Induced Cell Cytotoxicity Research Articles

DNA damage and cell death in human oral squamous cell carcinoma cells: The potential biological effects of cannabidiol

ObjectiveThe present study examined the in vitro effects on oral squamous cell carcinoma cells (HSC-3) of cannabidiol (CBD), the main chemical component of Cannabis, proposed as a novel adjuvant therapy in the treatment of cancers. DesignCell viability (MTT assay), morphology (SEM), apoptosis and cell cycle (flow cytometry), and DNA damage (phospho-γ-H2AX immunofluorescence) were evaluated. Cytotoxicity was evaluated with concentrations between 100 µM and 1 µM, and two concentrations were selected for subsequent analysis: 25 µM, as toxic dose, and 6.25 µM, as non-toxic. ResultsCBD caused a dose- and time-dependent reduction in viability of 64 %, 96 %, and 99 % with 25 µM, 50 µM and 100 µM, respectively, after 72 h (p < 0.001), cell cycle arrest in G0-G1 phase with increased apoptosis in particular at 72 h for 25 µM (p < 0.001), significant morphological alterations with 25 µM, still present even at 6.25 µM, and significantly increased cell damage considering a significant increase in the percentage of highly positive cells (5 phosphorylated γH2AX foci), which is around 29 % for 25 µM and 19 % for 6.25 µM after 24 h. ConclusionsCBD inhibits oral cancer growth causing DNA damage. In general, induced cell cytotoxicity appears to be dose- and time-related. Doses of CBD ≥25 μM showed a high reduction in viability. CBD could possibly represent a new therapeutic molecule for its cytotoxic effects against oral squamous cell carcinoma. The mechanism involved in the suppressive effect caused by CBD needs further investigation.

Optimizing Coreopsis tinctoria Flower Extraction and Inhibiting CML Activity: Box-Behnken Design.

Chronic myelogenous leukemia (CML) is an uncommon type of cancer of the bone marrow associated with high mortality. Although several effective therapies have been developed to reduce symptoms in patients with CML, many of these methods are associated with side effects. Coreopsis tinctoria Nutt. (C. tinctoria) is a natural medicinal material that possesses antioxidant and anticancer activities. Yet, its effect in treating leukemia has still not been fully explored. To optimize the C. tinctoria flower extraction process and investigate whether these extracts can impair CML cell survival. The extraction process of C. tinctoria was optimized by the Box-Behnken design response surface method. K562 cells were treated with different volumes (0, 10, 25, 50, and 100 μL) of C. tinctoria flower extracts. The effect of C. tinctoria extract on cell morphology and cell apoptosis was assessed by light microscopy, laser confocal microscopy, and flow cytometry. We established the following optimized C. tinctoria flower extraction conditions: temperature of 84.4°C, extraction period of 10 mins, solid-liquid ratio of 1:65, and times 4. These conditions were applied for C. tinctoria flower extraction. Pre-incubation of extracts prepared under the aforementioned optimal conditions with K562 cells induced cell cytotoxicity and cell apoptosis. C. tinctoria flower extracts exert obvious anti-leukemia effects in vitro and may be a potential drug candidate for leukemia treatment.

GNAS knockout potentiates HDAC3 inhibition through viral mimicry-related interferon responses in lymphoma

Despite selective HDAC3 inhibition showing promise in a subset of lymphomas with CREBBP mutations, wild-type tumors generally exhibit resistance. Here, using unbiased genome-wide CRISPR screening, we identify GNAS knockout (KO) as a sensitizer of resistant lymphoma cells to HDAC3 inhibition. Mechanistically, GNAS KO-induced sensitization is independent of the canonical G-protein activities but unexpectedly mediated by viral mimicry-related interferon (IFN) responses, characterized by TBK1 and IRF3 activation, double-stranded RNA formation, and transposable element (TE) expression. GNAS KO additionally synergizes with HDAC3 inhibition to enhance CD8+ T cell-induced cytotoxicity. Moreover, we observe in human lymphoma patients that low GNAS expression is associated with high baseline TE expression and upregulated IFN signaling and shares common disrupted biological activities with GNAS KO in histone modification, mRNA processing, and transcriptional regulation. Collectively, our findings establish an unprecedented link between HDAC3 inhibition and viral mimicry in lymphoma. We suggest low GNAS expression as a potential biomarker that reflects viral mimicry priming for enhanced response to HDAC3 inhibition in the clinical treatment of lymphoma, especially the CREBBP wild-type cases.

The comparison of the dynamics of Ca2+ and bleomycin intracellular delivery after cell sonoporation and electroporation in vitro

Ca2+, in combination with SP or EP, induces cell cytotoxicity much faster compared to BLM. The application of BLM in combination with, SP or EP, reaches the level of cell death, induced by similar combination with Ca2+, only after 72 h. The methods of SP and EP were calibrated according to the level of differential cytotoxicity, determined after 6 days (using cell clonogenic assay). The combination of Ca2+ SP induces cell death faster than Ca2+ EP – after Ca2+ SP it increases to a maximum level after 15 min and remains constant for up to 6 days, while the cytotoxic efficiency after Ca2+ EP increases to the level of Ca2+ SP only after 72 h. The combination of BLM SP shows a very similar dynamics to BLM EP – both reach maximal level of cytotoxicity after 48–72 h.Ca2+ and BLM in combination with SP have shown similar levels of cytotoxicity at higher acoustic pressures (≥250 kPa); therefore, Ca2+ SP can be used to induce immediate and maximal level of cytotoxic effect. The faster cytotoxic efficiency of Ca2+ in combination with SP than EP was determined to be due to the involvement of microbubble inertial cavitation.

Abstract 77: Real-time multiparametric assessment of immune cell-mediated cytotoxicity

Abstract A hallmark of effective immunotherapy is the reliance on the cytotoxic effects of immune cells, such as T-cells and natural killer (NK) cells, on tumor lesions. Cell cluster formation is an important sign of activation, and proliferation, as well as an indication of the killing capability of immune cells. Understanding and modulating the mechanisms underlying the activation, proliferation, and clustering of immune cells is essential for the development of innovative approaches to promote immune cell-induced cytotoxicity and address tumor progression. Here, we report a novel kinetic immuno-oncology assay using the Agilent xCELLigence RTCA eSight, which can simultaneously and quantitatively assess effector/immune cell clustering and proliferation through brightfield live cell time-lapse imaging and immune cell-mediated cytotoxicity of target/cancer cells through real-time monitoring cancer cell viability over a chronic treatment. In this study, we validated the assay using 2 cell models, including CD19 CAR T-cells against Raji cells and NK-92 cells against A549 cells, in a 96-well plate format. The activation, clustering, and proliferation of CD19 CAR T-cells and NK-92 at different Effector: Target (E: T) ratios, as well as the sequential killing of the target cancer cells, were plotted using imaging and cellular-impedance-based readouts throughout the entire assay, respectively. Our results showed 1) the clustering and proliferation of the effector cells could be accurately assessed by brightfield without the facilitation of fluorescent label using the image analysis module of the system; 2) the killing efficacy of the effector cells quantified by the impedance readout highly correlated to their clustering and proliferation assessed by the live-cell imaging; 3) cellular impedance provided a sensitive readout CAR-T killing of immobilized Raji cell count, independent of fluorescent labeling; 4) the kinetics of cluster formation/proliferation and killing efficacy of CD19 CAR T-cells were different from NK cells. The capability of multiparametric assessment of immune cell-medicated cytotoxicity from both effector and target cell perspectives of the assay provides a simple and real-time workflow to understand the correlation and interaction between effector and target cells, which could be an addition of immense value to the development of innovative immunotherapies. Citation Format: Grace Yang, Yan Lu, Tian Wang, Peifang Ye, Xiaoyu Zhang. Real-time multiparametric assessment of immune cell-mediated cytotoxicity [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 77.

CAR-NK cells for cancer immunotherapy: recent advances and future directions.

Natural Killer (NK) cells, intrinsic to the innate immune system, are pivotal in combating cancer due to their independent cytotoxic capabilities in antitumor immune response. Unlike predominant treatments that target T cell immunity, the limited success of T cell immunotherapy emphasizes the urgency for innovative approaches, with a spotlight on harnessing the potential of NK cells. Despite tumors adapting mechanisms to evade NK cell-induced cytotoxicity, there is optimism surrounding Chimeric Antigen Receptor (CAR) NK cells. This comprehensive review delves into the foundational features and recent breakthroughs in comprehending the dynamics of NK cells within the tumor microenvironment. It critically evaluates the potential applications and challenges associated with emerging CAR-NK cell therapeutic strategies, positioning them as promising tools in the evolving landscape of precision medicine. As research progresses, the unique attributes of CAR-NK cells offer a new avenue for therapeutic interventions, paving the way for a more effective and precise approach to cancer treatment.

Helical sulfonyl-γ-AApeptides modulating Aβ oligomerization and cytotoxicity by recognizing Aβ helix

In contrast to prevalent strategies which make use of β-sheet mimetics to block Aβ fibrillar growth, in this study, we designed a series of sulfonyl-γ-AApeptide helices that targeted the crucial α-helix domain of Aβ13-26 and stabilized Aβ conformation to avoid forming the neurotoxic Aβ oligomeric β-sheets. Biophysical assays such as amyloid kinetics and TEM demonstrated that the Aβ oligomerization and fibrillation could be greatly prevented and even reversed in the presence of sulfonyl-γ-AApeptides in a sequence-specific and dose-dependent manner. The studies based on circular dichroism, Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) spectra unambiguously suggested that the sulfonyl-γ-AApeptide Ab-6 could bind to the central region of Aβ42 and induce α-helix conformation in Aβ. Additionally, Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) was employed to rule out a colloidal mechanism of inhibitor and clearly supported the capability of Ab-6 for inhibiting the formation of Aβ aggregated forms. Furthermore, Ab-6 could rescue neuroblastoma cells by eradicating Aβ-mediated cytotoxicity even in the presence of pre-formed Aβ aggregates. The confocal microscopy demonstrated that Ab-6 could still specifically bind Aβ42 and colocalize into mitochondria in the cellular environment, suggesting the rescue of cell viability might be due to the protection of mitochondrial function otherwise impaired by Aβ42 aggregation. Taken together, our studies indicated that sulfonyl-γ-AApeptides as helical peptidomimetics could direct Aβ into the off-pathway helical secondary structure, thereby preventing the formation of Aβ oligomerization, fibrillation and rescuing Aβ induced cell cytotoxicity.

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.

Revealing profile of cancer-educated platelets and their factors to foster immunotherapy development

Among multiple hemostasis components, platelets hyperactivity plays major roles in cancer progression by providing surface and internal components for intercellular crosstalk as well as by behaving like immune cells. Since platelets participate and regulate immunity in homeostatic and disease states, we assumed that revealing platelets profile might help in conceiving novel anti-cancer immune-based strategies. The goal of this review is to compile and discuss the most recent reports on the nature of cancer-associated platelets and their interference with immunotherapy. An increasing number of studies have emphasized active communication between cancer cells and platelets, with platelets promoting cancer cell survival, growth, and metastasis. The anti-cancer potential of platelet-directed therapy has been intensively investigated, and anti-platelet agents may prevent cancer progression and improve the survival of cancer patients. Platelets can (i) reduce antitumor activity; (ii) support immunoregulatory cells and factors generation; (iii) underpin metastasis and, (iv) interfere with immunotherapy by expressing ligands of immune checkpoint receptors. Mediators produced by tumor cell-induced platelet activation support vein thrombosis, constrain anti-tumor T- and natural killer cell response, while contributing to extravasation of tumor cells, metastatic potential, and neovascularization within the tumor. Recent studies showed that attenuation of immunothrombosis, modulation of platelets and their factors have a good perspective in immunotherapy optimization. Particularly, blockade of intra-tumoral platelet-associated programmed death-ligand 1 might promote anti-tumor T cell-induced cytotoxicity. Collectively, these findings suggest that platelets might represent the source of relevant cancer staging biomarkers, as well as promising targets and carriers in immunotherapeutic approaches for combating cancer.

Unexpected hydroxyl radical production in brewed tea under sunlight.

Tea is one of the world's most popular and widely consumed beverages. It is a common pastime to enjoy a cup of tea in the sunshine. However, little attention has been given to understanding the possible photochemical reactions occurring beneath the calm surface of brewed tea. Epigallocatechin gallate (EGCG), which is widely used in food and beverages, is the most significant active ingredient found in tea. In this study, we investigated the presence of free radicals in both an aqueous EGCG solution and brewed tea under simulated sunlight conditions. To our surprise, we unexpectedly observed the production of hydroxyl radicals (•OH) in brewed tea. It was found that sunlight irradiation played a critical role in the formation of •OH, independent of the presence of metal ions. Furthermore, we demonstrated that the •OH generated from the EGCG aqueous solution induced cell cytotoxicity and DNA damage in vitro. Considering the crucial role of •OH in various fields, including human health and the environment, it is important to further explore the practical implications of •OH production in brewed tea under sunlight. In summary, our study unveils the unexpected formation of •OH in brewed tea and emphasizes the significance of sunlight-induced reactions. The observed cytotoxic and DNA-damaging effects of •OH emphasize the importance of understanding the potential health consequences associated with tea consumption. Further research in this area will contribute to a better understanding of the broader implications of •OH production in brewed tea under sunlight.

Targeted Hyperbranched Nanoparticles for Delivery of Doxorubicin in Breast Cancer Brain Metastasis.

Breast cancer brain metastases (BM) are associated with a dismal prognosis and very limited treatment options. Standard chemotherapy is challenging in BM patients because the high dosage required for an effective outcome causes unacceptable systemic toxicities, a consequence of poor brain penetration, and a short physiological half-life. Nanomedicines have the potential to circumvent off-target toxicities and factors limiting the efficacy of conventional chemotherapy. The HER3 receptor is commonly expressed in breast cancer BM. Here, we investigate the use of hyperbranched polymers (HBP) functionalized with a HER3 bispecific-antibody fragment for cancer cell-specific targeting and pH-responsive release of doxorubicin (DOX) to selectively deliver and treat BM. We demonstrated that DOX-release from the HBP carrier was controlled, gradual, and greater in endosomal acidic conditions (pH 5.5) relative to physiologic pH (pH 7.4). We showed that the HER3-targeted HBP with DOX payload was HER3-specific and induced cytotoxicity in BT474 breast cancer cells (IC50: 17.6 μg/mL). Therapeutic testing in a BM mouse model showed that HER3-targeted HBP with DOX payload impacted tumor proliferation, reduced tumor size, and prolonged overall survival. HER3-targeted HBP level detected in ex vivo brain samples was 14-fold more than untargeted-HBP. The HBP treatments were well tolerated, with less cardiac and oocyte toxicity compared to free DOX. Taken together, our HER3-targeted HBP nanomedicine has the potential to deliver chemotherapy to BM while reducing chemotherapy-associated toxicities.

Downregulation of Tumor Promotor Genes in Oryza Sativa Linn.-Induced Antiproliferative Activity of Human Squamous Carcinoma Cells.

Oral cancer represents the third leading cause of death in Southeast Asia and targeted therapy could prevent or delay disease etymology. Oryza sativa Linn. (OS) extract has been implicated as an antitumor agent in many cancer types, however none has been investigated in human squamous carcinoma-2 (HSC-2) cells, thus we aim to investigate the effects of OS on HSC-2 cells. Our study investigated the growth inhibitory effects of an ethanolic extract of OS on HSC-2 cells by BrdU ELISA and MTT assays, as well as changes in tumor promoter genes using RT-qPCR and western blotting. We found that OS was able to induce cell cytotoxicity and inhibit HSC-2 proliferation. OS also decreased the expression of genes involved in the TGF-β/Smads signaling pathway and genes involved in cell motility such as GPNMB, ITGB6, and E2F1 by RT-qPCR. Western blotting confirmed the downregulation of TGF-β1 by OS. Co-treatment of OS and 5-Flurouracil also reversed Snail and Slug overexpression caused by HSC-2 exposure to 5-Flurouracil. Together, these results indicate that OS can inhibit HSC-2 cell proliferation and this may involve TGF-β1 downregulation. Thus, this study shows OS could be useful for the treatment of patients with squamous carcinoma.

Immune evasion and therapeutic opportunities based on natural killer cells.

Natural killer (NK) cells can elicit an immune response against malignantly transformed cells without recognizing antigens, and they also exhibit cytotoxic effects and immune surveillance functions in tumor immunotherapy. Although several studies have shown the promising antitumor effects of NK cells in immunotherapy, their function is often limited in the tumor microenvironment because tumor cells can easily escape NK cell-induced death. Thus, for efficient tumor immunotherapy, the mechanism by which tumor cells escape NK cell-induced cytotoxicity must be fully understood. Various novel molecules and checkpoint receptors that mediate the disruption of NK cells in the tumor microenvironment have been discovered. In this review, we analyze and detail the major activating and inhibitory receptors on the surface of NK cells to delineate the mechanism by which tumor cells suppress NKG2D ligand expression and increase tumor receptor and inhibitory receptor expression [NKG2A, programmed cell death 1 (PD-1), and T-cell immunoglobulin and immunoreceptor tyrosine inhibitory motif (TIGIT)] on the NK cell surface, and thus inhibit NK cell activity. We also reviewed the current status of treatments based on these surface molecules. By comparing the therapeutic effects related to the treatment status and bypass mechanisms, we attempt to identify optimal single or combined treatments to suggest new treatment strategies for tumor immunotherapy.

Anti-cancerous effect of corn silk: a critical review on its mechanism of action and safety evaluation.

Cancer is a broad collection of diseases that can begin in almost any organ or tissue of the body. Corn silk is the hair-like stigmata of female maize flowers which is generally discarded as waste from maize cultivation. The current study targets the anti-cancer potential of corn silk and its bioactive compounds namely, polyphenols, flavonoids, and sterols. The polyphenols and flavonoids like quercetin, rutin, apigenin and beta-sitosterol are a range of compounds from corn silk which were investigated for their anticancer effect. Corn silk showed apoptotic and antiproliferative effects in cancer cells through different signalling pathways, essentially the serine/threonine kinases (Akt)/lipid kinases (PI3Ks) pathway. The study revealed that corn silk compounds target immune cell responses, induce cell cytotoxicity, and upregulate the expression of proapoptotic genes p53, p21, caspase 9, and caspase 3 in certain cancer cell lines including HeLa cervical cancer cells, MCF-7 breast cancer cells, PANC-02 pancreatic cancer cells and Caco-2 colon cancer cells. Flavonoids derived from corn silk enhance T cell mediated immune response and decrease inflammatory factors. Corn silk bioactive compounds were found to reduce the side effects of cancer therapy. Antioxidants of corn silk, quercetin and rutin help in reducing the nephrotoxicity of chemotherapeutic drugs. The study also suggests that corn silk has anti-cancerous potential as it targets tumour suppression and inhibits metastasis A dose of 500mg/kg body weight of corn silk has been found safe for human consumption. Corn silk extract can be used as a preventive or therapeutic step to cure cancer. The anti-cancer property, mechanism and role of corn silk in controlling cancer-related side effects have been critically reviewed providing new scope for the use of corn silk in cancer therapy.

Arabinogalactan ameliorates benzo[a]pyrene-induced intestinal epithelial barrier dysfunction via AhR/MAPK signaling pathway

Benzo[a]pyrene (B[a]P), a kind of pollutant, can disrupt the gut microbiota, but its effects on the function of intestinal epithelial barrier (IEB) is still unclear. Arabinogalactan (AG), a natural polysaccharide, can protect intestinal tract. Thus, the purpose of this study was to evaluate the effect of B[a]P on IEB function and the mitigation effect of AG on the IEB dysfunction induced by B[a]P using a Caco-2 cell monolayer model. We found B[a]P could damage the IEB integrity by inducing cell cytotoxicity, increasing lactate dehydrogenase leakage, decreasing the transepithelial electrical resistance, and increasing fluorescein isothiocyanate-dextran flux. The mechanism of B[a]P-induced IEB damage may through induction of oxidative stress, including increasing reactive oxygen species levels, decreasing glutathione levels, reducing the activity of superoxide dismutase, and increasing malonaldehyde levels. Moreover, it can be due to increasing secretion of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α), down-regulated expression of tight junction (TJ) proteins (claudin-1, zonula occludens [ZO]-1, and occludin), and induced activation of aryl hydrocarbon receptor (AhR)/mitogen activated protein kinase (MAPK) signaling pathway. Remarkably, AG ameliorated B[a]P-induced IEB dysfunction through inhibited oxidative stress and pro-inflammatory factor secretion. Our study demonstrated B[a]P could damage the IEB and AG could alleviate this damage.

Cassia fistula L. pod shell and leaf extracts induce cell cytotoxicity and suppress cell migration by downregulating EGFR in MCF-7 cells

Introduction: Cassia fistula Linn. (CF) is a well-known Thai medicinal plant and a source of many bioactive compounds. The present work designed to examine the anticancer effects of pod shell and leaf of CF extracts on a human breast cancer cell line via the suppression of tumor development and metastasis. The expression of an epidermal growth factor receptor (EGFR) expression was interested to explore. Methods: The in vitro anticancer activities of the CF pod shell and leaf extracts, including cell cytotoxicity, cell growth, cell migration, cell death, and reactive oxygen species (ROS) formation were evaluated using sulforhodamine B (SRB), colony forming, wound healing, and flow cytometric analysis, respectively. The EGFR protein expression was determined by western blot analysis, which is related to increased cancer cell growth and resistance to apoptosis. Results: CF pod shell and leaf extracts showed significant toxicities against MCF-7 cells and inhibited the cancer cells proliferation and migratory ability of breast cancer MCF-7 cells in concentration- and time-dependent manners. Both extracts induced late cell apoptosis and significantly generated ROS formation at a dose of 250 mg/mL. Western blotting data exhibited low levels of EGFR protein expression after treating with the extracts at a dose of 1000 mg/mL. Conclusion: CF pod shell and leaf extracts are able to reduce breast cancer cell proliferation, increase cell apoptosis, and suppress cell migration through the downregulation of EGFR expression indicating anticancer activities.

Unveiling the Antioxidant, Clinical Enzyme Inhibitory Properties and Cytotoxic Potential of Tambourissa peltata Baker-An Understudied Endemic Plant.

This study documents for the first time the phytochemical composition and biological activities of Tambourissa peltata Baker, an endemic plant from Mauritius. Phytochemical extraction was performed using ethyl acetate, methanol and distilled water as solvents. The phytochemical composition was determined through HPLC-MS and other standard assays. The DPPH, ABTS, FRAP, CUPRAC and phosphomolybdenum assays were employed for the determination of the antioxidant potential, whereas cell viability assays were used to determine the cytotoxicity. The highest phenolic and phenolic acid contents were obtained in the aqueous extract (179.91 ± 0.67 gallic acid equivalents/g and 55.74 ± 1.43 caffeic acid equivalents/g). The highest quantity of flavonoids was obtained in the ethyl acetate extract (28.97 ± 0.46 rutin equivalents/g). The methanolic extract was the highest source of flavonols (33.71 ± 0.13 mg catechin equivalents/g). A total of 34 phytochemicals were identified, mainly proanthocyanidins and flavonoid glycosides. The highest antioxidant activity in DPPH (973.40 ± 5.65 mg TE (Trolox equivalents)/g), ABTS (2030.37 ± 40.83 mg TE/g), FRAP (1461.39 ± 5.95 mg TE/g), CUPRAC (1940.99 ± 20.95 mg TE/g) and phosphomolybdenum (8.37 ± 0.23 mmol TE/g) assays was recorded for the aqueous extract. The ethyl acetate extract was the most active metal chelator. The highest acetylcholinesterase inhibitor was the methanolic extract, whereas the ethyl acetate extract was the most active against BChE. The tyrosinase enzyme was most inhibited by the methanolic extract. Alpha-amylase and glucosidase were most inhibited by the aqueous extract. The methanolic extract was capable of inducing cell cytotoxicity to the human colorectal carcinoma without damaging normal cells. T. peltata warrants further attention from the scientific community given its multifaceted biological properties.

Monalizumab efficacy correlates with HLA-E surface expression and NK cell activity in head and neck squamous carcinoma cell lines.

NKG2A, an inhibitory receptor expressed on NK cells and T cells, leads to immune evasion by binding to HLA-E expressed on cancer cells. Here, we investigated the relationship between HLA-E surface expression on head and neck squamous cell carcinoma (HNSCC) cell lines and the efficacy of monalizumab, an NKG2A inhibitor, in promoting NK cell activity. Six HNSCC cell lines were used as target cells. After exposure to IFN- γ, HLA-E surface expression on HNSCC cell lines was measured by flow cytometry. Peripheral blood mononuclear cells (PBMCs) from healthy donors and isolated NK cells were used as effector cells. NK cells were stimulated by treatment with IL-2 and IL-15 for 5days, and NK cell-induced cytotoxicity was analyzed by CD107a degranulation and 51Cr release assays. We confirmed that HLA-E expression was increased by IFN-γ secreted by NK cells and that HLA-E expression was different for each cell line upon exposure to IFN-γ. Cell lines with high HLA-E expression showed stronger inhibition of NK cell cytotoxicity, and efficacy of monalizumab was high. Combination with cetuximab increased the efficacy of monalizumab. In addition, stimulation of isolated NK cells with IL-2 and IL-15 increased the efficacy of monalizumab, even in the HLA-E low groups. Monalizumab efficacy was correlated with HLA-E surface expression and was enhanced when NK cell activity was increased by cetuximab or cytokines. These results suggest that monalizumab may be potent against HLA-E-positive tumors and that monalizumab efficacy could be improved by promoting NK cell activity.

Cytotoksyczność i biokompatybilność kompozytowych, światłoutwardzalnych żywic stomatologicznych

The physical, chemical, and biological properties of restorative dental materials influence the clinical outcome of dental treatment. Methacrylate-based resins are extensively used in everyday dental practice, but accumulating records point to their toxicity. This article reviews the most important experimental data on cell-induced cytotoxicity of composite dental resins, methacrylates and epoxy compounds used to induce polymerization and reduce resin shrinkage. Methacrylate monomers produce reactive oxygen species, induce apoptosis and/or genotoxicity, and affect cell proliferation in culture. There is a need for awareness about the biological risks of these materials. New sensitive methods introduced to the preclinical testing of dental resins may help to highlight specific mechanisms, quantify the biological risk and guide the dentist to select the right biomaterial for dental applications.

CD123 Directed IgM Antibody-based T-cell Engager, IGM-2537, Demonstrates Potent in vitro and in vivo Activity with Minimal Cytokine Release

Introduction: Despite recent progress, the outlook for patients with acute myeloid leukemia (AML) remains poor. Novel therapies are needed for effective treatment of AML, especially in the relapsed/refractory setting. Evidence suggests that incomplete depletion of leukemia stem cells (LSCs) causes relapse. CD123, the IL-3 receptor alpha chain, is highly expressed on both leukemic blasts and LSCs and thus is a suitable therapeutic target in AML. CD123-based immunotherapies including bispecific antibodies or CAR T cells have shown promise, but cytokine release syndrome (CRS) is a major safety concern with dose limiting toxicity (DLT). We are developing IGM-2537, a novel IgM antibody-based CD123xCD3 bispecific T cell engager (TCE), designed with potent anti-tumor activity and lower cytokine release. IGM-2537 has 10 binding sites for CD123, and single binding site for CD3 via single chain Fv domain (scFv) fused to a joining (J) chain, thus providing high affinity, high avidity binding to CD123-positive malignant cells while co-engagement of CD3 T cells leading to T cell induced cytotoxicity with reduced cytokine release. Here we report functional characterization of IGM-2537 in a broad range of in vitro, ex vivo and in vivo anti-tumor efficacy studies. The improved tolerability, good pharmacodynamic (PD) response and lower cytokine release profile of a benchmark IgM TCE comparator in cynomolgus monkeys will be discussed. Materials and Methods: IGM-2537 was constructed using the pentameric human IgM framework. Recombinant IgM heavy chains and light chains with high affinity variable domains derived from an anti-CD123 IgG were co-expressed with a J-chain fused to anti-CD3 scFv. Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity, and epitope mapping, respectively. Bispecific enzyme-linked immunosorbent assay (ELISA), T-cell NFAT reporter assay and T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 to co-engage both CD123 and CD3 antigens, to induce T cell activation and target cytotoxicity, respectively. Ex vivo AML colony formation assay was applied to assess its activity to eliminate AML blasts. Human MV4-11 xenograft tumor model in humanized NSGdKO mice were utilized to determine anti-tumor efficacy of IGM-2537. Lastly, to confirm that CD123 directed IgM-based TCE molecule can potentially provide better safety profile, a benchmark IgM TCE comparator was assessed in cynomolgus monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound to human CD123 with high affinity, avidity, and specificity. Furthermore, IGM-2537 bound a unique epitope of human CD123 in domain 2 which does not overlap with IL-3. IGM-2537 co-engaged with both CD123 and CD3 antigens, leading to T cell-redirected killing of AML cell lines with concomitant T cell activation. Moreover, IGM-2537 eliminated AML blast cells at physiologically relevant effector/target (E/T) ratios in an ex vivo assay. Most importantly, IGM-2537 demonstrated comparable maximal killing activity to a benchmark IgG TCE molecule but shows significant reduction of cytokine release exemplified by IFN-γ, TNF-α and IL-6, which provide initial evidence of better preclinical safety profile for a CD123 directed IgM-based T-cell engager. As expected, the efficient in vitro TDCC activity of IGM-2537 translated into in vivo anti-tumor efficacy with tumor growth inhibition observed at dose ranges from 1 to 10 mg/kg. To further support that the IgM TCE format can provide a better safety window, a benchmark IgM TCE comparator was evaluated for tolerability (including cytokine production), PK and PD responses in cynomolgus monkeys. All animals tolerated the benchmark comparator well at dose levels up to 10 mg/kg (the maximal dose level evaluated); however, the tolerability observed with IgM TCE was 100x greater compared to IgG TCE. Complete depletion of CD123-positive basophils and substantial reduction of plasmacytoid dendritic cells (pDC) were seen in blood and bone marrow with minimal to no cytokine induction. In summary, our IGM-2537 demonstrates potent in vitro and in vivo activity with minimal cytokine induction. IgM as the framework of a TCE molecule can potentially provide better safety profile and an improved therapeutic window.