MEF2 is an in vivo immune-metabolic switch.

Infections disturb metabolic homeostasis in many contexts, but the underlying connections are not completely understood. To address this, we use paired genetic and computational screens in Drosophila to identify transcriptional regulators of immunity and pathology and their associated target genes and physiologies. We show that Mef2 is required in the fat body for anabolic function and the immune response. Using genetic and biochemical approaches, we find that MEF2 is phosphorylated at a conserved site in healthy flies and promotes expression of lipogenic and glycogenic enzymes. Upon infection, this phosphorylation is lost, and the activity of MEF2 changes--MEF2 now associates with the TATA binding protein to bind a distinct TATA box sequence and promote antimicrobial peptide expression. The loss of phosphorylated MEF2 contributes to loss of anabolic enzyme expression in Gram-negative bacterial infection. MEF2 is thus a critical transcriptional switch in the adult fat body between metabolism and immunity.

Involvement of Both Extrinsic and Intrinsic Apoptotic Pathways in Tridecylpyrrolidine-Diol Derivative-Induced Apoptosis In Vitro.

Despite the decreasing trend in mortality from colorectal cancer, this disease still remains the third most common cause of death from cancer. In the present study, we investigated the antiproliferative and pro-apoptotic effects of (2S,3S,4R)-2-tridecylpyrrolidine-3,4-diol hydrochloride on colon cancer cells (Caco-2 and HCT116). The antiproliferative effect and IC50 values were determined by the MTT and BrdU assays. Flow cytometry, qRT-PCR and Western blot were used to study the cellular and molecular mechanisms involved in the induction of apoptotic pathways. Colon cancer cell migration was monitored by the scratch assay. Concentration-dependent cytotoxic and antiproliferative effects on both cell lines, with IC50 values of 3.2 ± 0.1 µmol/L (MTT) vs. 6.46 ± 2.84 µmol/L (BrdU) for HCT116 and 2.17 ± 1.5 µmol/L (MTT) vs. 1.59 ± 0.72 µmol/L (BrdU), for Caco-2 were observed. The results showed that tridecylpyrrolidine-induced apoptosis was associated with the externalization of phosphatidylserine, reduced mitochondrial membrane potential (MMP) accompanied by the activation of casp-3/7, the cleavage of PARP and casp-8, the overexpression of TNF-α and FasL and the dysregulation of Bcl-2 family proteins. Inhibition of the migration of treated cells across the wound area was detected. Taken together, our data show that the anticancer effects of tridecylpyrrolidine analogues in colon cancer cells are mediated by antiproliferative activity, the induction of both extrinsic and intrinsic apoptotic pathways and the inhibition of cell migration.

Derivatives Incorporating Acridine, Pyrrole, and Thiazolidine Rings as Promising Antitumor Agents.

Derivatives combining acridine, pyrrole, and thiazolidine rings have emerged as promising candidates in the field of antitumor drug discovery. This paper aims to highlight the importance of these three structural motifs in developing potent and selective anticancer agents. The integration of these rings within a single molecule offers the potential for synergistic effects, targeting multiple pathways involved in tumor growth and progression. Spiro derivatives were efficiently synthesized in a two-step process starting from isothiocyanates and 2-cyanoacetohydrazide. The thiourea side chain in spiro derivatives was utilized as a key component for the construction of the thiazolidine-4-one ring through regioselective reactions with bifunctional reagents, namely methyl-bromoacetate, dietyl-acetylenedicarboxylate, ethyl-2-bromopropionate, and ethyl-2-bromovalerate. These reactions resulted in the formation of a single regioisomeric product for each derivative. Advanced spectroscopic techniques, including 1D and 2D NMR, FT-IR, HRMS, and single-crystal analysis, were employed to meticulously characterize the chemical structures of the synthesized derivatives. Furthermore, the influence of these derivatives on the metabolic activity of various cancer cell lines was assessed, with IC50 values determined via MTT assays. Notably, derivatives containing ester functional groups exhibited exceptional activity against all tested cancer cell lines, boasting IC50 values below 10 µM. Particularly striking were the spiro derivatives with methoxy groups at position 3 and nitro groups at position 4 of the phenyl ring. These compounds displayed remarkable selectivity and exhibited heightened activity against HCT-116 and Jurkat cell lines. Additionally, 4-oxo-1,3-thiazolidin-2-ylidene derivatives demonstrated a significant activity against MCF-7 and HCT-116 cancer cell lines.

Homospisulosine induced apoptosis in cervical carcinoma cells is associated with phosphorylation of Bcl-2 and up-regulation of p27/Kip1.

Spisulosine (1-deoxysphinganine) is a sphingoid amino alcohol isolated from the sea clams that showed potent antiproliferative activity against a broad spectrum of solid tumors but failed in clinical trials due to neurotoxicity. However, its structural similarity to other bioactive sphingoids, interesting mode of action, and appreciable potency against cancer cells make it a suitable lead for future anticancer drug development. The present study was conducted to elucidate mechanisms of the antiproliferative/cytotoxic effects of newly synthesized spisulosine analog homospisulosine (KP7). The evaluation was performed on cervical carcinoma cells, representing an in vitro model of one of the most common cancer types and a significant worldwide cause of women's cancer mortality. Treatment with homospisulosine (2.0 µM) for 24, 48, and 72 h significantly inhibited the growth of HeLa cells in vitro and induced apoptosis detectable by DNA fragmentation, externalization of phosphatidylserine, dissipation of mitochondrial membrane potential, activation of caspase-3 and cleavage of PARP. In addition, treating HeLa cells with spisulosine increased p27 and Bcl-2 on protein levels and phosphorylation of Bcl-2 on Ser70 residue. These results support the potential for spisulosine analogs represented here by homospisulosine for future therapeutic development.

Synthesis and biological activity of sphingosines with integrated azobenzene switches.

A flexible synthetic approach towards biologically active sphingoid base-like compounds with an integrated azobenzene framework was achieved via installing a chiral amino-alcohol fragment into the azobenzene system by utilizing the Wittig olefination of substituted (E)-triphenyl[4-(phenyldiazenyl)benzyl]phosphonium salts and d-isoascorbic acid derived aldehydes. All the prepared derivatives underwent a series of experiments to probe their photochromic properties, including the reversible E/Z isomerisation, material fatigue and thermal relaxation rate. The targeted E- and Z-isomeric sphingoid analogues were screened in vitro for anticancer activity on a panel of seven human malignant cell lines. Cell viability experiments revealed outstanding antiproliferative/cytotoxic activities of all the tested compounds with IC50 values in the low micromolar range for the most active derivatives. The biological activity of E- and Z-isomeric forms is different. Their entirely accurate differentiation is prevented by the rapid thermal relaxation of the corresponding Z-isomers.

Benzylidenetetralones, cyclic chalcone analogues, induce cell cycle arrest and apoptosis in HCT116 colorectal cancer cells.

Colorectal cancer is the third most common cancer in the world, with 1.2 million new cancer cases annually. Chalcones are secondary metabolite precursors of flavonoids that exhibit diverse biological activities, including antioxidant and antitumor activities. The aim of this study was to investigate the antiproliferative effect of new synthetic chalcone derivatives on HCT116 cells. (E)-2-(2',4'-dimethoxybenzylidene)-1-tetralone (Q705) was found to be the most active (IC50 = 3.44 ± 0.25 µM). Based on these results, this compound was chosen for further analysis of its biochemical and molecular mechanisms. Our results showed that Q705 inhibited the growth and clonogenicity of HCT116 cells. The results of a flow cytometric analyses suggested that this compound caused a significant cell cycle arrest in G2/M phase and increased the proportion of cells in the subG0/G1 phase, marker of apoptosis. Q705-induced apoptosis was confirmed by TdT-mediated dUTP nick end labelling (TUNEL) assay. Treatment of HCT116 cells with this chalcone significantly increased the caspase-3,-7 activity and resulted in cleavage of poly-ADP-ribose polymerase (PARP). Changes in the nuclear morphology such as chromatin condensation were also observed. These effects were associated with a decreased expression of bcl-xL and increased overall ratio of bax/bcl-xL mRNA levels. Immunofluorescence and qRT-PCR analysis revealed that Q705 induced H2AX histone modifications characteristic of DNA damage, disruption of microtubule organization and downregulation of tubulins. In summary, these results suggest that the cyclic chalcone analogue Q705 has potential as a new compound for colorectal cancer therapy.

ROS-dependent antiproliferative effect of brassinin derivative homobrassinin in human colorectal cancer Caco2 cells.

This study was designed to examine the in vitro antiproliferative effect of brassinin and its derivatives on human cancer cell lines. Among seven tested compounds, homobrassinin (K1; N-[2-(indol-3-yl)ethyl]-S-methyldithiocarbamate) exhibited the most potent activity with IC50 = 8.0 µM in human colorectal Caco2 cells and was selected for further studies. The flow cytometric analysis revealed a K1-induced increase in the G2/M phase associated with dysregulation of α-tubulin, α1-tubulin and ß5-tubulin expression. These findings suggest that the inhibitory effect of K1 can be mediated via inhibition of microtubule formation. Furthermore, simultaneously with G2/M arrest, K1 also increased population of cells with sub-G1 DNA content which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V/PI double staining, DNA fragmentation assay and chromatin condensation assay. The apoptosis was associated with the loss of mitochondrial membrane potential (MMP), caspase-3 activation as well as intracellular reactive oxygen species (ROS) production. Moreover, the antioxidant Trolox blocked ROS production, changes in MMP and decreased K1 cytotoxicity, which confirmed the important role of ROS in cell apoptosis. Taken together, our data demonstrate that K1 induces ROS-dependent apoptosis in Caco2 cells and provide the rationale for further in vivo anticancer investigation.

Targeting hematological malignancies with isoxazole derivatives.

Compounds with a heterocyclic isoxazole ring are well known for their diverse biologic activities encompassing antimicrobial, antipsychotic, immunosuppressive, antidiabetic and anticancer effects. Recent studies on hematological malignancies have also shown that some of the isoxazole-derived compounds feature encouraging cancer selectivity, low toxicity to normal cells and ability to overcome cancer drug resistance of conventional treatments. These characteristics are particularly promising because patients with hematological malignancies face poor clinical outcomes caused by cancer drug resistance or relapse of the disease. This review summarizes the knowledge on isoxazole-derived compounds toward hematological malignancies and provides clues on their mechanism(s) of action (apoptosis, cell cycle arrest, ROS production) and putative pharmacological targets (c-Myc, BET, ATR, FLT3, HSP90, CARM1, tubulin, PD-1/PD-L1, HDACs) wherever known.

A 'Chiron' approach to novel phytosphingosine mimetics based on a cascade [3,3]-sigmatropic rearrangement.

Straightforward access to enantiomerically pure 3,4-diamino-3,4-dideoxyphytosphingosines, as novel analogues of natural d-ribo-phytosphingosine was accomplished, starting from two available chirons: dimethyl l-tartrate and d-isoascorbic acid. A sequential Overman rearrangement followed by late-stage introduction of the alkyl side chain moiety via olefin cross-metathesis is the cornerstone of this approach. The preliminary evaluation study of the synthesised sphingomimetics, based on their ability to inhibit a proliferation of human cancer cells, showed promising cytotoxicity against Jurkat and HeLa cells for (2R,3R,4S)-2,3,4-triaminooctadecan-1-ol trihydrochloride.

The synthesis and anticancer activity of analogs of the indole phytoalexins brassinin, 1-methoxyspirobrassinol methyl ether and cyclobrassinin.

An effective synthesis of analogs of the indole phytoalexin cyclobrassinin with NR1R2 group instead of SCH3 was developed starting from indole-3-carboxaldehyde. The target compounds were prepared by spirocyclization of 1-Boc-thioureas with the formation of isolable spiroindoline intermediates, followed by the trifluoroacetic acid-induced cascade reaction consisting of methanol elimination, deprotection and rearrangement of the iminium ion. The structures of novel products were elucided by the (1)H and (13)C NMR spectroscopy, including HMBC, HSQC, COSY, NOESY and DEPT measurements. Several newly synthesized compounds demonstrated significant antiproliferative/cytotoxic activity against human leukemia and solid tumor cell lines, as well as remarkable selectivity of these effects against cancer cells relative to the non-malignant HUVEC cells.

Novel naphthalimide polyamine derivatives as potential antitumor agents.

A novel series of naphthalimide polyamine conjugates were designed, synthesized and evaluated for in vitro antiproliferative activity against human leukemia (Jurkat), human cervical adenocarcinoma (HeLa), human breast adenocarcinoma (MCF-7) and human lung adenocarcinoma (A549) cell lines. From the six derivatives, the new I1 and A3 exhibited highest antiproliferative activity with the IC50 values of 5.67-11.02 µmol · L(-1). Cell cycle analysis of Jurkat cells exposed to I1 at a concentration of 30 µmol × L(-1) for 24 h exhibited a mild increase in S and G2/M fraction caused by accumulation of cells. This arrest was followed by an increase in sub-G0/G1 after 48 h of incubation. Jurkat cells exposed to A3 at a concentration of 30 µmol × L(-1) for 24 h showed an increase in G0/G1 fraction and after 48 h an increase in G2/M fraction followed by an increase in sub-G0/G1 after 72 h of incubation. Moreover, the A3 compound was observed to displace the intercalating agent ethidium bromide from calf thymus DNA using fluorescence spectroscopy. The apparent binding constant was estimated to be 3.1 × 10(6) M(-1) what indicates non-intercalating mode of DNA binding. On the other hand, we found no inhibitory effect of studied compounds on topoisomerase I and topoisomerase II activity. Finally, the localization of these compounds in the cells due to their inherent fluorescence was investigated with the fluorescence microscopy. Our results suggest that the naphthalimide polyamine conjugates rapidly penetrate to the cancer cells. Further studies are necessary to investigate the precise mechanism of action and to find out the relationship between the structure, character and position of substituents of naphthalimide polyamine conjugates and their biological activities.

Cyclic chalcone analogue KRP6 as a potent modulator of cell proliferation: an in vitro study in HUVECs.

In the present investigation a novel series of chalcone analogues were synthesized and evaluated for their anti-proliferative activity in human umbilical vein endothelial cells (HUVECs). Among 14 tested compounds, chalcone analogue (E)-3-(2'-methoxybenzylidene)-4-chromanone (KRP6) exhibited the most potent activity with IC50 19 µM. Moreover, HUVECs exhibited divergent, even opposing concentration-dependent responses to KRP6. This compound was the most potent inhibitor of cell proliferation and extracellular matrix formation (fibronectin and type IV collagen) at higher concentrations (20-50 µM). In contrast, KRP6 stimulated the compensatory increase in proliferative activity including extracellular matrix formation at low concentrations (1, 10 µM). KRP6 concentration-dependently modulated phosphorylation of Akt and mitogen-activated protein kinases such as extracellular signal-regulated kinase-1/-2 and p38 kinase, suggesting that these pathways play a role in the effect mediated by this compound. In addition, we found a selective effect on activated endothelial cells, in particular with resting endothelial cells. In conclusion, KRP6 is a potent modulator of selected steps of the angiogenic process in vitro. Accordingly, further in vivo research should be performed to facilitate its use in clinical practice.

Antiproliferative and antiangiogenic properties of horse chestnut extract.

This study was designed to examine the in vitro antiproliferative effect of the horse chestnut extract (HCE) on cancer cell lines. Furthermore, we have investigated the in vitro effect of HCE on some angiogenic events by using human umbilical vein endothelial cells. The cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and anchorage-independent growth by colony-forming assay. To understand the growth inhibitory effects, carcinoma cell lines (Jurkat, CEM, HeLa, and MCF-7) were treated with various concentrations of HCE. Incubation of Jurkat, CEM, HeLa, and MCF-7 cancer cells with HCE at 125 µg/mL for 72 h caused 93.7%, 32.3%, 20.4% and 40.4% reduction in cell survival. Colony-forming assay also confirmed growth-inhibitory effects of the compound studied. In HeLa HCE-treated cells, we found a significant increase in cells having sub-G(0) /G(1) DNA content which is considered to be a marker of apoptotic cell death. Apoptosis was also further confirmed by DNA fragmentation analysis.Furthermore, HCE inhibited migration of human umbilical vein endothelial cells as well as decreased secretion of matrix metalloproteinase and vascular endothelial growth factor.In conclusion, the present study has assessed the in vitro antiproliferative/antiangiogenic potential of HCE. These results generate a rationale for in vivo efficacy studies with horse chestnut in preclinical cancer models.

Ceramides and their roles in programmed cell death.

Programmed cell death plays a crucial role in maintaining the homeostasis and integrity of multicellular organisms, and its dysregulation contributes to the pathogenesis of many diseases. Programmed cell death is regulated by a range of macromolecules and low-molecular messengers, including ceramides. Endogenous ceramides have different functions, that are influenced by their localization and the presence of their target molecules. This article provides an overview of the current understanding of ceramides and their impact on various types of programmed cell death, including apoptosis, anoikis, macroautophagy and mitophagy, and necroptosis. Moreover, it highlights the emergence of dihydroceramides as a new class of bioactive sphingolipids and their downstream targets as well as their future roles in cancer cell growth, drug resistance and tumor metastasis.

Divergent access to a novel 3,4-diaminophytosphingosine-like ceramide via sequential Overman rearrangement.

A straightforward approach to a novel phytosphingosine-like ceramide has been accomplished. The cornerstone features of this divergent synthesis are a cascade Overman rearrangement of tris(imidate) to introduce three desired stereogenic centres via sequential chirality transfer and an effective olefin cross-metathesis to install a long side chain. The final unusual phytoceramides were evaluated for their capacity to inhibit the proliferation of cancer cell lines. The preliminary results revealed that compound 21 exhibits promising anticancer activity against HeLa and HCT-116 cells as well as the excellent selectivity in cytotoxicity (malignant vs non-malignant cell lines).

Straightforward access to novel cytotoxic phytosphingosine-like aminotriols from l-erythrose chiron.

A divergent approach to a small library of long-chain 6-amino-1,4,5-triols as novel phytosphingosine-type entities, together with their preliminary cytotoxic evaluation, was achieved. Construction of the target compounds addressed two key aspects. First, the installation of a carbon-nitrogen bond via two prototypes of [3,3]-sigmatropic rearrangements and second the introduction of an alkyl side chain unit by using a late stage olefin cross-metathesis process. As shown in cell viability experiments, the corresponding HCl salts proved to be the most cytotoxic derivatives among all the tested substances, with IC50 values in the lower micromolar range on the Jurkat, HeLa and HCT-116 cell lines.

Synthesis and in vitro anticancer activity of penaresidin-related stereoisomeric analogues.

A straightforward route to penaresidin-based derivatives with an unsubstituted alkyl side chain was developed. To construct these stereoisomeric azetidene-derived alkaloids, [3,3]-sigmatropic rearrangements followed by late stage olefin cross metathesis and an intramolecular nucleophilic type substitution were involved as the key transformations. The protected d-ribofuranose was chosen as the sole chiral source. The ability of target molecules to inhibit cancer cells proliferation was evaluated on a panel of five malignant cell lines.

Jaspine B Hydrochloride-induced Apoptosis in HeLa Cells Is Associated With Disrupted Sphingolipid Metabolism and Ceramide Overload.

BACKGROUND/AIM: A series of experiments on HeLa cells were conducted to provide new information concerning the anti-cancer properties of jaspine B hydrochloride (JBH). MATERIALS AND METHODS: HeLa cells treated with 0.5 µmol/l JBH for 24, 48, and 72 h underwent flow cytometric analysis of the cell cycle, and measurement of phosphatidylserine externalization, mitochondrial membrane potential (MMP), casp-3 activation, cleavage of PARP, ceramide levels, aSMase activity, and Bcl-2 release. nSMase activity was measured by a colorimetric assay. Gene expression was determined by qRT-PCR. Immunocytochemistry was performed to detect p21 and p27 expression. RESULTS: JBH-induced apoptosis in HeLa cells associated with externalization of phosphatidylserine, reduced MMP, activation of casp-3, and cleavage of PARP as well as up-regulation of TNF-α, FasL, and casp-8. Significant increase in nSMase activity, ceramide levels, Bcl-2 release (predominantly in the inactive form), and pro-apoptotic nuclear localization of p21 and p27 were also detected. CONCLUSION: JBH-induced apoptosis in HeLa cells is associated with disrupted sphingolipid homeostasis resulting in increased ceramide levels.

Cytotoxic effect of mistletoe (Viscum album L.) extract on Jurkat cells and its interaction with doxorubicin.

Mistletoe preparations are frequently used by cancer patients because of their ability to stimulate the immunity and to improve the quality of life. Moreover mistletoe and its active substances (especially lectins) possess cytotoxic effect on various cancer cell lines. However, only little is known about its interaction with anticancer drugs. Therefore the cytotoxic and apoptosis-inducing effects of aqueous mistletoe extract (VA) and its interaction with doxorubicin (DOXO) were investigated in Jurkat cells. The results show that VA extract as well as DOXO exert cytotoxic effects on Jurkat cells in a dose-dependent manner. Cytotoxicity of DOXO was much stronger (LC(50) = 11.68 ng/mL) than that of VA extract (LC(50) = 35.67 microg/mL). Their combination led to synergism only at those concentrations that were highly cytotoxic alone. Both substances (alone and in combination) induced DNA fragmentation in Jurkat cells. In conclusion, an aqueous extract prepared from mistletoe tops exerted cytotoxic and apoptosis-inducing effects on Jurkat cells alone as well as in combination with DOXO.

Effect of selected flavones on cancer and endothelial cells.

In our study we used quercetin (3,3 ,4 ,5,7-pentahydroxyflavone) as the reference standard to compare antiproliferative and antiangiogenic effects of chrysin (5,7-dihydroxyflavone) and 3-hydroxyflavone. Our data indicates that chrysin and 3-hydroxyflavone showed significantly higher cytotoxic effect than reference standard quercetin. These tested agents significantly decreased cell survival with the efficacy of 65-85% at the concentration 100 micromol/l for HUVEC, lung carcinoma and leukemic cells being the most sensitive. Cell cycle analysis indicates that quercetin and 3-hydroxyflavone might affect the cell cycle of Jurkat cells by a similar or the same mechanism of action which lead to G2/M arrest as well as to an increase in sub-G0/G1 fraction. Treatment of Jurkat cells with chrysin resulted only increase in the fraction of cells with sub-G0/G1 DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis was confirmed by DNA fragmentation and by staining with annexin V. All three tested flavones inhibited endothelial cell migration after 24 h of incubation at a concentration 100 micromol/l. At a lower concentration (10 micromol/l) only quercetin significantly inhibited migration of endothelial cells. Furthermore, in our experiments decreased secretion of matrix metalloproteinases (MMP-2 and MMP-9) was observed after a 72 h treatment with quercetin. No decrease in secretion of MMP-2 and MMP-9 was seen after chrysin and 3-hydroxyflavone treatment. On the other hand, our results showed that none of three flavonoids blocked microcapillary tube formation. Further studies are necessary to investigate the mechanism of action and to find out the relationship between the structure, character and position of substituents of natural substances and their biological activities.