Significant Reduction In Cell Viability Research Articles

Abstract 4634: Inhibitory-κB kinase alpha as a biomarker and therapeutic target in colorectal cancer

Abstract Introduction/Aims: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality. The canonical NF-κB pathway driven by IKKβ has been implicated in CRC development and progression, however less research has focused on non-canonical NF-κB signaling, regulated by inhibitory-κB kinase alpha (IKKα). This project aimed to assess IKKα, phospho-IKKα (pIKKαs176) and IKKβ expression in a retrospective CRC cohort to establish association with survival outcomes. Subsequently, this project aimed to determine the effect of abrogating IKKα activity without perturbing the canonical NF-κB pathway controlled by IKKβ using a first-in-class selective IKKα inhibitor in vitro/ex vivo. Methods: Immunohistochemical staining for IKKα/pIKKα s176/IKKβ was performed using tissue microarrays from a CRC patient cohort (n=787). QuPath® software was used to semi-quantitively assess expression levels and scores were analyzed for association with cancer-specific survival (CSS). CRC cell lines (n=3), patient-derived organoids (PDOs; n=5) and patient-derived explants (n=5) were treated with novel IKKα inhibitor SU1644 and assessed for cell viability and proliferation. Results: Positive staining for IKKα, pIKKαs176 and IKKβ was detected in tumor specimens. High cytoplasmic expression of IKKα within tumor cells was associated with reduced CSS in patients with right-sided colon cancer (HR=3.091, 95%CI; 1.128-9.467, log rank p=0.021). Conversely, high IKKβ expression was a marker of good prognosis (HR=0.627, 95%CI; 0.457-0.861, log rank p=0.004). A high ratio of IKKα to IKKβ was associated with reduced CSS in the full cohort (HR=1.404, 95%CI; 1.050-1.879, log rank p=0.021) and this was potentiated in right-sided colon cases (HR=1.772, 95%CI; 1.107-2.837, log rank p=0.016). Selective inhibition of IKKα using 1µM SU1644 in vitro caused a significant reduction in HT29 colony formation (p=0.012) and cell viability (p=0.039). PDOs showed altered cell morphology, reduced cell viability and proliferation when treated with SU1644. In the patient-derived explant model, Ki67 expression was reduced in tumor explants treated with SU1644. Conclusions: These data establish high cytoplasmic IKKα expression within tumor cells as a marker of poor prognosis in CRC and conversely high IKKβ expression as a marker of good prognosis. This highlights the importance of development of selective IKKα inhibitor SU1644. IKKα inhibition using SU1644 demonstrated anti-cancer activity in recapitulative CRC disease models. Citation Format: Kathryn A. Pennel, Molly McKenzie, Guang-Yu Lian, Jean A. Quinn, Sara Samir Foad Al-Badran, Campbell S. Roxburgh, Simon P. Mackay, Joanna Birch, Joanne Edwards. Inhibitory-κB kinase alpha as a biomarker and therapeutic target in colorectal 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 4634.

Abstract 4181: AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in acute myeloid leukemia

Abstract Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold prediction software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients. Citation Format: Niels Vandewalle, Hatice Satilmis, Emma M. Verheye, Rong Fan, Yanmeng Wang, Timo W. De Groof, Tessa Kerre, Nathan De Beule, Ann De Becker, Elke De Bruyne, Eline Menu, Karin Vanderkerken, Karine Breckpot, Nick Devoogdt, Kim De Veirman. AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in acute myeloid leukemia [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 4181.

Combination MEG3 lncRNA and Ciprofloxacin dramatically decreases cell migration and viability as well as induces apoptosis in GC cells in vitro.

Gastric cancer (GC) is a prominent cause of cancer-related mortality worldwide. Long noncoding RNA (lncRNA) maternal expression gene3 (MEG3) participates in numerous signaling pathways by targeting the miRNA-mRNA axis. Studies on human tumors have demonstrated that the antibiotic Ciprofloxacin induces cell cycle changes, programmed cell death, and growth suppression. In this study, we transfected MEG3 lncRNA and Ciprofloxacin into the MKN-45 GC cell line. qRT-PCR was employed to evaluate the effects on the specific microRNA and mRNA. The wound healing test, MTT assay, and flow cytometry were used to assess the impact of their administration on cell migration, viability, and apoptosis, respectively. Research showed that miR-147 expression fell even more after MEG3 lncRNA transfection, leading to an increase in B-cell lymphoma 2 (BCL-2) levels. Ciprofloxacin transfection did not significantly affect the axis, except for MEG3, which led to its slight upregulation. MEG3 lncRNA inhibited the migration of MKN-45 cells compared to the control group. When MEG3 lncRNA was coupled with Ciprofloxacin, there was a significant reduction in cell migration compared to untreated groups and controls. MTT assay and flow cytometry demonstrated that MEG3 lncRNA decreased cell viability and triggered apoptosis. Simultaneous administration of MEG3 lncRNA and Ciprofloxacin revealed a significant reduction in cell viability caused by increased apoptosis obtained from MTT or flow cytometry assays. Modulating the miR-147-BCL-2 axis decreases cell migration and survival while promoting cell death. In conclusion, combining MEG3 lncRNA with Ciprofloxacin may be an effective therapeutic approach for GC treatment by influencing the miR-14-BCl-2 axis, resulting in reduced cell viability, migration, and increased apoptosis.

Anti-cancer activity of Moringa oleifera Lam. seed oil on oral cancer

BackgroundDespite recent advances in the diagnosis and treatment of oral cancer, relative survival rates have not changed significantly. Moringa oleifera (M. oleifera) Lam. is one such plant with its anticancer properties being proved in its leaves, stem, flowers but no studies are yet reported proving the anticancer property of its seed oil on oral cancer. This study aimed to evaluate the anti-proliferative and cytotoxic effect of M. oleifera seed oil against two Oral squamous cell carcinoma cell lines CAL27 and SCC15 using MTT assay.Results2D GC-TOF Mass spectrometry revealed a total of 199 compounds, among which the majority were alkanes (68.2016%) and fatty acid esters (11.1399%). The MTT assay report showed good dose-dependent activity. A significant reduction in cell viability within 24 h with IC50 value of 17.78 µg/mL and 24.28 µg/mL for all treatment groups was observed for both the cell lines CAL27 and SCC15.ConclusionMTT assay showed a significant decrease in cell viability with an increase in the oil dose, thereby revealing the cytotoxic and anti-proliferative activity of M. oleifera seed oil on oral cancer cell lines namely CAL27and SCC15. The results of this study indicate that M. oleifera seed oil can be used as a potent anti-cancer agent in the treatment of Oral cancer.Graphical abstract

Dynamic Mechanical and Biological Characterization of New 3D-Printed Polymeric Dental Materials: A Preliminary Study

The literature shows evidence of the mechanical investigation of numerous polymeric dental biomaterials using a static approach. A more representative mechanical analysis of such materials must take into account the dynamic masticatory load of the oral cavity. The aim of this work is to study the dynamic mechanical proprieties and provide an in vitro characterization of 11 3D-printed new dental biomaterials to understand their clinical applications under physiological conditions. The analysis included Dynamical Mechanical Analysis (DMA) and an MTT cytotoxicity assay. The mechanical results at low frequencies (1–11 Hz) show high uncertainty, less fragility, and less strength. The biological results show a significant reduction in cell viability (p < 0.01) at both the 3 and 24 h timepoints, with a degree of recovery observed at 24 h. To assess the clinical potential of dental biomaterials, it is necessary to determine whether there are good dynamic mechanical properties and reduced adverse biological effects on oral cells. This may allow for the facile fabrication via 3D printing of prosthetic devices that can support masticatory loads over long periods of time. Further investigations of the presented polymeric materials are needed, exploring biological assessments for longer than 24 h.

Abstract B003: The OTX2 gene induces tumor growth and triggers leptomeningeal metastasis by regulating the mTOR signaling pathway in medulloblastomas

Abstract B003: The OTX2 gene induces tumor growth and triggers leptomeningeal metastasis by regulating the mTOR signaling pathway in medulloblastomas

Synthesis and Characterization of Amine and Aldehyde-Containing Copolymers for Enzymatic Crosslinking of Gelatine.

In tissue engineering (TE), the support structure (scaffold) plays a key role necessary for cell adhesion and proliferation. The protein constituents of the extracellular matrix (ECM), such as collagen, its derivative gelatine, and elastin, are the most attractive materials as possible scaffolds. To improve the modest mechanical properties of gelatine, a strategy consists of crosslinking it, as naturally occurs for collagen, which is stiffened by the oxidative action of lysyl oxidase (LO). Here, a novel protocol to crosslink gelatine has been developed, not using the commonly employed crosslinkers, but based on the formation of imine bonds or on aldolic condensation reactions occurring between gelatine and properly synthesized copolymers containing amine residues via LO-mediated oxidation. Particularly, we first synthesized and characterized an amino butyl styrene monomer (5), its copolymers with dimethylacrylamide (DMAA), and its terpolymer with DMAA and acrylic acid (AA). Three acryloyl amidoamine monomers (11a-c) and their copolymers with DMAA were then prepared. A methacrolein (MA)/DMAA copolymer already possessing the needed aldehyde groups was finally developed to investigate the relevance of LO in the crosslinking process. Oxidation tests of amine copolymers with LO were performed to identify the best substrates to be used in experiments of gelatine reticulation. Copolymers obtained with 5, 11b, and 11c were excellent substrates for LO and were employed with MA/DMAA copolymers in gelatine crosslinking tests in different conditions. Among the amine-containing copolymers, that obtained with 5 (CP5/DMMA-43.1) afforded a material (M21) with the highest crosslinking percentage (71%). Cytotoxicity experiments carried out on two cell lines (IMR-32 and SH SY5Y) with the analogous (P5) of the synthetic constituent of M21 (CP5/DMAA) had evidenced no significant reduction in cell viability, but proliferation promotion, thus establishing the biocompatibility of M21 and the possibility to develop it as a new scaffold for TE, upon further investigations.

Exploration of the protective mechanisms of Icariin against cisplatin-induced renal cell damage in canines.

This study delves into the protective mechanisms of Icariin (ICA) against cisplatin-induced damage in Madin-Darby canine kidney (MDCK) cells. Comprising two distinct phases, the investigation initially employed a single-factor randomized design to ascertain the minimal cisplatin concentration eliciting MDCK cell damage, spanning concentrations from 0 to 16 mmol/L. Concurrently, various concentrations of ICA (ranging from 5 to 50 mmol/L) were combined with 1 mmol/L cisplatin to determine the most efficacious treatment concentration. Subsequent investigations utilized four treatment groups: control, 1 mmol/L cisplatin, 1 mmol/L cisplatin + 20 mmol/L ICA, and 1 mmol/L cisplatin + 25 mmol/L ICA, aimed at elucidating ICA's protective mechanisms. Findings from the initial phase underscored a significant reduction in MDCK cell viability with 1 mmol/L cisplatin in comparison to the control (P < 0.01). Notably, the inclusion of 20 and 25 mmol/L ICA substantively ameliorated MDCK cell viability under 1 mmol/L cisplatin (P < 0.01). Moreover, cisplatin administration induced an elevation in inflammatory factors, malondialdehyde (MDA), reactive oxygen species (ROS), and Bax protein levels, while concurrently suppressing superoxide dismutase (SOD), catalase (CAT), and Bcl-2 expression (P < 0.01). Conversely, supplementation of 20 and 25 mmol/L ICA demonstrated a marked increase in mitochondrial membrane potential and levels of SOD, CAT, and Bcl-2 (P < 0.01). These interventions effectively attenuated inflammatory responses and suppressed Bax protein expression (P < 0.05), consequently mitigating cisplatin-induced apoptosis in MDCK cells (P < 0.01). In summary, these findings elucidate the role of ICA in impeding apoptosis in cisplatin-induced MDCK cells by regulating inflammatory responses, oxidative stress, and autophagic protein expression.

An Essential Role of c-Fos in Notch1-mediated Promotion of Proliferation of KSHV-Infected SH-SY5Y Cells.

This study aimed to investigate the influence of Notch1 on c-Fos and the effect of c-Fos on the proliferation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected neuronal cells. Real-time PCR and western blotting were used to determine c-Fos expression levels in KSHV-infected (SK-RG) and uninfected SH-SY5Y cells. C-Fos levels were measured again in SK-RG cells with or without Notch1 knockdown. Next, we measured c-Fos and p-c-Fos concentrations after treatment with the Notch1 γ-secretase inhibitor LY-411575 and the Notch1 activator Jagged-1. MTT and Ki-67 staining were used to evaluate the proliferation ability of cells after c-Fos levels downregulation. CyclinD1, CDK6, and CDK4 expression levels and cell cycle were analyzed by western blotting and flow cytometry, respectively. After the c-Fos intervention, the KSHV copy number and gene expression of RTA, LANA and K8.1 were analyzed by real-time TaqMan PCR. C-Fos was up-regulated in KSHV-infected SK-RG cells. However, the siRNA-mediated knockdown of Notch1 resulted in a significant decrease in the levels of c-Fos and p-c-Fos (P <0.01, P <0.001). Additionally, a decrease in Cyclin D1, CDK6, and CDK4 was also detected. The Notch1 inhibitor LY-411575 showed the potential to down-regulate the levels of c-Fos and p-c-Fos, which was consistent with Notch1 knockdown group (P <0.01), whereas the expression and phosphorylation of c-Fos were remarkably up-regulated by treatment of Notch1 activator Jagged-1 (P <0.05). In addition, our data obtained by MTT and Ki-67 staining revealed that the c-Fos down-regulation led to a significant reduction in cell viability and proliferation of the SK-RG cells (P <0.001). Moreover, FACS analysis showed that the cell cycle was arrested in the G0/G1 stage, and the expressions of Cyclin D1, CDK6, and CDK4 were down-regulated in the c-Fos-knockdown SK-RG cells (P <0.05). Reduction in total KSHV copy number and expressions of viral genes (RTA, LANA and K8.1) were also detected in c-Fos down-regulated SK-RG cells (P <0.05). Our findings strongly indicate that c-Fos plays a crucial role in the promotion of cell proliferation through Notch1 signaling in KSHV-infected cells. Furthermore, our results suggest that the inhibition of expression of key viral pathogenic proteins is likely involved in this process.

WO3-ZnO as an anti-gastric cancer agent and efficient photocatalyst in the synthesis of substituted benzimidazoles

Benzimidazoles have potential for developing important drugs and fighting cancerous gastric cells. In this study, an efficient and eco-friendly photocatalyst, WO3-ZnO, is used to synthesize some 2-substituted benzimidazoles by condensing aromatic aldehydes with o-phenylenediamine. The synthesis is achieved by irradiation with a commercial green laser. Various techniques like FT-IR, XRD, DLS, UV–Vis, and FE-SEM are used to analyze structure and properties of WO3-ZnO. The nanocatalyst shows excellent reusability and reproducibility. Additionally, cytotoxicity of WO3-ZnO nanoparticles are tested against KATO-III human gastric cancer cells, and MTT assay confirms their cytotoxicity with a significant reduction in cell viability. The cytotoxicity of WO3-ZnO nanoparticles against the performed cell line under in vivo conditions will be directed soon and the obtained results will be compared with the in vitro conditions.

Pan-EGFR Inhibitor Dacomitinib Resensitizes Paclitaxel and Induces Apoptosis via Elevating Intracellular ROS Levels in Ovarian Cancer SKOV3-TR Cells.

Paclitaxel is still used as a standard first-line treatment for ovarian cancer. Although paclitaxel is effective for many types of cancer, the emergence of chemoresistant cells represents a major challenge in chemotherapy. Our study aimed to analyze the cellular mechanism of dacomitinib, a pan-epidermal growth factor receptor (EGFR) inhibitor, which resensitized paclitaxel and induced cell cytotoxicity in paclitaxel-resistant ovarian cancer SKOV3-TR cells. We investigated the significant reduction in cell viability cotreated with dacomitinib and paclitaxel by WST-1 assay and flow cytometry analysis. Dacomitinib inhibited EGFR family proteins, including EGFR and HER2, as well as its downstream signaling proteins, including AKT, STAT3, ERK, and p38. In addition, dacomitinib inhibited the phosphorylation of Bad, and combination treatment with paclitaxel effectively suppressed the expression of Mcl-1. A 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay revealed a substantial elevation in cellular reactive oxygen species (ROS) levels in SKOV3-TR cells cotreated with dacomitinib and paclitaxel, which subsequently mediated cell cytotoxicity. Additionally, we confirmed that dacomitinib inhibits chemoresistance in paclitaxel-resistant ovarian cancer HeyA8-MDR cells. Collectively, our research indicated that dacomitinib effectively resensitized paclitaxel in SKOV3-TR cells by inhibiting EGFR signaling and elevating intracellular ROS levels.

Vascularized tissue on mesh-assisted platform (VT-MAP): a novel approach for diverse organoid size culture and tailored cancer drug response analysis.

This study presents the vascularized tissue on mesh-assisted platform (VT-MAP), a novel microfluidic in vitro model that uses an open microfluidic principle for cultivating vascularized organoids. Addressing the gap in 3D high-throughput platforms for drug response analysis, the VT-MAP can host tumor clusters of various sizes, allowing for precise, size-dependent drug interaction assessments. Key features include capability for forming versatile co-culture conditions (EC, fibroblasts and colon cancer organoids) that enhance tumor organoid viability and a perfusable vessel network that ensures efficient drug delivery and maintenance of organoid health. The VT-MAP enables the culture and analysis of organoids across a diverse size spectrum, from tens of microns to several millimeters. The VT-MAP addresses the inconsistencies in traditional organoid testing related to organoid size, which significantly impacts drug response and viability. Its ability to handle various organoid sizes leads to results that more accurately reflect patient-derived xenograft (PDX) models and differ markedly from traditional in vitro well plate-based methods. We introduce a novel image analysis algorithm that allows for quantitative analysis of organoid size-dependent drug responses, marking a significant step forward in replicating PDX models. The PDX sample from a positive responder exhibited a significant reduction in cell viability across all organoid sizes when exposed to chemotherapeutic agents (5-FU, oxaliplatin, and irinotecan), as expected for cytotoxic drugs. In sharp contrast, PDX samples of a negative responder showed little to no change in viability in smaller clusters and only a slight reduction in larger clusters. This differential response, accurately replicated in the VT-MAP, underscores its ability to generate data that align with PDX models and in vivo findings. Its capacity to handle various organoid sizes leads to results that more accurately reflect PDX models and differ markedly from traditional in vitro methods. The platform's distinct advantage lies in demonstrating how organoid size can critically influence drug response, revealing insights into cancer biology previously unattainable with conventional techniques.

Epstein-Barr Virus miR-BARTs 7 and 9 modulate viral cycle, cell proliferation, and proteomic profiles in Burkitt lymphoma

The Epstein-Barr Virus (EBV) encodes viral microRNAs (miRs) that have been implicated in the pathogenesis of nasopharyngeal and gastric carcinomas, yet their potential roles in lymphomas remain to be fully elucidated. This study evaluated the impact of CRISPR/Cas9-mediated knockdown of EBV miRs BART-7 and BART-9 in EBV-positive Burkitt lymphoma cells Akata. As anticipated, the Akata cells subjected to CRISPR/Cas9-mediated knockdown of either EBV BART-7 or BART-9 exhibited a significant reduction in the expression of these viral miRs compared to cells with wild-type (wt) EBV genomes. This outcome effectively validates the experimental model employed in this study. Knocking down either BART-7 or BART-9 resulted in a notable reduction in cell viability and proliferation rates, alongside an elevation in the expression of EBV lytic genes. Global proteomic analysis revealed that the knockdown of EBV BART-7 significantly decreased the expression of ubiquitin/proteasome proteins while concurrently increasing RNA binding proteins (RBPs). Conversely, BART-9 knockdown reduced proteins associated with oxidoreductase activity, particularly those involved in fatty acid metabolism. Our findings unveil previously undiscovered EBV miRs BARTs 7 and 9 roles in cellular pathways relevant to both viral biology and lymphomagenesis.

The evaluation of four nano-formulations loaded-Elsinochrome A on characteristics and in vitro cytotoxicity effect.

Elsinochrome A (EA) is a naturally occurring photosensitizer with potential applications in photodynamic therapy (PDT) for various malignancies. Despite its promising therapeutic properties, the poor solubility of EA hampers its effective utilization in clinical settings. To circumvent this limitation, we engineered four distinct nano-formulations: PLGA/EA nanoparticles (NPs), CMC-PLGA/EA NPs, mPEG-PCL/EA nanomicelles (NMs), and LHP-CHOL/EA nanoliposomes (NLs), all designed to enhance the solubility of EA. A comparative evaluation of these formulations, based on metrics such as particle size, Zeta potential, drug loading efficiency, and encapsulation efficiency, identified PLGA/EA NPs and mPEG-PCL/EA NMs as the most efficacious candidates. Subsequent in vitro investigations into the drug release kinetics under varying pH conditions and the impact on cell viability and apoptosis in A549 and MCF-7 cell lines were conducted. Remarkably, the maximum drug release for PLGA/EA NPs and mPEG-PCL/EA NMs was recorded at 62.5% and 70.8% in an acidic environment (pH 5.7), respectively. Upon exposure to 460nm light, PLGA/EA NPs induced a significant reduction in A549 cell viability to 13.8% and an apoptosis rate of 93.8%, whereas mPEG-PCL/EA NMs elicited a decrease in MCF-7 cell viability to 12.8% and an apoptosis rate of 73.0%.

Evaluation of anti-cancer potential of saffron extracts against kidney and bladder cancer cells

Crocus Sativus L. (saffron) has been used for centuries as a medicinal plant in traditional medicine. Its main bioactive secondary metabolites, which include crocins, crocetin, picrocrocin and safranal, are known to possess significant antioxidant as well as anti-proliferative activities. With the aim to test the activity of saffron on kidney and bladder cancer cell lines, we tested the effect of extracts obtained from dried stigmas derived from different Italian regions (Lombardy and Tuscany) in two different years, on the cell viability. The treatment with saffron extracts showed a significant reduction of cell viability in most of our tumoral cellular systems, while the standards crocin and safranal alone at the same concentrations did not exerted any cytotoxicity. When the extracts and standards were mixed together, they exerted a milder inhibition of cell viability, indicating that the antitumor activity is likely a result of the synergy of the various compounds. In addition, in a circular economy vision, we recovered Crocus Sativus flowers and tested them on cancer cells, demonstrating a consistent reduction of cell viability, while kaempferol (main compound present in flower extracts) did not show compatible results. Our results extend the interests around saffron-derived bioactive molecules for their potential in therapeutics.

Characterizing a novel antimicrobial molecule effective for drug-resistant pathogens and bacterial biofilm that augments keratinocyte migration

Hypervalent silver complexes, containing Ag(II) and Ag(III), have recently gained interest due to their potent antimicrobial efficacy. This work explores the synthesis and antimicrobial activity of a novel tribasic silver bisperiodate tetrahydrate (K3AgBP) complex containing both silver and iodine in hypervalent states: Ag(III) and I(VII). The antimicrobial efficacy of K3AgBP was evaluated and compared to hypervalent silver oxynitrate (Ag7NO11) and periodate salts, and conventional antibiotic drug compounds against methicillin-susceptible (MSSA) and resistant Staphylococcus aureus (USA-300 MRSA), susceptible (PAO1) and carbapenem-resistant blaVIM-2-positive (VIM-2) Pseudomonas aeruginosa, and drug-resistant strains of Candida. Minimum inhibitory concentrations for K3AgBP were equal or lower than silver oxynitrate and periodate salts for all species tested and significantly lower than antibiotics against MRSA, VIM-2 and antifungal efficacy against clinically isolated resistant C. albicans (Ca3917) was also shown. Hypervalent silvers, impregnated in a dressing at 0.4 mg Ag/cm2, were evaluated against 72-hour established biofilm. K3AgBP more effectively (P < 0.05) disrupted and eradicated S. aureus and P. aeruginosa biofilm, 6.72 ± 0.23 and 6.81 ± 0.23 log reduction within 4-hours and 2-hours respectively, versus 4.33 ± 0.23 and 4.97 ± 0.68 log CFU/ml respectively for silver oxynitrate. To assess possible cytotoxicity, viability of cultured HaCat keratinocytes treated with K3AgBP or hypervalent silver oxynitrate was determined using an MTT assay. Treatment with 1 or 10 µM K3AgBP or Ag7NO11 for 2 or 24 h did not cause a significant reduction in cell viability. Signficanty, 10 µM K3AgBP augmented HaCat cell migration. Absence of cytotoxicity along with the unexpected effects on migration, as well as stability and antimicrobial and antibiofilm efficacy support the potential application of K3AgBP as a topical antimicrobial.

New Insight into Enzyme Inhibitory and Anti-proliferative Activity of Echinops ritro L. (Asteraceae)

Echinops ritro L. is a perennial herbaceous plant in the tribe Cardueae, Asteraceae family, widely distributed, up to 1000 m a.s.l. In the present study, the enzyme inhibitory and anti-proliferative activity of E. ritro leaves and flowering heads extracts were assayed. Quantitative determination of 22 analytes, including eleven acylquinic and one hydroxycinnamic acids, and ten flavonoids was performed by Ultra high-performance liquid chromatography – diode array detector (UHPLC-DAD) analyses. The major compound was 1,5-dicaffeoylquinic acid, being presented at $$20.48\pm 0.22$$ µg/mg in the flowering heads, while the leaves demonstrated very high levels of chlorogenic acid ($$117.29\pm 14.20$$ µg/mg) and 3,5-dicaffeoylquinic acid ($$12.79\pm 0.282$$ µg/mg). The flavonoids were dominated by isoquercitrin and rutin ($$3.13\pm 0.033$$ and $$3.25\pm 0.057$$ µg/mg, respectively) together with metoxylated aglycones nepetin, hispidulin and jaceosidin (up to 0.51 µg/mg). The leaves extract revealed prominent acetylcholinesterase ($$2.33\pm 0.09$$ mg GALAE/g) and α-glucosidase inhibitory potential ($$0.37\pm 0.01$$ mmol ACAE/g). Nevertheless, the flowering heads extract showed distinct butyrylcholinesterase ($$1.41\pm0.35$$ mg GALAE/g), tyrosinase ($$45.66\pm0.84$$ mg KAE/g), amylase ($$0.37\pm0.02$$ mmol ACAE/g), and lipase ($$9.06\pm1.43$$ mg OE/g) inhibitory activity. The anti-proliferative assay was completed towards THP-1 monocyte leukemic cells, where at a concentration $$&gt;1000$$ µg/mL, a significant dose dependent reduction of cell viability was observed for the leaves extract. In conclusion, the data revealed a new insight into the taxon for its health benefit application.

Preclinical Evaluation of panobinostat and ONC201 for the treatment of diffuse intrinsic pontine glioma (DIPG)

Diffuse intrinsic pontine glioma (DIPG) also referred as paediatric high-grade glioma (pHGG) is a fast-growing and aggressive type of childhood brain cancer. Recent studies investigating the molecular pathogenesis of DIPG have identified new therapeutic targets, paving the way for a new line of drugs mainly HDAC inhibitors. However, despite long years of trials, no significant results have been generated yet. Panobinostat is a HDAC inhibitor that has shown promising preclinical cytotoxicity in DIPG but failed so far in clinical trials. This study aims to re-evaluate the efficacy of Panobinostat in DIPG in vitro using patient-derived DIPG cell cultures obtained directly from patients. ONC201 is another potentially effective drug in DIPG. This apoptotic agent has been considered in a few clinical trials in diffuse glioma including DIPG. Our results reveal a dose-dependent response to Panobinostat and ONC201 in DIPG cells. However, Panobinostat caused a significant reduction in the mean percentage cell viability at a lower concentration compared to ONC201. Panobinostat caused significant decreases in DIPG cell viability at concentrations greater than or equal to 0.002 μM (p<0.05), the response reached a plateau after 0.1 μM, which reduced cell viability to 32.81 % ± 0.25 % (p = 6.74E−06) when compared to control cells. ONC201 only significantly induced apoptosis at concentrations equal or higher than 0.01 μM (p<0.05), with its effect plateauing after 0.2 μM. This pre-clinical study supports the effectiveness of Panobinostat as a potential therapeutic agent for DIPG compared to ONC201, with no apparent synergistic effect observed in combination.

Abstract C096: Targeting the p53 pathway to treat pediatric Malignant Rhabdoid and Atypical Teratoid Rhabdoid Tumors

Abstract Background: Pediatric rhabdoid tumors, namely Malignant Rhabdoid Tumors (MRTs) and Atypical Teratoid Rhabdoid Tumors (ATRTs) in the central nervous system (CNS), are aggressive pediatric malignancies characterized by the loss of functional INI1 protein due to SMARCB1 gene mutations. Current treatment options for these tumors are limited and ineffective, necessitating the exploration of novel therapeutic strategies. Rhabdoid cells retain an intact p53 pathway, a critical tumor suppressor pathway involved in regulating cell cycle and apoptosis. Previous studies have shown that the MDM2 inhibitor, Idasanutlin, effectively eliminates flanked rhabdoid tumors, suggesting that targeting the p53 pathway holds promise for developing effective therapies against MRTs and ATRTs. Methods: In this study, we employed a combination approach using the MDM2 inhibitor, Idasanutlin, and the XPO1 inhibitor, Selinexor, to activate the p53 pathway. The impact of this combination treatment on tumor cell viability, proliferation, and apoptosis was assessed both in vitro and in vivo using MRT and ATRT models. Additionally, CRISPR screening and proteomic studies were conducted to identify specific dependencies in refractory models and investigate their influence on treatment response. Results: Treatment with the combination of Idasanutlin and Selinexor activated the p53 signaling pathway, leading to increased levels of MDM2 and p21 proteins, two direct targets of p53, and resulting in apoptosis in MRT and ATRT models. In vitro experiments demonstrated a significant reduction in cell viability and proliferation upon treatment. In vivo studies using MRT and ATRT xenograft models showed a remarkable decrease in tumor growth following the combination treatment. However, variable responses were observed among different models, indicating the presence of dependency patterns influencing treatment sensitivity. Proteomic studies and genetic manipulation provided further insights into the potential dependencies Conclusions: Our findings highlight the potential of the combination of Idasanutlin and Selinexor to activate the p53 pathway and induce apoptosis in MRT and ATRT models. Although the treatment strategy effectively activated p53, we identified dependencies that may contribute to incomplete treatment sensitivity. Further investigation into these dependency patterns is necessary to optimize the therapeutic approach. Overall, targeting the p53 pathway using combination therapies shows promise for the development of effective treatments for pediatric MRTs and ATRTs, warranting further preclinical and clinical investigations. Citation Format: Alaa Refaat, Hyekyung Cho, Jennifer Stripay, Charles Roberts, Christopher Tinkle, Anang Shelat, Martine Roussel. Targeting the p53 pathway to treat pediatric Malignant Rhabdoid and Atypical Teratoid Rhabdoid Tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C096.

Xpo-1 Antagonism Transcriptionally Reprograms and Is a Therapeutic Vulnerability in GATA-3 Driven T-Cell Lymphomas

Background: The transcription factor GATA-3, via the oncogenic transcriptional program it directs, is a bona fide proto-oncogene, and thus therapeutic target, in a subset of T-cell lymphomas (TCL). The eukaryotic translation initiation factor eIF4E, in complex with helicase and scaffolding factors, facilitates 40S ribosome and Met-tRNA recruitment, both of which are rate-limiting steps in the initiation of translation. Pathways that converge on eIF4E, including PI3K/AKT/mTOR-dependent phosphorylation of 4E-BP1, are frequently activated in GATA-3 PTCL. Within the nucleus, eIF4E specifically binds oncogenic mRNAs, and then forms a complex with a co-factor (LRPPRC) and the nuclear export receptor XPO1. In contrast to bulk mRNA, these eIF4E-bound mRNA are dependent upon XPO1 for nuclear export. Therefore, as we have shown that GATA-3 is oncogenic in T-cell lymphomas (TCL), we hypothesized that selective XPO1 inhibition may transcriptionally reprogram these TCL and is a novel therapeutic vulnerability. Methods: XPO1 expression was examined by immunohistochemistry or flow cytometry in TCL specimens and in TCL that emerge in multiple genetically engineered mouse (GEM) models. Transcripts that are eIF4e clients and dependent upon XPO-1 for nuclear export were identified in an unbiased manner by eIF4e RNA immunoprecipitation (eIF4e-RIP) and RNA-seq. The extent to which XPO1 antagonism transcriptionally reprograms TCL cells, and its value as a therapeutic strategy, was examined pharmacologically with selinexor in ex vivo and in vivo studies using primary TCL specimens and GEM/PDX models, respectively. Results: Among the PTCL cases (n=53) examined, XPO1 expression was observed in 62%, and was highly expressed in CTCL, particularly in patients with large cell transformation. We sought to further understand the extent to which XPO1 expression is associated with T-cell lymphomagenesis. We utilized multiple GEM TCL models in which Smarcb1 (SNF5) or PTEN were conditionally deleted in CD4 + T cells. In both GEM models, a significant increase in XPO-1 expression was associated with the emergence of a malignant clone. A significant reduction in cell viability and increased apoptosis was observed in TCL cell lines and primary specimens treated in vitro/ex vivo with selinexor. In both GEM models, and a PDX model, selinexor treatment similarly decreased TCL progression in vivo. Given these findings, we elected to examine XPO1's potential role in transporting oncogenic, and eIF4e client, transcripts. We performed eIF4e-RIP and RNA-seq in two representative cell lines. Gene set enrichment analysis utilizing the eIF4e client RNA identified demonstrated enrichment for pathways with an established role in TCL (e.g. TCR), including GATA-3. We subsequently performed eIF4e-RIP and quantified GATA-3 transcripts by qRT-PCR in cell lines (n=4), a PDX, and in primary SS specimens (n=2). Compatible with the RNA-seq data, significant enrichment in GATA-3 transcripts was observed. These findings imply that GATA-3 transcripts are dependent upon XPO1 for nuclear export. To directly examine this, the ratio of cytoplasmic to nuclear (C:N) GATA-3 transcripts were examined by qRT-PCR in cell lines treated with selinexor. A significant decrease in the C:N ratio was observed upon XPO1 inhibition, confirming that nuclear export of GATA-3 transcripts is XPO1 dependent. We similarly examined the extent to which GATA-3 target genes are eIF4e clients and XPO1 dependent for nuclear export. We had previously described GATA-3 target genes by integrated ChIP-seq/RNA-seq. Integration of this prior dataset with our eIF4e-RIP sequencing data demonstrated that ≈21-30% of previously described GATA-3 target genes were also eIF4e clients. We selected a few such targets, including ITK, for further validation. These GATA-3 targets were eIF4e clients and dependent upon XPO1 for nuclear export. Finally, we examined expression of these proteins in a diverse panel of T-cell lymphoma/leukemia cell lines and a primary SS specimen after brief exposure to selinexor, and compatible with our findings, a significant decrease in protein expression was observed. Conclusions: Collectively then, our findings demonstrate that XPO1 antagonism, by impairing the nuclear export of both GATA-3 and many of its target gene mRNA, is a novel strategy to transcriptionally reprogram and therapeutically target GATA-3 driven TCL.