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1.
iScience ; 27(4): 109366, 2024 Apr 19.
Article in English | MEDLINE | ID: mdl-38510127

ABSTRACT

Triple-negative breast cancer (TNBC) contributes greatly to mortality of breast cancer, demanding new targetable options. We have shown that TNBC patients have high ΔNp63 expression in tumors. However, the function of ΔNp63 in established TNBC is yet to be explored. In current studies, targeting ΔNp63 with inducible CRISPR knockout and Histone deacetylase inhibitor Quisinostat showed that ΔNp63 is important for tumor progression and metastasis in established tumors by promoting myeloid-derived suppressor cell (MDSC) survival through tumor necrosis factor alpha. Decreasing ΔNp63 levels are associated with decreased CD4+ and FOXP3+ T-cells but increased CD8+ T-cells. RNA sequencing analysis indicates that loss of ΔNp63 alters multiple MDSC properties such as lipid metabolism, chemotaxis, migration, and neutrophil degranulation besides survival. We further demonstrated that targeting ΔNp63 sensitizes chemotherapy. Overall, we showed that ΔNp63 reprograms the MDSC-mediated immunosuppressive functions in TNBC, highlighting the benefit of targeting ΔNp63 in chemotherapy-resistant TNBC.

2.
Chem Biol Interact ; 378: 110489, 2023 Jun 01.
Article in English | MEDLINE | ID: mdl-37059213

ABSTRACT

We assessed the mechanism of human androgen receptor-mediated endocrine-disrupting effect by a triazole fungicide, metconazole in this study. The internationally validated stably transfected transactivation (STTA) in vitro assay, which was established for determination of a human androgen receptor (AR) agonist/antagonist by using 22Rv1/MMTV_GR-KO cell line, alongside an in vitro reporter-gene assay to confirm AR homodimerization was used. The STTA in vitro assay results showed that metconazole is a true AR antagonist. Furthermore, the results from the in vitro reporter-gene assay and western blotting showed that metconazole blocks the nuclear transfer of cytoplasmic AR proteins by suppressing the homodimerization of AR. These results suggest that metconazole can be considered to have an AR-mediated endocrine-disrupting effect. Additionally, the evidence from this study might help identify the endocrine-disrupting mechanism of triazole fungicides containing a phenyl ring.


Subject(s)
Androgen Receptor Antagonists , Endocrine Disruptors , Fungicides, Industrial , Protein Multimerization , Receptors, Androgen , Transcriptional Activation , Triazoles , Triazoles/chemistry , Triazoles/toxicity , Fungicides, Industrial/chemistry , Fungicides, Industrial/toxicity , Protein Multimerization/drug effects , Humans , Receptors, Androgen/chemistry , Receptors, Androgen/metabolism , Endocrine Disruptors/chemistry , Endocrine Disruptors/pharmacology , Androgen Receptor Antagonists/chemistry , Androgen Receptor Antagonists/toxicity , Cell Line, Tumor , Transcriptional Activation/drug effects , Cytotoxins/chemistry , Cytotoxins/toxicity
3.
Environ Pollut ; 325: 121437, 2023 May 15.
Article in English | MEDLINE | ID: mdl-36907237

ABSTRACT

This study was carried out to provide the evidence with respect to the adverse potential of chlorpropham, a representative carbamate ester herbicide product, on the endocrine system by using in vitro testing methods in accordance with the Organization for Economic Cooperation and Development Test Guideline No. 458 (22Rv1/MMTV_GR-KO human androgen receptor [AR] transcriptional activation assay) and a bioluminescence resonance energy transfer-based AR homodimerization assay. Results revealed that chlorpropham had no AR agonistic effects, but it was determined to be a true AR antagonist without intrinsic toxicity against the applied cell lines. In the mechanism of chlorpropham-induced AR-mediated adverse effects, chlorpropham suppressed cytoplasmic AR translocation to the nucleus by inhibiting the homodimerization of the activated ARs. This suggests that chlorpropham exposure caused endocrine-disrupting effects through its interactions with human AR. Additionally, this study might help identify the genomic pathway of the AR-mediated endocrine-disrupting potential of N-phenyl carbamate herbicides.


Subject(s)
Chlorpropham , Herbicides , Humans , Chlorpropham/metabolism , Chlorpropham/toxicity , Herbicides/toxicity , Herbicides/metabolism , Receptors, Androgen , Androgens , Carbamates/toxicity , Endocrine System
4.
Biochem Biophys Res Commun ; 635: 52-56, 2022 12 20.
Article in English | MEDLINE | ID: mdl-36257192

ABSTRACT

Given our previous finding that certain tumor-suppressing functions of p53 are exerted by the p53/p21 complex, rather than p53 alone, cells may have a system to regulate the p53/p21 interaction. As p53 binds to p21 via its C-terminal domain, which contains acetylable lysine residues, we investigated whether the C-terminal acetylation of p53 influences the p53/p21 interaction. Indeed, the p53/p21 interaction was reduced when various types of cells (HCT116 colon cancer, A549 lung cancer, and MCF7 breast cancer cells) were treated with MS-275, an inhibitor of SIRT1 (a p53 deacetylase), or with SIRT1-targeting small interfering RNAs. These treatments also increased the acetylation levels of the five lysine residues (K370, K372, K373, K381, K382) in the C-terminal domain of p53. The p53/p21 interaction was also reduced when these lysine residues were substituted with glutamine (an acetylation memetic), but not arginine (an unacetylable lysine analog). While the inhibitory effect of the lysine-to-glutamine substitution was evident upon the substitution of all the five lysine residues, the substitution of only two (K381, K382) or three residues (K370, K372, K373) was less effective. Consistently, the five substitutions reduced the ability of p53 to regulate cell invasion and death by liberating Bax from Bcl-w. Overall, our data suggest that the acetylation, especially the hyperacetylation, of the p53 C-terminal domain suppresses the p53/p21-complex-dependent functions of p53 by inhibiting the p53/p21 interaction. We propose that cellular components involved in the acetylation or deacetylation of the p53 C-terminus are critical regulators of the formation of p53/p21 complex.


Subject(s)
Lysine , Tumor Suppressor Protein p53 , Tumor Suppressor Protein p53/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Lysine/metabolism , Sirtuin 1/genetics , Sirtuin 1/metabolism , Glutamine/metabolism , Cell Line, Tumor , Promoter Regions, Genetic , Acetylation
5.
Biochem Biophys Res Commun ; 621: 151-156, 2022 09 17.
Article in English | MEDLINE | ID: mdl-35834924

ABSTRACT

The p53 tumor suppressor regulates cell functions either by acting as a transcription factor or by interacting with other proteins. Previously, we reported that the non-transcriptional actions of p53 can be facilitated by the binding of p53 to p21. Herein, we investigated whether p53/p21 interaction influences the transcriptional activity of p53. We observed that the expression of the p53 promoter-based reporter gene is dependent on p21 levels. Moreover, using a p21 variant that is unable to bind p53, we showed that p53 promoter activity requires p53/p21 interaction. To investigate the possible role of p21 in regulating the expression of endogenous p53 targets, we analyzed mRNA levels of Puma, Mdm2, and Gadd45a in untreated control and γ-ray-irradiated cells. We observed that while Puma expression is dependent on p53 regardless of γ-irradiation, p53 mediates the expression of Mdm2 and Gadd45a only in irradiated cells. Notably, p53/p21 interaction is required only for the p53-dependent expression of the tested genes and not Mdm2 and Gadd45a in non-irradiated cells. Moreover, chromatin immunoprecipitation assay revealed that p21 is required for the binding of p53 to the promoters of Puma, Mdm2, and Gadd45a. Collectively, our data support the view that the p53/p21 complex is involved in regulating p53-dependent gene expression. These findings provide a new foundation for understanding the transcriptional action of p53.


Subject(s)
Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p53 , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Expression , Gene Expression Regulation , Promoter Regions, Genetic , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism
6.
Front Vet Sci ; 8: 587659, 2021.
Article in English | MEDLINE | ID: mdl-34604364

ABSTRACT

The gastrointestinal tract is the most common exposure route of xenobiotics, and intestinal toxicity can result in systemic toxicity in most cases. It is important to develop intestinal toxicity assays mimicking the human system; thus, stem cells are rapidly being developed as new paradigms of toxicity assessment. In this study, we established human embryonic stem cell (hESC)-derived enterocyte-like cells (ELCs) and compared them to existing in vivo and in vitro models. We found that hESC-ELCs and the in vivo model showed transcriptomically similar expression patterns of a total of 10,020 genes than the commercialized cell lines. Besides, we treated the hESC-ELCs, in vivo rats, Caco-2 cells, and Hutu-80 cells with quarter log units of lethal dose 50 or lethal concentration 50 of eight drugs-chloramphenicol, cycloheximide, cytarabine, diclofenac, fluorouracil, indomethacin, methotrexate, and oxytetracycline-and then subsequently analyzed the biomolecular markers and morphological changes. While the four models showed similar tendencies in general toxicological reaction, hESC-ELCs showed a stronger correlation with the in vivo model than the immortalized cell lines. These results indicate that hESC-ELCs can serve as a next-generation intestinal toxicity model.

7.
Oncol Lett ; 22(3): 681, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34345306

ABSTRACT

Our previous study revealed that the tumor suppressor/transcription factor p53 directly binds to its transcriptional target, p21, and that the p53/p21 complex binds to zinc finger protein SNAI2 (Slug), a tumor promoter/transcription factor; thereby promoting the degradation of Slug by Mdm2, an E3 ligase. The present study demonstrated that Slug reduced the cellular expression levels of p53 and p21 in HCT116 colon cancer by decreasing their protein stability. In parallel, Slug increased the mRNA and protein expression levels of Mdm2 in these cells. Moreover, knockdown of Mdm2 using specific small interfering RNAs abolished the ability of Slug to induce the degradation of p53 and p21. Considering the well-known function of Mdm2 in facilitating p53 and p21 degradation, these data suggested that Slug promoted p53 and p21 degradation by inducing Mdm2 expression. Moreover, Slug increased ubiquitination levels of p53 in HCT116 cells. This is consistent with the fact that Mdm2 induces p53 degradation by ubiquitinating p53, and further confirmed that Mdm2 acted downstream of Slug. Comparative studies using HCT116 cells and their p53- or p21-knockout variants have revealed that Slug requires p21 to induce p53 degradation. This result is consistent with our previous study, which revealed that Mdm2 acts more efficiently on p53 in the p53/p21 complex compared with on p53 alone. By contrast, Slug did not require p53 to induce p21 degradation, suggesting that p53 was dispensable in Mdm2-mediated p21 degradation. Notably, the ability of Slug to increase/decrease Mdm2/p53 and p21 levels, respectively, was not confined to HCT116 cells alone, but was also confirmed in A549 and H460 lung cancer cells. Collectively, the results of the present study suggested that Slug could counter p53 and p21. The balance between these two opposing groups (Slug vs. p53/p21) may depend on environmental stresses and the internal physiology of cells.

8.
J Environ Pathol Toxicol Oncol ; 40(2): 65-79, 2021.
Article in English | MEDLINE | ID: mdl-33822518

ABSTRACT

Environmental pollution (EP) is a well-known threat to wild animals, but its toxicological impact is poorly understood. In vitro toxicity evaluation using cells of lower predators could be a promising way to assess and monitor the effects of EPs on whole wildlife populations that are related in the food web. Here, we describe EPs' toxic effect and mechanism in the primary fibroblast derived from the embryo of the striped field mouse, Apodemus agrarius. Characterization of the primary fibroblast was via morphology, genetics, immunocytochemistry, and stable culture conditions for optimal toxicity screening. Cell viability assays-MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and lactate dehydrogenase (LDH)-were performed to observe cytotoxicity, and quantitative PCR was conducted to confirm gene alteration by EP exposure. MTT and LDH assays confirmed the cytotoxicity of transfluthrin (TF), benzyl butyl phthalate (BBP), and 17ß-estradiol (E2) with IC50 values of 10.56 µM, 10.82 µM, and 24.08 µM, respectively, following 48-h exposures. mRNA expression of androgen-binding protein, growth hormone receptor, cytochrome C oxidase, and cytochrome P450-1A1 was induced after exposure to TF, BBP, and E2. We unveiled new EP mechanisms at the mammalian cellular level and discovered potential biomarker genes for monitoring of EPs. Based on our findings, we propose the primary fibroblast of A. agrarius as a valuable model to assess the toxicological effects of EP on wildlife.


Subject(s)
Cyclopropanes/toxicity , Endocrine Disruptors/toxicity , Estradiol/toxicity , Estrogens/toxicity , Fibroblasts/drug effects , Fluorobenzenes/toxicity , Insecticides/toxicity , Phthalic Acids/toxicity , Androgen-Binding Protein/genetics , Animals , Cell Survival/drug effects , Cells, Cultured , Cyclooxygenase 1/genetics , Cytochrome P-450 CYP1A1/genetics , Embryo, Mammalian/cytology , Fibroblasts/metabolism , Murinae , Receptors, Somatotropin/genetics
9.
Oncol Lett ; 21(5): 395, 2021 May.
Article in English | MEDLINE | ID: mdl-33777218

ABSTRACT

Benzimidazole derivatives are used for their antihelmintic properties, but have also been reported to exert anticancer effects. In the present study, the anticancer effects of albendazole on prostate cancer cells were assessed using proliferation, clonogenic and migration assays. To investigate the anticancer mechanisms of albendazole, reactive oxygen species (ROS) levels were measured, and the expression of genes associated with oxidative stress and Wnt/ß-catenin signaling was confirmed by reverse transcription-quantitative PCR and western blotting. Albendazole selectively inhibited the proliferation of the PC3, DU145, LNCaP and AT2 prostate cancer cell lines at concentrations that did not affect the proliferation of a normal prostate cell line (RWPE-1). Albendazole also inhibited the colony formation and migration of PC3 and DU145 cells, as well as inducing ROS production. Diphenyleneiodonium chloride, an inhibitor of NADPH oxidase (NOX), one of the sources of ROS, decreased basal ROS levels in the PC3 and DU145 cells, but did not reduce albendazole-associated ROS production, suggesting that ROS production following albendazole treatment was NOX-independent. The anticancer effect was decreased when albendazole-induced ROS was reduced by treatment with antioxidants (glutathione and N-acetylcysteine). Furthermore, albendazole decreased the mRNA expression of CDGSH iron sulfur domain 2, which regulates antioxidant activity against ROS, as well as the antioxidant enzymes catalase, and glutathione peroxidase 1 and 3. Albendazole also decreased the mRNA expression of catenin ß1 and transcription factor 4, which regulate Wnt/ß-catenin signaling and its associated targets, Twist family BHLH transcription factor 1 and BCL2. The albendazole-related decrease in the expression levels of oxidative stress-related genes and Wnt/ß-catenin signaling proteins was thought to be associated with ROS production. These results suggest that the antihelmintic drug, albendazole, has inhibitory effects against prostate cancer cells in vitro. Therefore, albendazole may potentially be used as a novel anticancer agent for prostate cancer.

10.
Biochem Biophys Res Commun ; 543: 23-28, 2021 03 05.
Article in English | MEDLINE | ID: mdl-33503543

ABSTRACT

Downregulation of the p53 tumor suppressor in cancers is frequently accompanied by the upregulation of Wip1 (a phosphatase) and Mdm2 (an E3 ubiquitin ligase). Mdm2 binds and ubiquitinates p53, promoting its degradation by the proteasome. As the p53/Mdm2 interaction is alleviated by the phosphorylation of the serine-15 (S15) residue of p53, Wip1, which can directly dephosphorylate phospho-S15, facilitates the Mdm2-mediated degradation of p53. Here, we found that p21WAF1/CIP1, previously shown to bind p53 and Mdm2, reduces the cellular levels of p53 protein by decreasing its stability. This is accompanied by a decrease in p53-S15 phosphorylation levels. In agreement, p21 promotes the p53/Wip1 interaction. Additionally, p21 interacts with Wip1, forming a trimeric complex of p53, p21, and Wip1. Studies using a p21 deletion mutant that cannot bind p53 revealed that the p53/p21 complex is more efficient than p53 alone in facilitating the binding of p53 to Wip1 and Mdm2. These findings indicate that p21 is a novel negative regulator of p53 stability and therefore, may be used as a target to restore p53 activity by preventing the action of Wip1 and Mdm2 on p53.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Neoplasms/metabolism , Protein Phosphatase 2C/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Cell Line, Tumor , Humans , Neoplasms/pathology , Phosphorylation , Protein Interaction Domains and Motifs , Proteolysis , Signal Transduction
11.
Prostate ; 81(3): 182-193, 2021 02.
Article in English | MEDLINE | ID: mdl-33368416

ABSTRACT

BACKGROUND: Recent developments in gene editing, using transcriptional activator-like effector nucleases (TALENs), have greatly helped the generation of genetically engineered animal models. The NK3 homeobox 1 (NKX3.1) protein plays important roles in prostate development and protein production, and functions as a tumor suppressor. Recently, NKX3.1 was shown to be associated with breast cancer in humans. METHODS: Our aim was to create a new rat model to elucidate the functions of NKX3.1. To that end, we generated Nkx3.1 knockout rats using TALENs and analyzed their phenotype. TALEN-mediated Nkx3.1 knockout was confirmed by T7 endonuclease I (T7E1) assay and DNA sequencing. Prostate weight and fertility were evaluated in the knockout rats, besides determining the proportion of epithelial cells and messenger RNA (mRNA) expression of genes associated with carcinogenesis. Breast tumors were examined by histopathology. RESULTS: Results suggested Nkx3.1 knockout rats have reduced fertility, decreased prostate weights, and increased epithelial cell layers. The mRNA expression of genes related to prostate carcinogenesis, namely Ar, Akt, and Pi3k, also increased. Moreover, the Nkx3.1 knockout rats often developed malignant breast tumors. CONCLUSIONS: We, therefore, successfully created the first Nkx3.1 knockout rat model, using TALEN-mediated gene targeting, and used it to identify defects associated with Nkx3.1 deficiency, not previously observed in mice. Loss of Nkx3.1 in rats led to lower reproductive capacity, and decreased prostate weights, apart from the risk of developing breast cancer. We, thus, proposed Nkx3.1 knockout rats as reliable models for studying the role of NKX3.1 in decreased prostate weights, fertility, and breast cancer, as well as in prostate cancer.


Subject(s)
Gene Knockout Techniques/methods , Homeodomain Proteins/physiology , Transcription Activator-Like Effector Nucleases/genetics , Transcription Factors/physiology , Amino Acid Sequence , Animals , Base Sequence , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Fertility , Genes, Tumor Suppressor , Homeodomain Proteins/genetics , Male , Models, Animal , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Transcription Factors/genetics
12.
BMC Biotechnol ; 20(1): 45, 2020 08 26.
Article in English | MEDLINE | ID: mdl-32843026

ABSTRACT

BACKGROUND: Vitrification is the most promising technology for successful cryopreservation of living organisms without ice crystal formation. However, high concentrations (up to ~ 6-8 M) of cryoprotective agents (CPAs) used in stem cell induce osmotic and metabolic injuries. Moreover, the application of conventional slow-freezing methods to cultures of 3-D organoids of stem cells in various studies, is limited by their size. RESULTS: In this study, we evaluated the effect of high concentrations of CPAs including cytotoxicity and characterized human mesenchymal stem cell (MSC) at single cell level. The cell viability, cellular damage, and apoptotic mechanisms as well as the proliferation capacity and multipotency of cells subjected to vitrification were similar to those in the slow-freezing group. Furthermore, we identified the possibility of vitrification of size-controlled 3-D spheroids for cryopreservation of organoid with high survivability. CONCLUSIONS: Our results demonstrate successful vitrification of both single cell and spheroid using high concentration of CPAs in vitro without cytotoxicity.


Subject(s)
Cell Culture Techniques/methods , Cryopreservation/methods , Cryoprotective Agents/chemistry , Stem Cells/cytology , Vitrification , Cell Proliferation , Cell Survival , Freezing , Humans , Mesenchymal Stem Cells , Reactive Oxygen Species
13.
Andrology ; 8(5): 1486-1493, 2020 09.
Article in English | MEDLINE | ID: mdl-32450005

ABSTRACT

BACKGROUND: Glutathione peroxidase 3 (Gpx3) protects cells from oxidative stress, and its reduced expression in human prostate cancer has been reported. OBJECTIVES: We hypothesized that Gpx3 might play an important role in the development of prostatic intraepithelial neoplasia (PIN), a pre-cancerous state of the prostate, and aimed to highlight the underlying molecular mechanism. MATERIALS AND METHODS: The following double-knockout mice Nkx3.1-/-; Gpx3+/+, Nkx3.1-/-; Gpx3+/-, Nkx3.1-/-; Gpx3-/- were produced. Randomly divided animals were weighed, and their genitourinary tract (GUT) weights were determined after euthanasia at 4, 8, and 12 months. The mRNA expression of the genes involved in oxidative stress and Wnt signaling was analyzed in the prostate. Histopathology, ROS, and superoxide dismutase (SOD) activities were also measured. RESULTS: Loss of Gpx3 did not affect body weight and GUT weight in Nkx3.1 knockout mice. The mRNA expression of SOD3, iNOS, Hmox, and CISD2, which are associated with oxidative stress, was increased in Nkx3.1-/-; Gpx3-/- mice at 4 months but decreased at 8 and 12 months. There was no change in ß-catenin and its targets associated with Wnt signaling. Increased ROS and decreased SOD activity were observed in Nkx3.1-/-; Gpx3-/- mice at 12 months of age. The histopathologic score and epithelium thickness were increased, and lumen area was decreased in Gpx3 knockout mice. DISCUSSION AND CONCLUSIONS: Gpx3 loss increased the hyperplasia of PIN in the pre-cancerous stage of the prostate. Loss of Gpx3 induced oxidative stress. Histopathologically, no invasive carcinoma was identified, and Gpx3 loss did not increase Wnt/ß-catenin signaling. Further research on the role of GPX3 in the transition of PIN to invasive carcinoma is needed. We show, for the first time, that the antioxidant enzyme GPX3 plays a vital role in inhibiting hyperplasia in the PIN stage of the prostate gland in vivo.


Subject(s)
Glutathione Peroxidase/deficiency , Oxidative Stress/physiology , Prostatic Hyperplasia/pathology , Prostatic Intraepithelial Neoplasia/pathology , Reactive Oxygen Species/metabolism , Animals , Homeodomain Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Prostatic Hyperplasia/metabolism , Prostatic Intraepithelial Neoplasia/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Transcription Factors/deficiency , Transcription Factors/genetics
14.
BMB Rep ; 53(9): 466-471, 2020 Sep.
Article in English | MEDLINE | ID: mdl-32336318

ABSTRACT

Several humanized mouse models are being used to study humanspecific immune responses and diseases. However, the pivotal needs of fetal tissues for the humanized mice model have been huddled because of the demand for ethical and medical approval. Thus, we have verified the hematopoietic and immunomodulatory function of HepaRG and developed a new and easy humanized mouse model to replace the use of fetal liver tissue. HepaRG co-transplanted Hu-NSG mice significantly increased CD45+ lymphocytes and CD19+ B cells and CD3+ T cells than normal Hu-NSG, suggesting enhanced reconstitution of the human immune system. These results have improved the applicability of humanized mice by developing new models easily accessible. [BMB Reports 2020; 53(9): 466-471].


Subject(s)
Hematopoietic Stem Cell Transplantation , Animals , Antigens, CD19/immunology , B-Lymphocytes/immunology , CD3 Complex/immunology , Cells, Cultured , Disease Models, Animal , Humans , Leukocyte Common Antigens/immunology , Mice , Mice, Inbred NOD , Mice, SCID , T-Lymphocytes/immunology
15.
BMB Rep ; 53(8): 437-441, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32336319

ABSTRACT

In accordance with requirements of the ICH S7B safety pharmacology guidelines, numerous next-generation cardiotoxicity studies using human stem cell-derived cardiomyocytes (CMs) are being conducted globally. Although several stem cell-derived CMs are being developed for commercialization, there is insufficient research to verify if these CMs can replace animal experiments. In this study, in vitro high-efficiency CMs derived from human embryonic stem cells (hESC-CMs) were compared with Sprague-Dawley rats as in vivo experimental animals, and primary cultured in vitro rat-CMs for cardiotoxicity tests. In vivo rats were administrated with two consecutive injections of 100 mg/kg isoproterenol, 15 mg/kg doxorubicin, or 100 mg/kg nifedipine, while in vitro rat-CMs and hESC-CMs were treated with 5 µM isoproterenol, 5 µM doxorubicin, and 50 µM nifedipine. We have verified the equivalence of hESC-CMs assessments over various molecular biological markers, morphological analysis. Also, we have identified the advantages of hESC-CMs, which can distinguish between species variability, over electrophysiological analysis of ion channels against cardiac damage. Our findings demonstrate the possibility and advantage of high-efficiency hESC-CMs as next-generation cardiotoxicity assessment. [BMB Reports 2020; 53(8): 437-441].


Subject(s)
Cardiotoxicity/metabolism , Embryonic Stem Cells/metabolism , Myocytes, Cardiac/physiology , Animals , Cardiotoxicity/drug therapy , Cell Differentiation/drug effects , Cells, Cultured , Disease Models, Animal , Doxorubicin/pharmacology , Doxorubicin/toxicity , Humans , Induced Pluripotent Stem Cells , Isoproterenol/pharmacology , Isoproterenol/toxicity , Male , Nifedipine/pharmacology , Nifedipine/toxicity , Rats , Rats, Sprague-Dawley
16.
Pathol Oncol Res ; 26(2): 977-984, 2020 Apr.
Article in English | MEDLINE | ID: mdl-30937835

ABSTRACT

Phloretin is a flavonoid with known anticancer activities. However, we do not fully understand how phloretin mitigates prostate cancer on the molecular level. In the present study, we examined changes in proliferation, colony formation, and migration after phloretin treatment in human prostate cancer cells PC3 and DU145. We measured reactive oxygen species (ROS) and gene expression. Phloretin increased ROS and suppressed cell proliferation, migration, and colony formation in both cell lines. Additionally, phloretin treatment increased oxidative stress, as demonstrated through lower antioxidant enzymes (catalase, SOD2, Gpx1, Gpx3). In addition, their regulator CISD2 decreased in expression. We also found that increased ROS significantly downregulated multiple components of the Wnt/ß-catenin signaling pathway (ß-catenin, TCF4, FoxA2, c-Myc) and Twist1. Thus, anticancer activity of phloretin against human prostate cancer cells occurs through generating ROS to influence Wnt/ß-catenin signaling. The results of this study suggest that phloretin has a therapeutic effect on prostate cancer in vitro, inhibiting the proliferation and migration of cancer cell lines PC3 and DU145. The mechanism of phloretin appears to be increasing ROS production. We thus recommend phloretin as a promising anticancer therapeutic agent.


Subject(s)
Antineoplastic Agents/pharmacology , Oxidative Stress/drug effects , Phloretin/pharmacology , Prostatic Neoplasms/pathology , Reactive Oxygen Species/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Male , Prostatic Neoplasms/metabolism , Wnt Signaling Pathway/drug effects
17.
Pathol Oncol Res ; 26(3): 2011-2012, 2020 07.
Article in English | MEDLINE | ID: mdl-31115862

ABSTRACT

AbstractThe original version of this article unfortunately contained an error in Figs. 1, 5 and 6. The asterisks and bars indicating statistical significance were missing in the figures.

18.
Genes Genomics ; 41(6): 701-712, 2019 06.
Article in English | MEDLINE | ID: mdl-30989490

ABSTRACT

BACKGROUND: Knockout (KO) mice developed by homologous recombination (HR) have become useful tools to elucidate gene function. However, HR has low KO efficiency and is time-consuming, labor-intensive, and expensive. 'Gene editing' has received much attention for efficient genetic manipulation. OBJECTIVE: As generation of KO mice is simplified, KO mice produced by HR can be feasibly reproduced using gene editing. However, phenotyping analysis and comparison between KO mice produced by these two techniques is necessary. METHODS: We generated p53 KO mice through gene editing and compared their phenotype with the already reported HR-mediated p53 KO mice. RESULTS: Tumors occurred in 36 (73%) of 49 homozygous KO mice and the mean age of occurrence was 23 weeks, with lymphoma (64%) and sarcoma (23%) being the most common. Tumors were also developed in 12 heterozygous mice and the mean age of occurrence was 40 weeks, with sarcoma (54%) and lymphoma (46%) in high proportion. Homozygotes had a mean life span of 157 ± 52 days and developmental abnormalities were found in females compared to in males (P < 0.05, P < 0.001). CONCLUSION: We analyzed the basic phenotype of p53 KO mice and observed no significant difference from the conventional HR-mediated p53 KO mice.


Subject(s)
Gene Editing/methods , Gene Knockout Techniques/methods , Neoplasms, Experimental/genetics , Phenotype , Tumor Suppressor Protein p53/deficiency , Animals , Female , Male , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/pathology , Tumor Suppressor Protein p53/genetics
19.
Front Pharmacol ; 10: 1517, 2019.
Article in English | MEDLINE | ID: mdl-32009948

ABSTRACT

The United States Food and Drug Administration-approved antipsychotic drug, pimozide, has anticancer activities. However, the role of reactive oxygen species (ROS) in its effect on prostate cancer is not well-known. We examined cell proliferation, colony formation, migration, ROS production, and the expression of antioxidant-related genes after treatment of human prostate cancer PC3 and DU145 cells with pimozide. In addition, histopathology, ROS production, and superoxide dismutase (SOD) activity were analyzed after administering pimozide to TRAMP, a transgenic mouse with prostate cancer. Pimozide increased the generation of ROS in both cell lines and inhibited cell proliferation, migration, and colony formation. Oxidative stress induced by pimozide caused changes in the expression of antioxidant enzymes (SOD1, peroxiredoxin 6, and glutathione peroxidase 2) and CISD2. Co-treatment with glutathione, an antioxidant, reduced pimozide-induced ROS levels, and counteracted the inhibition of cell proliferation. Administration of pimozide to TRAMP mice reduced the progression of prostate cancer with increased ROS generation and decreased SOD activity. These results suggest that the antipsychotic drug, pimozide, has beneficial effects in prostate cancer in vivo and in vitro. The mechanism of pimozide may be related to augmenting ROS generation. We recommend pimozide as a promising anticancer agent.

20.
Sci Total Environ ; 653: 36-44, 2019 Feb 25.
Article in English | MEDLINE | ID: mdl-30399559

ABSTRACT

Trimethyltin chloride (TMT), one of the most widely used organotin compounds in industrial and agricultural fields, is widespread in soil, aquatic systems, foodstuffs and household items. TMT reportedly has toxic effects on the nervous system; however, there is limited information about its effects on eye development and no clear associated mechanisms have been identified. Therefore, in the present study, we investigated eye morphology, vison-related behavior, reactive oxygen species (ROS) production, apoptosis, histopathology, and gene expression to evaluate the toxicity of TMT during ocular development in zebrafish embryos. Exposure to TMT decreased the axial length and surface area of the eye and impaired the ability of zebrafish to recognize light. 2',7'-dichlorofluorescein diacetate and acridine orange assays revealed dose-dependent increases in ROS formation and apoptosis in the eye. Furthermore, pyknosis of retinal cells was confirmed through histopathological analysis. Antioxidative enzyme-related genes were downregulated and apoptosis-inducing genes were upregulated in TMT-treated zebrafish compared to expression in controls. Retinal cell-specific gene expression was suppressed mainly in retinal ganglion cells, bipolar cells, and photoreceptor cells, whereas amacrine cell-, horizontal cell-, and Müller cell-specific gene expression was enhanced. Our results demonstrate for the first time the toxicity of TMT during eye development, which occurs through the induction of ROS-mediated apoptosis in retinal cells during ocular formation.


Subject(s)
Apoptosis/drug effects , Embryonic Development/drug effects , Eye/drug effects , Reactive Oxygen Species/metabolism , Trimethyltin Compounds/adverse effects , Water Pollutants, Chemical/adverse effects , Zebrafish/embryology , Animals , Eye/embryology , Eye/growth & development , Gene Expression Profiling
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