Assess Cell Viability Research Articles

Penthorum chinense Pursh inhibits ferroptosis in cellular and Caenorhabditis elegans models of Alzheimer's disease

Background: Ferroptosis, a unique type of cell death triggered by iron-dependent lipid peroxidation, plays a critical role in the pathogenesis of Alzheimer's disease (AD), a debilitating condition marked by memory loss and cognitive impairment due to the accumulation of beta-amyloid (Aβ) and hyperphosphorylated Tau protein. Increasing evidence suggests that inhibitors of ferroptosis could be groundbreaking in the treatment of AD. Method: In this study, we established in vitro ferroptosis using erastin-, RSL-3-, hemin-, and iFSP1-induced PC-12 cells. Using MTT along with Hoechst/PI staining, we assessed cell viability and death. To determine various aspects of ferroptosis, we employed fluorescence probes, including DCFDA, JC-1, C11 BODIPY, Mito-Tracker, and PGSK, to measure ROS production, mitochondrial membrane potential, lipid peroxidation, mitochondrial morphology, and intracellular iron levels. Additionally, Western blotting, biolayer interferometry technology, and shRNA were utilized to investigate the underlying molecular mechanisms. Furthermore, p-CAX APP Swe/Ind- and pRK5-EGFP-Tau P301L overexpressing PC-12 cells, along with Caenorhabditis elegans (C. elegans) strains CL4176, CL2331, and BR5270, were employed to examine ferroptosis in AD models. Results: Here, we conducted a screening of our natural medicine libraries and identified the ethanol extract of Penthorum chinense Pursh (PEE), particularly its ethyl acetate fraction (PEF), displayed inhibitory effects on ferroptosis in cells. Specifically, PEF inhibited the generation of ROS, lipid peroxidation, and intracellular iron levels. Furthermore, PEF demonstrated protective effects against H2O2-induced cell death, ROS production, and mitochondrial damage. Mechanistic investigations unveiled PEF's modulation of intracellular iron accumulation, GPX4 expression and activity, and FSP1 expression. In p-CAX APP Swe/Ind and pRK5-EGFP-Tau P301L overexpressing PC-12 cells, PEF significantly reduced cell death, as well as ROS and lipid peroxidase production. Moreover, PEF ameliorated paralysis and slowing rate in Aβ and Tau transgenic C. elegans models, while inhibiting ferroptosis, as evidenced by decreased DHE intensity, lipid peroxidation levels, iron accumulation, and expression of SOD-3 and gst-4. Conclusion: Our findings highlight the suppressive effects of PEF on ferroptosis in AD cellular and C. elegans models. This study helps us better understand how ferroptosis affects AD and emphasizes the potential of PCP as a candidate for AD intervention.

Evaluation of Alginate Hydrogel Microstrands for Stromal Cell Encapsulation and Maintenance

Mesenchymal stromal cells (MSCs) have displayed potential in regenerating organ function due to their anti-fibrotic, anti-inflammatory, and regenerative properties. However, there is a need for delivery systems to enhance MSC retention while maintaining their anti-fibrotic characteristics. This study investigates the feasibility of using alginate hydrogel microstrands as a cell delivery vehicle to maintain MSC viability and phenotype. To accommodate cell implantation needs, we invented a Syringe-in-Syringe approach to reproducibly fabricate microstrands in small numbers with a diameter of around 200 µm and a porous structure, which would allow for transporting nutrients to cells by diffusion. Using murine NIH 3T3 fibroblasts and primary embryonic 16 (E16) salivary mesenchyme cells as primary stromal cell models, we assessed cell viability, growth, and expression of mesenchymal and fibrotic markers in microstrands. Cell viability remained higher than 90% for both cell types. To determine cell number within the microstrands prior to in vivo implantation, we have further optimized the alamarBlue assay to measure viable cell growth in microstrands. We have shown the effect of initial cell seeding density and culture period on cell viability and growth to accommodate future stromal cell delivery and implantation. Additionally, we confirmed homeostatic phenotype maintenance for E16 mesenchyme cells in microstrands.

The effect of 1-deoxynojirimycin isolated from logging residue of Bagassa guianensis on an in vitro cancer model

Introduction:Bagassa guianensis Aubl, a tree widely distributed in Brazil, significantly contributes to the furniture industry. Notably, it harbors the bioactive compound 1-deoxynojirimycin (1-DNJ), which is retrievable from timber residues and retains activity even days after wood extraction. This makes Bagassa guianensis a promising biological resource for anticancer therapy and pharmacology studies. This study delves into the in vitro antineoplastic actions of 1-DNJ, focusing on adenocarcinoma gastric cell lines (ACP02) and glioblastoma (A172).Methods: The effect of 1-DNJ on cell viability was evaluated after 72 hours of treatment in the ACP02 and A172 cell lines. We also assessed the effect of 1-DNJ on the pattern of cell migration, cell death, changes in the cell cycle by flow cytometry, the production of reactive oxygen, and its antioxidant capacity in the scavenging of free radicals.Results: Assessing cell viability after 72 h (about 3 days) of treatment reveals a remarkable reduction, particularly in glioblastoma cells (A172), exhibiting a lower IC50 compared to ACP02 and MRC5 (fibroblast derived from normal lung tissue) cell lines. This decreased viability correlates with reduced reactive oxygen species (ROS) production in both cell lines after the treatment with 1-DNJ. Furthermore, 1-DNJ induces cell cycle arrest, impedes cell migration, and prompts cell death in ACP02 and A172.Discussion: These findings support 1-DNJ as a potent antineoplastic agent, particularly efficacious against glioblastoma and gastric adenocarcinoma. Thus, unveiling the therapeutic potential of Bagassa guianensis Aubl for cancer treatment and expanding the horizons of bioeconomy applications.

Functionalization of PCL-Based Fiber Scaffolds with Different Sources of Calcium and Phosphate and Odontogenic Potential on Human Dental Pulp Cells

This study investigated the incorporation of sources of calcium, phosphate, or both into electrospun scaffolds and evaluated their bioactivity on human dental pulp cells (HDPCs). Additionally, scaffolds incorporated with calcium hydroxide (CH) were characterized for degradation, calcium release, and odontogenic differentiation by HDPCs. Polycaprolactone (PCL) was electrospun with or without 0.5% w/v of calcium hydroxide (PCL + CH), nano-hydroxyapatite (PCL + nHA), or β-glycerophosphate (PCL + βGP). SEM/EDS analysis confirmed fibrillar morphology and particle incorporation. HDPCs were cultured on the scaffolds to assess cell viability, adhesion, spreading, and mineralized matrix formation. PCL + CH was also evaluated for gene expression of odontogenic markers (RT-qPCR). Data were submitted to ANOVA and Student’s t-test (α = 5%). Added CH increased fiber diameter and interfibrillar spacing, whereas βGP decreased both. PCL + CH and PCL + nHA improved HDPC viability, adhesion, and proliferation. Mineralization was increased eightfold with PCL + CH. Scaffolds containing CH gradually degraded over six months, with calcium release within the first 140 days. CH incorporation upregulated DSPP and DMP1 expression after 7 and 14 days. In conclusion, CH- and nHA-laden PCL fiber scaffolds were cytocompatible and promoted HDPC adhesion, proliferation, and mineralized matrix deposition. PCL + CH scaffolds exhibit a slow degradation profile, providing sustained calcium release and stimulating HDPCs to upregulate odontogenesis marker genes.

A phenotypic screening approach to target p60AmotL2-expressing invasive cancer cells

BackgroundTumor cells have the ability to invade and form small clusters that protrude into adjacent tissues, a phenomenon that is frequently observed at the periphery of a tumor as it expands into healthy tissues. The presence of these clusters is linked to poor prognosis and has proven challenging to treat using conventional therapies. We previously reported that p60AmotL2 expression is localized to invasive colon and breast cancer cells. In vitro, p60AmotL2 promotes epithelial cell invasion by negatively impacting E-cadherin/AmotL2-related mechanotransduction.MethodsUsing epithelial cells transfected with inducible p60AmotL2, we employed a phenotypic drug screening approach to find compounds that specifically target invasive cells. The phenotypic screen was performed by treating cells for 72 h with a library of compounds with known antitumor activities in a dose-dependent manner. After assessing cell viability using CellTiter-Glo, drug sensitivity scores for each compound were calculated. Candidate hit compounds with a higher drug sensitivity score for p60AmotL2-expressing cells were then validated on lung and colon cell models, both in 2D and in 3D, and on colon cancer patient-derived organoids. Nascent RNA sequencing was performed after BET inhibition to analyse BET-dependent pathways in p60AmotL2-expressing cells.ResultsWe identified 60 compounds that selectively targeted p60AmotL2-expressing cells. Intriguingly, these compounds were classified into two major categories: Epidermal Growth Factor Receptor (EGFR) inhibitors and Bromodomain and Extra-Terminal motif (BET) inhibitors. The latter consistently demonstrated antitumor activity in human cancer cell models, as well as in organoids derived from colon cancer patients. BET inhibition led to a shift towards the upregulation of pro-apoptotic pathways specifically in p60AmotL2-expressing cells.ConclusionsBET inhibitors specifically target p60AmotL2-expressing invasive cancer cells, likely by exploiting differences in chromatin accessibility, leading to cell death. Additionally, our findings support the use of this phenotypic strategy to discover novel compounds that can exploit vulnerabilities and specifically target invasive cancer cells.

SiRNA-Mediated Knockdown of ABCB1 Enhances the Efficacy of Doxorubicin and Vinorelbine in Breast Cancer Cells.

Breast cancer remains a leading cause of cancer-related deaths among women, primarily attributed to the formidable challenge of multidrug resistance, often driven by the overexpression of the ABCB1 gene. This study aimed to assess the synergistic effects of siRNA, doxorubicin, and vinorelbine on ABCB1 gene expression and cell viability in doxorubicin-resistant MCF-7/ADR breast cancer cells, with siRNA targeting ABCB1 to reduce its expression and doxorubicin/ vinorelbine to eradicate cancer cells. Our methodology involved culturing MCF-7 and MCF-7/ADR cells in standard cell culture conditions. The synthesized siRNA sequences transfected cells with siRNA at final concentrations of 10, 20, and 30 nM and assessed cell viability using the MTT assay was performed. Real-time PCR was employed to quantify ABCB1 mRNA expression levels. Results indicated that MCF-7/ADR cells exhibited substantial resistance to vinorelbine and doxorubicin compared to MCF-7 cells, displaying resistance at 12.50 μM and 25.00 μM for vinorelbine and 6.25 μM and 25.00 μM for doxorubicin. Remarkably, siRNA treatment effectively reversed drug resistance in MCF-7/ADR cells across all concentrations of vinorelbine and doxorubicin tested. When combined, siRNA, doxorubicin, and vinorelbine yielded a significantly greater reduction in cell viability compared to individual drug treatments, particularly at a 20 μM siRNA concentration. This combination therapy also significantly suppressed ABCB1 gene expression by a factor of 41.48 in MCF-7 cells relative to MCF-7/ADR cells. these findings suggest that combining siRNA, doxorubicin, and vinorelbine holds promise as a therapeutic strategy to overcome ABCB1-mediated multidrug resistance in breast cancer. Further investigations and clinical trials are warranted to evaluate its clinical efficacy rigorously.

Indole-3-Carbinol Promotes Apoptosis and Inhibits the Metastasis of Esophageal Squamous Cell Carcinoma by Downregulating the Wnt/β-Catenin Signaling Pathway.

The incidence and mortality rates of esophageal squamous cell carcinoma (ESCC) have been significantly increasing in China. Indole-3-carbinol (I3C), a naturally occurring component in cruciferous vegetables, is an effective cancer therapy. Yet, its effect and action mechanism in ESCC are still not fully understood. This study explored the role of I3C in ESCC in vitro and in vivo by focusing on the Wnt/β-catenin signaling pathway. MTT and flow cytometry were used to assess cell viability and apoptosis in EC18 and TE1 cells, while wound healing and transwell assays were used to investigate cell migration and invasion in vitro. Expression of β-catenin, c-myc, and cyclin D1 was determined by Western blot; LiCl (an agonist of the canonical Wnt signaling that inhibits GSK3β activity) was used to assess the role of I3C on the Wnt/β-catenin signaling pathway. For in vivo experiments, nude BALB/c mice bearing EC18 xenografts were treated with I3C and/or LiCl. I3C promoted ESCC apoptosis and inhibited cell migration and invasion by downregulating β-catenin, c-myc, and cyclin D1 in vitro and decreased the tumor growth in vivo; this process was reversed by LiCl treatment. In summary, I3C inhibits ESCC malignant behavior by suppressing the Wnt/β-catenin signaling pathway, thus deeming it a promising drug for ESCC treatment.

4′-O-methylbavachalcone alleviates ischemic stroke injury by inhibiting parthanatos and promoting SIRT3

Cerebral ischemia-reperfusion injury (CIRI) can induce massive death of ischemic penumbra neurons via oxygen burst, exacerbating brain damage. Parthanatos is a form of caspase-independent cell death involving excessive activation of PARP-1, closely associated with intense oxidative stress following CIRI. 4′-O-methylbavachalcone (MeBavaC), an isoprenylated chalcone component in Fructus Psoraleae, has potential neuroprotective effects. This study primarily investigates whether MeBavaC can act on SIRT3 to alleviate parthanatos of ischemic penumbra neurons induced by CIRI. MeBavaC was oral gavaged to the middle cerebral artery occlusion-reperfusion (MCAO/R) rats after occlusion. The effects of MeBavaC on cerebral injury were detected by the neurological deficit score and cerebral infarct volume. In vitro, PC-12 cells were subjected to oxygen and glucose deprivation/reoxygenation (OGD/R), and assessed cell viability and cell injury. Also, the levels of ROS, mitochondrial membrane potential (MMP), and intracellular Ca2+ levels were detected to reflect mitochondrial function. We conducted western blotting analyses of proteins involved in parthanatos and related signaling pathways. Finally, the exact mechanism between the neuroprotection of MeBavaC and parthanatos was explored. Our results indicate that MeBavaC reduces the cerebral infarct volume and neurological deficit scores in MCAO/R rats, and inhibits the decreased viability of PC-12 cells induced by OGD/R. MeBavaC also downregulates the expression of parthanatos-related death proteins PARP-1, PAR, and AIF. However, this inhibitory effect is weakened after the use of a SIRT3 inhibitor. In conclusion, the protective effect of MeBavaC against CIRI may be achieved by inhibiting parthanatos of ischemic penumbra neurons through the SIRT3-PARP-1 axis.

Patient-derived tumor-like cell clusters for personalized chemo- and immunotherapies in non-small cell lung cancer.

Many patient-derived tumor models have emerged recently. However, their potential to guide personalized drug selection remains unclear. Here, we report patient-derived tumor-like cell clusters (PTCs) for non-small cell lung cancer (NSCLC), capable of conducting 100-5,000 drug tests within 10days. We have established 283 PTC models with an 81% success rate. PTCs contain primary tumor epithelium self-assembled with endogenous stromal and immune cells and show a high degree of similarity to the original tumors in phenotypic and genotypic features. Utilizing standardized culture and drug-response assessment protocols, PTC drug-testing assays reveal 89% overall consistency in prospectively predicting clinical outcomes, with 98.1% accuracy distinguishing complete/partial response from progressive disease. Notably, PTCs enable accurate prediction of clinical outcomes for patients undergoing anti-PD1 therapy by combining cell viability and IFN-γ value assessments. These findings suggest that PTCs could serve as a valuable preclinical model for personalized medicine and basic research in NSCLC.

MicroRNA-105-5p protects against chondrocyte injury, extracellular matrix degradation, and osteoarthritis progression by targeting SPARCL1.

Both microRNA (miR)-105-5p and SPARCL1 were discovered to be differentially expressed in osteoarthritis (OA), but their roles and exact mechanisms have not been entirely elaborated. This paper sets out to probe the impact of miR-105-5p/SPARCL1 on chondrocyte injury, extracellular matrix degradation, and osteoarthritis progression. C28/I2 cells were stimulated with IL-1β to construct an in vitro OA model. C28/I2 cells were transfected with sh-SPARCL1, oe-SPARCL1, or miR-105-5p mimic before IL-1β induction. CCK-8 assay, flow cytometry, and ELISA were adopted to assess cell viability, apoptosis, and inflammatory factor expression, respectively. The binding relationship of miR-105-5p to SPARCL1 was assessed using dual-luciferase reporter assay. After an OA rat model was established, rats underwent intra-articular injection with ago-miR-105-5p. TUNEL was applied to determine cell apoptosis in vivo. mRNA and protein levels were measured by qRT-PCR and western blot, respectively, in vitro and in vivo. IL-1β treatment diminished miR-105-5p expression and augmented SPARCL1 expression in C28/I2 cells. miR-105-5p decreased SPARCL1 expression by targeting SPARCL1. miR-105-5p overexpression or SPARCL1 silencing prominently reversed the decrease in viability and the promotion of inflammatory factor production, cartilage matrix degradation, and apoptosis in IL-1β-stimulated C28/I2 cells. Furthermore, upregulation of SPARCL1 nullified the influence of miR-105-5p overexpression on viability, apoptosis, inflammation, and cartilage matrix degradation in IL-1β-stimulated C28/I2 cells. miR-105-5p overexpression ameliorated knee cartilage tissue injury in OA rats. Conclusively, miR-105-5p exerted suppressive effects on chondrocyte injury, extracellular matrix degradation, and OA progression by targeting SPARCL1.

Low-molecular-weight fucoidan increases telomere length and immunostimulatory effects on NK-92 cells following inhaled anesthetic injury

Inhaled anesthetics, such as isoflurane, may cause side effects, including short-term immunosuppression and DNA damage. In contrast, low molecular weight fucoidan (LMF), derived from brown seaweed, exhibits promising immunomodulatory effects. In this study, we determined the effect of isoflurane on telomeres and examined the potential of LMF to ameliorate the harmful effects of isoflurane. Male Lewis rats, the mouse lymphoma cell line YAC-1, and the human nature killer cell line NK-92 MI were exposed to isoflurane. The relative telomere length (T/S) ratio and mRNA expression were determined by quantitative PCR. The viability assay was used to assess cell viability. In vivo, 2% isoflurane exposure, which is a clinically relevant concentration, reduced telomere length, and correlated with exposure frequency and duration. Isoflurane concentrations above 2% shortened YAC-1 telomeres, with minimal impact on cell viability. LMF pre-treatment enhanced NK-92 MI cell survival resulting from isoflurane exposure and exerted superior telomere protection compared with LMF post-treatment. Furthermore, adding LMF during isoflurane exposure resulted in a significant increase in IFN-γ, TNF-α, and IL-10 mRNA compared with the untreated group. LMF protected against isoflurane-induced telomere shortening, enhanced NK cell viability, and modulated cytokine expression, thus mitigating postoperative immune suppression and risk of tumor metastasis.

Turmeric Inhibits MDA-MB-231 Cancer Cell Proliferation, Altering miR-638-5p and Its Potential Targets.

Recent research suggests curcumin extracted from the turmeric plant may inhibit the proliferation of cancer cells by controlling the expression of microRNAs (miRNAs). The effect of phenolic curcumin on miR-638-5p and potential target gene expressions in the triple negative breast cancer (TNBC) cell line MDA-MB-231 was investigated in this study. GSE154255 and GSE40525 datasets were downloaded and analyzed using GEO2R to identify dysregulated miRNAs in TNBC. To find differently expressed genes in breast cancer (BRCA), The Cancer Genome Atlas Program data was examined. Utilizing in silico tools, KEGG, GO, and other enrichment analyses were performed. The databases miRNet, miRTarBase v8.0, and TarBase v.8 were used for miRNA and mRNA matching. Real-time quantitative reverse transcription polymerase chain reaction was used to examine the levels of miRNA and its targets in miRNA mimic transfected/curcumin-treated MDA-MB-231 cultures and controls. The cell viability detection kit-8 method was used to assess cell viability, and the scratch assay was used to conduct migration assessment. Bioinformatics analysis showed that miR-638-5p was significantly reduced in TNBC patients. Experimental results showed that miR-638-5p was upregulated in MDA-MB-231 treated with curcumin, while the potential target genes of miR-638-5p, CFL1, SIX4, MAZ, and CDH1 were downregulated. Mimic miR-638-5p transfection inhibited MDA-MB-231 cell proliferation and reduced migration and expression of CFL1, SIX4, and MAZ genes was decreased in mimic miR-638-5p transfected cells. These findings suggest that curcumin exerts its anticancer effects on MDA-MB-231 cells by modulating the expression of miR-638-5p and its possible target genes.

The effect of quercetin on adipogenesis, lipolysis, and apoptosis in 3T3-L1 adipocytes: The role of SIRT1 pathways.

Lipotoxicity, caused by adipocyte triglyceride over-accumulation, contributes to obesity-related comorbidities such as hypertension, type 2 diabetes, coronary heart disease, respiratory dysfunction, and osteoarthritis. This study focuses on determining how sirtuin-1 (SIRT-1) mediates quercetin's (QCT) effect on 3T3-L1 adipocytes. Key aspects of this study include preventing adipogenesis, inducing lipolysis, and stimulating adipocyte apoptosis. 3T3-L1 adipocytes underwent treatment with varying QCT doses, lipopolysaccharide (LPS), and the SIRT-1 inhibitor EX-527, followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide [MTT] assay for cell viability assessment. Furthermore, quantitative real-time polymerase chain reaction measured mRNA expression levels of adipogenesis markers (fatty acid synthase [FASN] and peroxisome proliferator-activated receptor gamma [PPARγ]), lipolysis markers (adipose triglyceride lipase [ATGL] and hormone-sensitive lipase [HSL]), and apoptosis markers (B-cell lymphoma2 [Bcl-2], Bcl-2 Associated -X-protein [BAX] and Caspase-3). The data showed that LPS+QCT significantly reduced cell viability in a dose- and time-dependent manner, unaffected by LPS+QCT+EX-527. Treatment with LPS+QCT did not affect FASN and PPARγ expression but significantly increased ATGL and HSL mRNA expression compared with LPS alone. Interestingly, EX-527 reversed the effects of LPS+QCT on lipogenesis and lipolysis markers completely. QCT enhanced apoptosis in a SIRT-1 independent pattern. The data suggest that QCT suppresses adipogenesis while increasing lipolysis via SIRT-1. However, QCT's effects on apoptosis appear to be independent of SIRT-1. These findings provide further evidence for QCT's effects on adipocytes, particularly its interaction with SIRT-1.

Printing GelMA bioinks: a strategy for buildingin vitromodel to study nanoparticle-based minocycline release and cellular protection under oxidative stress.

Owing to its thermoresponsive and photocrosslinking characteristics, gelatin methacryloyl (GelMA)-based biomaterials have gained widespread usage as a novel and promising bioink for three-dimensional bioprinting and diverse biomedical applications. However, the flow behaviors of GelMA during the sol-gel transition, which are dependent on time and temperature, present significant challenges in printing thick scaffolds while maintaining high printability and cell viability. Moreover, the tunable properties and photocrosslinking capabilities of GelMA underscore its potential for localized drug delivery applications. Previous research has demonstrated the successful incorporation of minocycline (MH) into GelMA scaffolds for therapeutic applications. However, achieving a prolonged and sustained release of concentrated MH remains a challenge, primarily due to its small molecular size. The primary aim of this study is to investigate an optimal extrusion printing method for GelMA bioink in extrusion bioprinting, emphasizing its flow behaviors that are influenced by time and temperature. Additionally, this research seeks to explore the potential of GelMA bioink as a carrier for the sustained release of MH, specifically targeting cellular protection against oxidative stress. The material properties of GelMA were assessed and further optimization of the printing process was conducted considering both printability and cell survival. To achieve sustained drug release within GelMA, the study employed a mechanism using metal ion mediation to facilitate the interaction between MH, dextran sulfate (DS), and magnesium, leading to the formation of nanoparticle complexes (MH-DS). Furthermore, a GelMA-basedin vitromodel was developed in order to investigate the cellular protective properties of MH against oxidative stress. The experimental results revealed that the printability and cell viability of GelMA are significantly influenced by the printing duration, nozzle temperature, and GelMA concentrations. Optimal printing conditions were identified based on a thorough assessment of both printability and cell viability. Scaffolds printed under these optimal conditions exhibited exceptional printability and sustained high cell viability. Notably, it was found that lower GelMA concentrations reduced the initial burst release of MH from the MH-dextran sulfate (MH-DS) complexes, thus favoring more controlled, sustained release profiles. Additionally, MH released under these conditions significantly enhanced fibroblast viability in anin vitromodel simulating oxidative stress.

Theoretical Evaluation of Fluorinated Resazurin Derivatives for In Vivo Applications.

Primarily owing to the pronounced fluorescence exhibited by its reduced form, resazurin (also known as alamarBlue®) is widely employed as a redox sensor to assess cell viability in in vitrostudies. In an effort to broaden its applicability for in vivo studies, molecular adjustments are necessary to align optical properties with the near-infrared imaging window while preserving redox properties. This study delves into the theoretical characterisation of a set of fluorinated resazurin derivatives proposed by Kachur et al., 2015 examining the influence of fluorination on structural and electrochemical properties. Assuming that the conductor-like polarisable continuum model mimics the solvent effect, the density functional level of theory combining M06-2X/6-311G* was used to calculate the redox potentials. Furthermore, (TD-)DFT calculations were performed with PBE0/def2-TZVP to evaluate nucleophilic characteristics, transition states for fluorination, relative energies, and fluorescence spectra. With the aim of exploring the potential of resazurin fluorinated derivatives as redox sensors tailored for in vivo applications, acid-base properties and partition coefficients were calculated. The theoretical characterisation has demonstrated its potential for designing novel molecules based on fundamental principles.

Abstract 4696: ERBB2 signaling and inhibition in bladder cancer

Abstract The role of human epidermal growth factor receptor-2 (HER2) protein and the inhibition of its oncogenic signaling pathway, ERBB2, is well defined and found to be beneficial in combination with standard chemotherapies in breast and certain gastro-intestinal cancers. Targeting the ERBB2 pathway has had unclear or inconsistent results in early trials in bladder cancer (BLCA) and the efficacy of its combination with cisplatin, a standard BLCA chemotherapy, is not known. In this study, we identify the transcriptomic oncogenic signatures that correlate with ERBB2 signaling in BLCA, explore how HER2 expression in BLCA cell lines correlates with treatment response in vitro, and evaluate the potential of ERBB2 inhibition in combination with cisplatin for BLCA. We analyzed the tumor transcriptome from the Cancer Genome Atlas (TCGA) study of BLCA with single sample Gene Set Enrichment Analyses (ssGSEA) for Hallmark and Oncogenic gene sets. We performed multiple Pearson correlation analyses of HER2 protein levels and ERBB2 signaling with other signaling pathways in the TCGA BLCA tumors and adjusted the significance for multiple comparisons using the Benjamini-Hochberg method. Immunoblotting was used to assess HER2 protein levels across a panel of bladder cancer cell lines. We then assessed cell viability and colony formation upon exposure to the specific ERBB2 inhibitor, tucatinib. Synergy between cisplatin and tucatinib in BLCA cell lines was evaluated using the Chou-Talalay method. In the TCGA BLCA dataset, HER2 protein positively correlates with ERBB2 signaling with a Pearson correlation coefficient of 0.45 and an adjusted p-value of 1.73E-17. ERBB2 signaling has multiple significant positive and negative correlations. ERBB2 signaling negatively correlates with the DNA repair gene set with a Pearson coefficient of -0.5 and an adjusted p-value of 1.40E-22. HER2 protein expression varies greatly between bladder cancer cell lines: HT-1197 and UMUC3 have lower HER2 expression, with HT-1376, T24, RT4, 5637, and SW780 showing higher HER2 expression. HT-1197 and RT4 had the lowest IC50s for tucatinib and showed a strong response to low doses of tucatinib in the colony formation assay. Cisplatin and tucatinib have synergetic growth inhibition (combination index <1) at low concentration levels in the HT-1197 and SW780 cell lines. Multiple oncogenic pathways coexist with ERBB2 signaling in BLCA. The varied response to specific ERBB2 inhibition and the lack of correlation of the response with HER2 protein level in cell lines alludes to alternative oncogenic pathways. A strong negative correlation of ERBB2 signaling with the DNA response pathway in the TCGA BLCA dataset, combined with preclinical evidence of synergy between tucatinib and cisplatin, indicates a potential for development of the combination as a clinical treatment strategy and warrants further study. Citation Format: Constance Caparas-Spaugy, Chris Stubben, Ken Boucher, Bingjian Feng, Aaron Atkinson, David Nix, Bradley Cairns, Sumati Gupta. ERBB2 signaling and inhibition in bladder 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 4696.

Abstract 991: Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology

Abstract Introduction: Pancreatic cancer has the lowest 5-year survival rates among all cancers because of the challenges with early detection, its aggressive nature, and resistance to chemotherapy; this makes prevention a promising approach for reducing related mortalities. The disease is predominantly diagnosed in individuals over 60, suggesting that age-related physiological decline impacts its onset; therefore, delaying aging hallmarks may delay its onset. Nicotinamide dinucleotide (NAD+) precursors may hold a unique advantage over other geroprotectors because they target multiple aging hallmarks. Heron, we investigated how NAD+ supplementation impacts normal human pancreatic ductal epithelial cells (HPDE) under stress as a surrogate for cancer prevention in vitro. We also examined the paradoxical impact of NAD+ precursor supplementation on pancreatic cancer growth during chemotherapy. Methods: In vitro, we mimicked age-related NAD+ decline in the media. We stressed HPDE cells under these conditions and evaluated the effect of supplementation with the NAD+ precursor, Nicotinamide mononucleotide (NMN). We assessed cell viability using the PicoGreen assay, mitochondrial function, and reactive oxygen species (ROS) using the Seahorse XF Analyzer and the mitoSox kit, respectively, DNA repair and DNA damage using a western blot for Poly ADP ribose (PAR) and gamma H2AX, respectively, and SIRT3 expression was determined using a western blot. To evaluate the impact of NAD+ supplementation on pancreatic cancer cells, we treated pancreatic cancer cells with NMN and oxaliplatin. We then assessed the mitochondrial function, ROS levels, DNA damage, and repair. We implanted MiaPaCa-2 xenografts into athymic nude mice and treated them daily with 300mg/Kg of NMN and biweekly 5mg/kg of Oxaliplatin. Results: Experiments revealed that NMN is not toxic to HPDE cells, even at high concentrations. There was increased cell survival of HPDE cells under stress (nutrient deprivation and alkylating agents) following NMN supplementation. We also noted reduced ROS levels, increased SIRT3 expression, and PARylation. Pancreatic cancer cells were less sensitive to Oxaliplatin following NMN supplementation and showed an increased oxygen consumption rate, increased PARylation, and reduced ROS levels. There was reduced sensitivity to oxaliplatin treatment in the mice with MiaPaCa-2 xenografts treated with NMN. Conclusion: These findings demonstrate a cytoprotective role of NAD+ precursor supplementation in normal pancreatic ductal epithelial cells, with implications for possible cancer prevention. NAD+ supplementation also rescued cancer cells under cytotoxic stress, meaning that geroprotectors like NMN, in conjunction with chemotherapy, may unintentionally promote cancer progression. Future studies will directly test NAD+ precursor supplementation in genetically engineered pancreatic cancer models. Citation Format: Faith Nakazzi, Mehrad Zarei, Hallie J. Graor, Alexander W. Loftus, Omid Hajihassanni, Jordan M. Winter. Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology [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 991.

Abstract 4531: The synergistic potential of PARP inhibitors and irinotecan in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is highly aggressive and associated with a poor prognosis. While effective treatment is limited after a relapse from platinum-based chemotherapy, recent research has identified novel pathway targets including DNA damage repair (DDR) pathway. Poly (ADP-ribose) polymerase inhibitors (PARPi) have been currently evaluated for their efficacy as a monotherapy and in combination with other agents, such as immunotherapy. Methods: We evaluated the efficacy of combining PARPi and irinotecan in ten SCLC cell lines with diverse genetic backgrounds in the DDR genes. After treating the cells with three different PARPi in combination with irinotecan, we assessed cell viability and the combination effects. Furthermore, we confirmed protein changes in the DDR pathway through western blotting and measured the number of apoptotic cells using Annexin V staining. Results: In our experiments, talazoparib had the most potent effect at low concentrations, while olaparib and venadaparib showed more pronounced synergistic effects (average fold change of IC50: olaparib, 1649±4049; talazoparib, 25±34.21; venadaparib, 336±596.01; see Table). The combination of talazoparib or venadaparib with irinotecan led to a reduction of IC50 values to below 10 nM in 7 out of 10 tested cell lines. Three cell lines carrying BRCA mutations displayed a strong sensitivity to PARPi, whereas no significant correlation found with alterations in other DDR genes. The combined use of both drugs increased p-chk1 and p-p53 proteins, along with apoptosis signaling. Additionally, venadaparib alone did not induce cell toxicity but significantly increased apoptosis when combined with irinotecan, even at very low concentrations. Conclusions: The combination of a PARPi with irinotecan significantly inhibited cell growth more than the use of either agent alone. Our findings indicate that clinical trials are warranted to confirm the effectiveness of combining PARPi with irinotecan in patients with SCLC. IC50 values of PARP inhibitors in SCLC cells w/or w/o irinotecan 50 Nm IC50 (nM) Olaparib Talazoparib Venadaparib BRCA or PTEN status Irinotecan 50 nM - + - + - + BRCA1 deletion NCI-H1694 5,933 148.9 52.38 2.19 >10,000 1.02 BRCA2 deletion NCI-H211 392.8 37.20 2.28 0.99 3.82 0.94 BRCA2 deletion NCI-H1048 1,704 282.7 9.74 2.36 62.01 3.02 PTEN deletion NCI-H1341 >10,000 6,880 452.9 83.9 >10,000 >10,000 PTEN deletion DMS 79 2,382 221.9 18.55 3.4 175.16 3.07 no mutation NCI-H841 5,099 6,202 529.5 380.5 >10,000 >10,000 no mutation NCI-H2029 >10,000 >10,000 478.2 56.16 >10,000 >10,000 no mutation NCI-H2227 3,407 827.5 52.03 2.17 >10,000 0.69 no mutation NCI-H146 5,094 3.31 56.11 0.86 1,295 0.93 no mutation NCI-H209 579.2 0.05 5.24 0.05 8.18 0.04 Citation Format: Songji Oh, Miso Kim, Soyeon Kim, Bhumsuk Keam, Jeonghwan Youk, Tae Min Kim, Dong-Wan Kim, Dae Seog Heo. The synergistic potential of PARP inhibitors and irinotecan in small cell lung 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 4531.

Abstract 7271: Proteasome inhibitor FHND6091: A potent therapeutic candidate forPTEN-deficient cholangiocarcinoma

Abstract Background: Cholangiocarcinoma (CCA), characterized by high genetic heterogeneity, poses a significant challenge due to limited therapeutic options. Notably, the tumor suppressor gene PTEN is frequently altered in more than 50% of CCA clinical specimens. PTEN deficiency enhances proteasome subunit expression and proteasome proteolytic activity, and facilitates the potential therapeutic efficacy of proteasome inhibitors in CCA. The proteasome inhibitor FHND6091, a novel drug developed by Jiangsu Chia Tai Fenghai Pharmaceutical Co., Ltd., has obtained IND approval from NMPA and is currently undergoing a first-in-human phase 1 trial for multiple myeloma (MM) and mantle cell lymphoma (MCL). Method: This study employed a comprehensive approach, encompassing both in vitro and in vivo pharmacology studies. The investigation involved purified human proteasome activity assays, cell viability assays, and in vivo anti-tumor activity studies utilizing a cholangiocarcinoma patient-derived xenograft (CCA PDX) model with PTEN deficiency. Results: FHND6091 exhibited potent inhibitory effects on the β5 subunit of the 20S proteasome, with IC50 value of 2.22 nM. In cell viability assessments, FHND6091 demonstrated selective efficacy in PTEN-deficient CCA cell lines, with IC50 value of 5.87 nM in HCCC-9810 (PTEN deletion, homozygous loss of exon 6 to 9) and 10.08 nM in RBE (PTEN intact expression). In a PDX xenograft model with PTEN deficiency, gavage administration of FHND6091 BIW at 1.0 mg/kg (first week)/1.5 mg/kg (subsequent two weeks) exhibited advanced anti-tumor activity (TGI = 52.32%) compared to intraperitoneal administration of Bortezomib at 0.5 mg/kg BIW (TGI = 31.88%). Tissue distribution studies in rats revealed the preference of FHND6091 for liver accumulation, with an exposure (AUC0-t) approximately 44 times that of plasma after a single dose. These data corroborated that FHND6091 could achieve a specific level of exposure in the target organ liver. Conclusion: FHND6091, characterized by its irreversibility, high potency and oral bioavailability, emerges as a promising proteasome inhibitor for the treatment of both hematologic and solid tumors. These findings strongly support FHND6091 as a potential candidate for the treatment of CCA in clinical settings. Furthermore, the regulating of sensitivity to proteasome inhibitors by the PTEN status extends beyond cholangiocarcinoma to encompass various human cancers, such as ovarian, prostate, and digestive system malignancies. This broader implication establishes a theoretical foundation supporting the potential efficacy of FHND6091 as a therapeutic agent for solid tumors. Citation Format: Yongqiang Zhu, Jingmiao Shi, Chenhui Li. Proteasome inhibitor FHND6091: A potent therapeutic candidate forPTEN-deficient cholangiocarcinoma [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 7271.

Abstract 594: ORIC-613, a potential first- and best-in-class, orally bioavailable, potent and selective PLK4 inhibitor with synthetic lethality in TRIM37 high cancer models

Abstract A synthetic lethal interaction between polo-like kinase 4 (PLK4) and elevated TRIM37 was discovered in breast cancer and neuroblastoma (Meitinger et al. 2020; Yeow et al. 2020). High TRIM37 renders cells dependent on PLK4, a kinase that controls centriole duplication. Amplification and copy number gains of the 17q23 amplicon, where TRIM37 resides, are common in breast cancer and neuroblastoma and associated with early relapse and poor prognosis, making PLK4 a promising new synthetic lethal target for these patients. The development candidate ORIC-613 is an orally bioavailable, potent and exquisitely selective small molecule inhibitor of PLK4, which is synthetic lethal in TRIM37 high cells. ORIC-613 is highly selective against the kinome, including against the closely related aurora and PLK families. ORIC-613 ADME/PK profile includes high cell permeability and low potential for drug-drug interactions based on CYP profiling. ORIC-613 blocked PLK4 trans-autophosphorylation of phosphodegrons, leading to stabilization of PLK4, thus directly demonstrating target engagement in cells. PLK4 protein stabilization correlated with cell viability for ORIC-613, providing a quantifiable pharmacodynamic (PD) association with antitumor activity. Cell viability assessment in cancer cell lines revealed that ORIC-613 showed greater potency in TRIM37 high versus TRIM37 low cell lines. Importantly, ORIC-613 induced significantly greater apoptosis in TRIM37 high versus low cancer cell lines as measured by caspase 3/7 assay. We confirmed that ORIC-613 activity is on-target using an engineered cell line system of PLK4 G95L, in which the leucine mutation maintains enzymatic function but blocks compound binding. In cell viability assays, ORIC-613 was potent in the parental cells and lost activity in G95L cells. Analysis of genomic data from tumors indicates that TRIM37 copy number amplification and gain is prevalent across numerous cancers. Oral dosing of ORIC-613 resulted in tumor growth inhibition and regressions in TRIM37 high xenograft breast tumors with no body weight loss. In summary, ORIC-613 is a potential first- and best-in-class development candidate, with exquisite selectivity for PLK4, which demonstrates synthetic lethality in TRIM37 high tumors and has the potential to benefit these patients. Citation Format: Kyle A. Edgar, Siobhan McRee, Gina Andretta, Chelsea Chen, Christophe Colas, Wei Fang, Wayne Kong, Jason Long, Fang Liu, Jared Moore, Aleskandr Pankov, Dan Shore, Joanne Tan, Robert Warne, Livia Ulicna, Rakesh Vekariya, Anneleen Daemen, F Romero, Melissa R. Junttila, Lori S. Friedman. ORIC-613, a potential first- and best-in-class, orally bioavailable, potent and selective PLK4 inhibitor with synthetic lethality in TRIM37 high cancer models [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 594.