), a moderate increase in the relative number of dead cells, and a moderate increase in cellular apoptosis. Group 2 was significantly more impactful, with a ~50% knockdown in cellular viability, a 100% reduction in colony formation capacity, a large increase in the relative number of dead cells, and a large increase in cellular apoptosis. Additionally, Group 2 induced a very small impact on the normal MCF10A cell line. Cumulatively, this work revealed the exciting impact of this combination therapeutic, indicating its potential for clinical application and further research.

Discovery of 5-{2-[5-Chloro-2-(5-ethoxyquinoline-8-sulfonamido)phenyl]ethynyl}-4-methoxypyridine-2-carboxylic Acid, a Highly Selective in Vivo Useable Chemical Probe to Dissect MCT4 Biology.

Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.