Normal Keratinocyte Cell Research Articles

Development of β‐Carboline‐Coumarin Based Hybrids as Potential Cytotoxic and Topoisomerase IIα Inhibitors

AbstractCancer remains a global health concern, prompting extensive efforts to develop novel inhibitors targeting the enzyme topoisomerase IIα as potential anti‐cancer agents. Herein, we strategically combined the pharmacophores from natural (β‐carboline) and (coumarin) sources, diversifying them at two distinct points. The in vitro evaluation of cytotoxic properties were estimated on human cancer cell lines, reveals that among the tested compounds, 13 r (isopropyl‐substituted β‐carboline) exhibited remarkable potency with IC50 value of 4.37 μM in the HCT116 cell line. Its selectivity for cancer cells was validated against normal keratinocyte cells, establishing a favourable selectivity index. Further, SAR studies indicate that no substitution (13 r) at C‐6 and C‐7 of β‐carboline, enhance anticancer activity. Assays confirmed its ability to intercalate with DNA, inhibit topoisomerase IIα, induce apoptosis, and disrupt mitochondrial membrane potential in HCT116 cells. Flow cytometric assays gauged the induction of apoptosis using Annexin‐V/PI dual staining and the disruption of mitochondrial membrane potential through JC staining. Results demonstrated that compound 13 r induced apoptosis and dose‐dependent depolarization of the mitochondrial membrane in HCT116 cells. Molecular docking studies further validated that compound 13 r binds within the active site of DNA when complexed with topo IIα, and this binding was stabilized through interactions with DNA base pairs and amino acid residues.

Preparation of Composite Hydrogels Based on Cysteine-Silver Sol and Methylene Blue as Promising Systems for Anticancer Photodynamic Therapy.

In this study, a novel supramolecular composite, "photogels", was synthesized by mixing of cysteine-silver sol (CSS) and methylene blue (MB). A complex of modern physico-chemical methods of analysis such as viscosimetry, UV spectroscopy, dynamic and electrophoretic light scattering, scanning electron microscopy and energy-dispersive X-ray spectroscopy showed that MB molecules are uniformly localized mainly in the space between fibers of the gel-network formed by CSS particles. Molecules of the dye also bind with the surface of CSS particles by non-covalent interactions. This fact is reflected in the appearance of a synergistic anticancer effect of gels against human squamous cell carcinoma even in the absence of light irradiation. A mild toxic influence of hydrogels was observed in normal keratinocyte cells. Photodynamic exposure significantly increased gel activity, and there remained a synergistic effect. The study of free-radical oxidation in cells has shown that gels are not only capable of generating reactive oxygen species, but also have other targets of action. Flow cytometric analysis allowed us to find out that obtained hydrogels caused cell cycle arrest both without irradiation and with light exposure. The obtained gels are of considerable interest both from the point of view of academics and applied science, for example, in the photodynamic therapy of superficial neoplasms.

Novel three-dimensional live skin-like in vitro composite for bioluminescence reporter gene assay.

We genetically manipulated HaCaTcells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL-8) and ubiquitin-C (UBC) promoters, respectively. Subsequently, we generated a three-dimensional (3D) skin-like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non-woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real-time bioluminescence monitoring, allowing the assessment of time- and dose-dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin.

Preparation and Characterization of Amoxicillin-Loaded Polyvinyl Alcohol/Sodium Alginate Nanofibrous Mat: Drug Release Properties, Antibacterial Activity, and Cytotoxicity

AbstractThis study aimed to prepare a polyvinyl alcohol/sodium alginate (PVSA) nanofibrous mat as an amoxicillin (AMOX) delivery system. AMOX was loaded to the PVSA nanofibers during electrospinning, and the AMOX-loaded PVSA (PVSA/AMOX) nanofibrous mat was cross-linked by glutaraldehyde (GA). The PVSA/AMOX nanofibrous mat was characterized by Fourier Transform infrared spectroscopy, scanning electron microscopy, Brunauer–Emmett–Teller, and mercury porosimetry analyses. The thickness, air permeability, and water vapor transmission rate of the PVSA/AMOX nanofibrous mat were 0.43 ± 0.08 mm, 17.2 ± 4.91 L/m2/s, and 1485 ± 13.6 g/m2/d, respectively, which were suitable for wound dressing applications. The tensile strength was 6.73 ± 0.48 MPa and elongation at a maximum load was 81.9 ± 17.0%, within the ranges of human skin’s values. The total porosity was 59.4%, enabling cell adhesion, migration, and proliferation. The PVSA/AMOX nanofibrous mat has high swelling (319 ± 4.2%) and low degradation (2.2 ± 0.1% in 10 days) ratios. The nanofibrous mat cross-linked with 0.25% GA solution for 20 min had a 73.07% cumulative release for 90 min. The drug release kinetics were obeyed to the Korsmeyer-Peppas model. The nanofibrous mat presented antibacterial activity on S. aureus ATCC 29213 and E. coli ATCC 25922, and there was no cytotoxic effect on the human normal keratinocyte cells, demonstrating the potential for use in wound dressing applications.

Unraveling the Mechanisms Involved in the Beneficial Effects of Magnesium Treatment on Skin Wound Healing.

The skin wound healing process consists of hemostatic, inflammatory, proliferative, and maturation phases, with a complex cellular response by multiple cell types in the epidermis, dermis, and immune system. Magnesium is a mineral essential for life, and although magnesium treatment promotes cutaneous wound healing, the molecular mechanism and timing of action of the healing process are unknown. This study, using human epidermal-derived HaCaT cells and human normal epidermal keratinocyte cells, was performed to investigate the mechanism involved in the effect of magnesium on wound healing. The expression levels of epidermal differentiation-promoting factors were reduced by MgCl2, suggesting an inhibitory effect on epidermal differentiation in the remodeling stage of the late wound healing process. On the other hand, MgCl2 treatment increased the expression of matrix metalloproteinase-7 (MMP7), a cell migration-promoting factor, and enhanced cell migration via the MEK/ERK pathway activation. The enhancement of cell migration by MgCl2 was inhibited by MMP7 knockdown, suggesting that MgCl2 enhances cell migration which is mediated by increased MMP7 expression. Our results revealed that MgCl2 inhibits epidermal differentiation but promotes cell migration, suggesting that applying magnesium to the early wound healing process could be beneficial.

Abstract 5904: Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways

Abstract Pancreatic cancer (PaCa) ranks as the fourth leading cause of cancer-related deaths in the United States and is among the top five worldwide. Its hallmark characteristics include early invasion, metastasis, resistance to chemotherapy and radiotherapy, and aggressive tumor progression. Evaluating new generation treatments with significant potential in this ongoing battle against cancer is crucial. Voltage-gated sodium channels (VGSC), long detected in various metastatic cell lines and tissue samples beyond breast, lung, and prostate cancer, are of particular interest. This study aimed to explore the therapeutic effects of inhibiting NaV1.7 and its neonatal variant, nNaV1.7, in pancreatic cancer cells. The study utilized human normal keratinocyte cell line HaCaT and pancreatic cancer cell lines PANC-1 and MiaPaCa-2. It investigated the roles and effects of adult and neonatal isoforms of the NaV1.7 channel in pancreatic cancer proliferation, invasion, and metastasis. The findings revealed significantly higher adult and neonatal NaV1.7 mRNA expressions in PANC-1 and MiaPaCa-2 cell lines compared to HaCaT cells. Specific siRNAs effectively downregulated channel mRNA and protein expressions. Notably, NaV1.7 siRNA treatments significantly inhibited cell proliferation and colony-forming capacity in PANC-1 and MiaPaCa-2 cells, without cytotoxic effects on HaCaT cells. Combining gemcitabine with specific NaV1.7 siRNAs enhanced drug efficacy in the PANC-1 cell line. Furthermore, NaV1.7 siRNAs significantly reduced cell invasion, migration, and wound healing in both cell lines. These siRNAs also induced apoptosis and G1 arrest in the cell cycle. They inhibited protein expressions involved in key signaling pathways, including P-Src/P-Fak/Integrin β1, EF2K, P-Akt, P-mTOR, PARP, Caspase-3/9, Cyclin D1/E1. This study is pioneering, as literature lacks research on the expression and function of NaV1.7 and nNaV1.7 in pancreatic cancer cells or tissue samples. In conclusion, NaV1.7 channel isoforms play a significant role in the development, progression, and metastasis of pancreatic cancer and present as a potential therapeutic target in PaCa. Citation Format: Mumin Alper Erdogan, Ayse Caner. Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways [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 5904.

Synthesis, characterization, antimicrobial and in vitro cytotoxic evaluation of trinuclear heterobimetallic complexes of Ni (II) derived by the reaction of [Ni(C10H10N2)2]Cl2 with dialkyl/diaryl organometallic dichlorides

The newly designed trinuclear heterobimetallic complexes [Ni(L)2(M)2R4Cl2] were synthesized by using methanolic solution of mononuclear Ni (II) complex and various organometallic dichlorides (where L = 1,8‐diaminaphthalene [C10H10N2], M = Sn [IV], Si [IV], Ti [IV] and Zr [IV] and R = Ph, Me, C5H5, etc.). The synthesized complexes were characterized on the basis of physico‐chemical and spectral (FTIR, UV‐visible, 1H‐NMR, 13C‐NMR, 29Si‐NMR, 119Sn‐NMR, Mass, and PXRD) studies. IR spectroscopy confirmed the coordination of ‐NH2 group to the metal centre. PXRD patterns showed the crystalline nature of all the synthesized complexes. Among the synthesized complexes, mononuclear metal complex adopted square planar geometry, while trinuclear heterobimetallic complexes adopted distorted octahedral geometry. This geometry is also supported by DFT calculations. The in vitro antimicrobial effectiveness of these resulting compounds has been scrutinized against a number of bacterial strains (Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, and Staphyloccocus aureus) by agar disc diffusion method and fungal strains (Candida albicans, Fusarium oxysporum and Aspergillus niger) by disc diffusion method, and promising results were attained. The in vitro cytotoxicity activity of the synthesized complexes has been scrutinized against human cervical cancer cells (HeLa) and human breast cancer cells (MCF‐7) using MTT assay and showed higher cytotoxicity than cis‐platin. Furthermore, their non‐toxic nature was confirmed when they tested against human normal keratinocyte cells (HaCaT). The results indicated better biological activity of heterobimetallic complexes than their monometallic complex.

Methanolic extract of Kigelia africana and wound healing: an in vitro study.

Kigelia africana (Lam.) Benth. (Bignoniaceae) syn. Kigelia pinnata (Jacq. DC) is a tropical plant that is native to tropical Africa. The aim of this study was to determine if a methanolic extract prepared from Kigelia africana (KAE) can promote wound healing in treated human normal epidermal keratinocyte (HaCaT) cells and human normal foreskin fibroblast cell line (BJ) cells compared with untreated cells. Experimental steps included: the methanolic extraction of the leaf and fruit of the Kigelia africana plant; the preparation of HaCaT and BJ cell lines; cell culture with a stable tetrazolium salt-based proliferation assay; and the evaluation of the wound healing effect of KAE (2μg/ml) in BJ and HaCaT cells. The phytochemical contents of KAE were determined using liquid chromatography quadrupole time-of-flight mass spectrometry. The following molecules were identified as being present in the KAE, among others: cholesterol sulfate; lignoceric acid; embelin; isostearic acid; linoleic acid; dioctyl phthalate; arg-pro-thr; 15-methyl-15(S)-PGE1; sucrose; benzododecinium (Ajatin); and 9-Octadecenamide (oleamide). KAE effected faster wound healing in treated cells compared with untreated cells for both cell lines. HaCaT cells that had been mechanically injured and treated with KAE healed completely in 48 hours compared with 72 hours for untreated HaCaT cells. Treated BJ cells healed completely in 72 hours compared with 96 hours for untreated BJ cells. Concentrations of KAE up to 300μg/ml had a very low cytotoxic effect on treated BJ and HaCaT cells. The experimental data in this study support the potential of KAE-based wound healing treatment to accelerate wound healing.

Machine learning-based signature of necrosis-associated lncRNAs for prognostic and immunotherapy response prediction in cutaneous melanoma and tumor immune landscape characterization.

Cutaneous melanoma (CM) is one of the malignant tumors with a relative high lethality. Necroptosis is a novel programmed cell death that participates in anti-tumor immunity and tumor prognosis. Necroptosis has been found to play an important role in tumors like CM. However, the necroptosis-associated lncRNAs' potential prognostic value in CM has not been identified. The RNA sequencing data collected from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) was utilized to identify differentially expressed genes in CM. By using the univariate Cox regression analysis and machine learning LASSO algorithm, a prognostic risk model had been built depending on 5 necroptosis-associated lncRNAs and was verified by internal validation. The performance of this prognostic model was assessed by the receiver operating characteristic curves. A nomogram was constructed and verified by calibration. Furthermore, we also performed sub-group K-M analysis to explore the 5 lncRNAs' expression in different clinical stages. Function enrichment had been analyzed by GSEA and ssGSEA. In addition, qRT-PCR was performed to verify the five lncRNAs' expression level in CM cell line (A2058 and A375) and normal keratinocyte cell line (HaCaT). We constructed a prognostic model based on five necroptosis-associated lncRNAs (AC245041.1, LINC00665, AC018553.1, LINC01871, and AC107464.3) and divided patients into high-risk group and low-risk group depending on risk scores. A predictive nomogram had been built to be a prognostic indicator to clinical factors. Functional enrichment analysis showed that immune functions had more relationship and immune checkpoints were more activated in low-risk group than that in high-risk group. Thus, the low-risk group would have a more sensitive response to immunotherapy. This risk score signature could be used to divide CM patients into low- and high-risk groups, and facilitate treatment strategy decision making that immunotherapy is more suitable for those in low-risk group, providing a new sight for CM prognostic evaluation.

Bioactivity and molecular docking of lactones isolated from Centaurea pseudosinaica Czerep

Two cytotoxic sesquiterpene lactones, 17-epichlorohyssopifolin A (1) and chlorjanerin (2), and a monoterpene lactone, loliolide (3) were isolated from Centaurea pseudosinaica. The cytotoxicity of the total extract and terpenoids 1–3 were evaluated against three human cancer cells (HepG2, PC-3, and HT-29), along with the human normal primary epidermal keratinocytes (HEKa) cells. With IC50 values ranging between 0.6 ± 0.04 and 5.0 ± 0.61 μg/mL against HepG2; 0.2 ± 0.01 and 11.9 ± 1.31 μg/mL against PC-3, and 0.04 ± 0.013 and 8.9 ± 0.97 μg/mL against HT-29, the total extract, and lactones 1–3 demonstrated cytotoxic effects. Compound 1 displayed the strongest impact on all cancer cells and a slightly safe effect on the normal cells HEKa. Compound 1 caused accumulation of HepG2 and HT-29 cells in G1 phase as displayed cell cycle analysis. On the other hand, the cell distributions were increased in the S phase in PC-3 cells. Furthermore, 1 caused apoptosis in PC-3 and HePG2 cells with 91.50%, and 79.72 %, respectively. A higher fraction of necrotic cells was observed in HT-29 cells amounting to 23.60%. These results suggested that the promising cytotoxicity exhibited by 1 is brought by the apoptosis induction in the cancer cells, which were evaluated. As the compounds showed antiproliferative effect against the HT-29 cells, the docking simulation was performed aiming at determining how they would interact with the EGFR enzyme, whose PDB: 4I23 is considered one of the two distinct wild types of EGFR enzymes. The antibacterial activity results revealed that 3 showed the most remarkable antibacterial effects, especially against the examined Gram-positive bacteria. The total extract exhibited potent activity against all examined bacteria. The total extract showed a potent antifungal effect against two Candida and two Aspergillus pathogens. The antioxidant activity revealed the potency of the total extract and 3 as antioxidant candidates. The obtained results refer to the importance of Centaurea pseudosinaica as a source of potent antiproliferative agents and the whole plant as an antipathogenic and antioxidant agent.

Sericin mediated gold/silver bimetallic nanoparticles and exploration of its multi-therapeutic efficiency and photocatalytic degradation potential

The current investigation aimed at bimetallic gold-silver nanoparticles (Au/Ag NPs), here called BM-GS NPs, synthesis using sericin protein as the reducing agent in an easy, cost-effective, and sustainable way. The obtained BM-GS NPs were characterized by UV–Visible spectroscopy, Transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDS), atomic force microscopy (AFM), Dynamic light scattering (DLS) and Zeta potential, X-ray Powder Diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), and Thermogravimetric analysis followed by evaluation of its multitherapeutic and photocatalytic degradation potentials. The TEM analysis revealed its spherical nature and the EDS result displayed the presence of both Ag and Au elements, confirming the synthesis of BM-GS NPs. The XRD pattern verified the crystalline nature of the nanoparticles (NPs). The DLS analysis showed an average size of 86.08 d nm and the zeta potential showed a highly negative value (−26.3 mV) which specifies that the generated bimetallic NPs are stable. The BM-GS NPs exhibited positive wound healing potential (with 63.38% of wound closure rate at 25 μg/ml, as compared to 54.42% by the untreated control) with very negligible toxicity effect on the cell viability of the normal keratinocyte cells. It also demonstrated promising antioxidant properties with 65.00%, 69.23%, and 63.03% activity at 100 μg/ml concentration for ABTS (2, 2-azinobis) (3-ethylbenzothiazoline-6-sulfonic acid)), DPPH (1, 1 diphenyl-2-picrylhydrazyl) and SOD (superoxide dismutase enzyme) assays respectively, antidiabetic potential (with a significantly high α-glucosidase inhibition potential of 99.69% at 10μg/ml concentration and 62.11% of α-amylase enzyme inhibition at 100 μg/ml concentration) and moderate tyrosinase inhibitory potential (with 17.09% at 100 μg/ml concentration). Besides, it displayed reasonable antibacterial potential with the diameter of zone of inhibition ranging between 10.89 and 12.39 mm. Further, its antibacterial mode of action reveals that its effects could be due to being very smaller, the NPs could have penetrated inside the cellular membrane thereby causing rupture and damage to the interior materials leading to cellular lysis. The photocatalytic evaluation showed that synthesized BM-GS NPs have the efficiency of degrading methylene blue dye by 34.70% within 3 h of treatment. The above findings revealed the multi-therapeutic efficacy of the sericin globular protein-mediated BM-GS NPs and its potential future applications in the cosmetics and food sector and environmental contamination management industries.

WITHAFERIN A CITOTOXICITY IN NORMAL KERATINOCYTES: AN IN VITRO STUDY

<h3>Objectives</h3> Epithelial tissue main function is the lining of connective tissue and is characterized by juxtaposition of epithelial cells. Keratinocytes are the main cells of epithelial tissue, and they are responsible for keratin synthesis. HaCat cells are a human keratinocyte lineage widely used for in vitro studies. Withaferin A (WA) is a highly oxygenated steroidal lactone with anti-angiogenic, proapoptotic, and antiproliferative properties, and several studies demonstrated its cytotoxic effect in different types of neoplasms. This study main objective was to explore WA's effect in normal epithelial cells lineage. <h3>Study Design</h3> HaCat cells were subjected to cell viability tests (The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, Neutral Red assay, and Picogreen assay) and measurement of intracellular reactive species dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay after 24 hours of treatment in different WA concentrations. <h3>Results</h3> WA was not able to induce oxidative stress in normal keratinocyte cells and only the higher dose tested (7 µM) was a capable of decreased cell viability, but not enough to induce cell death. <h3>Conclusions</h3> Although this drug has known therapeutic potential in the treatment of different types of cancer, we can conclude that WA does not disrupt the function of normal epithelium.

Abstract 98: The role of HORMAD1 in transformation of keratinocytes following vemurafenib treatment

Abstract BRAF inhibitor (BRAFi) therapies are highly effective treatments for metastatic melanoma. However, BRAFi monotherapies are associated with a significant risk of squamous cell carcinoma (SCC) development in melanoma patients. Consequently, BRAF inhibition results in an unanticipated activation of the MAPK pathway in surrounding cells that harbour wildtype BRAF. Fortunately, a combination approach with both a BRAFi and the selective MAPK inhibitor (MEKi), trametinib, attenuates BRAFi-induced risk of SCCs. Despite the fact that this combination therapy is widely used in clinical practice, the mechanisms associated with SCC induction and why this effect is attenuated with a MEK inhibitor remain poorly understood. HORMAD1, a meiosis specific gene, has been shown to have an impact on genomic instability and proliferation in the development of SCCs. Moreover, recent work in our lab has demonstrated that the MAPK pathway regulates HORMAD1 expression in SCC cell lines. Therefore, we sought to investigate a potential role of HORMAD1 in the risk and development of SCCs as a mechanism of BRAFi that is attenuated with the co-administration of the MEKi, trametinib. Using the BRAFi, vemurafenib, we inhibited the kinase activity of activated BRAF (V600E) and the MEKi, trametinib to evaluate the expression of HORMAD1 in melanoma and normal keratinocyte cell lines. To determine the effects of these two inhibitors, a cell proliferation assay was conducted followed by the evaluation of various components of cell cycle regulation and genomic instability. We found that HORMAD1 protein expression levels increased following treatment with the BRAFi, vemurafenib, in the normal keratinocyte cell line, HaCaT, but not in the melanoma cell line, A375. In contrast, HORMAD1 expression decreased following treatment with the MEKi, trametinib in both HaCaT and A375. Cell proliferation assays depict a vulnerability to both vemurafenib and trametinib in both normal and melanoma cell lines, however, HaCaT cells appear to be more susceptible to increased levels of genomic instability following treatment with vemurafenib when compared to A375. Our results suggest that HORMAD1 likely plays a key role in the development of SCCs by increasing genomic instability and proliferation in normal keratinocytes following BRAFi monotreatment. The increased risk of SCC formation is attenuated by dampening HORMAD1 levels with the co-administration of the MEKi, trametinib. Citation Format: Jennifer Gantchev, Amelia Martinez Villarreal, Brandon Ramchatesingh, Ivan V. Litvinov. The role of HORMAD1 in transformation of keratinocytes following vemurafenib treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 98.

Antimicrobial efficacy and biocompatibility of extracts from Cryptocarya species.

This study evaluated the efficacy of Cryptocarya spp extracts on biofilm of Candida albicans and its biocompatibility. Mature biofilm of C. albicans was formed on denture base acrylic resin samples and the fungicidal effect of the extracts was evaluated by Alamar Blue® assay, counting colony-forming units (CFU/mL) and confocal laser scanning microscopy (CLSM). Cytotoxicity of extracts from Cryptocarya species was evaluated by AlamarBlue® assay, using normal oral keratinocytes (NOK) cells. In additional, Analysis of plant extracts by ultra-high-performance liquid chromatography-diode array detector-tandem mass spectrometry (UPLC-DAD-MS) was performed. The results showed significant reduction in the cellular metabolism and in the number of CFU/mL of C. albicans (p<0.05). The concentration of 0.045 g/mL completely inhibited the number of CFU/mL. Regarding cytotoxicity, all extracts decreased cell viability compared to the control group. CLSM analysis showed predominance of live cells, but with a great difference between the groups. Antimicrobial activity of extracts from Cryptocarya on C. albicans biofilm was confirmed. However, all extracts showed toxicity on NOK cells.

Cytotoxicity, phytotoxicity, and photocatalytic assessment of biopolymer cellulose-mediated silver nanoparticles

The appearance of silver nanoparticles (Ag NPs), their exponential production and the consumer products that contain them have raised concerns about their potential risks. Hence, the monitoring of Ag NPs in environmental and biological systems is of vital importance. Furthermore, until now, the information about the toxicity of Ag NPs synthesized from green sources rather than polluting methods is scarce. Hence, in the current work, a simple and straightforward synthesis of Ag NPs mediated by cellulose biopolymer and the evaluation of their cytotoxicity, phytotoxicity, and photocatalytic activity were described for the first time. Initially, the green synthesized Ce-Ag NPs were fully physicochemically characterized. Ce-Ag NPs were sphere-shaped, had high crystallinity, and had a mean diameter of 5–100 nm. The in vitro cytotoxicity evaluation of the green synthesized Ce-Ag NPs was carried out in human normal keratinocyte (HaCaT) and human breast cancer (MCF-7) cells by MTT assay. After the 24 h-treatment with Ce-Ag NPs (0.1–500 µg/mL), the viability of both cell lines was significantly inhibited. Furthermore, the Ce-Ag NPs exhibited dose-dependent cytotoxicity with a median inhibitory concentration (IC 50 ) of 5.4 μg/mL and 4.9 μg/mL against HaCaT and MCF-7 cells, respectively. Moreover, in the phytotoxicity assay, the Ce-Ag NPs did not significantly affect seed germination, roots, and shoots length of Mung ( Vigna radiata ) but affect seed germination and shoots length of Wheat ( Triticum aestivum ). Besides, the photocatalytic activity of Ce-Ag NPs was evaluated by the decomposition of methylene blue (MB), detecting a great degradation from 120 min • Biopolymer cellulose was used for the green synthesis of silver NPs. • Ce-AgNPs shown dose-dependent cytotoxicity on human normal keratinocyte (HaCaT) cells. • Ce-AgNPs shown dose-dependent cytotoxicity on human breast cancer (MCF-7) cells. • Ce-AgNPs did not affect seed germination nor lengths of shoots and roots of Mung. • Ce-AgNPs affected seed germination and lengths of shoots of Wheat. • Ce-AgNPs exhibited a significant photocatalytic activity.

286 HORMAD1 Contributes to the Development of Squamous Cell Carcinomas in Melanoma Patients

BRAF inhibitor (BRAFi) therapies are effective treatments for metastatic melanoma. However, BRAFi monotherapies are associated with a significant risk of squamous cell carcinoma (SCC) in melanoma patients. Consequently, BRAF inhibition results in an activation of the MAPK pathway in cells that harbour wildtype BRAF. Fortunately, a combination approach with both a BRAFi and the selective MAPK inhibitor (MEKi), trametinib, attenuates BRAFi-induced risk of SCCs. Despite the wide use of this therapy, the mechanisms associated with SCC induction remain poorly understood. HORMAD1, a meiosis gene, has been shown to have an impact on genomic instability and proliferation in the development of SCCs. Recent work has shown that HORMAD1 expression is regulated by the MAPK pathway in SCC cell lines. Therefore, we sought to investigate a potential role of HORMAD1 in the risk and development of SCCs that is attenuated with the administration of the MEKi, trametinib. We used BRAFi, vemurafenib and the MEKi, trametinib to evaluate the expression of HORMAD1 in melanoma and normal keratinocyte cell lines. To determine the effects of the two inhibitors, a cell proliferation assay was conducted followed by the evaluation of various components of cell cycle regulation and genomic instability. We found that HORMAD1 protein expression increased following treatment with vemurafenib in normal keratinocytes but not in a melanoma cells. In contrast, HORMAD1 expression decreased following treatment with trametinib in both HaCaT and A375. Cell proliferation assays depict a vulnerability to both vemurafenib and trametinib in both normal and melanoma cell lines, however, HaCaT cells appear to be more susceptible to increased levels of genomic instability following treatment with vemurafenib. Our results suggest that HORMAD1 likely plays a key role in the development of SCCs by increasing genomic instability and proliferation in normal keratinocytes following BRAFi monotreatment. The increased risk of SCC formation is attenuated by dampening HORMAD1 levels with the co-administration of the MEKi, trametinib.

AN IN VITRO EXAMINATION OF OSCC-DERIVED EXOSOMES

<h3>Background</h3> Exosomes are membrane-bound nanovesicles released by cells into their extracellular environment. Exosomes influence cancer metastasis. The RNA levels of oncogenes within exosomes derived from cancer cells may thus provide a useful source for monitoring cancer progression and deploying early intervention. <h3>Objective</h3> To compare the RNA levels of FOXM1, DNMT1, HOXA7, CCNB1, and HSPA1A in exosomes from normal primary gingival keratinocyte (PGK) and oral squamous cell carcinoma (OSCC) cell lines. <h3>Methods</h3> Exosomes derived from PGK and OSCC cell lines (SCC4 and SCC25) were isolated using ultracentrifugation and their presence was confirmed by transmission electron cryomicroscopy. The exosomal RNA was extracted and subjected to reverse transcription quantitative polymerase chain reaction for mRNA analysis. <h3>Results</h3> Transmission electron cryomicroscopy revealed bilayered structures measuring 50 to 200 nm, consistent with the expected size of exosomes. Transcripts for the genes of interest were detected in the OSCC cells (mean 2^−ΔCT: 0.1563, 0.0111, 0.011, 0.1062, and 0.58, respectively) and exosomes (mean 2^−ΔCT: 0.3564, 0.0036, 0.0458, 0.0718, and 0.7252, respectively). HOXA7 was not detected in PGK. Of the remaining genes of interest, more RNA was detected in the OSCC cells (mean fold upregulations of 4.44, 2.80, 2.28, and 2.90, respectively) and OSCC exosomes (mean fold upregulations of 6.33, 2.20, 3.16, and 5.75, respectively) when compared with the PGK cells and exosomes. <h3>Conclusions</h3> The results provide the first evidence that there is more FOXM1 and HSPA1A RNA in OSCC exosomes compared with PGK exosomes. These results will be used for ex vivo studies comparing exosomes in saliva and blood samples from patients with and without OSCC.

Effects of Ni - substitution on structural, magnetic hyperthermia, photocatalytic and cytotoxicity study of MgFe2O4 nanoparticles

Over the last two decades, magnetic hyperthermia (MH) has become a significant capable additional method to well-recognized cancer treatments such as chemotherapy and radiotherapy. The Mg1−xNixFe2O4 (x = 0.1, 0.2, 0.3, 0.4 and 0.5) spinel ferrite nanoparticles (SFNPs) were successfully synthesized and assessed them as agents for MH affect. The synthesized NPs were well characterized for structure, morphology, and particle size, and weight loss using X-ray diffractometer (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectrophotometer (FT-IR), and thermogravimetric analysis (TGA). Prepared powder samples saturation mass magnetization (Ms) values were enhanced with the enhancing of the Ni2+ -concentration. The Ms values were found to be 40.72–48.18 emu g−1. The synthesized SFNPs as MH agents display well specific heat generation rate (SGHR) values 97–328 W g−1 at a constant frequency of 316 kHz, hence the present synthesized SFNPs could be useful for MH applications and can be used as a significant heating cause for thermal therapy MH treatment of cancer. The synthesized samples were exhibits good Ms, best morphology, monodispersed performance, significant Ms and best SHGR values. All these types of characteristics are vital, for their use in MH treatment of cancer application. The photocatalytic degradation efficiency (DE%) was examined by degrading methylene blue (MB) under visible light radiation in the multi-lamp photoreactor. Our results designate that, Ni2+-substituted MgFe2O4 SFNPs can be best employed for effective wastewater purification. Nearly monosized Ni2+- doped MgFe2O4 NPs exhibited better photocatalytic activity of MB under visible light radiation. These types of catalysts could be useful in wastewater treatment applications. The synthesized biocompatible magnetic semiconductor nanoparticles can be utilized as photocatalysts and could also be recycled and separated by applying an external magnetic field. The developments of monosize NPs with significant Ms are required for advanced biomedical as well as electronic applications. Cytotoxicity studies conducted in MCF-7, MDA-MB-231 breast cancer cells, and normal keratinocyte cells showed that the synthesized NPs are biocompatible but possessed a very toxic effect on MCF-7 and MDA-MB-231 cell lines may be owing to the generation of reactive oxygen species and/or ferroptosis.

Bioprospecting of Natural Compounds from Brazilian Cerrado Biome Plants in Human Cervical Cancer Cell Lines.

Cervical cancer is the third most common in Brazilian women. The chemotherapy used for the treatment of this disease can cause many side effects; then, to overcome this problem, new treatment options are necessary. Natural compounds represent one of the most promising sources for the development of new drugs. In this study, 13 different species of 6 families from the Brazilian Cerrado vegetation biome were screened against human cervical cancer cell lines (CCC). Some of these species were also evaluated in one normal keratinocyte cell line (HaCaT). The effect of crude extracts on cell viability was evaluated by a colorimetric method (MTS assay). Extracts from Annona crassiflora, Miconia albicans, Miconia chamissois, Stryphnodendron adstringens, Tapirira guianensis, Xylopia aromatica, and Achyrocline alata showed half-maximal inhibitory concentration (IC50) values < 30 μg/mL for at least one CCC. A. crassiflora and S. adstringens extracts were selective for CCC. Mass spectrometry (Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (ESI FT-ICR MS)) of A. crassiflora identified fatty acids and flavonols as secondary compounds. One of the A. crassiflora fractions, 7C24 (from chloroform partition), increased H2AX phosphorylation (suggesting DNA damage), PARP cleavage, and cell cycle arrest in CCC. Kaempferol-3-O-rhamnoside and oleic acid were bioactive molecules identified in 7C24 fraction. These findings emphasize the importance of investigating bioactive molecules from natural sources for developing new anti-cancer drugs.

Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells

ObjectivesVolume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. MethodsCell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. ResultsVRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. ConclusionsWe have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.