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1.
Int J Mol Sci ; 23(21)2022 Nov 03.
Article in English | MEDLINE | ID: mdl-36362215

ABSTRACT

Flow cytometry is the gold-standard laser-based technique to measure and analyze fluorescence levels of immunostaining and DNA content in individual cells. It provides a valuable tool to assess cells in the G0/G1, S, and G2/M phases, and those with polyploidy, which holds prognostic significance. Frozen section analysis is the standard intraoperative assessment for tumor margin evaluation and tumor resection. Here, we present flow cytometry as a promising technique for intraoperative tumor analysis in different pathologies, including brain tumors, leptomeningeal dissemination, breast cancer, head and neck cancer, pancreatic tumor, and hepatic cancer. Flow cytometry is a valuable tool that can provide substantial information on tumor analysis and, consequently, maximize cancer treatment and expedite patients' survival.


Subject(s)
Brain Neoplasms , Breast Neoplasms , Humans , Female , Flow Cytometry/methods , Brain Neoplasms/pathology , Cell Division , G2 Phase , Breast Neoplasms/pathology , Cell Cycle
2.
Sci Rep ; 9(1): 18512, 2019 12 06.
Article in English | MEDLINE | ID: mdl-31811174

ABSTRACT

The co-synthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences are still little studied. Here, we showed that the number of constitutive origins mapped in the Trypanosoma brucei genome is less than the minimum required to complete replication within S-phase duration. By the development of a mechanistic model of DNA replication considering replication-transcription conflicts and using immunofluorescence assays and DNA combing approaches, we demonstrated that the activation of non-constitutive (backup) origins are indispensable for replication to be completed within S-phase period. Together, our findings suggest that transcription activity during S phase generates R-loops, which contributes to the emergence of DNA lesions, leading to the firing of backup origins that help maintain robustness in S-phase duration. The usage of this increased pool of origins, contributing to the maintenance of DNA replication, seems to be of paramount importance for the survival of this parasite that affects million people around the world.


Subject(s)
DNA Replication , Replication Origin , S Phase , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism , Cell Cycle , Computer Simulation , DNA Damage , G2 Phase , Genomic Instability , Histones/metabolism , Microscopy, Fluorescence , Monte Carlo Method , Protein Domains , Stochastic Processes
3.
J Mol Endocrinol ; 63(3): 187-197, 2019 10.
Article in English | MEDLINE | ID: mdl-31416050

ABSTRACT

Testicular Leydig cells (LC) are modulated by several pathways, one of them being the histaminergic system. Heme oxygenase-1 (HO-1), whose upregulation comprises the primary response to oxidative noxae, has a central homeostatic role and might dysregulate LC functions when induced. In this report, we aimed to determine how hemin, an HO-1 inducer, affects LC proliferative capacity and whether HO-1 effects on LC functions are reversible. It was also evaluated if HO-1 interacts in any way with histamine, affecting its regulatory action over LC. MA-10 and R2C cell lines and immature rat LC were used as models. Firstly, we show that after a 24-h incubation with 25 µmol/L hemin, LC proliferation is reversibly impaired by cell cycle arrest in G2/M phase, with no evidence of apoptosis induction. Even though steroid production is abrogated after a 48-h exposure to 25 µmol/L hemin, steroidogenesis can be restored to control levels in a time-dependent manner if the inducer is removed from the medium. Regarding HO-1 and histamine interaction, it is shown that hemin abrogates histamine biphasic effect on steroidogenesis and proliferation. Working with histamine receptors agonists, we elucidated that HO-1 induction affects the regulation mediated by receptor types 1, 2 and 4. In summary, HO-1 induction arrests LC functions, inhibiting steroid production and cell cycle progression. Despite their reversibility, HO-1 actions might negatively influence critical phases of LC development and differentiation affecting their function as well as other androgen-dependent organs. What's more, we have described a hitherto unknown interaction between HO-1 induction and histamine effects.


Subject(s)
Heme Oxygenase-1/metabolism , Histamine/pharmacology , Leydig Cells/metabolism , Animals , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line , Cell Proliferation/drug effects , Enzyme Induction/drug effects , G2 Phase/drug effects , Hemin/pharmacology , Leydig Cells/cytology , Leydig Cells/drug effects , Male , Mice , Mitosis/drug effects , Rats, Sprague-Dawley , Steroids/biosynthesis
4.
Cell Cycle ; 17(14): 1721-1744, 2018.
Article in English | MEDLINE | ID: mdl-29995582

ABSTRACT

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs) show unique cell cycle characteristics, such as a short doubling time due to an abbreviated G1 phase. Whether or not the core cell cycle machinery directly regulates the stemness and/or the differentiation potential of hPSCs remains to be determined. To date, several scenarios describing the atypical cell cycle of hPSCs have been suggested, and therefore there is still controversy over how cyclins, master regulators of the cell cycle, are expressed and regulated. Furthermore, the cell cycle profile and the expression pattern of major cyclins in hESCs-derived neuroprogenitors (NP) have not been studied yet. Therefore, herein we characterized the expression pattern of major cyclins in hPSCs and NP. We determined that all studied cyclins mRNA expression levels fluctuate along cell cycle. Particularly, after a thorough analysis of synchronized cell populations, we observed that cyclin E1 mRNA levels increased sharply in G1/S concomitantly with cyclin E1 protein accumulation in hPSCs and NP. Additionally, we demonstrated that cyclin E1 mRNA expression levels involves the activation of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a mechanism that requires a functional CDK2 but not GSK3ß activity. ABBREVIATIONS: hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III ß-tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium.


Subject(s)
Cyclin E/genetics , Gene Expression Regulation , Neurons/cytology , Neurons/metabolism , Oncogene Proteins/genetics , Pluripotent Stem Cells/cytology , Cell Proliferation , Cells, Cultured , Cyclin E/metabolism , E2F Transcription Factors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/metabolism , G1 Phase Cell Cycle Checkpoints , G2 Phase , Humans , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitosis , Neural Stem Cells/metabolism , Oncogene Proteins/metabolism , Pluripotent Stem Cells/metabolism , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
5.
Oxid Med Cell Longev ; 2017: 7865073, 2017.
Article in English | MEDLINE | ID: mdl-28761624

ABSTRACT

Breast cancer is one of the most prevalent cancers in the world and is also the leading cause of cancer death in women. The use of bioactive compounds of functional foods contributes to reduce the risk of chronic diseases, such as cancer and vascular disorders. In this study, we evaluated the antioxidant potential and the influence of pitaya extract (PE) on cell viability, colony formation, cell cycle, apoptosis, and expression of BRCA1, BRCA2, PRAB, and Erα in breast cancer cell lines (MCF-7 and MDA-MB-435). PE showed high antioxidant activity and high values of anthocyanins (74.65 ± 2.18). We observed a selective decrease in cell proliferation caused by PE in MCF-7 (ER+) cell line. Cell cycle analysis revealed that PE induced an increase in G0/G1 phase followed by a decrease in G2/M phase. Also, PE induced apoptosis in MCF-7 (ER+) cell line and suppressed BRCA1, BRCA2, PRAB, and Erα gene expression. Finally, we also demonstrate that no effect was observed with MDA-MB-435 cells (ER-) after PE treatment. Taken together, the present study suggests that pitaya may have a protective effect against breast cancer.


Subject(s)
Breast Neoplasms/drug therapy , Cactaceae/chemistry , Gene Expression Regulation, Neoplastic/drug effects , Neoplasm Proteins/biosynthesis , Plant Extracts/pharmacology , Receptors, Estrogen/biosynthesis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Division/drug effects , Female , G2 Phase/drug effects , Humans , MCF-7 Cells , Plant Extracts/chemistry
6.
Exp Parasitol ; 173: 9-17, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27939813

ABSTRACT

Congenital transmission of Trypanosoma cruzi (T. cruzi) is partially responsible for the progressive globalization of Chagas disease. During congenital transmission the parasite must cross the placental barrier where the trophoblast, a continuous renewing epithelium, is the first tissue in contact with the parasite. The trophoblast turnover implies cellular proliferation, differentiation and apoptotic cell death. The epithelial turnover is considered part of innate immunity. We previously demonstrated that T. cruzi induces cellular differentiation and apoptosis in this tissue. Here we demonstrate that T. cruzi induces cellular proliferation in a trophoblastic cell line. We analyzed the cellular proliferation in BeWo cells by determining DNA synthesis by BrdU incorporation assays, mitotic index, cell cycle analysis by flow cytometry, as well as quantification of nucleolus organizer regions by histochemistry and expression of the proliferation markers PCNA and Ki67 by Western blotting and/or immunofluorescence. Additionally, we determined the ERK1/2 MAPK pathway activation by the parasite by Western blotting.


Subject(s)
Cell Proliferation , Trophoblasts/cytology , Trophoblasts/parasitology , Trypanosoma cruzi/physiology , Animals , Cell Division , Cell Line, Tumor , DNA/biosynthesis , Flow Cytometry , G2 Phase , Ki-67 Antigen/metabolism , MAP Kinase Signaling System , Mitotic Index , Nucleolus Organizer Region/ultrastructure , Proliferating Cell Nuclear Antigen/metabolism , S Phase , Trophoblasts/metabolism
7.
J Cell Sci ; 130(1): 164-176, 2017 01 01.
Article in English | MEDLINE | ID: mdl-27363990

ABSTRACT

The cytostome-cytopharynx complex is the main site for endocytosis in epimastigotes of Trypanosoma cruzi It consists of an opening at the plasma membrane surface - the cytostome - followed by a membrane invagination - the cytopharynx. In G1/S cells, this structure is associated with two specific sets of microtubules, a quartet and a triplet. Here, we used electron microscopy and electron tomography to build 3D models of the complex at different stages of the cell cycle. The cytostome-cytopharynx is absent in late G2 and M phase cells, whereas early G2 cells have either a short cytopharynx or no visible complex, with numerous vesicles aligned to the cytostome-cytopharynx microtubules. The microtubule quartet remains visible throughout cell division (albeit in a shorter form), and is duplicated during G2/M. In contrast, the microtubule triplet is absent during late G2/M. Cells in cytokinesis have an invagination of the flagellar pocket membrane likely to represent early stages in cytostome-cytopharynx assembly. Cells in late cytokinesis have two fully developed cytostome-cytopharynx complexes. Our data suggest that the microtubule quartet serves as a guide for new cytostome-cytopharynx assembly.


Subject(s)
Cell Division , Life Cycle Stages , Trypanosoma cruzi/cytology , Trypanosoma cruzi/growth & development , Cytokinesis , Flagella/metabolism , Flagella/ultrastructure , G2 Phase , Metaphase , Microtubules/metabolism , Microtubules/ultrastructure , Models, Biological , Trypanosoma cruzi/ultrastructure
8.
J Cancer Res Clin Oncol ; 142(10): 2119-30, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27520309

ABSTRACT

UNLABELLED: Multidrug resistance is the major obstacle for successful treatment of breast cancer, prompting the investigation of novel anticancer compounds. PURPOSE: In this study, we tested whether LQB-223, an 11a-N-Tosyl-5-deoxi-pterocarpan newly synthesized compound, could be effective toward breast cancer cells. METHODS: Human breast cell lines MCF-7, MDA-MB-231, HB4a and MCF-7 Dox(R) were used as models for this study. Cell culture, MTT and clonogenic assay, flow cytometry and Western blotting were performed. RESULTS: The LQB-223 decreased cell viability, inhibited colony formation and induced an expressive G2/M arrest in breast cancer cells. There was an induction in p53 and p21(Cip1) protein levels following treatment of wild-type p53 MCF-7 cells, which was not observed in the mutant p53 MDA-MB-231 cell line, providing evidence that the compound might act to modulate the cell cycle regardless of p53 status. In addition, LQB-223 resulted in decreased procaspase levels and increased annexin V staining, suggesting that the apoptotic cascade is also triggered. Importantly, LQB-223 treatment was shown to be less cytotoxic to non-neoplastic breast cells than docetaxel and doxorubicin. Strikingly, exposure of doxorubicin-resistant MCF-7-Dox(R) cells to LQB-223 resulted in suppression of cell viability and proliferation in levels comparable to MCF-7. Of note, MCF-7-Dox(R) cells have an elevated expression of the P-glycoprotein efflux pump when compared to MCF-7. CONCLUSION: Together, these results show that LQB-223 mediates cytotoxic effects in sensitive and resistant breast cancer cells, while presenting low toxicity to non-neoplastic cells. The new compound might represent a potential strategy to induce toxicity in breast cancer cells, especially chemoresistant cells.


Subject(s)
Breast Neoplasms/drug therapy , Carcinoma, Ductal, Breast/drug therapy , Pterocarpans/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Apoptosis/drug effects , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/metabolism , Cell Division/drug effects , Cell Line, Tumor , Docetaxel , Doxorubicin/adverse effects , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Female , G2 Phase/drug effects , Humans , MCF-7 Cells , Phenotype , Pterocarpans/adverse effects , Taxoids/adverse effects , Taxoids/pharmacology
9.
Chromosome Res ; 23(4): 719-32, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26152239

ABSTRACT

Etoposide (ETO), a drug used for the treatment of human tumors, is associated with the development of secondary malignancies. Recently, therapeutic strategies have incorporated chemosensitizing agents to improve the tumoral response to this drug. ETO creates DNA double-strand breaks (DSB) via inhibition of DNA topoisomerase II (Top2). To repair DSB, homologous recombination (HR) and non-homologous end-joining (NHEJ), involving D-NHEJ (dependent of the catalytic subunit of DNA-dependent protein kinase, DNA-PKcs) and B-NHEJ (backup repair pathway) are activated. We evaluated the progression of the DNA damage induced by the Top2 poison ETO in G2 phase of human HeLa cells after chemical inhibition of DNA-PKcs with NU7026. Compared to ETO treatment alone, this combined treatment resulted in a twofold higher rate of chromatid breaks and exchanges when analysis was performed in the following metaphase. Moreover, when analysis was performed in the second metaphase following treatment, increases in the percentage of micronuclei with H2AX (biomarker for DSB) foci in binucleated cells and dicentric chromosomes were seen. In post-mitotic G1 phase, a close association between unresolved DSB and meiotic recombination 11 homolog A (MRE11) signals was observed, demonstrating the contribution of MRE11 in the DSB repair by B-NHEJ. Hence, chemical inhibition of DNA-PKcs impaired both D-NHEJ and HR repair pathways, altering the maintenance of chromosomal integrity and cell proliferation. Our results suggest that the chemosensitizing effectiveness of the DNA-PKcs inhibitor and the survival rate of aberrant cells may contribute to the development of therapy-related tumors.


Subject(s)
Antineoplastic Agents, Phytogenic/toxicity , Chromosome Aberrations/chemically induced , DNA-Activated Protein Kinase/deficiency , Etoposide/toxicity , G2 Phase/drug effects , G2 Phase/genetics , DNA Breaks, Double-Stranded/drug effects , DNA End-Joining Repair/drug effects , DNA-Binding Proteins/metabolism , Gene Rearrangement , HeLa Cells , Histones/metabolism , Homologous Recombination , Humans , MRE11 Homologue Protein , Mitotic Index , Protein Binding , Rad51 Recombinase/metabolism , Sister Chromatid Exchange/genetics
10.
PLoS One ; 8(11): e79364, 2013.
Article in English | MEDLINE | ID: mdl-24223931

ABSTRACT

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.


Subject(s)
Protein-Tyrosine Kinases/genetics , Trypanosoma brucei brucei/cytology , Trypanosoma brucei brucei/genetics , Amino Acid Sequence , Cell Division , Databases, Genetic , Down-Regulation , G2 Phase , Genome, Protozoan/genetics , Molecular Sequence Data , Phenotype , Phosphorylation , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/metabolism , Schizosaccharomyces/genetics , Sequence Homology, Nucleic Acid , Survival Analysis , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/growth & development
11.
Nutr Cancer ; 65(7): 1076-85, 2013.
Article in English | MEDLINE | ID: mdl-24053141

ABSTRACT

Prostate cancer is the most common malignancy in men and the second leading cause of cancer-related mortality in men of the Western world. Lycopene has received attention because of its expcted potential to prevent cancer. In the present study, we evaluated the influence of lycopene on cell viability, cell cycle, and apoptosis of human prostate cancer cells and benign prostate hyperplastic cells. Using MTT assay, we observed a decrease of cell viability in all cancer cell lines after treatment with lycopene, which decreased the percentage of cells in G0/G1 phase and increased in S and G2/M phases after 96 h of treatment in metastatic prostate cancer cell lineages. Flow citometry analysis of cell cycle revealed lycopene promoted cell cycle arrest in G0/G1 phase after 48 and 96 h of treatment in a primary cancer cell line. Using real time PCR assay, lycopene also induced apoptosis in prostate cancer cells with altered gene expression of Bax and Bcl-2. No effect was observed in benign prostate hyperplasia cells. These results suggest an effect of lycopene on activity of human prostate cancer cells.


Subject(s)
Apoptosis/drug effects , Carotenoids/pharmacology , Cell Survival/drug effects , G1 Phase/drug effects , G2 Phase/drug effects , Prostatic Hyperplasia/pathology , Prostatic Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Gene Expression , Humans , Lycopene , Male , Prostatic Hyperplasia/metabolism , Prostatic Neoplasms/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism
12.
PLoS One ; 8(8): e72582, 2013.
Article in English | MEDLINE | ID: mdl-23991123

ABSTRACT

We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3(Ras) mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner.


Subject(s)
Cell Division/physiology , Fibroblast Growth Factor 2/physiology , G2 Phase/physiology , Proto-Oncogene Proteins p21(ras)/physiology , Proto-Oncogene Proteins pp60(c-src)/physiology , Animals , DNA Replication , Mice , Phosphatidylinositol 3-Kinases/metabolism
13.
Toxicol Appl Pharmacol ; 272(1): 117-26, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-23756174

ABSTRACT

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC50 values in the nanomolar range. Cell cycle arrest in G2/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G2/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential.


Subject(s)
Apoptosis/drug effects , Benzophenones/pharmacology , Cell Division/drug effects , G2 Phase/drug effects , Tubulin Modulators , Tubulin/biosynthesis , Annexins/metabolism , Antimetabolites , Benzophenones/chemical synthesis , Bromodeoxyuridine , Caspases/metabolism , Cell Death/drug effects , Cell Membrane/physiology , Cell Membrane/ultrastructure , Cell Survival/drug effects , Coloring Agents , Comet Assay , DNA Fragmentation/drug effects , HL-60 Cells , Humans , Membrane Potential, Mitochondrial/drug effects , Models, Molecular , Polymerization , Tetrazolium Salts , Thiazoles
14.
Toxicol In Vitro ; 27(5): 1458-66, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23542210

ABSTRACT

Sesquiterpene lactones (SLs) are natural products with a variety of biological activities. Previously, we demonstrated the cytotoxic effects of three new α-santonin derivatives on different tumor cell lines with low toxic effects upon peripheral human leukocytes. Here, we evaluated the mechanism of action triggered by these derivatives. HL-60 cell cycle determined after 24h treatment revealed a significant inhibition on cell-cycle progression and leading to an increasing of cells in G2/M [7.6% and 9.0% for compound 3% and 9.0% and 8.6% for compound 4 (1 and 2 µM, respectively)]. However, after 48 h exposure, all compounds caused G2/M reduction and a significant DNA fragmentation. Compounds 2, 3 and 4 were able to induce apoptosis on leukemia cells, which was corroborated by phosphatidyserine externalization and activation of caspases-3 and -7 after 24h exposure. None of the derivatives analyzed caused depolarization of mitochondrial membrane within 24h of incubation, suggesting the involvement of the extrinsic apoptotic pathway in the death process. The antiproliferative action of these compounds is related to the DNA synthesis inhibition and cell cycle arrest, which probably lead to apoptosis activation. Therefore, these santonin derivatives are promising lead candidates for development of new cytotoxic agents.


Subject(s)
Cytotoxins/pharmacology , Santonin/analogs & derivatives , Santonin/pharmacology , Apoptosis/drug effects , Caspase 3/metabolism , Caspase 7/metabolism , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , DNA Fragmentation , G2 Phase , HL-60 Cells , Humans , Membrane Potential, Mitochondrial/drug effects
15.
PLoS One ; 8(1): e53517, 2013.
Article in English | MEDLINE | ID: mdl-23301080

ABSTRACT

PI3K/Akt is an important pathway implicated in the proliferation and survival of cells in the CNS. Here we investigated the participation of the PI3K/Akt signal pathway in cell cycle of developing retinal progenitors. Immunofluorescence assays performed in cultures of chick embryo retinal cells and intact tissues revealed the presence of phosphorylated Akt and 4E-BP1 in cells with typical mitotic profiles. Blockade of PI3K activity with the chemical inhibitor LY 294002 (LY) in retinal explants blocked the progression of proliferating cells through G2/M transition, indicated by an expressive increase in the number of cells labeled for phosphorylated histone H3 in the ventricular margin of the retina. No significant level of cell death could be detected at this region. Retinal explants treated with LY for 24 h also showed a significant decrease in the expression of phospho-Akt, phospho-GSK-3 and the hyperphosphorylated form of 4E-BP1. Although no change in the expression of cyclin B1 was detected, a significant decrease in CDK1 expression was noticed after 24 h of LY treatment both in retinal explants and monolayer cultures. Our results suggest that PI3K/Akt is an active pathway during proliferation of retinal progenitors and its activity appears to be required for proper CDK1 expression levels and mitosis progression of these cells.


Subject(s)
Gene Expression Regulation, Developmental , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Retina/embryology , Retina/metabolism , Stem Cells/cytology , Animals , Cell Cycle , Cell Division , Cell Proliferation , Cells, Cultured , Chick Embryo , Chromones/pharmacology , Cyclin B1/metabolism , G2 Phase , Histones/metabolism , Microscopy, Fluorescence , Mitosis , Morpholines/pharmacology , Phosphorylation , Retina/cytology , Signal Transduction , Time Factors
16.
Mar Drugs ; 10(9): 2002-2022, 2012 Sep.
Article in English | MEDLINE | ID: mdl-23118717

ABSTRACT

Sulfated fucans comprise families of polydisperse natural polysaccharides based on sulfated L-fucose. Our aim was to investigate whether fucan nanogel induces cell-specific responses. To that end, a non toxic fucan extracted from Spatoglossum schröederi was chemically modified by grafting hexadecylamine to the polymer hydrophilic backbone. The resulting modified material (SNFuc) formed nanosized particles. The degree of substitution with hydrophobic chains was close to 100%, as estimated by elemental analysis. SNFfuc in aqueous media had a mean diameter of 123 nm and zeta potential of -38.3 ± 0.74 mV, as measured by dynamic light scattering. Nanoparticles conserved their size for up to 70 days. SNFuc cytotoxicity was determined using the MTT assay after culturing different cell lines for 24 h. Tumor-cell (HepG2, 786, H-S5) proliferation was inhibited by 2.0%-43.7% at nanogel concentrations of 0.05-0.5 mg/mL and rabbit aorta endothelial cells (RAEC) non-tumor cell line proliferation displayed inhibition of 8.0%-22.0%. On the other hand, nanogel improved Chinese hamster ovary (CHO) and monocyte macrophage cell (RAW) non-tumor cell line proliferation in the same concentration range. The antiproliferative effect against tumor cells was also confirmed using the BrdU test. Flow cytometric analysis revealed that the fucan nanogel inhibited 786 cell proliferation through caspase and caspase-independent mechanisms. In addition, SNFuc blocks 786 cell passages in the S and G2-M phases of the cell cycle.


Subject(s)
Polyethylene Glycols/chemistry , Polyethylene Glycols/pharmacology , Polyethyleneimine/chemistry , Polyethyleneimine/pharmacology , Polysaccharides/chemistry , Polysaccharides/pharmacology , Amines/chemistry , Animals , Aorta/drug effects , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Endothelial Cells/drug effects , G2 Phase/drug effects , Hep G2 Cells , Humans , Hydrocarbons/chemistry , Nanogels , Nanoparticles/chemistry , Particle Size , Plant Extracts/chemistry , Plant Extracts/pharmacology , Polymers/chemistry , Rabbits , S Phase/drug effects , Seaweed/chemistry
17.
Environ Mol Mutagen ; 53(8): 608-18, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22987276

ABSTRACT

The role of DNA double strand break (DSB) repair pathways, non-homologous end joining (NHEJ), and homologous recombination (HR) was evaluated to prevent the chromosome instability induced by the topoisomerase II (Top2) poisons, idarubicin, and etoposide in Chinese hamster cell lines. XR-C1 (DNA-PKcs deficient) and V-C8 (BRCA2 deficient) showed higher sensitivity to increased concentrations of Top2 poisons compared with their normal counterparts, CHO9 and V79. Both proficient and deficient cells exhibited a marked DSB induction in all phases of the cell cycle. Additionally, deficient cells showed persistent DNA damage 24 hr post-treatment. Chromosomal aberrations increased in the first mitosis following Top2 poison-treatments in G1 or G2 in proficient and deficient cells. CHO9 and V79 demonstrated chromosome and chromatid exchanges following treatments in G1 and G2 phases, respectively. Deficient cells showed high frequencies of chromatid exchanges following treatments in G1 and G2. Simultaneously, we analyzed the micronuclei (MN) induction in interphase cells after treatments in G1, S, or G2 of the previous cell cycle. Both Top2 poisons induced an important increase in MN in CHO9, V79, and V-C8 cells. XR-C1 exhibited an increased MN frequency when cells were treated in G1 phase but not in S or G2. This MN reduction was due to a cell accumulation at G2/M and death in G2-treated cells. Our data suggest that NHEJ and HR operate differentially throughout the cell cycle to protect from Top2 poison-induced chromosome instability, and that DNA-PKcs-dependent NHEJ pathway allows the survival of chromosome damaged cells during S/G2 to the next interphase.


Subject(s)
Chromosome Aberrations/drug effects , DNA Breaks, Double-Stranded/drug effects , DNA Topoisomerases, Type II/metabolism , Animals , CHO Cells , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line , Cricetinae , Cricetulus , Etoposide/toxicity , G1 Phase/drug effects , G1 Phase/genetics , G2 Phase/drug effects , G2 Phase/genetics , Idarubicin/toxicity , Micronuclei, Chromosome-Defective/chemically induced
18.
Genet Mol Res ; 11(3): 2814-22, 2012 Aug 24.
Article in English | MEDLINE | ID: mdl-23007976

ABSTRACT

Anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase in cell division, which catalyses ubiquitination of cell-cycle regulators. Studying this complex could reveal important information regarding its application in cancer research and therapy. In this study, 4 synthesized small interfering RNAs (siRNAs) were transfected into HEK293T cells to suppress messenger RNA (mRNA) of Apc11; 2 of these reduced the amount of Apc11 mRNA by over 50%. Further experiments showed that rather than causing apoptosis, siRNA transfection led to cell-cycle distributions characterized by less time spent in G2/M phase and more time spent in G1 phase. This phenomenon was specifically induced by Apc11 silencing, as co-transfection of siRNA and an Apc11 plasmid could reverse this distribution bias. Our results suggested that siRNA targeted against Apc11 could hamper entry into G2/M phase. Current efforts are focused on elucidating the function and utility of the APC complex for clinical applications.


Subject(s)
Cell Division , G2 Phase , Gene Knockdown Techniques , Ubiquitin-Protein Ligase Complexes/metabolism , Anaphase-Promoting Complex-Cyclosome , Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome , HEK293 Cells , Humans , RNA, Small Interfering/metabolism , Transfection
19.
Mutat Res ; 731(1-2): 133-9, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-22230195

ABSTRACT

We analyzed the behavior of interstitial telomeric sequences (ITSs) in the progeny of Chinese Hamster Ovary (CHO) cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if ITSs play some role in the long-term clastogenic effect of this antibiotic. To this end, CHO cells were treated with a single concentration of BLM (2.5µg/ml), and the frequency of unstable chromosomal aberrations was determined at several times after treatment (18h, and 6, 15 and 34/36 days) by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations decreased on average five times 6 days after treatment compared with the one induced 18h after treatment. Moreover, no significant differences in the frequency of aberrations were observed between untreated and BLM-exposed cells at 15 or 34/36 days after treatment. These data indicate that, in terms of unstable aberrations, the in vitro clastogenic effect of BLM on CHO cells persists for at least 6 days but less than 15 days after exposure. In addition, we found that BLM induces ITSs instability, cytogenetically detectable as acentric fragments (18h after treatment) or additional (new) FISH signals (6 days after treatment). We propose that the delayed effect of BLM on ITSs mainly results from breakage of heterochromatic ITSs blocks and further insertion of these sequences at the sites of monochromatid breaks occurring at G2 phase of the cell cycle, since most of the additional FISH signals were present as single dots and located at interstitial sites of the involved chromosomes.


Subject(s)
Antibiotics, Antineoplastic/toxicity , Bleomycin/toxicity , Chromosomal Instability/drug effects , Mutagens/toxicity , Telomere/drug effects , Animals , CHO Cells , Chromosome Aberrations/drug effects , Cricetinae , Cricetulus , G2 Phase/drug effects , Humans , In Situ Hybridization, Fluorescence , Time Factors
20.
PLoS One ; 6(11): e27707, 2011.
Article in English | MEDLINE | ID: mdl-22110734

ABSTRACT

The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26-27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70-80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p<0.05). When myeloma cells were treated with bortezomib, we observed a 48% reduction of cells in the G2/M phase among inhibited cells while controls showed 13% (empty vector) and 9% (ineffective shRNA) reduction, respectively (p<0.01). Furthermore, inhibited cells treated with bortezomib showed an increased percentage of apoptotic cells (Annexin V+/PI-) in comparison with bortezomib-treated controls (p<0.001). We found that MAGE-C1/CT7 protects SKO-007 cells against bortezomib-induced apoptosis. Therefore, we could speculate that MAGE-C1/CT7 gene therapy could be a strategy for future therapies in MM, in particular in combination with proteasome inhibitors.


Subject(s)
Antigens, Neoplasm/genetics , Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Multiple Myeloma/pathology , Neoplasm Proteins/deficiency , Neoplasm Proteins/genetics , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Pyrazines/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Bortezomib , Cell Division/drug effects , Cell Division/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , G2 Phase/drug effects , G2 Phase/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Gene Silencing , Humans , Multiple Myeloma/genetics , Neoplasm Invasiveness , RNA, Small Interfering/genetics
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