Dose-dependent Cell Viability Research Articles

Abstract LB027: Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository

Abstract Second-generation androgen ablation therapies (ADT), enzalutamide, and abiraterone are initially effective on metastatic castration-resistant prostate cancer (mCRPC). However, nearly 30-40% of patients eventually become resistant to these treatments due to the presence of androgen-receptor variants (AR-V), which lack a ligand-binding domain. The grim reality that metastatic CRPC is virtually incurable for most patients poses a significant challenge for clinicians striving to enhance these individuals' quality of life and lifespan. Considering drug discovery efforts, we partnered with the National Cancer Institute's (NCI) Natural Product Repository, which possesses a diverse collection of natural product extracts derived from plant, marine, and microbial organisms. We developed high-throughput phenotypic screening assays to screen the crude natural product extract libraries on AR and AR-variants CRPC cell lines (C4-2B and 22Rv1). Several extracts inhibited the growth of both CRPC cell lines and were further confirmed for dose-dependent cell viability inhibition assays. Once we determined the IC50 dose, we performed a Western blot analysis that confirmed the inhibition of AR and AR-V7 expression in CRPC cells.In subsequent studies, we narrowed down by fractionating the most potent extracts and confirmed their ability to inhibit AR expression and signaling on CRPC cell lines. Currently, we are characterizing the potent compounds and their inhibitory activity on AR and AR-variants, in vitro and in vivo CRPC models. Many nutraceuticals have been shown to target AR signaling, synthesis, or degradation which effectively inhibited the growth of CRPC. Our collaborative efforts with NCI will permit us find N-terminal AR degrading molecules (AF-1), which are essential for the AR transcriptional activities of CRPC. In sum, discovery of ovel small molecules from our drug development program have the potential to translate these effective natural products into drugs with clinical benefits to treat CRPC. Citation Format: Neha Tyagi, Sepideh Hosseiniporgham, Ashish Tyagi, Balaji Chandrasekaran, Katherine Bonilla, Inna Kriger, James Saccchettini, Chendil Damodaran. Identifying natural inhibitors of androgen receptor (AR) and its variants from NCI Natural Product Repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB027.

Synergistic Effects of Type I PRMT1 and Type II PRMT5 Inhibitors Against Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by the extensive expansion of monoclonal aberrant plasma cells in the bone marrow and the second most prevalent hematological malignancies worldwide. Despite the promising outcomes of advanced therapies, relapsed/refractory (RR) diseases remain incurable, highlighting an unmet clinical need to identify novel therapeutic targets. An emerging field of investigation is the role of aberrant post-translational modifications in multiple myeloma pathology. Arginine methylation is one of the most predominant post-translational modifications in cells, catalyzed by protein arginine methyltransferases (PRMTs) that plays a crucial role in multiple cellular processes. Type I PRMTs catalyze mono- (MMA) and asymmetric demethylation (ADMA) of arginine; Type II PRMTs catalyze mono- and symmetric demethylation (SDMA) of arginine on proteins; Type III PRMT mediates MMA only. Aberrant expression of PRMTs is strongly associated with poor prognosis of solid cancers and hematologic malignancies. Recently, we identified PRMT1 as a novel therapeutic vulnerability in MM cells. Among PRMTs, PRMT1 is the highest expressed gene in MM cell lines and patient cells. The elevation of PRMT1 expression is strongly associated with relapsed/refractory patients and poor clinical outcomes. Deletion of PRMT1 and suppression of the PRMT1's enzymatic activity exhibited a strong anti-MM in vitro via suppressing cell cycle and elevating the apoptosis. We also found that inhibition of PRMT1 boosts the T cell activation in the co-culture setup, indicating a potential enhancement of immunotherapy. Using a subcutaneous xenograft model, we showed that suppressing PRTM1 in vivo by either genetic or pharmaceutical approaches robustly inhibits MM tumor growth and extends survival of tumor-bearing mice. Our findings demonstrated that PRMT1 is indispensable in MM. PRMT5, a PRMT type II, is prognostically relevant and a known druggable target in MM. PRMT1 and PRMT5 inversely regulate post-transcription and have been implicated in human cancers. Recent studies in solid tumor showed a strong anti-tumor effect in vivo using the combinational inhibition of PRMT1 and PRMT5. We hypothesize that PRMT1 and PRMT5 might have a complementary function in MM cells. The combinational inhibition PRMT1 and PRTM5 will exhibit a synergistic anti-MM effect. To address this hypothesis, we treated MM cell lines with two clinical trial inhibitors: GSK3368715 (GSK) and EPZ 015666 (EPZ), suppressing PRMT type I and type II inhibitor, respectively. We first performed dose-dependent cell viability inhibition and selected effective doses for drug combination treatments in three different MM cell lines. The combination of GSK and EPZ demonstrated a strong synergistic effect in suppressing the viability of MM cells. Using the SynergyFinder algorithm, we found that the synergistic indexes (>10, synergism) for 3 MM cell lines JJN3, MM1S, and KMS11 were 33.5, 32.8, and 14.3, respectively. We next assessed the effects of combined treatment on the cell cycle and apoptosis of MM cells. We found that combination of GSK and EPZ strikingly elevated the apoptosis and suppressed cell cycle of MM cell lines compared single treatment and untreated control. Intriguingly, the combination treatment of two inhibitors significantly decreased MMA, SDMA, and ADMA, suggesting a robust suppression of global arginine methylation. Of note, we found a similar anti-MM effect when treating bortezomib-resistant MM cells with GSK and EPZ, indicating that PRMT type I and type II inhibitors potentially have a substantial clinical impact on relapsed MM. Of note, the same dose of GSK and EPZ that exhibits the synergistic anti-MM effect, didn't suppress the viability of healthy peripheral blood cells. In summary, our preliminary data suggest that suppression of both PRMT type I and type II might provide a potential effective combination treatment for both newly diagnosed and relapsed MM patients. We're currently investigating the underling mechanisms of how PRMTs regulate MM pathology and validating the in vivo efficacy of combination treatment using patient-derived xenograft.

Mahanine mediated therapeutic inhibition of estrogen receptor-α and CDK4/6 expression, decipher the chemoprevention-signaling cascade in preclinical model of breast cancer

Ethnopharmacological relevanceMahanine (MH), a naturally occurring carbazole alkaloid, isolated from Ayurvedic medicinal plant Murraya koenigii (L.) Spreng, has been shown to have various pharmacological properties, including its inhibitory activity against different breast cancers (BC) subtypes. Aim of the studyWhile MH triggers apoptosis in BC cells regardless of subtype, the specific mechanism of MH action is not fully understood. In this study, we show the effect of MH in preventing BC progression by inducing apoptosis in relation to estrogen receptor-α (ERα) and cell cycle regulatory proteins. Materials and methodsTo assess the pharmacological activity in various in vitro and in vivo tests, isolated and pure MH was used. To conclude the study, cutting edged molecular biology techniques including Western blot analysis, enzyme-linked immunosorbent assay (ELISA), molecular simulation study, and other related software analysis were employed. ResultsMH demonstrated dose dependent cell viability against drug sensitive (MCF-7 and MDA-MB-231) and paclitaxel resistant (MCF-7TR and MDA-MB-231TR) BC cells. MH also exhibited synergistic activity with tamoxifen (TAM) against estrogen receptor positive (ER+) BC cells by inhibiting ERα expression in MCF-7 cells and N-Methyl-N-nitrosourea (MNU)-induced mammary tumor in a dose-dependent manner while having no effect on vinculin expression. In addition, MH inhibited cell cycle regulatory genes namely CDK1/CDK4/CDK6/CDC25A and neo-angiogenesis through downregulation of CD31/PECAMs in MCF-7, MDA-MB-231 cells and mammary tumors from MNU-induced rats. MH therapy has been shown to be significantly able to lower the serum leptin level and to be beneficial against the initiation of tumor development in SD rats for up to 12 weeks. Molecular modeling study revealed that MH has antagonized the effectiveness of several types of estrogen those bind to the ERα and has comparable binding efficacy to TAM. ConclusionOverall, the current investigation showed the ability of MH to modify cell cycle genes especially CDK4 and CDK6 might be responsible for its anticancer activity against different breast cancer subtypes. Additionally, this study will aid in advancing MH translational research to the clinical trial stage.

Magnetically Separable Mg−Zn Ferrite Nanoparticles Synthesized Using Ocimum Basilicum Leaf Extract Potentiates Cytotoxicity: In vitro

AbstractThe present manuscript portrays environmental benign, cost‐effective and energy efficient green route way to synthesize Mg−Zn ferrite nanoparticles [Mg0.05Zn0.05Fe2O4 NPs] using Ocimum Basilicum leaf extract [OBLE]. X‐ray diffraction (XRD) analysis revealed that Ocimum Basilicum leaf extract mediated Mg−Zn ferrite nanoparticles [OBLE@Mg−Zn NPs] exhibit face centred cubic crystallinity with space group as confirmed by Rietveld refinement studies. Microstructural analysis illustrated uniform surface morphology with well interlinked grain with an average grain size of 35.6±0.12 nm. FTIR spectra showed existence of hydroxyl −OH group at 3403 cm−1 and C=C vibration at 2345 cm−1. Magnetic hysteresis loop (M−H loop) confirmed the superparamagnetic behaviour of OBLE@Mg−Zn NPs. Cytotoxic activity of OBLE@Mg−Zn NPs was carried out against breast cancer cell line MDA‐MB‐231. Dose dependent cell viability of cancerous cells depicted cytotoxic potential of OBLE@Mg−Zn NPs.

Use of Cold Atmospheric Plasma in the Treatment of Squamous Cell Carcinoma: in vitro Effects and Clinical Application in Feline Tumors: A Pilot Study

Cold atmospheric plasma (CAP) has shown promising results against squamous cell carcinoma (SCC) in both in vivo and in vitro assays, mainly in humans and mice. Its applicability for treatment of feline tumors, however, remains unknown. This study aimed to evaluate the anticancer effects of CAP on a head and neck squamous cell carcinoma (HNSCC) cell lineage and against a clinical case of cutaneous SCC in a cat. Control and treatment groups employing the HNSCC cell line (SCC-25) were used, the latter exposed to CAP for 60 seconds, 90 seconds, or 120 seconds. The cells were subjected to the MTT assay nitric oxidation assay and thermographic in vitro analyses. The clinical application was performed in one cat with cutaneous SCC (3 sites). The lesions were treated and evaluated by thermographic, histopathological, and immunohistochemical examinations (caspase-3 and TNF-alpha). Treatment of the SCC-25 cells for 90 seconds and 120 seconds resulted in a significant nitrite concentration increase. Decreased cell viability was observed after 24 hours and 48 hours, regardless of exposure time. However, the cell viability reduction observed at 72 hours was significant only in the 120 seconds treatment. In vitro, the temperature decreased for all treatment times, while the plasma induced a slight increase in mean temperature (0.7°C) in the in vivo assay. Two of the 3 clinical tumors responded to the treatment: one with a complete response and the other, partial, while the third (lower lip SCC) remained stable. Both remaining tumors displayed apoptotic areas and increased expression of caspase-3 and TNF-alpha. Adverse effects were mild and limited to erythema and crusting. The CAP exhibited an in vitro anticancer effect on the HNSCC cell line, demonstrated by a dose-dependent cell viability reduction. In vivo, the therapy appears safe and effective against feline cutaneous SCC. The treatment did not result in a clinical response for 1 of 3 lesions (proliferative lower lip tumor), however, a biological effect was still demonstrated by the higher expression of apoptosis indicators.

Novel Erlotinib–Chalcone Hybrids Diminish Resistance in Head and Neck Cancer by Inducing Multiple Cell Death Mechanisms

In a search for novel therapeutic options for head and neck squamous cell carcinomas (HNSCCs) generally treated with limited therapeutic success, we synthesized a series of novel erlotinib-chalcone molecular hybrids with 1,2,3-triazole and alkyne linkers and evaluated them for their anticancer activity on Fadu, Detroit 562 and SCC-25 HNSCC cell lines. Time- and dose-dependent cell viability measurements disclosed a significantly increased efficiency of the hybrids compared to the 1:1 combination of erlotinib and a reference chalcone. The clonogenic assay demonstrated that hybrids eradicate HNSCC cells in low micromolar concentrations. Experiments focusing on potential molecular targets indicate that the hybrids trigger the anticancer effect by a complementary mechanism of action that is independent of the canonical targets of their molecular fragments. Confocal microscopic imaging and real-time apoptosis/necrosis detection assay pointed to slightly different cell death mechanisms induced by the most prominent triazole- and alkyne-tethered hybrids (6a and 13, respectively). While 6a featured the lowest IC50 values on each of the three HNSCC cell lines, in Detroit 562 cells, this hybrid induced necrosis more markedly compared to 13. The therapeutic potential indicated by the observed anticancer efficacy of our selected hybrid molecules validates the concept of development and justifies further investigation to reveal the underlying mechanism of action.

Insights into the per- and polyfluoroalkyl substances-contaminated paper mill processing discharge: Detection, phytotoxicity, bioaccumulative profiling, and health risk verification

Per- and polyfluoroalkyl substances (PFASs), a new class of contaminants of emerging concern in different water sources and industrial discharges, have been highlighted, mainly ascribed to the high mobility, persistent, and bioaccumulative behaviors. In this work, the analytical detection of PFASs in the paper mill processing discharge (PMPD), physicochemical parameters, tannin and lignin, and phenolic content were carried out. The phytotoxicity evaluation and bioaccumulative profiles of PMPD-assisted irrigation have been conducted using a soilless cultivation system. The cytotoxicity, genotoxicity and human exposure risk were ascertained via in vitro assay on the human hepatocellular carcinoma (HepG2) cell and the estimation of daily intake, respectively. PFASs were quantitatively detected at the mean concentration of 2712.72 ng/L, consisted of mainly perfluoroalkyl carboxylic acids (PFCAs), perfluoroalkane sulfonic acids (PFSAs), perfluoroalkane sulfonamidoacetic acids (FASAAs), perfluoroalkane sulfonamides (FASAs), and fluorotelomer sulfonic acids (FTSAs). Significant reductions of the physical elongation, dry weight, and chlorophylls up to 33.49%, 31.71%, and 52.17% were recorded. Profound increase in the proline (101.81%), hydrogen peroxide (55.56%), superoxide anion (67.86%), lipid peroxidation (43.91%), and protein oxidation (32.31%), and up-regulation of antioxidant enzymes activities have been quantified. Bioaccumulation and translocation of PFASs revealed a concentration-, chain length- and functional group-dependent trend within different plant compartments. PFASs-contaminated PMPD treatments induced dose-dependent cell viability reduction and DNA damage in the HepG2 cell. The findings have highlighted that PMPD could be an underestimated source of PFASs, which represents a sharp threat to plants and public health via the water-food crop-human continuum. Future studies gearing towards the genomic profiling of PFASs in different plant species are suggested.

Eumelanin from the Black Soldier Fly as Sustainable Biomaterial: Characterisation and Functional Benefits in Tissue-Engineered Composite Scaffolds.

An optimized extraction protocol for eumelanins from black soldier flies (BSF-Eumel) allows an in-depth study of natural eumelanin pigments, which are a valuable tool for the design and fabrication of sustainable scaffolds. Here, water-soluble BSF-Eumel sub-micrometer colloidal particles were used as bioactive signals for developing a composite biomaterial ink for scaffold preparation. For this purpose, BSF-Eumel was characterized both chemically and morphologically; moreover, biological studies were carried out to investigate the dose-dependent cell viability and its influence on human mesenchymal stem cells (hMSCs), with the aim of validating suitable protocols and to find an optimal working concentration for eumelanin-based scaffold preparation. As proof of concept, 3D printed scaffolds based on methacrylated hyaluronic acid (MEHA) and BSF-Eumel were successfully produced. The scaffolds with and without BSF-Eumel were characterized in terms of their physico-chemical, mechanical and biological behaviours. The results showed that MEHA/BSF-Eumel scaffolds had similar storage modulus values to MEHA scaffolds. In terms of swelling ratio and stability, these scaffolds were able to retain their structure without significant changes over 21 days. Biological investigations demonstrated the ability of the bioactivated scaffolds to support the adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells.

Apoptotic effect of crude scorpion venom on head and neck squamous cell carcinoma cell line versus normal human epithelial cell line

Aim: The aim of this study was to investigate and compare the apoptotic effect of Leiurus quinquestriatus (LQ) scorpion venom on oral squamous cell carcinoma (OSCC) and normal human epithelial cell lines, in an attempt to introduce a novel selective therapeutic modality for OSCC. Methodology: we explored the effect of LQ scorpion venom on OSCC cell line (HNO-97) compared to normal oral epithelial (OEC) cell line. The cell viability by MTT assay and the expression of PUMA by ELISA were analyzed. Cell cycle analysis was studied for both groups using flow cytometry. Finally, we compared the expression of ROS by ELISA. Results LQ scorpion venom showed selective cytotoxic and apoptotic effect on OSCC cells. In comparison to normal cells, significant dose dependent cell viability reduction, cell cycle arrest and increased expression of apoptotic marker PUMA as well as ROS were observed in cancer cell group in comparison to the control group. Conclusions: LQ crude venom exerts a strong cytotoxic effect against OSCC with minimal effect on normal epithelial cells, thus, providing a selective, effective and less toxic possible chemotherapeutic agent for such a malignancy.

Deep learning prediction of chemical-induced dose-dependent and context-specific multiplex phenotype responses and its application to personalized alzheimer's disease drug repurposing.

Predictive modeling of drug-induced gene expressions is a powerful tool for phenotype-based compound screening and drug repurposing. State-of-the-art machine learning methods use a small number of fixed cell lines as a surrogate for predicting actual expressions in a new cell type or tissue, although it is well known that drug responses depend on a cellular context. Thus, the existing approach has limitations when applied to personalized medicine, especially for many understudied diseases whose molecular profiles are dramatically different from those characterized in the training data. Besides the gene expression, dose-dependent cell viability is another important phenotype readout and is more informative than conventional summary statistics (e.g., IC50) for characterizing clinical drug efficacy and toxicity. However, few computational methods can reliably predict the dose-dependent cell viability. To address the challenges mentioned above, we designed a new deep learning model, MultiDCP, to predict cellular context-dependent gene expressions and cell viability on a specific dosage. The novelties of MultiDCP include a knowledge-driven gene expression profile transformer that enables context-specific phenotypic response predictions of novel cells or tissues, integration of multiple diverse labeled and unlabeled omics data, the joint training of the multiple prediction tasks, and a teacher-student training procedure that allows us to utilize unreliable data effectively. Comprehensive benchmark studies suggest that MultiDCP outperforms state-of-the-art methods with unseen cell lines that are dissimilar from the cell lines in the supervised training in terms of gene expressions. The predicted drug-induced gene expressions demonstrate a stronger predictive power than noisy experimental data for downstream tasks. Thus, MultiDCP is a useful tool for transcriptomics-based drug repurposing and compound screening that currently rely on noisy high-throughput experimental data. We applied MultiDCP to repurpose individualized drugs for Alzheimer’s disease in terms of efficacy and toxicity, suggesting that MultiDCP is a potentially powerful tool for personalized drug discovery.

Responses of dinoflagellate cells to ultraviolet-C irradiation.

Dinoflagellates are important aquatic microbes and major harmful algal bloom (HAB) agents that form invasive species through ship ballast transfer. UV-C installations are recommended for ballast treatments and HAB controls, but there is a lack of knowledge in dinoflagellate responses to UV-C. We report here dose-dependent cell cycle delay and viability loss of dinoflagellate cells irradiated with UV-C, with significant proliferative reduction at 800 Jm-2 doses or higher, but immediate LD50 was in the range of 2400-3200 Jm-2 . At higher dosages, some dinoflagellate cells surprisingly survived after days of recovery incubation, and continued viability loss, with samples exhibiting DNA fragmentations per proliferative resumption. Sequential cell cycle postponements, suggesting DNA damages were repaired over one cell cycle, were revealed with flow cytometric analysis and transcriptomic analysis. Over a sustained level of other DNA damage repair pathways, transcript elevation was observed only for several components of base pair repair and mismatch repair. Cumulatively, our findings demonstrated special DNA damage responses in dinoflagellate cells, which we discussed in relation to their unique chromo-genomic characters, as well as indicating resilience of dinoflagellate cells to UV-C.

Cytocompatibility of Pluronics F-127 on adenocarcinomic human alveolar basal epithelial cells (A549 cells).

Pluronics, due to its high water-soluble and thermoreversible ability, attracted much in biomedical applications. They are mainly utilized in drug delivery, gene therapy, and tissue remodeling. The study aims to explore the cytocompatibility of Pluronics F-127, which has gained much popularity due to its various properties. The cells were exposed to varying concentrations of Pluronics F-127 in A549 cells for 24h. According to the MTT and neutral red assay, A549 cells displayed dose-dependent cell viability. The cell's morphology was preserved after treatment, as seen in phase-contrast and Giemsa staining. When exposed to PF-127, lysosomal, cytoskeletal, and nuclear integrity were maintained. The percentage of live cells in all the treated groups was more significant than 90%, according to the live/dead flow cytometric analyses. The study identified the cytocompatibility of Pluronics F-127 required for the breakthrough in biomedical applications.

The Biological Effects of Polystyrene Nanoplastics on Human Peripheral Blood Lymphocytes.

Environmental exposure to microplastics (MPs) and nanoplastics (NPs) is an increasing concern from human health perspectives. Little information on the genotoxic and cytotoxic potential of NP particles in human cells is available. We aimed to assess the cytotoxic and genotoxic potential of polystyrene nanoplastics (PSNPs) at different concentrations (2000μg/mL, 1000μg/mL, and 500μg/mL) by using chromosomal aberration (CA) and cytokinesis-block micronucleus assays (CBMN) on human peripheral lymphocytes. Dose-dependent hemolytic activity and cell viability were observed against the PSNPs exposure. Increased chromosomal aberrations, such as chromosomal breaks and dicentric chromosomes, and an increase in nucleoplasmic bridge (NBP) formation and nuclear budding (NBUD) were observed. The frequency of mitotic index (MI) decreased significantly in the PSNP-exposed groups from lower to higher concentrations. A significant increase in micronuclei (MN) formation and cytostasis% and a dose-dependent reduction in nuclear division index (NDI) in PSNP-exposed groups indicated oxidative stress-mediated cytotoxicity, DNA damage, and genomic instabilities due to PSNP exposure in human lymphocyte cells. This study highlights the importance of understanding the toxic mechanisms and associated chronic and acute health effects on humans due to exposure to this pervasive environmental pollutant.

Association of Risperidone With Gastric Cancer: Triangulation Method From Cell Study, Animal Study, and Cohort Study.

Purpose: To examine the effects of risperidone, an atypical antipsychotic agent, on gastric cancer. Methods: A triangulation method comprising bench studies, including cell and animal experiments, and a retrospective cohort study, was subsequently performed. Results: The bench study indicated that risperidone exerted more prominent tumor inhibition effects than other atypical antipsychotics on the proliferation of KATO-III cells, a human gastric cancer cell line. Significant and dose-dependent cell viability was observed in Hs27 cells (control cells) in the presence of risperidone compared with in KATO-III cells. Both in vivo and in vitro results indicated that risperidone significantly inhibited the proliferation of KATO-III cells by inducing ROS and apoptosis, and that it suppressed the growth of xenografted KATO-III tumors in nude mice. In addition, the population-based cohort study found that risperidone users had reduced risks of gastric cancer compared with non-users, with lowered adjusted hazard ratios (HRs) for two induction periods (HR = 0.75; 95% confidence interval [CI] 0.68–0.83 for the one-year induction period, and HR = 0.68; 95% CI 0.61–0.75 for the two-year induction period). Conclusion: The findings are consistent with anticancer effects associated with risperidone, but further research and evaluations are warranted.

E-Cigarette Aerosols Promote Oral S. aureus Colonization by Delaying an Immune Response and Bacterial Clearing.

E-cigarette (e-cig) vapor has been shown to play a pathological role in oral health and alter the oral microbiota, providing growth advantages for opportunistic pathogens. Enrichment of Staphylococcus aureus, a commensal resident in the oral cavity, correlates with the progression of periodontal disease, suggesting a role as an opportunistic pathogen. Environmental conditions, such as cigarette smoke, are known to increase S. aureus virulence, yet the role of S. aureus in periodontitis and oral preneoplasia is unknown. We exposed oral epithelial cells to e-cig aerosols and showed a dose-dependent cell viability reduction, regardless of nicotine content, in a possible attempt to repair DNA damage, as measured by pH2AX. S. aureus attachment to oral epithelial cells and bacterial biofilm formation were enhanced upon e-cig exposure, indicating an increased capacity for oral colonization. Mechanistically, e-cig aerosol exposure resulted in an immunosuppression, as determined by a reduction in IL8, IL6, and IL1β secretion by oral epithelial cells during co-culture with S. aureus. Consistent with this, e-cig vape reduced the oral epithelial cell clearance of S. aureus. Furthermore, we observed an increased expression of the inflammatory regulator COX2. This work suggests that e-cigs promote S. aureus colonization and modulate the oral inflammatory response, possibly promoting oral periodontitis and preneoplasia.

Differential in vitro anti-leukemic activity of resveratrol combined with serine palmitoyltransferase inhibitor myriocin in FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) carrying AML cells.

Treatment of FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) AML is restricted due to toxicity, drug resistance and relapse eventhough targeted therapies are clinically available. Resveratrol with its multi-targeted nature is a promising chemopreventive remaininglimitedly studied in FLT3-ITD AMLregarding toceramide metabolism. Here, its cytotoxic, cytostatic and apoptotic effects are investigated in combination with serine palmitoyltransferase (SPT), the first enzyme of de novo pathway of ceramide production, inhibitor myriocin on MOLM-13 and MV4-11 cells. We assessed dose-dependent cell viability, flow cytometric cell death and cell cycle profiles of resveratrol in combination with myriocin by MTT assay, annexin-V/PI staining and PI staining respectively. Resveratrol's dose-dependent effect on SPT protein expression was also checked by western blot. Resveratrol decreased cell viability in a dose- dependent manner whereas myriocin did not affect cell proliferation effectively in both cell linesafter 48h treatments. Although resveratrol induced both apoptosis and asignificant S phase arrest in MV4-11 cells, it triggered apoptosisand non-significant S phase accumulation in MOLM-13 cells. Co-administrations reduced cell viability. Increased cytotoxic effect of co-treatments was further proved mechanistically through induction of apoptosis via phosphatidylserine relocalization. The cell cycle alteration in co-treatment was significant with an S phase arrest in MV4-11 cells, however, it was not effective on cell cycle progression of MOLM-13 cells. Resveratrol also increased SPT expression. Overall, modulation of SPT together with resveratrol might be the possible explanation for resveratrol's action. It could be an integrative medicine for FLT3-ITD AML after investigating its detailed mechanism of action in relation to de novo pathway of ceramide production.

The attenuation of antibiotic resistant non-albicans Candida species, cytotoxicity, anti-inflammatory effects and phytochemical profiles of five Vachellia species by FTIR and UHPLC–Q/Orbitrap/MS

This work investigated the antifungal, cytotoxic and LPS-induced anti-inflammatory effects of five Vachellia species (V. karroo, V. kosiensis, V. sieberiana, V. tortalis and V. xanthophloea). The antifungal activity of the aqueous-methanolic extracts were performed using the broth dilution method against four non-albicans Candida species (C. glabrata, C. auris, C. tropicalis and C. parapsilosis). The cytotoxic and anti-inflammatory effects of the extracts were evaluated on African green monkey Vero kidney cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and the 2ʹ,7ʹ-dichlorofluorescin diacetate (H2DCF-DA) method. The fourier-transform infrared spectroscopy (FTIR) and Q Exactive plus orbitrap™ Ultra-high-performance liquid chromatography-mass spectrometer (UHPLC-MS) analysis was conducted to evaluate phytochemical constituents of the extracts. The plant extracts selected in this study displayed potency against the Candida species tested, with MIC values ≤0.62 mg/mL for V. karroo, V. kosiensis and V. xanthophloea. A dose-dependent cell viability was observed on Vero cells with all extracts showing LC50 values >20 μg/mL. Extracts tested at 10 μg/mL elicited a significant decrease in lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) in Vero cells with V. sieberiana, V. tortilis, V. karroo, V. kosiensis and V. xanthophloea displaying inhibitory percentages of 35%, 32%, 55%, 52% and 49%, respectively. Characterisation of functional groups representing compounds in the extracts demonstrated the presence of different classes of compounds of the aliphatic, sugar and aromatic types. The Q Exactive plus orbitrap™ mass spectrometer enabled tentative identification of three major compounds in the extracts, including epigallocatechin, methyl gallate and quercetin amongst others. Based on the mass spectrometer results, it is postulated that quercetin found mostly in active extracts of V. karroo, V. xanthophloea, and V. kosiensis may be responsible for the observed antifungal and anti-inflammatory activity. This data demonstrates that the Vachellia species that were investigated could potentially be promising candidates for the management of fungal infections and related inflammation.

Resistance Mechanisms Underlying Venetoclax Resistance in Mantle Cell Lymphoma

Background: BCL-2 is highly expressed in mantle cell lymphoma (MCL), and targeting BCL-2 with venetoclax (ABT-199) has resulted in positive clinical outcomes in MCL patients. However, resistance to various targeted agents often emerges; therefore, we generated venetoclax-resistant MCL cell lines to uncover the mechanisms mediating acquired venetoclax resistance and performed high-throughput drug screening to identify novel anti-MCL agents that can overcome venetoclax resistance.Methods: We generated 3 venetoclax-resistant cell lines (Mino, Rec-1 and Granta519) by long-term venetoclax exposure, and the anti-MCL activity of venetoclax against these acquired resistance lines was measured with dose-dependent cell viability and apoptosis assays compared with the parental lines. Aberrant protein expression between the parental and resistant cell lines was detected using Reverse Phase Protein Array (RPPA) analysis. A drug library (> 1000 drugs; Selleck Chemicals Ltd) was screened to identify agents that inhibit the cell growth of both the parental and resistant cells. The candidate drugs that showed anti-MCL activity were confirmed by dose-dependent cell viability and Annexin V apoptosis assays. To determine the mechanisms underlying the anti-MCL activity of the candidate agents in the parent and venetoclax-resistant MCL cell lines, we conducted RPPA analysis before and after drug treatment.Results: Chronic exposure to venetoclax resulted in markedly less sensitivity to venetoclax. The IC50 values of the venetoclax-resistant cell lines (Mino-Re, Rec-1-Re and Granta519-Re) were determined to be 603 nM, 676 nM and 456 nM, respectively, which are 600-fold, 500-fold and 25-fold higher than the IC50 values of the parental cells lines. RPPA analysis showed that proteins such as PTEN, PDCD4, AKT, ATM, BCL-2, BAX and BAK were down-regulated and p-AKT, p21, STAT3, Cyclin B1 and MCL-1 were up-regulated in the venetoclax-resistant MCL cell lines. The results were confirmed by Western blotting, and further Western blotting experiments revealed expression and modification differences among anti-apoptotic and pro-apoptotic protein family members between the parental and venetoclax-resistant MCL cell lines. Specifically, both MCL-1 and BCL-xL were elevated; however, BIM and BAX were absent in the venetoclax-resistant cells. High-throughput drug screening showed striking anti-MCL activity with two PI3K inhibitors, PIK-75 (PI3K α inhibitor) and GSK1059615 (pan PI3K inhibitor), in dose-dependent cell viability and apoptosis assays. RPPA analysis of the parental and venetoclax-resistant MCL cell lines with and without PI3K inhibitor treatment suggested that both drugs inhibited the cell viability of the MCL cell lines by blocking the activated PI3K/AKT pathway.Conclusion:The PI3K/AKT pathway is activated in venetoclax-resistant MCL cell lines, and targeting this pathway may be a potential approach to overcoming venetoclax resistance. DisclosuresWang:Pharmacyclics: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Research Funding; BeiGene: Research Funding; Asana Biosciences: Research Funding; Proteolix: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Acerta Pharma: Consultancy, Research Funding.

FoxM1: A New Therapeutic Target for Mantle Cell Lymphoma

IntroductionMantle cell lymphoma (MCL) is an aggressive B-cell lymphoma subtype with elevated B-cell receptor activity. Ibrutinib (IBN), the Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have an overall response rate of 68% in relapsed or refractory MCL patients. However, with the emergence of IBN resistance, novel therapies to thwart resistance are urgently needed. FOXM1 (Forkhead box M1) is a proliferation-associated transcription factor that stimulates cell proliferation and exhibits a proliferation-specific expression pattern. FOXM1 has recently been classified as a human proto-oncogene and we previously found this gene to be associated with IBN resistance in our gene expression analysis; therefore, the prognostic significance of FOXM1 and its potential as a new MCL therapeutic target was investigated.MethodsBone marrow aspirates and peripheral blood clinical specimens were obtained from MCL patients. FOXM1 gene expression was assessed in 50 primary MCL patient samples using Quantitative real-time PCR (Q-PCR). To conduct dose-dependent cell viability assays, MCL cell lines (ATCC) were cultured in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Millipore). The drug screening was performed in a 96-well format in which MCL cells were seeded at 10,000 cells per well and were treated with the FOXM1 inhibitor thiostrepton or ibrutinib at the following concentrations: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 uM. Cell viability was tested using the CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation period. To determine the effects of inhibiting FOXM1 on apoptosis, MCL cells (200,000 cells) were incubated with IBN or thiostrepton at varying doses (1, 3, 5 uM) for 24 hours, and apoptosis was detected using the Annexin V-binding assay. Furthermore, caspase-3/7 activity was assessed in MCL cells with the Caspase-Glo-3/7 Assay (Promega) after 24 hours of treatment with thiostrepton or IBN.ResultsTo evaluate the possibility that FOXM1 is important for MCL, we conducted an analysis of FOXM1 expression in MCL patient samples at the time of diagnosis using Q-PCR. FOXM1 expression was detected in 50 MCL patient samples, and FOXM1 expression levels were significantly higher (82.3%, fold change=2) than the levels observed in peripheral blood mononuclear cells (PBMCs). The association between FOXM1 expression in MCL and clinical pathology was also analyzed by examining the relationship between FOXM1 and Ki-67 expression (a cell-proliferation marker) in 40 diagnosed MCL patient samples, and a significant correlation was noted between the two markers (p<0.05).The effects of inhibiting FOXM1 on MCL cell lines (Jeko-1, Jeko-1-BTK-KO-#1, Jeko-1-BTK-KO-#2, Mino, Maver-1, Z-138, JVM-2, and JVM-13) in vitro were examined by transfection of siRNA targeting FOXM1 or treatment with the FOXM1 inhibitor thiostrepton. Thiostrepton reduced the cell viability of the MCL cell lines in a dose-dependent manner compared with the DMSO control. Likewise, siFOXM1-transfected MCL cell lines showed reduced cell viability compared with the control siRNA-transfected cells (P < 0.01). Annexin V/PI dual staining showed that thiostrepton induced apoptosis in the MCL cell lines in a dose-dependent manner. Furthermore, activated-caspase3-7 expression was increased in a dose-dependent manner in MCL cell lines after treatment with thiostrepton.ConclusionWe have shown that FOXM1 inhibition may be a potential candidate treatment for MCL based on the results of our clinicopathological assessment and in vitro studies. Clinical studies are warranted to further validate FOXM1 as a potential therapeutic target in high-risk MCL. DisclosuresWang:Celgene: Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Oncternal: Research Funding; BeiGene: Research Funding; Juno Therapeutic: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Honoraria, Research Funding; Oncoceutics: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Asana: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Karus: Research Funding.

HBCD, TBECH, and BTBPE exhibit cytotoxic effects in human vascular endothelial cells by regulating mitochondria function and ROS production.

Brominated flame retardants (BFRs), such as, 1,2,5,6-tetrabromocyclooctane (HBCD), 1,2-dibromo-4-(1,2-dibromopropyl)cyclohexane (TBECH), and 1 1,2-bis-(2,4,6-tribromophenoxy)ethane (BTBPE), have garnered increasing attention due to their potent biological effects. In the present study, the toxicity of HBCD, TBECH, and BTBPE in human vascular endothelial cells (ECs) was explored. The data showed that HBCD, TBECH, and BTBPE induced cytotoxicity, namely dose-dependent cell viability reduction, cell membrane permeability and apoptosis increase, migration, and lumen formation inhibition. Moreover, HBCD was found to be more toxic than BTBPE or TBECH. Exposure to HBCD, TBECH, and BTBPE led to the production of reactive oxygen species, mitochondrial superoxide generation, and mitochondrial membrane potential collapse, implying that reactive stress caused the cytotoxicity. The ATP content, glutathione content, superoxide dismutase, and MDA activities were reduced, indicating that mitochondrial dysfunction may be the key mechanisms responsible for apoptosis. The present study suggested that mitochondria are a new target of BFRs in ECs and further deepened our understanding of the developmental toxicity of BFRs.