IC20 Values Research Articles

Mutagenicity and DNA damage assessment of locally produced nano-hydroxyapatite-silica-glass ionomer cement on human periodontal ligament fibroblasts using Ames and Comet assays

AimThis research assessed the mutagenicity and DNA damage of a novel type of nano-hydroxyapatite-silica glass ionomer cement (nano-HA-SiO2-GIC) and a conventional GIC (cGIC) using Ames and Comet assays. MethodsCell viability was tested on human periodontal ligament fibroblasts (HPLFs) using 3.125 mg/ml, 6.25, 12.5, 25, 50, 100 and 200 mg/ml, on both types of GICs employing MTT assay. For the Comet assay, HPLFs were treated with IC50, IC25 and IC10 of test materials and the tail moments were measured. In the Ames test, four genotypic variants of strains of Salmonella typhimurium (TA100, TA98, TA1537 and TA1535) and a strain of Escherichia coli (WP2 uvrA) were employed. The material tested was extracted using sterile distilled water (0.2 g per ml) at 37 °C for 72 h. This was considered as 100 %, which was diluted to 50, 25, 12.5 and 6.25 % utilizing sterile distilled water. These five concentrations were incubated with the bacterial strains with and without metabolic activation (S9), along with appropriate positive controls. The number of revertant colonies was used to evaluate the outcome. ResultsThe highest cell viability (159.4 %) for nano-HA-SiO2-GIC was noticed at 3.125 mg/ml, while the lowest (24.26 %) was observed at 200 mg per ml. IC50, IC25 and IC10 values were 95.27, 51.4 and 20.1 mg/ml for cGIC, 106.9, 55.8 and 22.9 mg/ml for nano-HA-SiO2-GIC, respectively. The IC10 of both test materials showed no significant DNA damage compared to that of the negative control based on the Comet assay. The plate treated with nano-HA-SiO2-GIC showed less than double the average number of revertant colonies compared to that of negative control with regard to the Ames test. ConclusionsIt can be concluded that nano-HA-SiO2-GIC is non-mutagenic based on the Ames test and did not cause DNA damage at the lowest concentration of IC10 based on the Comet assay.

Design and synthesis of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives as potential Werner-dependent antiproliferative agents.

To discover new Werner (WRN) helicase inhibitors, a series of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives were designed and synthesized through a structural optimization strategy, and the anticancer activities of 25 new target compounds against PC3, K562, and HeLa cell lines were evaluated by the MTT assay. Some of these compounds exhibited excellent inhibitory activity against three different cancer cell lines. Compounds 6a, 8i, and 13a showed better antiproliferative activity against K562 cells, with IC50 values of 3871.5, 613.6 and 134.7nM, respectively, than did paclitaxel (35.6nM), doxorubicin (2689.0nM), and NSC 617145 (20.3nM). To further verify whether the antiproliferative activity of these compounds is dependent on WRN, PC3 cells overexpressing WRN (PC3-WRN) were constructed to further study their antiproliferative potency in vitro, and the inhibition ratio and IC20 values showed that compounds 6a, 8i, and 13a were more sensitive to PC3-WRN than were the control group cells (PC3-NC). The IC20 ratios of compounds 6a, 8i, and 13a to PC3-NC and PC3-WRN were 94.3, 153.4 and 505.5, respectively. According to the docking results, the compounds 6a, 8i, and 13a overlapped well with the binding pocket of 6YHR. Further study demonstrated that among the tested compounds, 13a was the most sensitive to PC3-WRN. In summary, our research identified a series of N-aryl-2-trifluoromethyl-quinazoline-4-amine derivatives as potential WRN-dependent anticancer agents.

Cytotoxic and apoptotic effects of Prunus spinosa fruit extract on HT-29 colon cancer line

Colon cancer holds the position of the third most common type of cancer and stands as the third leading cause of cancer-related deaths for both men and women. Modern strategies in cancer prevention center around the use of natural compounds, which demonstrate a range of effects, including preventive, inhibitory, and latency-inducing impacts on the progression of cancer. In the present study, aqueous extracts derived from the fruits of Prunus spinosa L. (blackthorn, Rosaceae) are employed to assess their cytotoxic potential against the HT-29 colon cancer cell line. The fruit extract is administered to the HT29 cell line in different concentrations over 24 and 48-hours to evaluate the induction of apoptosis. The MTT cell viability test is employed to quantify the cytotoxic effect, indicating the extent of the impact. Additionally, the EB/AO (ethidium bromide/acridine orange) dual staining method is utilized to gather supplementary information regarding the cytotoxic effects. Observations after 24 hours of exposure showed no significant cytotoxic effect; however, 48-hour exposure revealed IC20, IC50, and IC80 values of 1.27, 173.7, and > 1000 µg/ml, respectively, as determined by MTT analysis. Correspondingly, values of 5.06, 123.8, and > 1000 µg/ml were recorded by the EB/AO dual staining method. Our results show that P. spinosa fruit water extract has an inhibitory effect on the HT-29 cell viability by exerting cytotoxic and apoptotic effects in a concentration-dependent and time-dependent manner. Toxicity studies have shown that MTT and EB/AO support each other and achieve similar results. Further extensive research into the metabolic and functional effects of P. spinosa could illuminate its potential and increase its economic importance in the field of anticancer treatments as a natural drug.

Melittin, A Potential Natural Toxin with Anticancer Properties: Regulating IL-1β, COX-2 and TNF-α in Human Colorectal Cancer Cells WiDr

Despite decades of study, the antiproliferation and molecular processes of the biomolecular elements of honeybee venom (i.e., melittin) as anticancer agents remain largely unclear. This study illustrated the antiproliferation effect of melittin by regulating IL-1β, COX-2 and TNF-α in human colorectal cancer cells WiDr. In order to assess the antiproliferation, various concentrations of melittin were contacted to the WiDr cell through MTT assay. Moreover, the ability of melittin to regulate the expression of IL-1β, COX-2 and TNF-α was determined by ELISA. The cytotoxic effect showed the inhibition of the WiDr cell in a dose-dependent manner, with the IC10, IC25 and IC50 values of 0.095 ± 0.001, 0.147 ± 0.002 and 0.207 ± 0.004, respectively. Additionally, in further investigations, melittin markedly inhibited the IL-1β and COX-2 expression but induced the TNF-α, demonstrating significant (p < 0.05) elevations compared to the control group. These findings point to the antiproliferative properties of melittin in WiDr cells. Therefore, as a unique natural treatment for colorectal cancer, melittin may have a lot of potential. HIGHLIGHTS In this study, we confirmed melittin has very strong anticancer activity against colorectal cell cancer cells with IC50 values of 0.207 ± 0.004 µM Melittin regulates colorectal cell cancer by inhibiting the IL-1β and COX-2 expression Melittin increases the expression of TNF-α to inhibit colorectal cell cancer GRAPHICAL ABSTRACT

Abstract 2690: In vivo CRISPR screen identifies SOAT1 as a chemotherapy chemosensitizing target for non-small cell lung cancer (NSCLC)

Abstract Purpose: To identify druggable gene targets that would sensitize NSCLCs to available chemotherapy agents. Experimental Procedures: We developed a CRISPR-Cas9 lentivirus library of 12,474 sgRNAs targeting 660 FDA proved ‘druggable’ putative proteins (18 guides/gene) and investigated their potential to chemosensitize (“drop out” in the presence of taxane) the NSCLC lung adenocarcinoma (LUAD) line NCI-H2009 (TP53 and KRAS mutant) in parallel studies in vitro (tissue culture) & in vivo (xenografts). Key elements of our experiments were: the use low doses of paclitaxel (IC10 values for in vitro and in vivo doses that barely affected tumor growth) compared to control treatment; treatment schedules in vitro and in vivo that mirrored those used in patients; multiple biologic replicates for each transfection and drug selection; and large representation (2,000 cells/sgRNA) for each guide. At the end time point (1 month) we harvested multiple replicates of the taxane and control treatments and sequenced each to identify each guide that “remained” or “dropped out.” Data Summary: We were looking for guides that selectively dropped out in the setting of low dose taxane exposure comparing taxane to control treatment and in vitro to in vivo results. Of importance, we found little overlap between guides that dropped out during taxane exposure in tissue culture vs those that dropped out in xenografts. From the top 10 gene “sensitizers” that selectively dropped out only with taxane treatment and selectively dropped out in xenografts compared to 2D mass tissue culture, we focused on SOAT1 (Sterol O-Acyltransferase 1). SOAT1 is a key enzyme for lipid metabolism which mediates conversion of intracellular free cholesterol to cholesteryl esters which are then stored as lipid droplets. SOAT1 is a potential cancer therapeutic target with a clinically available inhibitor, avasimibe. H2009 cells with SOAT1 hemizygously removed (CRISPR) grew well in vitro and in vivo in the absence of chemotherapy treatment, but were dramatically sensitized to taxanes compared to parental H2009 cells. While avasimibe treatment, at concentrations achievable in patients, sensitized NSCLC to taxanes, this sensitization was not as dramatic as hemizygous removal by CRISPR. Of equal importance, SOAT1 H2009 hemizygous knockout (KO) cells were also dramatically sensitized to etoposide, pemetrexed, and gemcitabine, other chemotherapy agents used in NSCLC treatment. Mechanistically, RNAseq analysis identified G2/M cell cycle related gene sets as enriched in the H2009 SOAT1 KO xenografts compared to SOAT1 wildtype xenografts with paclitaxel treatment. Conclusions: Our results using CRISPR screening in vivo, identified SOAT1 as a critical therapeutic chemosensitizing target requiring only 50% inhibition of activity for sensitizing NSCLC to several chemotherapy agents routinely used in the clinic. Citation Format: Long Shan Li, Kenneth Huffman, Huiyu Li, Michael Peyton, Hyunsil Park, Kimberley Avila, Luc Girard, Mathew Augustine, Joshua T. Mendell, John D. Minna. In vivo CRISPR screen identifies SOAT1 as a chemotherapy chemosensitizing target for non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2690.

Evaluation of Lipid Extracts from the Marine Fungi Emericellopsis cladophorae and Zalerion maritima as a Source of Anti-Inflammatory, Antioxidant and Antibacterial Compounds.

Marine environments occupy more than 70% of the earth's surface, integrating very diverse habitats with specific characteristics. This heterogeneity of environments is reflected in the biochemical composition of the organisms that inhabit them. Marine organisms are a source of bioactive compounds, being increasingly studied due to their health-beneficial properties, such as antioxidant, anti-inflammatory, antibacterial, antiviral, or anticancer. In the last decades, marine fungi have stood out for their potential to produce compounds with therapeutic properties. The objective of this study was to determine the fatty acid profile of isolates from the fungi Emericellopsis cladophorae and Zalerion maritima and assess the anti-inflammatory, antioxidant, and antibacterial potential of their lipid extracts. The analysis of the fatty acid profile, using GC-MS, showed that E. cladophorae and Z. maritima possess high contents of polyunsaturated fatty acids, 50% and 34%, respectively, including the omega-3 fatty acid 18:3 n-3. Emericellopsis cladophorae and Z. maritima lipid extracts showed anti-inflammatory activity expressed by the capacity of their COX-2 inhibition which was 92% and 88% of inhibition at 200 µg lipid mL-1, respectively. Emericellopsis cladophorae lipid extracts showed a high percentage of inhibition of COX -2 activity even at low concentrations of lipids (54% of inhibition using 20 µg lipid mL-1), while a dose-dependent behaviour was observed in Z. maritima. The antioxidant activity assays of total lipid extracts demonstrated that the lipid extract from E. cladophorae did not show antioxidant activity, while Z. maritima gave an IC20 value of 116.6 ± 6.2 µg mL-1 equivalent to 92.1 ± 4.8 µmol Trolox g-1 of lipid extract in the DPPH• assay, and 101.3 ± 14.4 µg mL-1 equivalent to 106.6 ± 14.8 µmol Trolox g-1 of lipid extract in the ABTS•+ assay. The lipid extract of both fungal species did not show antibacterial properties at the concentrations tested. This study is the first step in the biochemical characterization of these marine organisms and demonstrates the bioactive potential of lipid extracts from marine fungi for biotechnological applications.

Direct inhibition of bisphenols on human and rat 11β-hydroxysteroid dehydrogenase 2: Structure-activity relationship and docking analysis

Bisphenols (BPs) as endocrine-disrupting compounds have drawn attention to their health hazards. Whether a BP interferes with glucocorticoid metabolism remains unclear. 11β-Hydroxysteroid dehydrogenase 2 (11β-HSD2) is a key glucocorticoid-metabolizing enzyme that controls fetal glucocorticoid levels across the placental barrier and mineralocorticoid receptor specificity in the kidney. In this study, 11 BPs were tested to inhibit human placental and rat renal 11β-HSD2 and were analyzed for inhibitory potency, mode action, and docking parameters. BPs had inhibitory potency against human 11β-HSD2: BPFL>BPAP>BPZ>BPB>BPC>BPAF>BPA>TDP and the IC10 values were 0.21, 0.55, 1.04, 2.04, 2.43, 2.57, 14.43, and 22.18 μM, respectively. All BPs are mixed inhibitors except BPAP, which is a competitive inhibitor for human 11β-HSD2. Some BPs also inhibited rat renal 11β-HSD2, with BPB (IC50, 27.74 ± 0.95) > BPZ (42.14 ± 0.59) > BPAF (54.87 ± 1.73) > BPA (77.32 ± 1.20) > other BPs (about 100 μM). Docking analysis showed that all BPs bound to the steroid-binding site, interacting with the catalytic residue Tyr232 of both enzymes and the most potent human 11β-HSD2 inhibitor BPFL acts possibly due to its large fluorene ring that has hydrophobic interaction with residues Glu172 and Val270 and π-stacking interaction with catalytic residue Tyr232. The increase in the size of substituted alkanes and halogenated groups in the methane moiety of the bridge of BPs increases its inhibitory potency. Regressions of the lowest binding energy with inhibition constant indicated that there was an inverse regression. These results indicated that BPs significantly inhibited human and rat 11β-HSD2 activity and that there were species-dependent differences.

Determination of Nicotinic Acid in Food and Pharmaceuticals by a Simple and Rapid Fluorescence Polarization Immunoassay (FPIA)

A simple and rapid fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies is reported for the first time for the determination of nicotinic acid. First, six nicotinic acid analogues were coupled with fluorescein isothiocyanate isomer I (FITC) to obtain tracers. The influences of tracer structure on the performance of FPIA as well as the structure-activity relationships were explored. Subsequently, the tracer concentration and antibody dilution ratio on the performance of FPIA were investigated. For the optimal conditions, the detection range of FPIA was from 1.1 to 145.6 μg mL−1 and its IC20 value as the limit of detection (LOD) was 1.1 μg mL−1. The cross-reactivity of antibodies with common vitamins was evaluated. The intra-assay recovery was from 92.0% to 101.2% in food and tablets, with inter-assay recoveries between 92.0% and 103.8%. The developed FPIA provided good sensitivity and reproducibility and therefore provided efficient and economic screening for nicotinic acid in food and tablets.

Targeting the anaphase-promoting complex/cyclosome (APC/C) enhanced antiproliferative and apoptotic response in bladder cancer

Improving the chemotherapy sensitivity of bladder cancer is a current clinical challenge. It is critical to seek out effective combination therapies that include low doses of cisplatin due to its dose-limiting toxicity. This study aims to investigate the cytotoxic effects of the combination therapy including proTAME, a small molecule inhibitor, targeting Cdc-20 and to determine the expression levels of several APC/C pathway-related genes that may play a role in the chemotherapy response of RT-4 (bladder cancer) and ARPE-19 (normal epithelial) cells. The IC20 and IC50 values were determined by MTS assay. The expression levels of apoptosis-associated (Bax and Bcl-2) and APC/C-associated (Cdc-20, Cyclin-B1, Securin, and Cdh-1) genes were assessed by qRT-PCR. Cell colonization ability and apoptosis were examined by clonogenic survival experiment and Annexin V/PI staining, respectively. Low-dose combination therapy showed a superior inhibition effect on RT-4 cells by increasing cell death and inhibiting colony formation. Triple-agent combination therapy further increased the percentage of late apoptotic and necrotic cells compared to the doublet-therapy with gemcitabine and cisplatin. ProTAME-containing combination therapies resulted in an elevation in Bax/Bcl-2 ratio in RT-4 cells, while a significant decrease was observed in proTAME-treated ARPE-19 cells. Cdc-20 expression in proTAME combined treatment groups were found to be decreased compared to their control groups. Low-dose triple-agent combination induced cytotoxicity and apoptosis in RT-4 cells effectively. It is essential to evaluate the role of APC/C pathway-associated potential biomarkers as therapeutic targets and define new combination therapy regimens to achieve improved tolerability in bladder cancer patients in the future.

Development of indirect competitive ELISA and CLEIA for quantitative analysis of melatonin in health products.

Melatonin (MT), as a hormone regulating the rhythm of sleep, is widely used in health products. However, illicit and excessive use of MT might cause undesirable effects. Therefore, it is essential to establish highly sensitive and specific rapid methods for MT analysis in health products. In this study, we established indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for sensitive and selective detection of MT in health products. Under optimal conditions, half-maximum inhibitory concentration (IC50 value) and limit of detection (LOD, IC10 value) for MT by ic-ELISA were 0.25 ng mL-1 and 0.03 ng mL-1 respectively, while the IC50 and LOD of ic-CLEIA were lower at 0.17 ng mL-1 and 0.03 ng mL-1 respectively. Three MT-free Chinese patent medicines were spiked with MT and the recovery rates ranged from 71.89% to 117% (ic-ELISA) and 83.66% to 107.17% (ic-CLEIA). The level of MT in six MT-containing health products was assessed in parallel using the developed methods and HPLC. Both ic-ELISA and ic-CLEIA showed good consistencies (R2 = 0.999 and 0.993, respectively) with HPLC, indicating that the two methods developed were sensitive, fast, and reliable for application in MT analysis.

Cytotoxic and Cytostatic Effects of Targeting mTOR and Hedgehog Pathways in Acute Myeloid Leukemia

Objectives: Acute myeloid leukemia (AML) is a highly aggressive heterogeneous hematopoietic malignancy characterized by a rapid and abnormal proliferation of immature myeloid leukemia cells in the bone marrow and peripheral blood. Aberrant alterations in signal transduction pathways are strongly associated with the progression of AML. This study aimed to investigate cell viability and the cell cycle in AML cells by targeting the Hedgehog and mTOR signaling pathways with rapamycin and GANT61. Materials and Method: The antiproliferative effect of rapamycin and GANT61 was assessed by the MTT cell viability assay in two AML cell lines: CMK and MOLM-13. The effect of the inhibitors on cell-cycle distribution was determined using propidium iodide staining and measured with flow cytometry. Results: Rapamycin, an mTOR inhibitor, and GANT61, a Gli-1 inhibitor, decreased the cell proliferation of CMK and MOLM-13 cells. The IC20 values, which is the drug concentration that inhibits cell growth by 20%, were combined and administered to the cells. The results show the drugs to have a combinatorial inhibitory effect on CMK cells but not on MOLM-13 cells. In addition, the combination of drugs arrested the cells during the G0/G1 phase. Conclusion: This study suggests a novel combination therapy approach for AML via mTOR and Hedgehog signaling pathway inhibition using rapamycin and GANT61, respectively. It also suggest further studies be performed to reveal the mechanism of action.

Mixture effects of tetrodotoxin (TTX) and drugs targeting voltage-gated sodium channels on spontaneous neuronal activity in vitro

Tetrodotoxin (TTX) potently inhibits TTX-sensitive voltage-gated sodium (NaV) channels in nerve and muscle cells, potentially resulting in depressed neurotransmission, paralysis and death from respiratory failure. Since a wide range of pharmaceutical drugs is known to also act on NaV channels, the use of medicines could predispose individuals to a higher susceptibility towards TTX toxicity. We therefore first assessed the inhibitory effect of selected medicines that act on TTX-sensitive (Riluzole, Chloroquine, Fluoxetine, Valproic acid, Lamotrigine, Lidocaine) and TTX-resistant (Carbamazepine, Mexiletine, Flecainide) NaV channels on spontaneous neuronal activity of rat primary cortical cultures grown on microelectrode arrays (MEA). After establishing concentration-effect curves, binary mixtures of the medicines with TTX at calculated NOEC, IC20 and IC50 values were used to determine if pharmacodynamic interactions occur between TTX and these drugs on spontaneous neuronal activity. At IC20 and IC50 values, all medicines significantly increased the inhibitory effect of TTX on spontaneous neuronal activity of rat cortical cells in vitro. Subsequent experiments using human iPSC-derived neuronal co-cultures grown on MEAs confirmed the ability of selected medicines (Carbamazepine, Flecainide, Riluzole, Lidocaine) to inhibit spontaneous neuronal activity. Despite the need for additional experiments using human iPSC-derived neuronal co-cultures, our combined data already highlight the importance of identifying and including vulnerable risk groups in the risk assessment of TTX.

Application study of RGB color extraction in water toxicity detection

In this work, a colorimetric biosensor based on the rapid color change of Escherichia coli DH 5α (E. coli) and p-benzoquinone (BQ) was introduced to evaluate water toxicity. Here, dark brown quinhydroquinone (QHQ) was obtained from the reaction between E. coli and BQ. The inhibition ratios were calculated by RGB (red, green, blue) values of samples or oxidation current of hydroquinone (HQ). Here, RGB technology was used for biotoxicity assessment for the first time. The results suggested that the biosensor-based RGB has an obvious dose-dependent response. The IC20 values of Hg2+ and Cu2+ obtained based on RGB values were 1.0 and 1.5 mg L-1, respectively. This biosensor can be used to detect quickly toxicity of the Cu2+ and Hg2+ in water without complicated operation steps. Therefore, the biosensor has potential application value in the field of water toxicity detection.

Effects of Tetracycline on Scenedesmus obliquus Microalgae Photosynthetic Processes.

Tetracycline (TC) antibiotics can be detected worldwide in the aquatic environment due to their extensive use and low utilization efficiency, and they may affect the physiological processes of non-target organisms. In this study, the acute and sub-acute toxicities of TC on the freshwater microalga Scenedesmus obliquus were investigated with an emphasis on algal photosynthesis and transcription alterations during an 8 d TC exposure. The results showed that the IC10, IC30 and IC50 values were 1.8, 4.1 and 6.9 mg/L, respectively. During sub-acute exposure, the microalgae of the IC10 treatment was able to recover comparable growth to that of the control by day 7, while significantly lower cell densities were observed in the IC30 and IC50 treatments at the end of the exposure. The photosynthetic efficiency Fv/FM of S. obliquus first decreased as the TC concentration increased and then returned to a level close to that of the control on day 8, accompanied by an increase in photosynthetic activities, including light harvesting, electron transport and energy dissipation. Transcriptomic analysis of the IC10 treatment (1.8 mg/L TC) revealed that 2157 differentially expressed genes were up-regulated and 1629 were down-regulated compared with the control. KEGG and GO enrichments demonstrated that 28 photosynthesis-related genes involving light-harvesting chlorophyll protein complex, photosystem I, photosystem II, photosynthetic electron transport and enzymes were up-regulated, which may be the factor responsible for the enhanced photosynthesis and recovery of the microalgae. Our work may be helpful not only for gaining a better understanding of the environmental risk of TC at concentrations close to the real levels in natural waters, but also for explaining photosynthesis and related gene transcription induced by antibiotics.

A Multicomponent Butyrylcholinesterase Preparation for Enzyme Inhibition-Based Assay of Organophosphorus Pesticides

A new method of producing butyrylcholinesterase (BChE) preparations, stable in storage and use, has been proposed. The BChE preparation is the enzyme co-immobilized with 0.2 M 5-5′-dithiobis (2-nitrobenzoic acid) in starch or gelatin gel. All experimental preparations retain enzyme activity for at least 300 d. The preparations based on gelatin gel show higher activity but lower sensitivity to the toxicants tested in this study compared to the starch gel-based preparations. A method has been proposed for integrated detection of anti-cholinesterase substances in aqueous solutions using the experimental preparation with immobilized BChE. After the additional incubation of the preparation with the immobilized enzyme in the solution of the analyte, the detection limits of malathion and pirimiphos-methyl determined using the IC20 values were below their maximum allowable concentrations—0.005 µM and 0.03 µM, respectively.

Antipsoriatic Potential of Quebecol and Its Derivatives.

Psoriasis is a chronic inflammatory skin disease mainly characterized by the hyperproliferation and abnormal differentiation of the epidermal keratinocytes. An interesting phenolic compound, namely quebecol (2,3,3-tri-(3-methoxy-4-hydroxyphenyl)-1-propanol) (compound 1, CPD1), was isolated from maple syrup in 2011 and was recently synthesized. Quebecol and its derivatives ethyl 2,3,3-tris(3-hydroxy-4-methoxyphenyl)propenoate (compound 2, CPD2) and bis(4-hydroxy-3-methoxyphenyl)methane (compound 3, CPD3) have shown antiproliferative and anti-inflammatory potential, making them promising candidates for the treatment of psoriasis. This study aimed to evaluate the antipsoriatic potential of quebecol and its derivatives on psoriatic skin substitutes produced according to the self-assembly method. A sulforhodamine B (SRB) assay determining the concentration that inhibits 20% of cell growth (IC20) was performed for CPD1, CPD2 and CPD3, and their IC20 values were 400, 150 and 350 μM, respectively. At these concentrations, cell viability was 97%, 94% and 97%, respectively. The comparative control methotrexate (MTX) had a cell viability of 85% at a concentration of 734 μM. Histological analyses of psoriatic skin substitutes treated with CPD1, CPD2 and CPD3 exhibited significantly reduced epidermal thickness compared with untreated psoriatic substitutes, which agreed with a decrease in keratinocyte proliferation as shown by Ki67 immunofluorescence staining. The immunofluorescence staining of differentiation markers (keratin 14, involucrin and loricrin) showed improved epidermal differentiation. Taken together, these results highlight the promising potential of quebecol and its derivatives for the treatment of psoriasis.

Determination of the chemical composition and antioxidant, anticancer, and antibacterial properties of essential oil of Pulicaria crispa from Saudi Arabia

Essential oils obtained from plants play critical roles in food and medicine. In this study, the phytochemical composition of Pulicaria crispa essential oil, and its antibacterial, antioxidant and anticancer properties were determined in vitro. The essential oil was extracted from the aerial parts of P. crispa through hydro-distillation using a Clevenger-type apparatus, and it was analyzed with GC-MS. The most dominant chemical constituents of the essential oil were sesquiterpenes (78.26%). The higher constituents were β-caryophyllene oxide (33.97%), modephene (23.34%), geranyl propionate (6.32%), geranyl isovalerate (6.74%), 4-cadinadiene (5%), humulene (4.05%), and β-caryophyllene (2.73%). The essential oil exhibited DPPH radical activity, and it exerted antibacterial effect against gram positive bacteria (Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. However, it had no antibacterial effect on gram negative bacteria (Pseudomonas aeruginosa, Shigella sonnei, Klebsiella pneumoniae and Escherichia coli). The P. crispa essential oil produced significant cytotoxic effects against Hep-G2, MCF-7, Coca-2, and HT-29 ​cells. The oil was most toxic to Hep-G2 cells, based on its IC20 and IC50 values. These results indicate that the essential oil from P. crispa has potent biological properties which can be useful in the food and pharmaceutical industries.

Acetylcholinesterase from the charru mussel Mytella charruana: kinetic characterization, physicochemical properties and potential as in vitro biomarker in environmental monitoring of mollusk extraction areas

Acetylcholinesterase (AChE; EC 3.1.1.7) from aquatic organisms have been used to evaluate the exposure of specimens to pesticides and heavy metals at sublethal levels in environmental samples. AChE of Mytella charruana was extracted to characterize its physicochemical and kinetic properties as well as the effect of organophosphate (dichlorvos, diazinon, chlorpyrifos, methyl-parathion and temephos), carbamates (carbaryl, carbofuran and aldicarb), benzoylureas (diflubenzuron and novaluron), pyrethroid (cypermethrin) and juvenile hormone analog - JHA (pyriproxyfen) and the effect of metal ions: Hg2+, Cd2+, Pb2+, As3+, Cu2+ and Zn2+, in order to evaluate the potential of the enzyme as biomarker. The optimum pH of M. charruana AChE was 8.5 and the maximum activity peak occurred at 48 °C, being highly thermostable maintaining 97.8% of its activity after incubation at 60 °C. The Michaelis-Menten constants (km) for the substrates acetylthiocholine and propionylthiocholine were 2.8 ± 1.26 and 4.94 ± 6.9 mmol·L−1, respectively. The Vmax values for the same substrates were 22.6 ± 0.90 and 10.2 ± 4.94 mU·mg−1, respectively. Specific inhibition results suggest an AChE presenting active site with dimensions between those of AChE and butyrylcholinesterase (BChE). The IC20 values related to the effect of the pesticides on the enzyme showed higher inhibitory power of temephos (0.17 μmol·L−1), followed by aldicarb (0.19 μmol·L−1) and diflubenzuron (0.23 μmol·L−1). Metal ions inhibited M. charruana enzyme in the following order: Hg2+ > Pb2+ > Cd2+ > As3+ > Cu2+ > Zn2+. These data suggest that the enzyme showed potential as in vitro biomarker of the exposure to temephos, mercury, zinc and copper.

Co-Treatments of Edible Curcumin from Turmeric Rhizomes and Chemotherapeutic Drugs on Cytotoxicity and FLT3 Protein Expression in Leukemic Stem Cells.

This study aims to enhance efficacy and reduce toxicity of the combination treatment of a drug and curcumin (Cur) on leukemic stem cell and leukemic cell lines, including KG-1a and KG-1 (FLT3+ LSCs), EoL-1 (FLT3+ LCs), and U937 (FLT3− LCs). The cytotoxicity of co-treatments of doxorubicin (Dox) or idarubicin (Ida) at concentrations of the IC10–IC80 values and each concentration of Cur at the IC20, IC30, IC40, and IC50 values (conditions 1, 2, 3, and 4) was determined by MTT assays. Dox–Cur increased cytotoxicity in leukemic cells. Dox–Cur co-treatment showed additive and synergistic effects in several conditions. The effect of this co-treatment on FLT3 expression in KG-1a, KG-1, and EoL-1 cells was examined by Western blotting. Dox–Cur decreased FLT3 protein levels and total cell numbers in all the cell lines in a dose-dependent manner. In summary, this study exhibits a novel report of Dox–Cur co-treatment in both enhancing cytotoxicity of Dox and inhibiting cell proliferation via FLT3 protein expression in leukemia stem cells and leukemic cells. This is the option of leukemia treatment with reducing side effects of chemotherapeutic drugs to leukemia patients.

Silencing Oncogene Translation Using Pateamine a Analogues As a Novel Therapeutic Strategy for c-Myc Driven Lymphoma

IntroductionThe oncogene c-Myc is a master transcription factor that is associated with pathogenesis and chemo-resistance in a vast array of human cancers including aggressive lymphoma. Although the therapeutic benefit of targeting c-Myc has been demonstrated in many preclinical studies, direct inhibitors of c-Myc have not been successfully developed for the clinic. On the other hand, translation of c-Myc is highly dependent on the eukaryotic translation initiation factor 4F (eIF4F), a complex comprised of the eIF4A, eIF4G, and eIF4E subunits. These results suggest that silencing the translation of c-Myc via inhibiting eIF4F or its subunits may be a valid alternative strategy for indirectly targeting the “undruggable” c-Myc oncogene. In the current project, we have investigated the pharmacological activity and mechanism of Pateamine A (PatA) analogues in lymphoma cells in vitro . PatA was originally purified as a marine natural product and demonstrated anti-proliferation activity. One of the analogues, Biotin-DMDA-PatA conjugate (B-DMDA-PatA) pulled down eIF4A, specifically inhibits de novo protein synthesis with much less effect on de novo RNA synthesis, and specifically inhibits cap dependent translation. DMDA-PatA acts by decreasing the interaction between eIF4A and eIF4G. Our hypothesis is the following: If PatA analogues preferentially inhibit the translation of oncogenes such as c-Myc, then PatA analogues will be highly effective for c-Myc driven cancers and potentially safe for animals and humans.MethodsMZ-735 is a recently developed and optimized PatA analogue, based on insights from DMDA-PatA. Both compounds were synthesized at the CPRIT Synthesis and Drug-Lead Discovery Laboratory at Baylor University. Cytotoxicity was evaluated in lymphoma cell lines using CellTiter-Glo (Promega®). The cells were exposed to the PatA analogues or the vehicle control on either continuous exposure or on a treatment (progressively decreasing exposure)-washout schedule. To evaluate the toxicity of PatA analogues to normal cells, cytotoxicity in peripheral blood mononuclear cells (PBMC) of healthy donors were tested. Immunoblot was used to study the molecular effects of MZ-735 on translation of oncogenes, particularly that of c-Myc, in lymphoma cell lines representing DLBCL, DHL, mantle cell lymphoma (MCL), cutaneous T cell lymphoma (CTCL), anaplastic large cell lymphoma (ALCL), and adult T cell lymphoma/leukemia (ATL). To further investigate the broad translational downregulation of poorly understood network of oncogenes by MZ-735, we are conducting in parallel proteomic and RNAseq studies in 3 cell lines representing DLBCL (no MYC translocation), DHL (with MYC and BCL2 translocation), and MCL (with CCND1 translocation). Finally, the efficacy and safety of MZ-735 will be investigated in xenograft mouse models of DHL.ResultsDMDA-PatA and MZ-735 were studied in 13 lymphoma cell lines. DMDA-PatA and MZ-735 exhibited concentration- and time-dependent cytotoxicity in lymphoma cell lines at low nanomolar concentrations (Figure 1A). The IC20 and IC50 values of each lymphoma cell lines are shown in Table 1. MZ-735 was more effective in B cell lymphoma than in T cell lymphoma cells. MZ-735 was significantly more potent than DMDA-PatA in all tested cell lines. Remarkably, a brief treatment by MZ-735 for as short as 3 hours followed by washout of the compound produced comparable cytotoxicity to that of continuous exposure of the compound without washout. Importantly, DMDA-PatA and MZ-735 did not show significant cytotoxic effect in PBMC from healthy donors (Figure 1B). MZ-735 markedly repressed the protein level of c-Myc in all tested lymphoma cell lines, and effectively suppressed the protein level of cyclin D1 in MCL cells (Figure 2). The expression of the MDM2 oncogene was suppressed in a way similar to C-MYC .ConclusionThese results suggest that the PatA analogue MZ-735 has potent pharmacological activity in lymphoma cell lines, and acts by suppressing the production of critical oncogenes, including C-MYC, CCND1, and MDM2 , but is not toxic to normal PBMC. MZ-735 may cause rapid and irreversible modification of the translation apparatus, which is not affected by drug washout, leading to rapid commitment to growth arrest and death of cancer cells. MZ-735 is a promising and novel treatment for C-MYC- and CCND1 -driven aggressive lymphoma. [Display omitted] DisclosuresO'Connor:Trillium Therapeutics: Research Funding; Celgene: Honoraria, Research Funding.