In vitro evaluation of cytotoxic potential of essential oil extracted from leaves of Croton heliotropiifolius Kunth in human tumor cells.

Croton heliotropiifolius Kunth, popularly known as "velame," is a shrub that resides in northeastern Brazil. The essential oil of C. heliotropiifolius contains high concentrations of volatile compounds in the leaves and is widely used in folk medicine for many purposes as an antiseptic, analgesic, sedative, and anti-inflammatory agent. Due to the apparent limited amount of information, the aim of this study was to determine the cytotoxic potential of essential oil extracted from leaves of C. heliotropiifolius, utilizing different human cancer cell lines (HL-60, leukemia; HCT-116, colon; MDA-MB435, melanoma; SF295, glioblastoma) and comparison to murine fibroblast L929 cell line. The chemical characterization of the essential oil revealed the presence of large amounts of monoterpenes and sesquiterpenes, the majority of which were aristolene (22.43%), germacrene D (11.38%), ɣ-terpinene (10.85%), and limonene (10.21%). The essential oil exerted significant cytotoxicity on all cancer cells, with low activity on murine L929 fibroblasts, independent of disruption of cell membranes evidenced by absence of hemolytic activity. The cytotoxicity identified was associated with oxidative stress, which culminated in mitochondrial respiration dysfunction and direct or indirect DNA damage (strand breaks and oxidative damage), triggering cell death via apoptosis. Our findings suggest that extracts of essential oil of C. Heliotropiifolius may be considered as agents to be used therapeutically in treatment of certain cancers.

Ethanolic extract from leaves of tithonia diversifolia induces apoptosis in HCT-116 cells through oxidative stress.

Tithonia diversifolia is a perennial bushy plant found in South America with significant ethnopharmacological importance as an antimalarial, antidiabetic, antibacterial, and anticancer agent. The aim of the present study was to determine the cytotoxicity of the ethanolic extract from leaves of T. diversifolia (TdE) on human cancer cell lines (HCT-116, SNB-19, NCIH-460 and MCF-7), as well as the mechanism of action involved in cell death and cellular modulation of oxidative stress. The TdE exhibited significant activity with IC50 values ranging from 7.12 to 38.41 µg/ml, with HCT-116 being the most sensitive cell line. Subsequent experiments were conducted with HCT-116 cell line. TdE decreased the number of viable cells, followed by induction of apoptotic events, increase in mitochondrial membrane permeabilization, and enhanced G2/M phase of the cell cycle. Pro-oxidative effects including elevated acidic vesicular organelle formation, lipid peroxidation, and nitric oxide by-products, as well as reduced levels of intracellular glutathione and reactive oxygen species production were also observed following incubation with TdE, which may lead to DNA damage followed by apoptotic cell death. These results demonstrate the potential of TdE ethanolic leaf extraction for biological activity and enhance the importance of continuing to study natural sources of plants for the development of anticancer agents.

Action mechanism of naphthofuranquinones against fluconazole-resistant Candida tropicalis strains evidenced by proteomic analysis: The role of increased endogenous ROS.

The increased incidence of candidemia in terciary hospitals worldwide and the cross-resistance frequency require the new therapeutic strategies development. Recently, our research group demonstrated three semi-synthetic naphthofuranquinones (NFQs) with a significant antifungal activity in a fluconazole-resistant (FLC) C. tropicalis strain. The current study aimed to investigate the action's preliminary mechanisms of NFQs by several standardized methods such as proteomic and flow cytometry analyzes, comet assay, immunohistochemistry and confocal microscopy evaluation. Our data showed C. tropicalis 24 h treated with all NFQs induced an expression's increase of proteins involved in the metabolic response to stress, energy metabolism, glycolysis, nucleosome assembly and translation process. Some aspects of proteomic analysis are in consonance with our flow cytometry analysis which indicated an augmentation of intracellular ROS, mitochondrial dysfunction and DNA strand breaks (neutral comet assay and γ-H2AX detection). In conclusion, our data highlights the great contribution of ROS as a key event, probably not the one, associated to anti-candida properties of studied NFQs.

Chromomycin A2 induces autophagy in melanoma cells.

The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins.

Natural products as new antimitotic compounds for anticancer drug development.

Cell cycle control genes are frequently mutated in cancer cells, which usually display higher rates of proliferation than normal cells. Dysregulated mitosis leads to genomic instability, which contributes to tumor progression and aggressiveness. Many drugs that disrupt mitosis have been studied because they induce cell cycle arrest and tumor cell death. These antitumor compounds are referred to as antimitotics. Vinca alkaloids and taxanes are natural products that target microtubules and inhibit mitosis, and their derivatives are among the most commonly used drugs in cancer therapy worldwide. However, severe adverse effects such as neuropathies are frequently observed during treatment with microtubule-targeting agents. Many efforts have been directed at developing improved antimitotics with increased specificity and decreased likelihood of inducing side effects. These new drugs generally target specific components of mitotic regulation that are mainly or exclusively expressed during cell division, such as kinases, motor proteins and multiprotein complexes. Such small molecules are now in preclinical studies and clinical trials, and many are products or derivatives from natural sources. In this review, we focused on the most promising targets for the development of antimitotics and discussed the advantages and disadvantages of these targets. We also highlighted the novel natural antimitotic agents under investigation by our research group, including combretastatins, withanolides and pterocarpans, which show the potential to circumvent the main issues in antimitotic therapy.

Selective cytotoxicity of withaphysalins in myeloid leukemia cell lines versus peripheral blood mononuclear cells.

Withaphysalins are C(28)-steroidal lactones structurally based on the ergostane skeleton that possess antiproliferative activity against tumor cell lines. In the present study, the antileukemic actvity of withaphysalin O (1), M (2), and N (3) isolated from Acnistus arborescens, against two leukemic cell lines, HL-60 and K562, was evaluated, and the cytotoxicity compared with the effects on peripheral blood mononuclear cells (PBMC). All tested compounds reduced the number of viable cells of the tumor cell lines after 24 h of exposure, except for compound 2 against the K562 cell line. The reduction was time-and concentration-dependent, and the IC(50) values ranged from 0.7 to 3.5 microM after 72 h of incubation. In addition to the growth inhibitory properties, the drugs decreased DNA synthesis after 24 h of drug exposure evaluated by the 5-bromo-2 -deoxyuridine incorporation method. None of the tested compounds reduced the number of PBMC (IC(50)>20 microM) after 72 h of incubation, in contrast to doxorubicin that decreased viable cells and increased non-viable cells even after 24 h of incubation. Morphological analysis of treated cells using hematoxylin/eosin staining indicated the presence of necrotic cells for all tested compounds in HL-60, confirmed by the use of acridine orange/ethidium bromide staining. In addition to necrotic cells, K562 cells showed morphological alterations consistent with apoptosis.

Molecular mechanism of action of 2-ferrocenyl-1,1-diphenylbut-1-ene on HL-60 leukemia cells.

The aim of this work was to investigate the mechanism of action of 2-ferrocenyl-1,1-diphenylbut-1-ene (1) on HL-60 human leukemia cells. While inactive against noncancerous cells, 1 provoked a concentration-dependent decrease in viable tumor cells, primarily via apoptosis, as evidenced by analysis of cell morphology, activation of caspases 3 and 7, increased DNA fragmentation, and externalization of phosphatidylserine. Necrosis was observed only at the highest tested concentration (4 µM). Compound 1 interfered with the cell cycle, causing an accumulation of cells in the G1 /G0 phase. Interaction of 1 with dsDNA and ssDNA was observed by differential pulse voltammetry and confirmed by hyperchromicity in the UV/Vis spectra of dsDNA, with an interaction constant of 2×10(4) M(-1). Both the organic analogue 1,1,2-triphenylbut-1-ene (2) and ferrocene were inactive against cancer and noncancer cell lines and did not react with DNA. These results reinforce the idea that the hybrid strategy of conjugating ferrocene to the structure of tamoxifen derivatives is advantageous in finding new substances with antineoplastic activity.

Natural products as new antimitotic compounds for anticancer drug development

Cell cycle control genes are frequently mutated in cancer cells, which usually display higher rates of proliferation than normal cells. Dysregulated mitosis leads to genomic instability, which contributes to tumor progression and aggressiveness. Many drugs that disrupt mitosis have been studied because they induce cell cycle arrest and tumor cell death. These antitumor compounds are referred to as antimitotics. Vinca alkaloids and taxanes are natural products that target microtubules and inhibit mitosis, and their derivatives are among the most commonly used drugs in cancer therapy worldwide. However, severe adverse effects such as neuropathies are frequently observed during treatment with microtubule-targeting agents. Many efforts have been directed at developing improved antimitotics with increased specificity and decreased likelihood of inducing side effects. These new drugs generally target specific components of mitotic regulation that are mainly or exclusively expressed during cell division, such as kinases, motor proteins and multiprotein complexes. Such small molecules are now in preclinical studies and clinical trials, and many are products or derivatives from natural sources. In this review, we focused on the most promising targets for the development of antimitotics and discussed the advantages and disadvantages of these targets. We also highlighted the novel natural antimitotic agents under investigation by our research group, including combretastatins, withanolides and pterocarpans, which show the potential to circumvent the main issues in antimitotic therapy.

Biological evaluation of twenty-eight ferrocenyl tetrasubstituted olefins: cancer cell growth inhibition, ROS production and hemolytic activity.

The antiproliferative effects of twenty-eight tetrasubstituted olefins bearing a ferrocenyl group, including six never-reported compounds, were evaluated against SF-295 (human glioblastoma), HCT-8 (human colon cancer), MDA-MB-435 (human melanoma) and HL-60 (human promyelocytic leukemia) using the MTT test. IC(50) values were determined for twenty-three active compounds and of these, ten compounds had IC(50) values lower than 2 µM on one or more cell lines. Of all the compounds, only two produced significant amounts of ROS on HL-60 cells, and ROS production and growth inhibition could not be correlated. The ten most antiproliferative compounds were tested for their hemolytic activity on mouse erythrocytes. Five compounds showing high antiproliferative activity and low hemolytic activity were thus identified for further study.

Toxicidad del veneno de Crotalus durissus cascavella en células de mamíferos / Crotalus durissus cascavella venom toxicity to mammalian cells / Toxicidade do veneno de Crotalus durissus cascavella em células de mamíferos

Las serpientes del género Crotalus ganaron espacio significativo en el campo científico desde el desarrollo de investigaciones con su veneno que involucran la actividad citotóxica. Crotalus durissus cascavella es la única subespecie grabadas del bioma de la Caatinga del nordeste de Brasil y los estudios carecen en sus propiedades del veneno. El presente estudio evaluó el veneno crudo de C. d. cascavella por su actividad citotóxica in vitro contra líneas celulares tumorales y las células normales. El mecanismo de la muerte celular inducida por este veneno también se investigó. C. d. cascavella mostró una alta citotoxicidad (valores de IC50 que oscila desde 2,7 hasta 6,9 g/mL) contra cinco líneas de células tumorales, entre las cuales: OVCAR - 8 y SKOV3 (carcinoma de ovario), PC-3M (carcinoma metastásico de próstata), MCF-7 (carcinoma de mama), SF - 268 (glioblastoma) y PBMC también. Las células tratadas con C. d. cascavella disparó la cascada apoptótica, según lo confirmado por diversos métodos que se muestran en este trabajo. Estos resultados refuerzan así el potencial biomédico y farmacológico de las toxinas del veneno de esta especie.

Snakes from the genus Crotalus gained significant space in the scientific field since the development of researches with their venom involving cytotoxic activity. Crotalus durissus cascavella is the only subspecies recorded of the Caatinga biome of Northeastern Brazil and its noticed a lack of studies on its venom properties. The present study evaluated the crude venom of C. d. cascavella for its cytotoxic activity in vitro against both tumor cell lines and normal cells. The mechanism of cell death induced by this venom was also investigated. C. d. cascavella venom presented high cytotoxicity (IC50 values ranging from 2.7 to 6.9 µg/mL) against five tumor cells lines: OVCAR-8 and SKOV3 (ovarian carcinomas), PC-3M (metastatic prostate carcinoma), MCF-7 (breast carcinoma), and SF-268 (glioblastoma) and PBMC as well. The cells treated with C. d. cascavella venom triggered the apoptotic pathway as confirmed by several methods. These results underline the biomedical potential of toxins from the venom from this species.

Serpentes do Gênero Crotalus ganhou espaço significativo no campo científico, desde o desenvolvimento de pesquisas com seu veneno envolvendo atividade citotóxica. A Crotalus durissus cascavella (C.d.cascavella) é uma subespécie característica da Caatinga do Nordeste do Brasil e faltam estudos sobre as propriedades de seu veneno. O presente estudo avaliou o veneno bruto de C. d. cascavella para a sua atividade citotóxica in vitro contra as linhagens de células tumorais e células normais. O mecanismo de morte celular induzida por este veneno também foi investigado. Observou-se alta citotoxicidade (valores de IC50 entre 2,7-6,9 µg/mL) contra cinco linhagens de células tumorais, dentre elas: OVCAR - 8 e SKOV3 (carcinoma ovariano), PC-3M (carcinoma metastático de próstata), MCF-7 (carcinoma mamário), SF - 268 (glioblastoma) e PBMC. As células tratadas dispararam a cascata apoptótica, como confirmado por vários métodos demonstrados neste trabalho. Esses resultados reforçam, assim, o potencial biomédico e farmacológico das toxinas do veneno dessa espécie.

Toxicidad del veneno de Crotalus durissus cascavella en células de mamíferos / Crotalus durissus cascavella venom toxicity to mammalian cells / Toxicidade do veneno de Crotalus durissus cascavella em células de mamíferos

Las serpientes del género Crotalus ganaron espacio significativo en el campo científico desde el desarrollo de investigaciones con su veneno que involucran la actividad citotóxica. Crotalus durissus cascavella es la única subespecie grabadas del bioma de la Caatinga del nordeste de Brasil y los estudios carecen en sus propiedades del veneno. El presente estudio evaluó el veneno crudo de C. d. cascavella por su actividad citotóxica in vitro contra líneas celulares tumorales y las células normales. El mecanismo de la muerte celular inducida por este veneno también se investigó. C. d. cascavella mostró una alta citotoxicidad (valores de IC50 que oscila desde 2,7 hasta 6,9 g/mL) contra cinco líneas de células tumorales, entre las cuales: OVCAR - 8 y SKOV3 (carcinoma de ovario), PC-3M (carcinoma metastásico de próstata), MCF-7 (carcinoma de mama), SF - 268 (glioblastoma) y PBMC también. Las células tratadas con C. d. cascavella disparó la cascada apoptótica, según lo confirmado por diversos métodos que se muestran en este trabajo. Estos resultados refuerzan así el potencial biomédico y farmacológico de las toxinas del veneno de esta especie.(AU)

Snakes from the genus Crotalus gained significant space in the scientific field since the development of researches with their venom involving cytotoxic activity. Crotalus durissus cascavella is the only subspecies recorded of the Caatinga biome of Northeastern Brazil and its noticed a lack of studies on its venom properties. The present study evaluated the crude venom of C. d. cascavella for its cytotoxic activity in vitro against both tumor cell lines and normal cells. The mechanism of cell death induced by this venom was also investigated. C. d. cascavella venom presented high cytotoxicity (IC50 values ranging from 2.7 to 6.9 µg/mL) against five tumor cells lines: OVCAR-8 and SKOV3 (ovarian carcinomas), PC-3M (metastatic prostate carcinoma), MCF-7 (breast carcinoma), and SF-268 (glioblastoma) and PBMC as well. The cells treated with C. d. cascavella venom triggered the apoptotic pathway as confirmed by several methods. These results underline the biomedical potential of toxins from the venom from this species.(AU)

Serpentes do Gênero Crotalus ganhou espaço significativo no campo científico, desde o desenvolvimento de pesquisas com seu veneno envolvendo atividade citotóxica. A Crotalus durissus cascavella (C.d.cascavella) é uma subespécie característica da Caatinga do Nordeste do Brasil e faltam estudos sobre as propriedades de seu veneno. O presente estudo avaliou o veneno bruto de C. d. cascavella para a sua atividade citotóxica in vitro contra as linhagens de células tumorais e células normais. O mecanismo de morte celular induzida por este veneno também foi investigado. Observou-se alta citotoxicidade (valores de IC50 entre 2,7-6,9 µg/mL) contra cinco linhagens de células tumorais, dentre elas: OVCAR - 8 e SKOV3 (carcinoma ovariano), PC-3M (carcinoma metastático de próstata), MCF-7 (carcinoma mamário), SF - 268 (glioblastoma) e PBMC. As células tratadas dispararam a cascata apoptótica, como confirmado por vários métodos demonstrados neste trabalho. Esses resultados reforçam, assim, o potencial biomédico e farmacológico das toxinas do veneno dessa espécie.(AU)

Cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-O-methyl derivative.

The cytotoxicity of nine naphthoquinones (NQ) was assayed against HL-60 (leukaemia), MDA-MB-435 (melanoma), SF-295 (brain) and HCT-8 (colon), all human cancer cell lines, and peripheral blood mononuclear cells (PBMC), as representatives of normal cells, after 72h of incubation. 5-Methoxy-1,4-naphthoquinone was the most active compound, showing IC(50) values in the range of 0.31 (1.7microM) in HL-60 to 0.88microg/mL (4.7microM) in SF-295 and IC(50) of 0.69microg/mL (3.7microM) against PBMC. With the introduction of a bromo-substituent in position 2 or 3 of juglone, the IC(50) significantly decreased, regardless of the position on the NQ moiety. However, compared with juglone methyl ether, the halogen substitution decreased the activity. To further understand the mechanism underlying the cytotoxicity of 5-methoxy-1,4-naphthoquinone, studies involving DNA fragmentation, cell cycle analysis, phosphatidyl serine externalization, mitochondrial depolarization and activation of caspases 8 and 3/7 were performed in HL-60 cell line, using doxorubicin as a positive control. The results indicate that the cytotoxic 5-methoxy-1,4-naphthoquinone activates caspases 8 and 3/7 and thus induces apoptosis independent of mitochondria.

Morphology and alfa-SMA, F-actin and JC1 protein expression in the mammary gland of goats in early hormonal lactation

Background: Mammary remodeling is determined by a combination of cell differentiation, proliferation and programmeddeath controlled not only by systemic hormones, but also by proteins produced either in the stromal or in the epithelialcompartments. However, few works were undertaken to use phenotypic markers for cellular components of the lactatingcaprine mammary gland and to determine structural–functional relationships. Thus, the aim of this research was to evaluate the morphology and α-SMA, F-actin and JC1 protein expression in the mammary gland of Canindé goats in earlyhormonal lactation.Materials, Methods & Results: Fourteen 2 years old female Canindé goats were used and distributed into two groups: nonlactating (n = 4) and lactating animals (n = 10). Lactation was induced by using estrogen, progesterone and prednisoloneaccording to previous protocol. All subjects were housed indoors and had four hours of daily access to solarium. Mammarygland biopsies were obtained at days 5 (D5) and 26 (D26) of the early stage of lactation and were assessed by histologicaland immunohistochemical analysis. The microstructure of mammary gland in lactating goats was analyzed by conventionalhistologic techniques. Immunohistochemistry was used by identify α-SMA in myoepithelial cells and JC1 in epithelialcells. α-SMA and F-actin expression were assessed by confocal microscopy. Concerning microscopic features, the alveolistructure was evaluated in respect to number, size and cell population. The structural units of the lactating mammary glandconsisted of variably shaped lobules. When comparing to lactating tissue, sections of the non-lactating mammary glandshowed a lower number and size of alveoli, separated by wide connective tissue. Lactating tissues demonstrated numerousand well-developed alveoli, separated by thin trabeculae of connective tissue. In relation to lactating goats, no difference (P> 0.05)...(AU)

Morphology and alfa-SMA, F-actin and JC1 protein expression in the mammary gland of goats in early hormonal lactation

Background: Mammary remodeling is determined by a combination of cell differentiation, proliferation and programmeddeath controlled not only by systemic hormones, but also by proteins produced either in the stromal or in the epithelialcompartments. However, few works were undertaken to use phenotypic markers for cellular components of the lactatingcaprine mammary gland and to determine structural–functional relationships. Thus, the aim of this research was to evaluate the morphology and α-SMA, F-actin and JC1 protein expression in the mammary gland of Canindé goats in earlyhormonal lactation.Materials, Methods & Results: Fourteen 2 years old female Canindé goats were used and distributed into two groups: nonlactating (n = 4) and lactating animals (n = 10). Lactation was induced by using estrogen, progesterone and prednisoloneaccording to previous protocol. All subjects were housed indoors and had four hours of daily access to solarium. Mammarygland biopsies were obtained at days 5 (D5) and 26 (D26) of the early stage of lactation and were assessed by histologicaland immunohistochemical analysis. The microstructure of mammary gland in lactating goats was analyzed by conventionalhistologic techniques. Immunohistochemistry was used by identify α-SMA in myoepithelial cells and JC1 in epithelialcells. α-SMA and F-actin expression were assessed by confocal microscopy. Concerning microscopic features, the alveolistructure was evaluated in respect to number, size and cell population. The structural units of the lactating mammary glandconsisted of variably shaped lobules. When comparing to lactating tissue, sections of the non-lactating mammary glandshowed a lower number and size of alveoli, separated by wide connective tissue. Lactating tissues demonstrated numerousand well-developed alveoli, separated by thin trabeculae of connective tissue. In relation to lactating goats, no difference (P> 0.05)...