Synthesis and anti-tumor activities of N'-benzylidene-2-(4-oxothieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazone derivatives

A compound with a cyclic thienopyrimidine moiety and an aceto-hydrazone moiety in its chemical structure was discovered in a cell-based screening to have noticeable cytotoxicity on several tumor cell lines. A total of 38 derivatives of this compound were synthesized at five steps with high yields. These compounds were tested in standard MTT assays, and several compounds exhibited improved cytotoxic activities. The most potent compounds have IC(50) values of 10-20 μM on A549, HeLa, and MBA-MD-231 tumor cells. Flow cytometry analysis of several active compounds and subsequent examination of caspase activation indicate that they induce caspase-dependent apoptosis in tumor cells. In addition, these compounds do not have obvious effect on a normal cell line HEK-293T, demonstrating the desired selectivity against tumor cells. Results from a fluorescence polarization-based in vitro binding assay indicate that this class of compounds does not significantly interrupt the interactions between Mcl-1 and Bid. Their cytotoxicity is achieved presumably through other mechanisms.     

Smoke,pheromone and kairomone olfactory receptor neurons in males and females of the pine sawyer Monochamus galloprovincialis (Olivier) (Coleoptera: Cerambycidae)

The response of antennal olfactory receptor neurons (ORNs) of Monochamus galloprovincialis to several odourants was tested using single sensillum electrophysiology. Behaviourally active pheromone, and kairomone (host and sympatric bark beetle pheromone) odours were tested alongside smoke compounds released by burnt wood that are potentially attractive to the insect. The antennae bore several types of sensilla. Two plate areas in the proximal and distal ends of each antennal segment were covered with basiconic sensilla that responded to the odour stimuli. Sensilla basiconica contained one or two cells of different spike amplitude. The 32 male and 38 female ORNs tested responded with excitations or inhibitions to the different plant odours. In general the response of male and female receptors was very similar so they were pooled to perform a cluster analysis on ORN responses. Six ORNs were clearly specialised for pheromone reception. Responses to kairomone and smoke odours were less specific than those of pheromone, but a group of 9 cells was clearly excited by smoke compounds (mainly eugenol and 4-methyl 2-methoxyphenol), a group of 8 cells was very responsive to α-pinene, β-pinene and cis-verbenol, and a group of 14 cells responded to a wider range of compounds. The rest of the cells (47%) were either non-responsive or slightly inhibited by smoke compounds. Dose–response curves were obtained for several compounds. Different compounds induced significantly different latencies and these appeared to be unrelated to their boiling point.     

Differential activation of wild-type estrogen receptor alpha and C-terminal deletion mutants by estrogens, antiestrogens and xenoestrogens in breast cancer cells

17beta-Estradiol (E2), diethylstilbestrol (DES) and several synthetic (or xenoestrogenic) compounds induced transactivation in MCF-7 or MDA-MB-231 cells transfected with wild-type estrogen receptor alpha (ERalpha) and a construct (pERE(3)) containing three tandem estrogen responsive elements (EREs) linked to a luciferase gene. In contrast, the antiestrogens ICI 182,780 and 4-hydroxytamoxifen (4-OHT) were inactive in this assay. We have investigated the effects of these compounds and several structurally-diverse estrogenic compounds on transactivation in cells transfected with pERE(3) and wild-type ERalpha, mutant ERalpha (1-553), and ERalpha (1-537) containing deletions of amino acids 595-554 and 595-538, respectively. These constructs were used to develop an in vitro assay to distinguish between different structural classes of estrogenic compounds. The results obtained using these constructs were highly cell context- and structure-dependent. Neither E2- nor diethylstilbestrol-induced transactivation in MCF-7 (or MDA-MB-231) cells transfected with pERE(3)/ERalpha (1-537) due to partial deletion of helix 12; however, octylphenol and nonlylphenol, resveratrol (a phytoestrogen), kepone and 2',3',4',5'-tetrachloro-4-biphenylol were "estrogenic" in MCF-7 cells transfected with pERE(3)/ERalpha (1-537). Moreover, the structure-dependent estrogenic activities of several synthetic estrogens (xenoestrogens) in MDA-MB-231 cells were different than those observed in MCF-7 cells. These results demonstrate that the estrogenic activity of many synthetic compounds do not require activation function 2 (AF-2) of ERalpha and are mechanistically different from E2. These data suggest that xenoestrogens are selective ER modulators (SERMs).     

Synthesis of 26-hydroxy-22-oxocholestanic frameworks from diosgenin and hecogenin and their in vitro antiproliferative and apoptotic activity on human cervical cancer CaSki cells

Certain steroidal compounds have demonstrated an antiproliferative effect against several tumor cell lines; however, their complete role on cancer cells is not currently established. Herein, we report the synthesis and evaluation of two new 26-hydroxy-22-oxocholestanic steroids on cervical cancer CaSki cells. The title compounds were prepared from diosgenin and hecogenin in excellent yields. We determined their effect on cell proliferation, cell cycle, and cell death. The cytotoxic effect of the title compounds on CaSki and human lymphocytes was also evaluated, indicating that the main cell death process is not necrosis; the null effect on lymphocytes implies that they are not cytotoxic. The observation of apoptotic bodies as well as the increase in the expression of active caspase-3 along with the fragmentation of DNA confirmed that such new cholestanic frameworks induced apoptosis in tumor cells. Significantly, their antiproliferative activity on tumor cells did not affect the proliferative potential of normal fibroblasts from cervix and peripheral blood lymphocytes. The title compounds show selective antitumor activity and therefore serve as promising lead candidates for further optimization.     

Phosphoproteomic analysis of leukemia cells under basal and drug-treated conditions identifies markers of kinase pathway activation and mechanisms of resistance

Protein kinase signaling is fundamental to cell homeostasis and is deregulated in all cancers but varies between patients. Understanding the mechanisms underlying this heterogeneity is critical for personalized targeted therapies. Here, we used a recently established LC-MS/MS platform to profile protein phosphorylation in acute myeloid leukemia cell lines with different sensitivities to kinase inhibitors. The compounds used in this study were originally developed to target Janus kinase, phosphatidylinositol 3-kinase, and MEK. After further validation of the technique, we identified several phosphorylation sites that were inhibited by these compounds but whose intensities did not always correlate with growth inhibition sensitivity. In contrast, several hundred phosphorylation sites that correlated with sensitivity/resistance were not in general inhibited by the compounds. These results indicate that markers of pathway activity may not always be reliable indicators of sensitivity of cancer cells to inhibitors that target such pathways, because the activity of parallel kinases can contribute to resistance. By mining our data we identified protein kinase C isoforms as one of such parallel pathways being more active in resistant cells. Consistent with the view that several parallel kinase pathways were contributing to resistance, inhibitors that target protein kinase C, MEK, and Janus kinase potentiated each other in arresting the proliferation of multidrug-resistant cells. Untargeted/unbiased approaches, such as the one described here, to quantify the activity of the intended target kinase pathway in concert with the activities of parallel kinase pathways will be invaluable to personalize therapies based on kinase inhibitors.     

Aminosquaraines as potential photodynamic agents: Synthesis and evaluation of in vitro cytotoxicity

The synthesis of several aminosquaraine cationic dyes displaying strong absorption within the so-called phototherapeutic window (650–850 nm) is described. Their cytotoxicity, under dark and illuminated conditions, was tested against several human tumor cell lines (breast, lung, cervical and hepatocellular carcinomas) and non-tumor porcine liver primary cells. All compounds showed to inhibit the growth of the tumor cells upon irradiation more than in the absence of light, in more or less extension, clearly exhibiting photodynamic activity. The photosensitizing ability against some cell lines, together with the low toxicity for the non-tumor primary PLP2 cells displayed by some of the compounds synthetized, turns them into potential candidates as photosensitizers for PDT.     

Novel nitroheterocyclic hypoxic markers for solid tumor: synthesis and biological evaluation

Based on the principle that the nitro-group can quench the fluorescence and can be reduced under hypoxic conditions, several novel nitroheterocyclic compounds without 2-nitroimidazole as potential hypoxic markers were prepared. Although they were synthesized from the same matrix, nitrosubstituted acenaphtho[1,2-b]quinoxaline, these compounds exhibited quite different fluorescence changes when they were differently nitrosubstituted. Their evaluation for imaging tumor hypoxia was carried out in V79 cells in vitro by Fluorescence Microplate Reader. After 3.5 h, the hypoxic-oxic fluorescence differential incubated with A1, A4, and A5 in V79 cells could reach 6, 9, and 11 times differential fluorescence between oxic and hypoxic cells separately, which are suitable for further evaluation as probes for hypoxic cells in tumors in vivo.     

Allyl sulfur compounds and cellular detoxification system: effects and perspectives in cancer therapy

Natural organosulfur compounds (OSCs) have been shown to have chemopreventive effects and to suppress the proliferation of tumor cells in vitro through the induction of apoptosis. The biochemical mechanisms underlying the antitumorigenic and anti-proliferative effects of garlic-derived OSCs are not fully understood. Several modes of action of these compounds have been proposed, and it seems likely that the rate of clearance of allyl sulfur groups from cells is a determinant of the overall response. The aim of this review is to focus attention on the effects of natural allyl sulfur compounds on the cell detoxification system in normal and tumor cells. It has been already reported that several natural allyl sulfur compounds induce chemopreventive effects by affecting xenobiotic metabolizing enzymes and inducing their down-activation. Moreover, different effects of water- and oil-soluble allyl sulfur compounds on enzymes involved in the detoxification system of rat tissues have been observed. A direct interaction of the garlic allyl sulfur compounds with proteins involved in the detoxification system was studied in order to support the hypothesis that proteins possessing reactive thiol groups and that are involved in the detoxification system and in the cellular redox homeostasis, are likely the preferential targets of these compounds. The biochemical transformation of the OSCs in the cell and their adducts with thiol functional groups of these proteins, could be considered relevant events to uncover the anticancer properties of the allyl sulfur compounds. Although additional studies, using proteomic approaches and transgenic models, are needed to identify the molecular targets and modes of action of these natural compounds, the allyl sulfur compounds can represent potential ideal agents in anticancer therapy, either alone or in association with other antitumor drugs.     

Molecular cloning, characterization, and downregulation of an acyltransferase that catalyzes the malonylation of flavonoid and naphthol glucosides in tobacco cells

Tobacco cells (Nicotiana tabacum L. Bright Yellow T-13) exposed to harmful naphthols accumulate them as glucosylated and further modified compounds [Taguchi et al. (2003a) Plant Sci. 164, 231-240]. In this study, we identified the accumulated compounds to be 6'-O-malonylated glucosides of naphthols. Cells treated with various phenolic compounds accumulated the flavonoids mainly as malonylglucosides. To clarify the function of this malonylation in tobacco, we isolated the cDNA encoding a malonyltransferase (NtMaT1) from a cDNA library derived from tobacco cells. The heterologous expression of the gene in Escherichia coli revealed that the recombinant enzyme had malonyltransferase activity against several phenolic glucosides such as flavonoid 7-O-glucosides, flavonoid 3-O-glucosides and naphthol glucosides. The substrate preference of the enzyme was similar to that of the tobacco cell extract. Malonylation activity in the transgenic cells markedly decreased with the suppression of the expression of NtMaT1 mRNA in tobacco BY-2 cells by RNA interference. The compounds administered to the transgenic cells were accumulated in the cells as glucosides or other modified compounds in place of malonylglucosides. These results show that NtMaT1 is the main catalyst of malonylation on glucosides of xenobiotic flavonoids and naphthols in tobacco plants.     

Peripheral coding of bitter taste in Drosophila

Taste receptors play a crucial role in detecting the presence of bitter compounds such as alkaloids, and help to prevent the ingestion of toxic food. In Drosophila, we show for the first time that several taste sensilla on the prothoracic legs detect bitter compounds both through the activation of specific taste neurons but also through inhibition of taste neurons activated by sugars and water. Each sensillum usually houses a cluster of four taste neurons classified according to their best stimulus (S for sugar, W for Water, L1 and L2 for salts). Using a new statistical approach based on the analysis of interspike intervals, we show that bitter compounds activate the L2 cell. Bitter-activated L2 cells were excited with a latency of at least 50 ms. Their sensitivity to bitter compounds was different between sensilla, suggesting that specific receptors to bitter compounds are differentially expressed among L2 cells. When presented in mixtures, bitter compounds inhibited the responses of S and W, but not the L1 cell. The inhibition was effective even in sensilla where bitter compounds did not activate the L2 cell, indicating that bitter compounds directly interact with the S and W cells. Interestingly, this inhibition occurred with latencies similar to the excitation of bitter-activated L2 cells. It suggests that the inhibition in the W and S cells shares similar transduction pathways with the excitation in the L2 cells. Combined with molecular approaches, the results presented here should provide a physiological basis to understand how bitter compounds are detected and discriminated.     

Bioactive triterpene derivatives from latex of two Euphorbia species

We have investigated the antifeedant and toxic effects of 23 semisynthetic terpenoid derivatives obtained through chemical modifications of the major components of Euphorbia resinifera (alpha-euphol and alpha-euphorbol) and E. officinarum (obtusifoliol and 31-norlanostenol) latex on several insect species (Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi), their selective cytotoxicity on insect Sf9 and mammalian CHO cells and their phytotoxic effects on Lactuca sativa. The conversions focused mainly on positions 3,7,11, and 24 with several oxidizing agents. A total of 18 compounds affected S. littoralis growth (IGR). Our results support the importance of the C-3 substituent, suggest the involvement of the C-7 substituent and indicate that the C-3 hydroxyl is not essential for the IGR effect. Overall, Sf9 cells were more sensitive to the active compounds than CHO cells. All of these compounds had non selective moderate phytotoxic effects on radicle elongation of L. sativa.     

Retained platinum uptake and indifference to p53 status make novel transplatinum agents active in platinum-resistant cells compared to cisplatin and oxaliplatin

Despite the clinical success of platinum-containing drugs in the treatment of solid tumors, acquired resistance remains a major obstacle. We previously identified a group of novel transplanaramine or transplatinum compounds based on distinct activity profiles in the NCI-60 panel. In the present study, parental KB-3.1 cells with wild-type p53 and its cisplatin- and oxaliplatin-resistant sublines harboring mutant p53 proteins were used to contrast several transplatinum compounds with cisplatin and oxaliplatin. The transplatinum compounds retained cytotoxic activity in the resistant cell lines. While intracellular accumulation and DNA platination of cisplatin and oxaliplatin was decreased in the resistant cells, the transplatinum compounds both accumulated intracellularly and platinated DNA at comparable levels in all cell lines. Cytoflow analysis confirmed that cisplatin and oxaliplatin alter the cell cycle distribution and result in apoptosis; however, at comparably toxic concentrations, the transplatinum compounds did not alter the cell cycle distribution. Analysis of the cytoplasmic fraction treated with acetone showed that cisplatin and oxaliplatin readily bound to macromolecules in the pellet, whereas a larger percentage of the transplatinum compounds remained in the supernatant. We concluded that, distinct from platinum compounds currently in use, transplatinum compounds accumulate intracellularly in resistant cells at levels comparable to those in drug-sensitive cells, do not affect the cell cycle and thus retain cytotoxicity independent of p53 status and likely have cytoplasmic targets that are important in their activity.     

Tandem screening of toxic compounds on GFP-labeled bacteria and cancer cells in microtiter plates

A 96-well fluorescence-based assay has been developed for the rapid screening of potential cytotoxic and bacteriocidal compounds. The assay is based on detection of green fluorescent protein (GFP) in HeLa human carcinoma cells as well as gram negative (Escherichia coli) and gram positive bacteria (Mycobacterium avium). Addition of a toxic compound to the GFP marked cells resulted in the loss of the GFP fluorescence which was readily detected by fluorometry. Thirty-nine distinct naphthoquinone derivatives were screened and several of these compounds were found to be toxic to all cell types. Apart from differences in overall toxicity, two general types of toxic compounds were detected, those that exhibited toxicity to two or all three of the cell types and those that were primarily toxic to the HeLa cells. Our results demonstrate that the parallel screening of both eukaryotic and prokaryotic cells is not only feasible and reproducible but also cost effective.     

Retained platinum uptake and indifference to p53 status make novel transplatinum agents active in platinum-resistant cells compared to cisplatin and oxaliplatin

Despite the clinical success of platinum-containing drugs in the treatment of solid tumors, acquired resistance remains a major obstacle. We previously identified a group of novel transplanaramine or transplatinum compounds based on distinct activity profiles in the NCI-60 panel. In the present study, parental KB-3.1 cells with wild-type p53 and its cisplatin- and oxaliplatin-resistant sublines harboring mutant p53 proteins were used to contrast several transplatinum compounds with cisplatin and oxaliplatin. The transplatinum compounds retained cytotoxic activity in the resistant cell lines. While intracellular accumulation and DNA platination of cisplatin and oxaliplatin was decreased in the resistant cells, the transplatinum compounds both accumulated intracellularly and platinated DNA at comparable levels in all cell lines. Cytoflow analysis confirmed that cisplatin and oxaliplatin alter the cell cycle distribution and result in apoptosis; however, at comparably toxic concentrations, the transplatinum compounds did not alter the cell cycle distribution. Analysis of the cytoplasmic fraction treated with acetone showed that cisplatin and oxaliplatin readily bound to macromolecules in the pellet, whereas a larger percentage of the transplatinum compounds remained in the supernatant. We concluded that, distinct from platinum compounds currently in use, transplatinum compounds accumulate intracellularly in resistant cells at levels comparable to those in drug-sensitive cells, do not affect the cell cycle and thus retain cytotoxicity independent of p53 status and likely have cytoplasmic targets that are important in their activity.     

Antiparasitic activity of flavonoids and isoflavones against Cryptosporidium parvum and Encephalitozoon intestinalis

Flavonoids, polyphenolic compounds found in plants, have demonstrated activity against several parasites and can augment the efficacy of other drugs by either increasing the uptake or decreasing the efflux of these drugs. We evaluated 11 of these compounds alone or in combination in order to test the hypothesis that flavonoids are effective against Cryptosporidium parvum and Encephalitozoon intestinalis. Using in vitro cell culture assays, HCT-8 cells or E6 cells were infected with C. parvum and E. intestinalis, respectively, and treated with compounds at doses ranging from 1 to 200 microM. We found that six compounds were active against C. parvum. Naringenin and genistein had the greatest activities with EC(50) of 15 and 25 microM, respectively. Two compounds, quercetin and apigenin, had activity against E. intestinalis at EC(50) of 15 and 50 microM, respectively. The EC(50) of trifluralin, a dinitroaniline compound, was decreased significantly when combined with genistein in an in vitro assay, suggesting that compounds may be used alone on in combination with other moderately active drugs to increase efficacy. In addition, induction of apoptosis by these compounds was studied but not observed to be a significant mechanism of action.     

Isolation and inhibitory activity against ERK phosphorylation of hydroxyanthraquinones from rhubarb

Five hydroxyanthraquinone compounds were purified from Chinese medicinal herb rhubarb by microwave-assisted extraction and silica gel column isolation. Their structures were identified by spectroscopic analysis. One of them, emodin, was found to block the ERK phosphorylation of PC3 cells at 20 microM. And two other compounds had potent proliferation inhibitory activity on A431 and PC3 cells at 20 microM. Furthermore, crude extracts revealed an antifungal activity stronger than that of purified compounds against several plant pathogens tested. Together, our study suggests that these compounds are biologically active and may lead to development of anticancer medicine.     

Biosynthesis and utilization of aromatic compounds by Mycobacterium smegmatis with particular reference to the origin of salicylic acid

1. Although Mycobacterium smegmatis could utilize a number of aromatic compounds as sole sources of carbon for growth, it did not appear to be able to use salicylic acid for growth or to metabolize it to any great extent. 2. When M. smegmatis was grown on shikimic acid as sole source of carbon, salicylic acid, anthranilic acid and 3,4-dihydroxybenzoic acid were released into the medium. When it was grown on quinic acid these compounds, together with p-hydroxybenzoic acid, p-hydroxyphenylacetic acid and a number of unidentified compounds, were formed. When it was grown on glucose only small amounts of salicylic acid could be detected. 3. When a washed suspension of cells with a normal iron content was incubated with shikimic acid, only small amounts of aromatic compounds were formed in the medium. When the cells were iron-deficient, substantial amounts of salicylic acid, 3,4-dihydroxybenzoic acid and catechol were formed, together with several other compounds not definitely identified. 4. When washed suspensions of cells, whether iron-sufficient or iron-deficient, were incubated with tryptophan no evidence of formation of salicylic acid, anthranilic acid or phenolic compounds was obtained. Washed suspensions did not convert anthranilic acid into salicylic acid. 5. When cell-free extracts of M. smegmatis were incubated with shikimic acid, or shikimic acid 5-phosphate, traces of anthranilic acid were formed under certain conditions. No formation of salicylic acid or other phenolic compound was observed even when a number of combinations of cofactors and coenzymes were tried.     

Interaction of organic chemicals with P-glycoprotein in the adrenal gland, kidney, and a multidrug-resistant KB cell.

P-glycoprotein (P-gp) is thought to mediate the transport of anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype in tumor cells. However, the function of P-gp in normal tissues is still not well understood. We present evidence indicating that the active efflux of several structurally unrelated organic compounds is mediated by P-gp in multidrug-resistant KB (KB-C2) cells and that these compounds interact with P-gp in the kidney and adrenal gland. The photoactive radioactive calcium channel blocker [3H]azidopine labels a protein of approximately 140 kDa in crude membrane fractions from human kidney and adrenal gland and a 130-kDa protein from bovine adrenal gland. These photolabeled proteins are immunoprecipitated with an anti-P-gp antibody. Photolabeling is inhibited by vinblastine, reserpine, and several other organic chemicals. These data indicate that the kidney and adrenal gland express P-gp (or a protein closely related to P-gp) that can interact with several organic compounds and that the P-gp expressed in these tissues has a drug-binding site similar to that of P-gp in KB-C2 cells. Our findings thus strongly support the hypothesis that P-gp can transport a wide variety of organic chemicals as well as anti-cancer drugs and that one of the physiological functions of P-gp is the excretion of certain classes of organic compounds.     

Cytotoxic and antibacterial activity of 2-oxopurine derivatives

Initial screening of the cytotoxic and antibacterial properties of 6-substituted 2-oxopurines and dihydro-2-oxopurines revealed that several compounds exhibited cytotoxicity against K-562 cells in the same range as the well known antileukemic drug 6-mercaptopurine. Most compounds were also tested for inhibitory effect on a Gram-positive bacterium, Lactobacillus casei, as well as the mycobacterium Mycobacterium tuberculosis. Generally the 2-oxopurines exhibited low antibacterial effect.