Diclofenac N-Derivatives as Therapeutic Agents with Anti-Inflammatory and Anti-Cancer Effect

A series of diclofenac N-derivatives (2, 4, 6, 8c, 9c, 10a-c) were synthesized in order to test their anti-cancer and anti-inflammatory effects. The anticarcinogen activity has been assayed against three cancer cell lines: HT29, human colon cancer cells; Hep-G2, human hepatic cells; and B16-F10, murine melanoma cells. First, we determined the cytotoxicity of the different compounds, finding that the most effective compound was compound 8c against all cell lines and both compounds 4 and 6 in human Hep-G2 and HT29 cell lines. Compounds 4 and 8c were selected for the percentage of apoptosis determination, cell cycle distribution, and mitochondrial membrane potential measure because these products presented the lowest IC₅₀ values in two of the three cancer cell lines assayed (B16-F10 and HepG2), and were two of the three products with lowest IC₅₀ in HT29 cell line. Moreover, the percentages of apoptosis induction were determined for compounds 4 and 8c, showing that the highest values were between 30 to 60%. Next, the effects of these two compounds were observed on the cellular cycle, resulting in an increase in the cell population in G2/M cell cycle phase after treatment with product 8c, whereas compound 4 increased the cells in phase G0/G1, by possible differentiation process induction. Finally, to determine the possible apoptosis mechanism triggered by these compounds, mitochondrial potential was evaluated, indicating the possible activation of extrinsic apoptotic mechanism. On the other hand, we studied the anti-inflammatory effects of these diclofenac (DCF) derivatives on lipopolysaccharide (LPS) activated RAW 264.7 macrophages-monocytes murine cells by inhibition of nitric oxide (NO) production. As a first step, we determined the cytotoxicity of the synthesized compounds, as well as DCF, against these cells. Then, sub-cytotoxic concentrations were used to determine NO release at different incubation times. The greatest anti-inflammatory effect was observed for products 2, 4, 8c, 10a, 10b, and 9c at 20 µg·mL⁻¹ concentration after 48 h of treatment, with inhibition of produced NO between 60 to 75%, and a concentration that reduces to the 50% the production of NO (IC_{50 NO}) between 2.5 to 25 times lower than that of DCF. In this work, we synthesized and determined for the first time the anti-cancer and anti-inflammatory potential of eight diclofenac N-derivatives. In agreement with the recent evidences suggesting that inflammation may contribute to all states of tumorigenesis, the development of these new derivatives capable of inducing apoptosis and anti-inflammatory effects at very low concentrations represent new effective therapeutic strategies against these diseases.     

Design,Synthesis, and Biological Evaluation of New Urushiol Derivatives as Potent Class I Histone Deacetylase Inhibitors

In the present study, a novel series of 11 urushiol-based hydroxamic acid histone deacetylase (HDAC) inhibitors was designed, synthesized, and biologically evaluated. Compounds 1 – 11 exhibited good to excellent inhibitory activities against HDAC1/2/3 (IC₅₀: 42.09–240.17 nM) and HDAC8 (IC₅₀: 16.11–41.15 nM) in vitro, with negligible activity against HDAC6 (>1409.59 nM). Considering HDAC8, docking experiments revealed some important features contributing to inhibitory activity. According to Western blot analysis, select compounds could notably enhance the acetylation of histone H3 and SMC3 but not-tubulin, indicating their privileged structure is appropriate for targeting class I HDACs. Furthermore, antiproliferation assays revealed that six compounds exerted greater in vitro antiproliferative activity against four human cancer cell lines (A2780, HT-29, MDA-MB-231, and HepG2, with IC₅₀ values ranging from 2.31–5.13 μM) than suberoylanilide hydroxamic acid; administration of these compounds induced marked apoptosis in MDA-MB-231 cells, with cell cycle arrest in the G2/M phase. Collectively, specific synthesized compounds could be further optimized and biologically explored as antitumor agents.     

Access to New Cytotoxic Triterpene and Steroidal Acid-TEMPO Conjugates by Ugi Multicomponent-Reactions

Multicomponent reactions, especially the Ugi-four component reaction (U-4CR), provide powerful protocols to efficiently access compounds having potent biological and pharmacological effects. Thus, a diverse library of betulinic acid (BA), fusidic acid (FA), cholic acid (CA) conjugates with TEMPO (nitroxide) have been prepared using this approach, which also makes them applicable in electron paramagnetic resonance (EPR) spectroscopy. Moreover, convertible amide modified spin-labelled fusidic acid derivatives were selected for post-Ugi modification utilizing a wide range of reaction conditions which kept the paramagnetic center intact. The nitroxide labelled betulinic acid analogue 6 possesses cytotoxic effects towards two investigated cell lines: prostate cancer PC3 (IC₅₀ 7.4 ± 0.7 μM) and colon cancer HT29 (IC₅₀ 9.0 ± 0.4 μM). Notably, spin-labelled fusidic acid derivative 8 acts strongly against these two cancer cell lines (PC3: IC₅₀ 6.0 ± 1.1 μM; HT29: IC₅₀ 7.4 ± 0.6 μM). Additionally, another fusidic acid analogue 9 was also found to be active towards HT29 with IC₅₀ 7.0 ± 0.3 μM (CV). Studies on the mode of action revealed that compound 8 increased the level of caspase-3 significantly which clearly indicates induction of apoptosis by activation of the caspase pathway. Furthermore, the exclusive mitochondria targeting of compound 18 was successfully achieved, since mitochondria are the major source of ROS generation.     

Modulation of Cyclins,p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxy)benzoic Acid

Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxy)benzoic acid (TMPBA) and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC₅₀ values of 5.9 and 7.9 μM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP) kinases, 5′ adenosine monophosphate-activated protein kinase (AMPK), and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK) signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy.     

Synthesis and Biological Evaluation of Novel Dehydroabietic Acid Derivatives Conjugated with Acyl-Thiourea Peptide Moiety as Antitumor Agents

A series of dehydroabietic acid (DHAA) acyl-thiourea derivatives were designed and synthesized as potent antitumor agents. The in vitro pharmacological screening results revealed that the target compounds exhibited potent cytotoxicity against HeLa, SK-OV-3 and MGC-803 tumor cell lines, while they showed lower cytotoxicity against HL-7702 normal human river cells. Compound 9n (IC₅₀ = 6.58 ± 1.11 μM) exhibited the best antitumor activity against the HeLa cell line and even displayed more potent inhibitory activity than commercial antitumor drug 5-FU (IC₅₀ = 36.58 ± 1.55 μM). The mechanism of representative compound 9n was then studied by acridine orange/ethidium bromide staining, Hoechst 33,258 staining, JC-1 mitochondrial membrane potential staining, TUNEL assay and flow cytometry, which illustrated that this compound could induce apoptosis in HeLa cells. Cell cycle analysis indicated that compound 9n mainly arrested HeLa cells in the S phase stage. Further investigation demonstrated that compound 9n induced apoptosis of HeLa cells through a mitochondrial pathway.     

Synthesis and Biological Evaluation of Pyrazoline and Pyrrolidine-2,5-dione Hybrids as Potential Antitumor Agents

In search of novel and effective antitumor agents, pyrazoline-substituted pyrrolidine-2,5-dione hybrids were designed, synthesized and evaluated in silico, in vitro and in vivo for anticancer efficacy. All the compounds exhibited remarkable cytotoxic effects in MCF7 and HT29 cells. The excellent antiproliferative activity toward MCF7 (IC₅₀=0.78±0.01 μM), HT29 (IC₅₀=0.92±0.15 μM) and K562 (IC₅₀=47.25±1.24 μM) cell lines, prompted us to further investigate the antitumor effects of the best compound S2 (1-(2-(3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione). In cell-cycle analysis, S2 was found to disrupt the growth phases with increased cell population in G₁/G₀ phase and decreased cell population in G₂/M phase. The excellent in vitro effects were also supported by inhibition of anti-apoptotic protein Bcl-2. In vivo tumor regression studies of S2 in HT29 xenograft nude mice, exhibited equivalent and promising tumor regression with maximum TGI, 66 % (i. p. route) and 60 % (oral route) at 50 mg kg⁻¹ dose by both the routes, indicating oral bioavailability and antitumor efficacy. These findings advocate that hybridization of pyrazoline and pyrrolidine-2,5-dioes holds promise for the development of more potent and less toxic anticancer agents.     

A Series of New Ligustrazine-Triterpenes Derivatives as Anti-Tumor Agents: Design,Synthesis, and Biological Evaluation

A series of novel ligustrazine-triterpenes derivatives was designed, synthesized and screened for their cytotoxicity against five cancer cell lines (Bel-7402, HepG2, HT-29, Hela, and MCF-7) and Madin-Darby canine kidney (MDCK). Current study suggested that most of the ligustrazine-triterpenes conjunctions showed better cytotoxicity than the starting materials. In particular, compound 4a exhibited better cytotoxic activity (IC₅₀ < 5.23 μM) against Bel-7402, HT-29, MCF-7, Hela, and HepG2 than the standard anticancer drug cisplatin (DDP). The cytotoxicity selectivity detection revealed that 4a exhibited low cytotoxicity (IC₅₀ > 20 μM) towards MDCK cells. A combination of fluorescence staining observation and flow cytometric analysis indicated that 4a could induce HepG2 cell apoptosis. Further studies suggested that 4a-induced apoptosis is mediated through depolarization of the mitochondrial membrane potential and increase of intracellular free Ca²⁺ concentration. In addition, the structure-activity relationships of these derivatives were briefly discussed.     

Oxazole‐Bridged Combretastatin A Analogues with Improved Anticancer Properties

Three new oxazole‐bridged combretastatin A analogues with additional functional groups at the B‐ring [‐SMe, ‐OH, p‐quinone] were tested for antiproliferative activity and specificity on human HL‐60 leukemia, 518A2 melanoma, and colon carcinomas HCT‐116 (wt)/(p53^?/?) and HT‐29 cells. While all oxazoles, except quinone 8 , were efficacious against HCT‐116 cells at submicromolar IC₅₀ values (48 h incubation), only thioanisole 5 achieved this potency in combretastatin‐refractory HT‐29 cells by significant upregulation of p21^cip1/waf1 associated with an S/G₂ cell‐cycle arrest.     

Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC₅₀) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC₅₀ value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC₅₀ values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.     

Comparative Anti-Inflammatory Effects of Salix Cortex Extracts and Acetylsalicylic Acid in SARS-CoV-2 Peptide and LPS-Activated Human In Vitro Systems

The usefulness of anti-inflammatory drugs as an adjunct therapy to improve outcomes in COVID-19 patients is intensely discussed in this paper. Willow bark (Salix cortex) has been used for centuries to relieve pain, inflammation, and fever. Its main active ingredient, salicin, is metabolized in the human body into salicylic acid, the precursor of the commonly used pain drug acetylsalicylic acid (ASA). Here, we report on the in vitro anti-inflammatory efficacy of two methanolic Salix extracts, standardized to phenolic compounds, in comparison to ASA in the context of a SARS-CoV-2 peptide challenge. Using SARS-CoV-2 peptide/IL-1β- or LPS-activated human PBMCs and an inflammatory intestinal Caco-2/HT29-MTX co-culture, Salix extracts, and ASA concentration-dependently suppressed prostaglandin E2 (PGE₂), a principal mediator of inflammation. The inhibition of COX-2 enzyme activity, but not protein expression was observed for ASA and one Salix extract. In activated PBMCs, the suppression of relevant cytokines (i.e., IL-6, IL-1β, and IL-10) was seen for both Salix extracts. The anti-inflammatory capacity of Salix extracts was still retained after transepithelial passage and liver cell metabolism in an advanced co-culture model system consisting of intestinal Caco-2/HT29-MTX cells and differentiated hepatocyte-like HepaRG cells. Taken together, our in vitro data suggest that Salix extracts might present an additional anti-inflammatory treatment option in the context of SARS-CoV-2 peptides challenge; however, more confirmatory data are needed.     

New Environmentally Friendly Oxidative Scission of Oleic Acid into Azelaic Acid and Pelargonic Acid

Oleic acid (OA) is a renewable monounsaturated fatty acid obtained from high oleic sunflower oil. This work was focused on the oxidative scission of OA, which yields a mono-acid (pelargonic acid, PA) and a di-acid (azelaic acid, AA) through an emulsifying system. The conventional method for producing AA and PA consists of the ozonolysis of oleic acid, a process which presents numerous drawbacks. Therefore, we proposed to study a new alternative process using a green oxidant and a solvent-free system. OA was oxidized in a batch reactor with a biphasic organic-aqueous system consisting of hydrogen peroxide (H₂O₂, 30 %) as an oxidant and a peroxo–tungsten complex Q₃{PO₄[WO(O₂)₂]₄} as a phase-transfer catalyst/co-oxidant. Several phase-transfer catalysts were prepared in situ from tungstophosphoric acid, H₂O₂ and different quaternary ammonium salts (Q⁺, Cl^–). The catalyst [C₅H₅N(n-C₁₆H₃₃)]₃{PO₄[WO(O₂)₂]₄} was found to give the best results and was chosen for the optimization of the other parameters of the process. This optimization led to a complete conversion of OA into AA and PA with high yields (>80 %) using the system OA/H₂O₂/[C₅H₅N(n-C₁₆H₃₃)]₃{PO₄[WO(O₂)₂]₄} (1/5/0.02 molar ratio) at 85 °C for 5 h. In addition, a new treatment was developed in order to recover the catalyst.     

Galactosylated Chitosan Oligosaccharide Nanoparticles for Hepatocellular Carcinoma Cell-Targeted Delivery of Adenosine Triphosphate

Nanoparticles composed of galactosylated chitosan oligosaccharide (Gal-CSO) and adenosine triphosphate (ATP) were prepared for hepatocellular carcinoma cell-specific uptake, and the characteristics of Gal-CSO/ATP nanoparticles were evaluated. CSO/ATP nanoparticles were prepared as a control. The average diameter and zeta potential of Gal-CSO/ATP nanoparticles were 51.03 ± 3.26 nm and 30.50 ± 1.25 mV, respectively, suggesting suitable properties for a drug delivery system. Subsequently, the cytotoxicity of Gal-CSO/ATP nanoparticles were examined by the methyl tetrazolium (MTT) assay, and the half maximal inhibitory concentration (IC₅₀) values were calculated with HepG2 (human hepatocellular carcinoma cell line) cells. The results showed that the cytotoxic effect of nanoparticles on HepG2 cells was low. In the meantime, it was also found that the Gal-CSO/ATP nanoparticles could be uptaken by HepG2 cells, due to expression of the asialoglycoprotein receptor (ASGP-R) on their surfaces. The presented results indicate that the Gal-CSO nanoparticles might be very attractive to be used as an intracellular drug delivery carrier for hepatocellular carcinoma cell targeting, thus warranting further in vivo or clinical investigations.     

Design and Synthesis of Imidazo[2,1‐b]thiazole–Chalcone Conjugates: Microtubule‐Destabilizing Agents

A series of chalcone conjugates featuring the imidazo[2,1‐b]thiazole scaffold was designed, synthesized, and evaluated for their cytotoxic activity against five human cancer cell lines (MCF‐7, A549, HeLa, DU‐145 and HT‐29). These new hybrid molecules have shown promising cytotoxic activity with IC₅₀ values ranging from 0.64 to 30.9 μM . Among them, (E)‐3‐(6‐(4‐fluorophenyl)‐2,3‐bis(4‐methoxyphenyl)imidazo[2,1‐b]thiazol‐5‐yl)‐1‐(pyridin‐2‐yl)prop‐2‐en‐1‐one ( 11 x ) showed potent antiproliferative activity with IC₅₀ values ranging from 0.64 to 1.44 μM in all tested cell lines. To investigate the mechanism of action, the detailed biological aspects of this promising conjugate ( 11 x ) were carried out on the A549 lung cancer cell line. The tubulin polymerization assay and immunofluoresence analysis results suggest that this conjugate effectively inhibits microtubule assembly in A549 cells. Flow cytometric analysis revealed that this conjugate induces cell‐cycle arrest in the G2/M phase and leads to apoptotic cell death. This was further confirmed by Hoechst staining, activation of caspase‐3, DNA fragmentation analysis, and Annexin V–FITC assay. Moreover, molecular docking studies indicated that this conjugate ( 11 x) interacts and binds efficiently with the tubulin protein.     

Sustainable Process for Production of Azelaic Acid Through Oxidative Cleavage of Oleic Acid

This work describes two sustainable methods for production and purification of azelaic acid (AA) to replace the current process of ozonolysis of oleic acid (OA). The first proceeds in two steps, coupling smooth oxidation of OA to 9,10‐dihydroxystearic acid (DSA) with subsequent oxidative cleavage by sodium hypochlorite. An alternative methodology is also proposed, using a chemocatalytic system consisting of H₂O₂/H₂WO₄ for direct oxidative cleavage of the double bond of OA at 373 K. A convenient technique for separation and purification of azelaic acid is also proposed.     

Comparison of the effect of six compactin-related compounds on cholesterol synthesis in five human cell types

We have investigated the effect of six compactin-related compounds—mevinolin, compactin, ML-236A, monacolin X, monacolin L and dihydromonacolin L—on cholesterol synthesis in human umbilical vein endothelial cells, human small intestine epithelial cells, human hepatoma cell line HEP G2, normal human skin fibroblasts and in skin fibroblasts from a patient with familial homozygous hypercholesterolemia. The inhibition of cholesterol synthesis was found to depend on both the cell type and the type of compound used. The most effective compounds were mevinolin and compactin. Monacolin X, monacolin L and ML-236A were less effective, and dihydromonacolin L was the least efficacious. Endothelial and epithelial cells were sensitive to very low concentrations of inhibitors (IC₅₀=1.0–30 pg/mL), HEP G2 cells required higher concentrations (IC₅₀=0.01–66 ng/mL) and fibroblasts needed even higher concentrations (IC₅₀=0.1–200 ng/mL). Lactone and acid forms of the inhibitors were equally active. None of the inhibitors had any effect on either protein or fatty acid synthesis in any of the cell types studied. It can be concluded that different compactin-related compounds show a range of potencies as cholesterol synthesis inhibitors and a dose-dependent tissue-selectivity.     

A kinetic study of the electrochemical oxidation of maleic acid on boron doped diamond

Maleic acid (MA) is one of the main intermediates formed during mineralization, by electrooxidation, of aromatic compounds contained in aqueous wastes. This work investigates oxidation of maleic acid with or without the presence of oxalic acid (OA) and formic acid (FA) in aqueous solution by using boron-doped diamond (BDD) electrodes. OA and FA are the main products formed in MA electrooxidation. Voltammetric studies conducted with a BDD electrode of small surface (0.196 cm²) show that MA oxidation takes place at a potential very close to that of the discharge of water. But, under potentiostatic conditions and at concentrations higher than 0.001 M, adsorption of MA blocks its own oxidation. Oxalic and formic acids are before the discharge of water. Again, the presence of maleic acid blocks the oxidation of formic and oxalic acids. Galvanostatic electrolyses of aqueous solutions of MA, OA, FA and mixtures of theses acids were conducted on a BDD electrode. Electrolyses were controlled by measurements of Total Organic Carbon, Chemical Oxygen Demand and by Liquid Chromatography. Results showed that MA was totally mineralized; FA and OA were very low concentration intermediaries. Electrolyses of solutions containing MA, initially in the presence of OA or FA, showed that the OA was oxidized at the same rate as the MA, whereas the FA oxidation began only when the MA had completely disappeared. These results suggest that OA oxidizes by a mass transport limited process coupled with a direct electron transfer with the anode. Under galvanostatic conditions, maleic acid and formic acid are probably oxidized via OH^· radicals generated by water discharge.     

Insights into the in vitro Anticancer Effects of Diruthenium‐1

The in vitro anticancer activity of the dinuclear trithiolato‐bridged arene ruthenium complex diruthenium‐1 (DiRu‐1) was evaluated against a panel of human cancer cell lines used as in vitro models for hepatocellular carcinoma (HepG2 cells), estrogen‐responsive breast adenocarcinoma (MCF‐7 cells), and triple‐negative breast adenocarcinoma (MDA‐MB‐231 cells). DiRu‐1 is highly cytotoxic to these cell lines, demonstrating half‐maximal inhibitory concentrations (IC₅₀) in the low‐nanomolar range (77±1.4 to 268.2±4.4 nm ). The main molecular mechanisms responsible for the high cytotoxicity of DiRu‐1 against the most responsive MCF‐7 cell line (IC₅₀=77±1.4 nm) were investigated on the basis of the capacity of DiRu‐1 to induce oxidative stress, apoptosis, and DNA damage, and to inhibit the cell cycle and proliferation. The results show that DiRu‐1 triggers caspase‐dependent apoptosis in MCF‐7 cells on both the intrinsic and extrinsic pathways. Moreover, the Ru complex also causes necrosis, mitotic catastrophe, and autophagy. DiRu‐1 increases the intracellular levels of reactive oxygen species (ROS), which play a significant role in its cytotoxicity and pro‐apoptotic activity. An important mechanism of the anticancer activity of DiRu‐1 appears to be the induction of DNA lesions, mainly due to apoptotic DNA fragmentation and cell‐cycle arrest at the G₂/M checkpoint. These changes are correlated with the concentration of DiRu‐1, the duration of the cell treatment, and the post‐treatment time.     

Anti-Inflammatory Activity of Three Triterpene from Hippophae rhamnoides L. in Lipopolysaccharide-Stimulated RAW264.7 Cells

Oleanolic acid (OA), asiatic acid (AA), and maslinic acid (MA) are ubiquitous isomeric triterpene phytochemicals with many pharmacological effects. To improve their application value, we used lipopolysaccharide (LPS) to induce RAW264.7 cells and studied the differences in the anti-inflammatory effects of the triterpenes according to their structural differences. MTT, Griess, and immunofluorescence assays, ELISA, flow cytometry, and Western blotting, were performed. The release of LPS-induced pro-inflammatory mediators, such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), and interleukin (IL-6), was significantly inhibited by OA, AA, and MA at the same concentration, and AA and MA promoted the production of anti-inflammatory factor IL-10. OA, AA, and MA inhibited LPS-induced NF-κB nuclear translocation in RAW264.7 cells. OA and AA inhibited the phosphorylation of ERK1/2, P38, and JNK1/2 in LPS-stimulated RAW264.7 cells. Moreover, OA increased LPS-induced Nrf2 expression and decreased Keap1 expression in RAW264.7 cells. OA, AA, and MA inhibited LPS-stimulated intracellular reactive oxygen species (ROS) production and alleviated mitochondrial membrane potential depletion. Overall, our data suggested that OA, AA, and MA exhibited significant anti-inflammatory effects in vitro. In particular, OA and AA take effects through the MAPKs, NF-κB, and Nrf2 signaling pathways.     

Inhibitory Effects of Cholesterol Derivatives on DNA Polymerase and Topoisomerase Activities, and Human Cancer Cell Growth

This paper describes the inhibitory activities of cholesterol derivatives such as cholesterol, sodium cholesteryl sulfate, cholesteryl-5α, 6α-epoxide, cholesteryl chloride, cholesteryl bromide, and cholesteryl hemisuccinate (compounds 1–6, respectively) against DNA polymerase (pol), DNA topoisomerase (topo), and human cancer cell growth. Among the compounds tested, compounds 2 and 6 revealed themselves to be potent inhibitors of animal pols, and the IC₅₀ values for pols were 0.84–11.6 and 2.9–148 μM, respectively. Compounds 2, 3 and 6 inhibited the activity of human topo II, with IC₅₀ values of 5.0, 12.5 and 120 μM, respectively. Compounds 2, 3 and 6 also suppressed human cancer cell (promyelocytic leukemia cell line, HL-60) growth, and LD₅₀ values were 8.8, 20.2 and 72.3 μM, respectively, suggesting that cell growth inhibition had the same tendency as the inhibition of topos rather than pols. Compounds 2 and 6 arrested the cells in S and G2/M phases, compound 3 arrested the cells in the G2/M phase, and these compounds also increased sub-G1 phase in the cell cycle. These results suggested that the effect of cell cycle arrest might be effective on both pols and topos activities. From these findings, the action mode of cholesterol derivatives as anti-cancer compounds is discussed.     

Novel Very Long-Chain α-Methoxylated Δ5,9 Fatty Acids from the Sponge Asteropus niger Are Effective Inhibitors of Topoisomerases IB

The novel fatty acids (2R,5Z,9Z)‐2‐methoxy‐25‐methyl‐5,9‐hexacosadienoic acid ( 1a ) and (2R,5Z,9Z)‐2‐methoxy‐24‐methyl‐5,9‐hexacosadienoic acid ( 1b ) were isolated in 80 % purity from the Caribbean sponge Asteropus niger by chloroform/methanol extraction followed by solvent partitioning and silica gel column chromatography. The compounds were characterized by utilizing a combination of gas chromatography‐mass spectrometry, nuclear magnetic resonance, and circular dichroism. Acids 1a and 1b were not detected in the phospholipids (PtdCho and PtdIns) of the sponge, but rather as free FA and possibly in glycosylceramides. The mixtures of 1a and 1b displayed cytotoxicity towards THP‐1 and HepG2 cells with EC_50's between 41 and 35 μg/mL. Apoptosis was not the preferred mode of cell death induced by 1a – 1b in the THP‐1 cells. This implies other types of cytotoxicity mechanisms, such as membrane disruption and/or the inhibition (EC₅₀ = 1.8 μg/mL) of the human topoisomerase IB enzyme (hTopIB), with a mechanism of inhibition different from the one displayed by camptothecin (CPT). In a separate experiment, the mixture of 1a and 1b also displayed cytotoxicity towards ex vivo mouse splenocytes infected with Leishmania infantum amastigotes (IC₅₀ = 0.17 mg/mL) and free living promastigotes (IC₅₀ = 0.34 mg/mL). It was also found that the FA were inhibitory of the Leishmania topoisomerase IB (LTopIB) with an EC₅₀ = 5.1 μg/mL. Taken together, 1a and 1b represent a new class of FA with potential as TopIB inhibitors that preferentially inhibit hTopIB over LTopIB.