Design, synthesis, and cytotoxicity screening of new synthesized imidazolidine-2-thiones as VEGFR-2 enzyme inhibitors.

A series of imidazolin-2-thione derivatives was synthesized and structurally confirmed through the use of different spectroscopic techniques such as infrared, nuclear magnetic resonance, and mass spectrometry along with elemental analyses. The breast cancer cell line MCF-7 was utilized in the evaluation of the cytotoxic activity of the prepared molecules. The tested molecules 3 and 7 exhibited the best results on MCF-7 cells, with mean IC50 values of 3.26 and 4.31 µM, respectively. The results of the VEGFR-2 assay indicated that compounds 3 and 7 displayed a good inhibition of the VEGFR-2 kinase enzyme. Additionally, DNA flow cytometry of compounds 3 and 7 showed cell cycle arrest at the G0/G1 phase, cell apoptosis, and marked DNA fragmentation in MCF-7 cells. Finally, compounds 3 and 7 were proved to upregulate the activation of effector caspase-3/7, as presented by the caspase-3/7 green flow cytometry assay.

Abnormal expression of TBX4 during anorectal development in rat embryos with ethylenethiourea-induced anorectal malformations

BACKGROUND: To assess the expression of T-box transcription factor 4 (TBX4) during the anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformations (ARM) rat embryos. METHODS: Anorectal malformations was induced by ETU on the 10th gestational day (E10) in rat embryos. Spatiotemporal expression of TBX4 was evaluated in normal (n = 490) and ETU-induced ARM rat embryos (n = 455) from E13 to E16 by immunohistochemical staining, Western blot analysis and real-time RT-PCR. RESULTS: In the normal embryos, immunohistochemical staining revealed that TBX4 expression was detected in the epithelium of hindgut and urorectal septum (URS) on E13. TBX4-immunopositive cells were increased significantly in the epithelium of hindgut and URS, the future anal orifice part of cloacal membrane on E14. On E15, abundant stained cells were observed in the rectum, URS and dorsal cloacal membrane and the expression of positive cells reached its peak. On E16, only sporadic positive cells were distributed in the epithelium of the distal rectum. In the ARM embryos, the hindgut/rectum, URS and dorsal cloacal membrane were faint for TBX4 immunohistochemical staining. In the normal group, TBX4 protein and mRNA expression showed time-dependent changes in the hindgut/rectum from E13 to E16 on Western blot and real-time RT-PCR. On E13 and E15, the expression level of TBX4 mRNA in the ARM group was significantly lower than that in the normal group (P < 0.05). On E15, the expression level of TBX4 protein in the ARM group was significantly lower than that in the normal group (P < 0.05). CONCLUSIONS: The expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.

Synthetic Lethality of a Novel Small Molecule Against Mutant KRAS-Expressing Cancer Cells Involves AKT-Dependent ROS Production.

AIMS: We recently reported the death-inducing activity of a small-molecule compound, C1, which triggered reactive oxygen species (ROS)-dependent autophagy-associated apoptosis in a variety of human cancer cell lines. In this study, we examine the ability of the compound to specifically target cancer cells harboring mutant KRAS with minimal activity against wild-type (WT) RAS-expressing cells. RESULTS: HCT116 cells expressing mutated KRAS are susceptible, while the WT-expressing HT29 cells are resistant. Interestingly, C1 triggers activation of mutant RAS, which results in the downstream phosphorylation and activation of AKT/PKB. Gene knockdown of KRAS or AKT or their pharmacological inhibition resulted in the abrogation of C1-induced ROS production and rescued tumor colony-forming ability. We also made use of HCT116 mutant KRAS knockout (KO) cells, which express only a single WT KRAS allele. Exposure of KO cells to C1 failed to increase mitochondrial ROS and cell death, unlike the parental cells harboring mutant KRAS. Similarly, mutant KRAS-transformed prostate epithelial cells (RWPE-1-RAS) were more sensitive to the ROS-producing and death-inducing effects of C1 than the vector only expressing RWPE-1 cells. An in vivo model of xenograft tumors generated with HCT116 KRAS(WT/MUT) or KRAS(WT/-) cells showed the efficacy of C1 treatment and its ability to affect the relative mitotic index in tumors harboring KRAS mutant. INNOVATION AND CONCLUSION: These data indicate a synthetic lethal effect against cells carrying mutant KRAS, which could have therapeutic implications given the paucity of KRAS-specific chemotherapeutic strategies. Antioxid. Redox Signal. 24, 781-794.

Expression of sodium-iodide symporter mRNA in the thyroid gland of Xenopus laevis tadpoles: developmental expression, effects of antithyroidal compounds, and regulation by TSH.

The uptake of iodide represents the first step in thyroid hormone synthesis by thyroid follicular cells and is mediated by the sodium-iodide symporter (NIS). In mammals, expression of NIS is stimulated by TSH and transcription of the NIS gene involves regulation by the thyroid-specific transcription factors Pax8 and Nkx2.1. In this study, we examined the mRNA expression of NIS, Pax8 and Nkx2.1 in the thyroid gland of Xenopus laevis tadpoles by semi-quantitative reverse transcriptase (RT)-PCR. During spontaneous metamorphosis, NIS mRNA expression was low in premetamorphic tadpoles, increased throughout prometamorphosis, and peaked at climax stage 60. Analysis of TSH beta-subunit (TSHbeta) mRNA in the pituitary of the same tadpoles revealed a close temporal relationship in the expression of the two genes during metamorphosis, suggesting a regulatory role of TSH in the developmental expression of NIS. Treatment of tadpoles with goitrogenic compounds (sodium perchlorate and ethylenethiourea) increased TSHbeta mRNA expression (approximately twofold) and caused thyroid gland hyperplasia, confirming that feedback along the pituitary-thyroid axis was operative. Analysis of gene expression in the thyroid gland revealed that goitrogen treatment was correlated with increased expression of NIS mRNA (approximately 20-fold). In the thyroid gland organ culture experiments, bovine TSH (bTSH; 1 mU/ml) strongly induced NIS mRNA expression. This effect was mimicked by co-culture of thyroid glands with pituitaries from stage 58 tadpoles and by agents that increase intracellular cAMP (forskolin, dibutyryl-cAMP). In addition, it could be shown that thyroid glands of X. laevis tadpoles express Pax8 and Nkx2.1 mRNA in a developmentally regulated manner and that ex vivo treatment of thyroid glands with bTSH, forskolin, and cAMP analogs increased the expression of Pax8 and Nkx2.1 mRNA. This is the first report on developmental profiles and hormonal regulation of thyroid gland gene expression in amphibian tadpoles and, together, results reveal a critical role of TSH in the regulation of NIS mRNA expression in the thyroid gland of X. laevis tadpoles.

Using small molecules to overcome drug resistance induced by a viral oncogene.

We used small molecule screening to discover compounds and mechanisms for overcoming E6 oncogene-mediated drug resistance. Using high-throughput screening in isogenic cell lines, we identified compounds that potentiate doxorubicin's lethality in E6-expressing colon cancer cells. Such compounds included quaternary ammonium salts, protein synthesis inhibitors, 11-deoxyprostaglandins, and two additional classes of compounds-analogs of 1,3-bis(4-morpholinylmethyl)-2-imidazolidinethione (a thiourea) and acylated secondary amines that we named indoxins. Indoxins upregulated topoisomerase IIalpha, the target of doxorubicin, thereby increasing doxorubicin lethality. We developed a photolabeling strategy to identify targets of indoxin and discovered a nuclear actin-related protein complex as a candidate indoxin target.

Relationship of mitochondrial function and cellular adenosine triphosphate levels to pMC540 and merodantoin cytotoxicity in MCF-7 human breast cancer cells.

In previous studies we have reported that preactivated merocyanine 540 (pMC540) and its chemically synthesized isolates merocil and merodantoin mediate their preferential cytotoxicity towards certain types of malignant cells including human breast cancer cells in vitro and in vivo. The mechanism of cytotoxic action appears to be, in part, via initial interaction with topoisomerase II leading to apoptosis. To further build upon these findings we now show that pMC540 and merodantoin disrupt mitochondrial morphology and function in intact MCF-7 human breast cancer cells as seen by their causing the release of rhodamine 123 from prestained cells, a rapid reduction in ATP levels, inhibition of succinate dehydrogenase activity and oxygen consumption. These data suggest that mitochondria may also be an important target for the cytotoxic action of pMC540 and merodantoin mediated through disruption of the energy balance.

Growth inhibitory effects of pMC540 and merodantoin on established MCF-7 human breast tumor xenografts.

The effect of preactivated merocyanine 540 (pMC540) and one of its chemically synthesized active isolate merodantoin on established human MCF-7 human breast tumor xenografts was investigated. Preactivation is a novel photochemical method for the production of chemotherapeutic compounds that exert their biological effects independent of light. These compounds thus produced, are cytotoxic to human breast cancer cells in vitro and in vivo but only minimally cytotoxic towards normal cells. Nude mice bearing established breast tumors (with or without exogenous estradiol) received injections of pMC540 (250 mg/kg) or merodantoin (75 mg/kg) with or without concurrent treatment with tamoxifen. Treatment with pMC540 and merodantoin caused a 74% and 84% inhibition of tumor growth respectively. Combination of these drugs with tamoxifen did not produce a significant enhancement of growth inhibition. In the absence of exogenous estradiol, identical treatment with pMC540 and merodantoin resulted in 41% and 25% inhibition of tumor growth respectively. Both agents caused a significant (59%) inhibition of growth of estrogen independent human breast tumors established from MDA-MB-435 cells. These results show that photochemically generated novel compounds in pMC540 are effective in suppressing the growth of established human MCF-7 and MDA-MB-435 breast tumor xenografts.

Correlation between DNA topoisomerase II activity and cytotoxicity in pMC540 and merodantoin sensitive and resistant human breast cancer cells.

We have shown previously that preactivated merocyanine 540 (pMC540) and merodantoin appear to mediate their cytotoxic effects via interaction with Topo II. Now, we demonstrate a correlation between DNA Topo II activity and drug-sensitive (MCF-7) and -insensitive (MDA-MB-231) breast cancer cell lines. Further studies indicate that MDA-MB-231 cells are insensitive to the cytotoxic and DNA cleavage effects of pMC540 and merodantoin. This loss of sensitivity is not associated with M(r) 170,000 P-glycoprotein over expression. However, in drug insensitive cells, the Topo II catalytic activity in crude nuclear extract was reduced two- to-three-fold and in cellular extracts was virtually absent as determined by decatenation of kDNA. Topoisomerase I activities appeared similar in extracts from MCF-7 and MDA-MB-231 cell lines. Drug-induced DNA cleavage was reduced two-to-threefold in nuclear extracts from MDA-MB-231. m-AMSA was more effective in inhibiting the decatenation activity in the nuclear extracts from MDA-MB-231 as compared to MCF-7 cells. Western blot analysis of whole-cell lysates revealed undetectable immunoreactivity of Topo II in the drug-insensitive cells. These data indicate that insensitivity of MDA-MB-231 to pMC540 and merodantoin is in part due to the reduced drug-induced formation of the cleavage complex and Topo II (170 kD) enzyme content.

Topoisomerase II-dependent novel antitumor compounds merocil and merodantoin induce apoptosis in Daudi cells.

Three photoproducts of merocyanine 540 have been isolated, chemically characterized and synthesized. Two of these photoproducts, merocil and merodantoin, show significant antitumor activity in vitro and in vivo while demonstrating minimal toxicity to normal cells and tissues. Treatment of lymphoma cells with these compounds resulted in a rapid decline in macromolecular synthesis, DNA fragmentation inhibitable by actinomycin D and cycloheximide, and a marked rise in intracellular calcium. In vitro analysis revealed that activity of these compounds is dependent on topoisomerase II. These results are discussed in terms of the novel class of topoisomerase II-dependent compounds for potential use in chemotherapy.

In vitro and in vivo growth suppression of MCF-7 human breast cancer by novel photoproducts and tamoxifen.

BACKGROUND: Preactivation is a novel photochemical method for the production of chemotherapeutic compounds that exert their biologic effects independent of light. The compounds that are produced, preactivated merocyanine 540 (pMC540) and merodantoin, are cytotoxic to cultured human breast cancer cells but are only minimally cytotoxic toward normal cells. Their effects against breast cancer have not been studied in vivo. METHODS: Estrogen-stimulated human MCF-7 breast adenocarcinoma cells were grown as solid tumors in athymic carrier mice. Animals bearing defined sizes of subcutaneously transplanted solid breast tumors received injections of pMC540 (250 mg/kg) with or without concurrent treatment with tamoxifen. Growth inhibitory effects of merodantoin (N,N'-dibutyl-2-thio-4,5-imidazolidion) on the breast tumor growth were determined. RESULTS: Direct injection of established tumors with eight doses of pMC540 (250 mg/kg) administered on alternate days resulted in significant tumor regression (P = 0.002). In three of seven animals, palpable tumors could not be detected after this treatment (16 days). Treatment through intramuscular injections (20 doses) with pMC540 (250 mg/kg) also caused a significant suppression of tumor area (P = 0.004; P = 0.0882; P = 0.0903) and a marginally significant suppression of tumor weight and volume, respectively. Combined treatment with tamoxifen and pMC540 (100 mg/kg) caused a 67% suppression of breast tumor growth. Treatment with 20 doses of merodantoin (75 mg/kg) suppressed the growth of breast tumors by 98%. CONCLUSION: To the authors' knowledge, these results show for the first time that photochemically generated novel compounds in pMC540 alone and in combination with tamoxifen are effective in suppressing in vivo growth of xenografted human MCF-7 breast tumors.

Rat hepatic microsomal metabolism of ethylenethiourea. Contributions of the flavin-containing monooxygenase and cytochrome P-450 isozymes.

The contributions of the rat hepatic flavin-containing monooxygenase (FMO) and cytochrome P-450 isozymes (P-450) in the ethylenethiourea (ETU) mediated inactivation of P-450 isozymes and covalent binding of the compound to microsomal proteins were investigated. In vitro, ETU was found to inhibit P-450 marker activities in microsomes obtained from untreated (UT) and phenobarbital (PB), beta-naphthoflavone (BNF), and dexamethasone (DEX) pretreated rats. This inhibition was dependent on the presence of NADPH and was completely abolished by coincubation with glutathione (GSH). Heat treatment of microsomes prior to ETU-mediated P-450 inactivation led to diminished loss of P-450 marker activities in microsomes obtained from UT and PB-pretreated, but not BNF- or DEX-pretreated rats, suggesting FMO involvement in the inactivation of some P-450 isozymes. Covalent binding of [14C]ETU to microsomal proteins was found to be NADPH-dependent and enhanced with BNF or DEX pretreatment of rats. This binding was completely inhibited by coincubation with GSH. Heat treatment of microsomes and P-450 inactivation studies indicated a predominant role of FMO in the observed covalent binding. Addition of the sulfhydryl reagents dithiothreitol (DTT) or GSH after the incubation of microsomes, [14C]ETU, and NADPH resulted in the complete release of bound ETU, suggesting the reduction of disulfide bonds between oxidized ETU and protein sulfhydryls. Microsomal heme content was not decreased following incubation of microsomes with ETU and NADPH, and P-450 appeared to be converted to P-420.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of ethylene thiourea administration upon rat liver cells.

The effect of ethylene thiourea (ETU) upon liver cells was evaluated in male Sprague-Dawley rats. ETU was administered ad libitum in drinking water at concentrations of 1, 5, 50, and 500 ppm for time intervals of 1, 2, and 5 days, 1 and 2 weeks, 1, 2, 4, and 8 months. Two additional groups of control animals received ETU-free drinking water or a diet supplemented with 0.06% 3-MeDAB. Electron microscopic evaluation of tissue samples could detect no changes in liver cell morphology of rats receiving 1, 5, or 50 ppm ETU for up to 8 months. By contrast, rats receiving 500 ppm ETU exhibited alterations in hepatic cell morphology after 4 months of exposure. These alterations included a dramatic increase in the amount of smooth endoplasmic reticulum (SER) with a concomitant reduction in rough endoplasmic reticulum (RER), and a relocation of microbodies and mitochondria to the periphery of the SER. No alterations were seen at the shorter time intervals. These changes probably represent a response to the sustained ingestion of high concentrations of highly toxic materials and most likely do not represent a specific response to ETU. No tumors were detected in any of the samples examined or in controls receiving ETU-free drinking water. Animals receiving 3-MeDAB in their diet all developed hepatic tumors within 4 months.

Efeitos da etileno-tiouréia (ETU) e da etileno-uréia (EU) sobre os níveis de lipoperoxidaçäo hepática em ratos / Effects of ethylen-thiourea (ETU) and of ethylene-urea (EU) on hepatic lipid peroxidation levels

A etileno-tiouréia (ETU) administrada a ratos exerce um efeito protetor contra a peroxidaçäo de lipídios hepáticos, medida pela produçäo de malonildialdeído. Esse efeito é dose e tempo dependente e deve-se provavelmente a presença de um grupo tiol na molécula da ETU, uma vez que a etileno-uréia (EU) um composto semelhante a ETU, mas que näo contém S näo altera a peroxidaçäo de lipídios em fígado. O maneb, um fungicida do grupo dos etilenobisditiocarbamatos na dose empregada (5.000 ppm na dieta), näo é täo eficaz quanto a ETU na proteçäo da peroxidaçäo de ácidos graxos polinsaturados de membranas, em fígado, embora esta seja produzida "in vivo" durante a biotransformaçäo do maneb

The effect of maneb, zineb, and ethylenethiourea on the humoral activity of the pituitary-thyroid axis in rat.

Ethylenebisdithiocarbamates (EBDCs) maneb and zineb are widely used fungicides the final metabolite of which is ethylenethiourea (ETU). EBDCs distort the humoral activity of the thyroid gland, and ETU is especially active in this respect. Male Wistar rats were exposed either to exogenous (100 ng i.p.) or endogenous (+4 degrees C) TRH stimulation. ETU (100-500 mg/kg i.p.) caused no changes in serum TSH levels whereas zineb (70-500 mg/kg i.p.) significantly decreased the bursts induced by cold or exogenous TRH. Maneb (20-200 mg/kg i.p.) significantly decreased the cold-induced TSH response while it had no effect on the TRH-stimulated TSH secretion. None of the agents caused significant changes in serum T3 or T4 levels. It seems that maneb, and zineb, but not ETU, inhibit rat TSH secretion through an action on the endogenous TRH at the hypothalamic or pituitary level. The mechanism behind this action may be the inhibition of dopamine-beta-hydroxylase.

The effect of oxidation of the sulfur atom on the mutagenicity of ethylenethiourea.

Ethylenethiourea is metabolized in mice by oxidation of the sulfur atom to form 2-imidazolin-2-yl sulfenate. Ethylenethiourea itself is a carcinogen, goitrogen, teratogen and a weak bacterial mutagen. The mutagenicities of ethylenethiourea, 2-imidazolin-2-yl sulfenate and their nitroso derivatives were compared in direct bacterial tests and in the host-mediated assay. In all the test systems applied, 2-imidazolin-2-yl sulfenate was less mutagenic than the parent compound.

Antimitochondrial effects of thioacetamide and ethylenethiourea in human and yeast cell cultures.

Cytological studies in the light microscope showed that thioacetamide (TAA) depressed the mitotic index in cultures of skin fibroblasts at the lowest concentrations used (100 µg/ml). At high concentration (1 mg/ml), TAA tended to cause aberration in nuclear morphology. Ethylenethiourea (ETU) had no effect on either mitotic index or nuclear morphology at 1 mg/ml. Fibroblast cultures treated with 1 mg/ml TAA and cultures grown in the presence of 2 mg/ml ETU were studied by electron microscopy. In some TAA-treated cells there was unfolding of the nuclear membrane and enlargement and granulation of the nucleolus, but these effects were not correlated. In all cells, TAA caused severe and characteristic damage to the majority of mitochondria, whether or not there were nuclear aberrations. The organelle showed extensive swelling of the cristae of the inner membrane and an increase in matrix density. Ultrastructure of other cell components appeared to be unaffected by this treatment. In ETU-treated cells some less severe swelling of inner mitochondrial membranes was seen and only in a minority of cells, whilst all other cell structures appeared normal. Similar membrane swelling and increase in matrix density was seen in isolated rat liver mitochondria after incubation with TAA, indicating a direct antimitochondrial effect of the carcinogen.When yeast cells were treated with TAA and ETU, primary antimitochondrial activity of these compounds was apparent from (1) inhibition of growth in non-fermentable medium, (2) selective blockage of mitochondrial protein synthesis and (3) induction of mitochondrial mutations. TAA was much more effective than ETU in all these respects.

Hepatic RNA synthesis in rats treated with ethylene thiourea.

The ability of ethylene thiourea (ETU) to inhibit RNA synthesis in rat liver was investigated. This compound, a possibly carcinogenic metabolite to the ethylene-bis-dithiocarbamate fungicides, was administered to rats by intraperitoneal injection, by nasogastric tube and in the diet, and rates of RNA synthesis were determined. By contrast with the carcinogens thioacetamide and acetylaminofluorene, high doses of ETU administered by any of these routes failed to inhibit the synthesis of nuclear or cytoplasmic RNA. In this respect ETU appears to differ from most hepatocarcinogens in its effect on cellular metabolism.

[Mutagenic activity of basfungin in its combined action with sodium nitrite]. / Prouchvane na mutagennata aktivnost na basfungin pri kombiniranoto mu deistvie s natriev nitrit.

The mutagenic activity of basfungin, when acting in combination with sodium nitrite, was studied in a subacute experiment, using the method of bone marrow cytogenetic analysis. The results were compared with those in groups in which basfungin and sodium nitrite were applied separately, as well as with the results of the parallel control. The mutagenic effect in the combined action group did not essentially differ from the effect of application of basfungin alone, both qualitatively and diaulitatively. Interesting results were obtained in the group receiving sodium nitrite alone - the percentage of cells with chromosomal aberrations was significantly than the control values.

[Difference in the sensitivity of the hamster and the rat to the effects of long-term administration of ethylenethiourea]. / Différence de sensibilité du hamster et du rat vis-à-vis des effets de l'administration à long terme de l'éthylène thiourée

Rats and hamsters were fed ETU at levels of 0, 5, 17, 60, 200 mg/kg in the diet. Body weights, food consumption, seric enzyme activities (GPT, alkaline phosphatase), hepatic enzyme activities (GPT, alkaline phosphatase G6PDH), cholesterolemia, thyroid weight and others organs, histology were the criteria studied. ETU was found causing hypercholesterolemia for the 2 species at 5 mg/kg dietary levels. Thyroid impairement is predominant in rat and hepatic impairment is predominant in hamster. ETU was found to be not carcinogenic for hamsters even at 200 mg/kg level and carcinogenic for rats at 60 mg/kg level for males and 200 mg/kg level for females.