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1.
Fitoterapia ; 157: 105108, 2022 Mar.
Article in English | MEDLINE | ID: mdl-34954263

ABSTRACT

This research aimed to investigate the estrogen-like effects of Leonurine hydrochloride (Leo). First, we developed a total synthesis of Leo from 3,4,5-trimethoxy-benzoic acid and the structure was confirmed through 1H NMR and mass spectrometry (MS). Then the estrogenic activity of Leo in vitro and in vivo was studied. The proliferation and proliferation inhibitory effects of Leo on MCF-7 cells and MDA-MB-231 cells indicate that Leo exerts estrogen-like effects through estrogen receptor α (ERα) and estrogen receptor ß((ERß) in vitro. Uterotrophic assay in juvenile mice showed that Leo has an estrogen-like effect in vivo, as it can promote the development of the uterus of juvenile mice, increase its uterine coefficient and the size of the uterine cavity, as well as the increased number of uterine glands and the thickened uterine wall. For further research, cyclophosphamide (CTX) was used to establish a mouse model of ovarian function decline. Through this model, we found that Leo can restore the estrous cycle of mice, increase the number of primordial and primary follicles in the ovaries of mice, and regulate the disordered hypothalamic-pituitary-ovarian (HPOA) axis of mice. Finally, the pharmacokinetics of Leo was studied and oral bioavailability of Leo was calculated to be 2.21%. Leo was synthesized and the estrogen-like effect in vitro and in vivo was confirmed as well as its pharmacokinetics.


Subject(s)
Gallic Acid , Menopause , Animals , Female , Humans , Male , Mice , Rats , Biological Availability , Blotting, Western , Body Weight/drug effects , Estrus/drug effects , Gallic Acid/analogs & derivatives , Gallic Acid/chemical synthesis , Gallic Acid/metabolism , Gallic Acid/pharmacokinetics , Gallic Acid/pharmacology , Gallic Acid/therapeutic use , Hydroxybenzoates/chemical synthesis , Menopause/drug effects , Mice, Inbred ICR , Ovary/pathology , Random Allocation , Sincalide/analysis , Uterus/pathology , Vagina/cytology
2.
Article in English | WPRIM (Western Pacific) | ID: wpr-927630

ABSTRACT

OBJECTIVE@#To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.@*METHODS@#Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.@*RESULTS@#WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.@*CONCLUSION@#Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.


Subject(s)
Animals , Humans , Annexin A5 , Apoptosis , Blotting, Western , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Cell Line , Cell Proliferation , Cells, Cultured , Flow Cytometry , Genotype , In Situ Hybridization , Larva/physiology , Phenotype , RNA/isolation & purification , Real-Time Polymerase Chain Reaction/standards , Rho Guanine Nucleotide Exchange Factors/metabolism , Sincalide/analysis , Spectrophotometry/methods , Zebrafish/physiology
3.
An Acad Bras Cienc ; 92(2): e20181165, 2020.
Article in English | MEDLINE | ID: mdl-32556047

ABSTRACT

This study describes the histological characteristics and distribution of gastrointestinal tract endocrine cells (ECs) of Prochilodus lineatus (detritivorous fish) using immunohistochemical procedures. The digestive tract of P. lineatus was divided into seven portions: stomach (cardial and pyloric), pyloric caeca, and intestine (anterior, glandular, middle and posterior). A pool of specific antisera against cholecystokinin (CCK-8), -neuropeptide Y (NPY), -ghrelin (Ghre) and -leu-enkephalin (Leu-ENK) to identify ECs were used. According to the morphological characteristics of ECs, two different types were identified and classified as open or closed-type. The number of ECs varied throughout the gastrointestinal tract, though a high abundance was found in the anterior intestine and pyloric caeca. A large number of ECs immunoreactive to CCK-8 and NPY were recorded in the anterior, glandular and middle intestine. ECs immunopositive to Leu-ENK were distributed in the stomach and pyloric caeca. For Ghre, immunopositive ECs were restricted to the glandular intestine. The results of the present study indicate that P. lineatus presents an ECs distribution pattern with species-specific particularities. However, CCK showed a distribution similar to that of omnivores, which is possibly related to local signaling functions in order to achieve the correct digestion of the various organisms found in the detritus.


Subject(s)
Characiformes/classification , Enkephalin, Leucine/analysis , Gastrointestinal Tract/chemistry , Ghrelin/analysis , Neuropeptides/analysis , Sincalide/analysis , Animals , Immunohistochemistry
4.
Int. braz. j. urol ; 45(3): 549-559, May-June 2019. tab, graf
Article in English | LILACS | ID: biblio-1012314

ABSTRACT

ABSTRACT Objective: To study the expression patterns of long noncoding RNA (lncRNA) colon cancer-associated transcript 1 (CCAT1) and the changes in cell proliferation, apoptosis, migration and invasion induced by silencing CCAT1 in bladder cancer cells. Materials and Methods: The expression levels of CCAT1 were determined using realtime quantitative polymerase chain reaction in cancerous tissues and paired normal tissues from 34 patients with bladder cancer. The relationship between clinical characteristics and CCAT1 expression was analyzed. And then we conducted cell experiments. Bladder urothelial carcinoma cell lines T24 and 5637 cells were transfected with CCAT1 small interfering RNA (siRNA) or scramble siRNA. Cell proliferation and apoptosis changes were determined using a Cell Counting Kit-8 (CCK-8) assay and a flow cytometry assay. Migration and invasion changes were measured using a wound healing assay and a trans-well assay. microRNAs (miRNAs) were predicted by Starbase 2.0, and their differential expression levels were studied. Results: CCAT1 was significantly upregulated in bladder cancer (P < 0.05). CCAT1 upregulation was positively related to tumor stage (P = 0.004), tumor grade (P = 0.001) and tumor size (P = 0.042). Cell proliferation, migration and invasion were promoted by abnormally expressed CCAT1. miRNAs miR-181b-5p, miR-152-3p, miR-24-3p, miR-148a-3p and miR-490-3p were potentially related to the aforementioned functions of CCAT1. Conclusion: CCAT1 plays an oncogenic role in urothelial carcinoma of the bladder. In addition, CCAT1 may be a potential therapeutic target in this cancer.


Subject(s)
Humans , Male , Female , Aged , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , RNA, Long Noncoding/analysis , Sincalide/analysis , Time Factors , Wound Healing/genetics , Down-Regulation , Gene Expression , Gene Expression Regulation, Neoplastic , Up-Regulation , Cell Movement/genetics , MicroRNAs/genetics , RNA, Small Interfering , Cell Line, Tumor , Cell Proliferation/genetics , Real-Time Polymerase Chain Reaction , Flow Cytometry
5.
Braz J Med Biol Res ; 51(7): e7220, 2018.
Article in English | MEDLINE | ID: mdl-29742265

ABSTRACT

An abnormality in the Lin28/let-7a axis is relevant to the progression of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), which could be a novel therapeutic target for this malignant tumor. The present study aimed to investigate the antiproliferative and anti-invasive effects of urolithin A in a stable full-length HBV gene integrated cell line HepG2.2.15 using CCK-8 and transwell assays. The RNA and protein expressions of targets were assessed by quantitative PCR and western blot, respectively. Results revealed that urolithin A induced cytotoxicity in HepG2.2.15 cells, which was accompanied by the cleavage of caspase-3 protein and down-regulation of Bcl-2/Bax ratio. Moreover, urolithin A suppressed the protein expressions of Sp-1, Lin28a, and Zcchc11, and elevated the expression of microRNA let-7a. Importantly, urolithin A also regulated the Lin28a/let-7a axis in transient HBx-transfected HCC HepG2 cells. Furthermore, urolithin A decelerated the HepG2.2.15 cell invasion, which was involved in suppressing the let-7a downstream factors HMGA2 and K-ras. These findings indicated that urolithin A exerted the antiproliferative effect by regulating the Lin28a/let-7a axis and may be a potential supplement for HBV-infected HCC therapy.


Subject(s)
Carcinoma, Hepatocellular/drug therapy , Coumarins/pharmacology , Liver Neoplasms/drug therapy , MicroRNAs/drug effects , RNA-Binding Proteins/drug effects , Analysis of Variance , Blotting, Western , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , Cell Proliferation/drug effects , Cell Survival/drug effects , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/virology , MicroRNAs/analysis , RNA-Binding Proteins/analysis , Real-Time Polymerase Chain Reaction , Reference Values , Reproducibility of Results , Sincalide/analysis , Time Factors , Virus Replication/drug effects
6.
Braz. j. med. biol. res ; 51(7): e7220, 2018. tab, graf
Article in English | LILACS | ID: biblio-889115

ABSTRACT

An abnormality in the Lin28/let-7a axis is relevant to the progression of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), which could be a novel therapeutic target for this malignant tumor. The present study aimed to investigate the antiproliferative and anti-invasive effects of urolithin A in a stable full-length HBV gene integrated cell line HepG2.2.15 using CCK-8 and transwell assays. The RNA and protein expressions of targets were assessed by quantitative PCR and western blot, respectively. Results revealed that urolithin A induced cytotoxicity in HepG2.2.15 cells, which was accompanied by the cleavage of caspase-3 protein and down-regulation of Bcl-2/Bax ratio. Moreover, urolithin A suppressed the protein expressions of Sp-1, Lin28a, and Zcchc11, and elevated the expression of microRNA let-7a. Importantly, urolithin A also regulated the Lin28a/let-7a axis in transient HBx-transfected HCC HepG2 cells. Furthermore, urolithin A decelerated the HepG2.2.15 cell invasion, which was involved in suppressing the let-7a downstream factors HMGA2 and K-ras. These findings indicated that urolithin A exerted the antiproliferative effect by regulating the Lin28a/let-7a axis and may be a potential supplement for HBV-infected HCC therapy.


Subject(s)
Humans , RNA-Binding Proteins/drug effects , Carcinoma, Hepatocellular/drug therapy , Coumarins/pharmacology , MicroRNAs/drug effects , Liver Neoplasms/drug therapy , Reference Values , Sincalide/analysis , Time Factors , Virus Replication/drug effects , Cell Survival/drug effects , Blotting, Western , Reproducibility of Results , Analysis of Variance , RNA-Binding Proteins/analysis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , MicroRNAs/analysis , Cell Proliferation/drug effects , Hep G2 Cells , Real-Time Polymerase Chain Reaction , Liver Neoplasms/genetics , Liver Neoplasms/virology
7.
Clin. transl. oncol. (Print) ; 19(6): 735-741, jun. 2017. tab, graf
Article in English | IBECS | ID: ibc-162831

ABSTRACT

Background. Acting as a proto-oncogene, long noncoding RNAs (lncRNAs) urothelial carcinoembryonic antigen 1 (UCA1) plays a key role in the occurrence and development of several human tumors. However, the expression and biological functions of UCA1 in glioma are less known. This study discussed the expression of UCA1 in glioma and its effect on the proliferation and cell cycle of glioma cells. Method. LncRNA UCA1 expressions in 64 glioma samples (Grade I-II in 22 cases and Grade III-IV in 42 cases, according to WHO criteria) and 10 normal brain samples were detected using real-time fluorescence quantitative PCR. On this basis, the correlations of UCA1 to clinicopathological characteristics and prognosis of glioma were assessed. Then, using qPCR, the lncRNA UCA1 expressions in glioma cell lines and astrocytes were detected. UCA1-overexpressing glioma cell lines U87 and U251 were further detected after siRNA transfection of these two cell lines, and the impact on cell proliferation and cell cycle was assessed with CCK-8 (cell counting kit-8) assay and flow cytometry method (FCM), respectively. The expression of cyclin D1, a cell cycle-related protein, was detected using Western Blot. Result. LncRNA UCA1 expression in the glioma samples was obviously higher as compared with the normal brain samples (P < 0.001), and the expression was correlated significantly with grading of the tumors (P < 0.05). However, lncRNA UCA1 expression was not correlated with age, gender, tumor size and KPS score (P > 0.05). After interference of UCA1 expression by siRNA transfection, the proliferation of both U251 and SHG-44 cells was inhibited (P < 0.05), with more cells arrested in G0/G1 (P < 0.05). Moreover, cyclin D1 expression was also downregulated considerably. Conclusion. LncRNA UCA1 can promote the proliferation and cell cycle progression of glioma cells by upregulating cyclin D1 transcription. So UCA1 may serve as an independent prognostic indicator and a novel therapeutic target for glioma (AU)


No disponible


Subject(s)
Humans , RNA, Long Noncoding/analysis , RNA, Long Noncoding/metabolism , Glioma/diagnosis , Gene Expression , Cell Proliferation , Sincalide/analysis , Prognosis , Glioma/pathology , Cyclin D1/analysis , Transfection , Blotting, Western , Polymerase Chain Reaction
8.
Mol Cells ; 40(2): 109-116, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28222496

ABSTRACT

Soybean agglutinin (SBA) is an anti-nutritional factor of soybean, affecting cell proliferation and inducing cytotoxicity. Integrins are transmembrane receptors, mediating a variety of cell biological processes. This research aims to study the effects of SBA on cell proliferation and cell cycle progression of the intestinal epithelial cell line from piglets (IPEC-J2), to identify the integrin subunits especially expressed in IPEC-J2s, and to analyze the functions of these integrins on IPEC-J2 cell cycle progression and SBA-induced IPEC-J2 cell cycle alteration. The results showed that SBA lowered cell proliferation rate as the cell cycle progression from G0/G1 to S phase (P < 0.05) was inhibited. Moreover, SBA lowered mRNA expression of cell cycle-related gene CDK4, Cyclin E and Cyclin D1 (P < 0.05). We successfully identified integrins α2, α3, α6, ß1, and ß4 in IPEC-J2s. These five subunits were crucial to maintain normal cell proliferation and cell cycle progression in IPEC-J2s. Restrain of either these five subunits by their inhibitors, lowered cell proliferation rate, and arrested the cells at G0/G1 phase of cell cycle (P < 0.05). Further analysis indicated that integrin α2, α6, and ß1 were involved in the blocking of G0/G1 phase induced by SBA. In conclusion, these results suggested that SBA lowered the IPEC-J2 cell proliferation rate through the perturbation of cell cycle progression. Furthermore, integrins were important for IPEC-J2 cell cycle progression, and they were involved in the process of SBA-induced cell cycle progression alteration, which provide a basis for further revealing SBA anti-proliferation and anti-nutritional mechanism.


Subject(s)
Epithelial Cells/drug effects , Integrins/metabolism , Plant Lectins/pharmacology , Soybean Proteins/pharmacology , Animals , Cell Cycle/drug effects , Cell Line , Cell Proliferation/drug effects , Epithelial Cells/cytology , Epithelial Cells/metabolism , Flow Cytometry , G1 Phase/drug effects , Integrins/antagonists & inhibitors , Resting Phase, Cell Cycle/drug effects , Sincalide/analysis , Sincalide/metabolism , Swine
9.
Zhonghua Nan Ke Xue ; 23(6): 503-509, 2017 Jun.
Article in Chinese | MEDLINE | ID: mdl-29722941

ABSTRACT

OBJECTIVE: To search for the methods of isolating, purifying and culturing corpus cavernosal endothelial cells (CCECs) from SD rats, observe their growth characteristics, and providing seed cells for the study of erectile dysfunction (ED). METHODS: The corpus cavernosal tissue from the SD rat was digested with 0.1% elastase, followed by purification of CCECs with immunomagnetic beads. After further amplification, monoclonal CCECs were sorted out with the cloning cylinder and their morphological and proliferative characteristics were observed. The von Willebrand factor (VWF) in the CCECs was identified by immunofluorescence staining, the CD31 molecule detected by immumohistochemistry, the purity of the CCECs determined by flow cytometry, and the proliferation of the cells measured with CCK-8 and growth curves. RESULTS: After 7 days of purification and culture, the CCECs were fused into a monolayer under the inverted phase-contrast microscope, arranged like flagstones. The growth curves showed that the CCECs were in latency with a low growth rate at 1-2 days, in the logarithmic growth phase with a rapid rate at 3-4 days, and into the platform phase around the 6th day. VWF was positively expressed in the CCECs with much green fluorescence, and so was CD31 with a large number of brownish particles. The positive rate of the CCECs which were labelled with the VWF purified with magnetic beads combined with cloning cylinders was up to (91.9±3.75)%. CONCLUSIONS: High-purity rat CCECs can be cultured successfully using immunomagnetic beads combined with cloning cylinders, with stable proliferation and passage in the endothelial cell medium.


Subject(s)
Cell Culture Techniques , Endothelial Cells/physiology , Erectile Dysfunction/pathology , Penis/cytology , Animals , Cell Movement , Cell Proliferation , Cells, Cultured , Endothelial Cells/chemistry , Endothelial Cells/cytology , Flow Cytometry , Humans , Immunomagnetic Separation , Male , Platelet Endothelial Cell Adhesion Molecule-1/analysis , Rats , Rats, Sprague-Dawley , Sincalide/analysis , von Willebrand Factor/analysis
10.
Oncol Rep ; 33(4): 1753-62, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25634241

ABSTRACT

To evaluate the inhibitory effect and mechanism of capecitabine metronomic chemotherapy on gastric cancer cells. In vitro, the effects of 5-fluorouracil (Fu) metronomic chemotherapy on proliferation, apoptosis, tube formation ability, and angiogenesis were detected. In vivo, Ki-67, CD34 and VEGF were detected by immunohistochemical staining (IHC). Flow cytometry was used to detect the percentage of circulating endothelial progenitors (CEPs), and VEGF and PDGF were detected by ELISA in the peripheral blood of nude mice. The proliferation of the SGC-7901 and AGS gastric cancer cell lines in the metronomic 5-Fu group was decreased compared with the control group in vitro. The total length of the small tubes and tubular junction numbers were significantly lower in the metronomic group than the control group. The VEGF and PDGF levels in the cell culture supernatants were lower in the metronomic group than the control group. Compared with the control group, the CEP percentage was decreased in the peripheral blood of tumor-bearing nude mice following treatment with metronomic 5-Fu or capecitabine chemotherapy. No significant changes were found in the conventional or control group. In the peripheral blood of tumor-bearing nude mice, the VEGF and PDGF levels were decreased in the metronomic groups. Metronomic 5-Fu inhibited the proliferation of gastric cancer cells in vitro and in vivo, and their antitumor effects were non-inferior to those of conventional dose chemotherapy, with mild side effects. Thus, tumor inhibition may be attributed to anti-angiogenesis.


Subject(s)
Adenocarcinoma/pathology , Angiogenesis Inhibitors/pharmacology , Antimetabolites, Antineoplastic/pharmacology , Capecitabine/pharmacology , Stomach Neoplasms/pathology , Adenocarcinoma/blood , Adenocarcinoma/blood supply , Administration, Metronomic , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/therapeutic use , Animals , Antimetabolites, Antineoplastic/administration & dosage , Antimetabolites, Antineoplastic/therapeutic use , Biomarkers, Tumor , Blood Cell Count , Capecitabine/administration & dosage , Capecitabine/therapeutic use , Cell Line, Tumor , Culture Media , Drug Screening Assays, Antitumor , Endothelial Progenitor Cells/drug effects , Fluorouracil/administration & dosage , Fluorouracil/pharmacology , Fluorouracil/therapeutic use , Human Umbilical Vein Endothelial Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Morphogenesis/drug effects , Platelet-Derived Growth Factor/analysis , Sincalide/analysis , Stomach Neoplasms/blood , Stomach Neoplasms/blood supply , Vascular Endothelial Growth Factor A/analysis , Xenograft Model Antitumor Assays
11.
Chem Biol Interact ; 219: 28-36, 2014 Aug 05.
Article in English | MEDLINE | ID: mdl-24704559

ABSTRACT

Artesunate (ART) is a semi-synthetic derivative of artemisinin extracted from Artemisia annua (sweet wormwood) that is conventionally used in anti-malarial drugs and more recently in medications that induce tumor cell apoptosis. Here, we investigated the effects and mechanistic pathways of ART in human myelodysplastic syndrome (MDS), a condition that commonly progresses to acute myeloid leukemia (AML). Human MDS SKM-1 cells, primary bone marrow (PBM) mononuclear cells from patients with refractory anemia with excess blasts (RAEB) or MDS-AML (MDS cell group), and PBM stromal cells from three patients without hematological diseases (non-MDS cell group) were cultured for 24, 48, or 72 h with or without various ART concentrations. CCK-8, western blot, JC-1 fluorescence, and Annexin-V/Propidium iodide (PI) labeling were used to assess cell proliferation, protein levels, mitochondrial membrane potentials (MMPs) and apoptosis, respectively. ART administration dose- and time-dependently inhibited SKM-1 proliferation. At 24, 48, and 72 h, ART IC50 values were 89.92, 4.24, and 1.28 µmol/L, respectively. ART only significantly inhibited proliferation in the MDS cell group, but it has little impact on proliferation of non-MDS cells. ART decreased MMPs, and dose-dependently induced SKM-1 cell apoptosis, peaking at 82.9% when treated with 200 µmol/L ART for 24h. Caspase-3 and -9 activation, poly(ADP-ribose) polymerase cleavage, decreased Bcl-2/Bax ratio and apoptosis inducing factor nuclear localization were implicated in apoptosis. Our results indicate that ART effectively induces apoptosis in SKM-1 cells through both caspase-dependent and -independent mitochondrial pathways.


Subject(s)
Artemisinins/pharmacology , Cell Proliferation/drug effects , Myelodysplastic Syndromes/drug therapy , Apoptosis/physiology , Artemisinins/administration & dosage , Artemisinins/therapeutic use , Artesunate , Benzimidazoles/analysis , Benzimidazoles/metabolism , Blotting, Western , Bone Marrow Cells , Carbocyanines/analysis , Carbocyanines/metabolism , Caspases/analysis , Caspases/metabolism , Cell Line, Tumor , Humans , Inhibitory Concentration 50 , Membrane Potential, Mitochondrial/physiology , Poly(ADP-ribose) Polymerases/analysis , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins c-bcl-2/analysis , Proto-Oncogene Proteins c-bcl-2/metabolism , Sincalide/analysis , Sincalide/metabolism , bcl-2-Associated X Protein/analysis , bcl-2-Associated X Protein/metabolism
12.
Oncol Rep ; 31(5): 2035-42, 2014 May.
Article in English | MEDLINE | ID: mdl-24626955

ABSTRACT

Microrna-143 (miR-143) has been suggested to be a tumor suppressor, yet its role in hematological tumors has not been determined. Thus, we aimed to explore the expression and function of miR-143 in leukemia cells. miR-143 expression was assessed in bone marrow samples from 63 leukemia patients and 15 healthy controls using q-PCR, and its correlation with DNMT3A expression was determined. In addition, after lentiviral-mediated miR-143 overexpression, K562 cell proliferation was evaluated using CCK-8 analysis; cell cycle progression and apoptosis were determined using flow cytometry. The expression of Bcl-2 and pro-caspase-3 and -9 was assessed by q-PCR and western blot analysis, respectively. Leukemia patients had significantly lower relative miR-143 expression than healthy controls (P=0.004), and the expression levels of miR143 and DNMTA3A were negatively correlated (r=-0.663, P=0.001). Overexpression of miR-143 decreased DNMT3A mRNA and protein expression, and significantly reduced K562 cell proliferation at 72 and 96 h (both P ≤ 0.018). In addition, reduced colony formation and cell cycle progression were observed upon miR-143 overexpression. Flow cytometric analysis revealed that the early apoptosis rate was higher in the miR-143 group than the rate in the NC group. Bcl-2 mRNA expression and pro-caspase-3 and -9 protein expression were reduced in the miR-143-expressing cells. These findings suggest that miR-143 plays an important role in leukemia cell proliferation and apoptosis, possibly through silencing of DNMT3A. Further studies are necessary to determine the prognostic value and therapeutic potential of targeting miR-143.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/biosynthesis , Leukemia, Myeloid, Acute/genetics , MicroRNAs/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Adolescent , Adult , Aged , Apoptosis/genetics , Bone Marrow Cells/pathology , Caspase 3/biosynthesis , Caspase 9/biosynthesis , Cell Line, Tumor , Cell Proliferation , Child , DNA Methyltransferase 3A , Female , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Humans , Male , MicroRNAs/biosynthesis , Middle Aged , Proto-Oncogene Proteins c-bcl-2/biosynthesis , RNA, Messenger/biosynthesis , Sincalide/analysis , Young Adult
13.
Asian Pac J Cancer Prev ; 13(3): 821-5, 2012.
Article in English | MEDLINE | ID: mdl-22631655

ABSTRACT

PURPOSE: Centromere protein H (CENP-H) and Ki67 are overexpressed in some malignancies, but whether they are predictors of survival after primary resection for hypopharyngeal squamous cell carcinoma (HSCC) remains unknown. METHODS: We assessed immunohistochemical expression of CENP-H and Ki67 in 112 HSCC specimens collected between March 2003 and March 2005 for analysis by clinical characteristics. The Kaplan-Meier method was used to analyze relapse-free survival and logistic multivariate regression to determine risk factors of relapse-free survival. Cholecystokinin octapeptide assays and flow cytometry were used to examine cell proliferation and apoptosis after siRNA inhibition of CENP-H in HSCC cells. RESULTS: Overall, 50 (44.6%) HSCC specimens showed upregulated CENP-H expression and 69 (61.6%) upregulated Ki67. An increased CENP-H protein level was associated with advanced cancer stage and alcohol history (P=0.012 and P=0.048, respectively) but an increased Ki67 protein level only with advanced cancer stage (P=0.021). Increased CENP-H or Ki67 were associated with short relapse-free survival (P<0.001 or P=0.009, respectively) and were independent predictors of relapse-free survival (P=0.001 and P=0.018, respectively). siRNA knockdown of CENP-H mRNA inhibited cell proliferation and promoted cancer cell apoptosis in vitro. CONCLUSIONS: Upregulated CENP-H and Ki67 levels are significantly associated with short relapse-free survival in HSCC. These factors may be predictors of a relapsing phenotype in HSSC cases.


Subject(s)
Carcinoma, Squamous Cell/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Hypopharyngeal Neoplasms/metabolism , Ki-67 Antigen/metabolism , Neoplasm Recurrence, Local/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis , Carcinoma, Squamous Cell/surgery , Cell Line, Tumor , Cell Proliferation , Chromosomal Proteins, Non-Histone/genetics , Disease-Free Survival , Female , Flow Cytometry , Humans , Hypopharyngeal Neoplasms/surgery , Ki-67 Antigen/genetics , Male , Middle Aged , Neoplasm Recurrence, Local/genetics , RNA Interference , RNA, Small Interfering , Sincalide/analysis
14.
J Endod ; 38(3): 330-8, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22341070

ABSTRACT

INTRODUCTION: Matrix extracellular phosphoglycoprotein (MEPE), a new member of the small integrin binding ligand N-glycosylated (SIBLING) family, is believed to play multifunctional roles in regulation of cell signaling, mineral homeostasis, and mineralization. METHODS: To study how MEPE affects the downstream genes involved in regulation of the proliferation and osteogenesis differentiation of dental pulp cells (DPCs), we explored the proliferation and osteogenesis differentiation capability of DPCs stimulated with recombinant MEPE or transfected with adenoviral-mediated human MEPE gene and used a systematic approach by osteogenesis real-time polymerase chain reaction arrays to profile osteogenesis-related gene expression after MEPE gene transfection. RESULTS: Our results indicated higher proliferation capability in a time- and dose-dependent pattern by cholecystokinin octapeptide assay, and gene/protein expression of osteogenic markers bone sialoprotein, dentin sialophosphoprotein, osteocalcin, and collagen I were up-regulated dependent on time points showed by real-time polymerase chain reaction and Western blot. Moreover, a total of 3 genes, including enamelin, transforming growth factor-ß2, and integrin α2, were significantly up-regulated. CONCLUSIONS: These results indicated that MEPE appeared to play an important positive role in proliferation and osteogenesis differentiation of DPCs through interaction with downstream signals.


Subject(s)
Dental Pulp/cytology , Extracellular Matrix Proteins/genetics , Glycoproteins/genetics , Osteogenesis/genetics , Phosphoproteins/genetics , Adenoviridae/genetics , Adolescent , Adult , Blotting, Western , Cell Differentiation/genetics , Cell Proliferation , Cells, Cultured , Child , Collagen Type I/analysis , Dental Enamel Proteins/genetics , Dental Pulp/physiology , Extracellular Matrix Proteins/analysis , Gene Expression Profiling , Gene Expression Regulation/genetics , Humans , Integrin alpha2/genetics , Integrin-Binding Sialoprotein/analysis , Osteocalcin/analysis , Phosphoproteins/analysis , Real-Time Polymerase Chain Reaction , Recombinant Proteins , Sialoglycoproteins/analysis , Sincalide/analysis , Transfection , Transforming Growth Factor beta2/genetics , Up-Regulation/genetics , Young Adult
15.
Pharm Biol ; 49(10): 1091-6, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21595574

ABSTRACT

CONTEXT: Polygonum amplexicaule D. Don var. sinense Forb. (Polygonaceae) (PAF) is a well known traditional herb used to treat some diseases, such as fractures, rheumatoid arthritis, muscle injury, and pain. However, its pharmacological mechanism of promoting the healing of fractures is still unknown. OBJECTIVE: The present study was designed to investigate the effects of PAF ethanol extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cell in vitro, thereby to illuminate the pharmacological mechanism to promote the healing of fractures. MATERIALS AND METHODS: The effects of PAF ethanol extracts on MC3T3-E1 cell proliferation and differentiation were detected by using CCK-8, cell cycle, alkaline phosphatase (ALP), and prostaglandin E(2) (PGE(2)) assays in vitro. RESULTS: The results showed that PAF ethanol extracts significantly stimulated cell proliferation at 0.1-100 µg/mL and the proportion of cells in S-phase increased from 16.33 to 27.29% in osteoblastic MC3T3-E1 cells. Moreover, PAF ethanol extracts increased ALP expression in MC3T3-E1 cells at the concentration from 0.1 to 100 µg/mL and inhibited PGE(2) production induced by TNF-α in osteoblasts at the concentrations ranging from 10 to 100 µg/mL in MC3T3-E1 osteoblasts. DISCUSSION AND CONCLUSION: These results indicated that PAF directly stimulates cell proliferation and differentiation of osteoblasts; therefore, this study preliminarily explored the pharmacological mechanism of PAF to promote the healing of bone rheumatism and various fractures.


Subject(s)
Bone and Bones/drug effects , Drugs, Chinese Herbal/pharmacology , Plant Extracts/pharmacology , Polygonum/chemistry , Rheumatic Diseases/metabolism , 3T3 Cells , Alkaline Phosphatase/analysis , Animals , Bone and Bones/physiology , Cell Cycle/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Dinoprostone/analysis , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Fractures, Spontaneous/metabolism , Fractures, Spontaneous/physiopathology , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolism , Phytotherapy , Plant Extracts/metabolism , Plant Tubers , Polygonum/cytology , Polygonum/metabolism , Sincalide/analysis
16.
Neuropeptides ; 41(5): 293-9, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17688943

ABSTRACT

The distribution and relative frequency of neuroendocrine cells in the small and large intestines of one-humped camel were studied using antisera against 5-hydroxytryptamine (5-HT), cholecystokinin (CCK-8), somatostatin (SOM), peptide tyrosine tyrosine (PYY), gastric inhibitory polypeptide (GIP), neuronal nitric oxide synthase (nNOS), gastrin releasing peptide (GRP), substance P (SP), and neurokinin A (NKA). Among these cell types, CCK-8 immunoreactive (IR) cells were uniformly distributed in the mucosa, while others showed varied distribution in the villi or crypts of the small intestine. Immunoreactive cells like 5HT, CCK-8, and SOM showed peak density in the villi and crypts of the small intestine and in the colonic glands of the large intestine, while cells containing SP were discerned predominately in the crypts. 5-HT, CCK-8 and SOM cells were mainly flask-shaped and of the open-variety, while PYY and SP immunoreactive cells were mainly rounded or basket-shaped and of the closed variety. Basically the distribution pattern of the endocrine cells in the duodenum, jejunum and colon of the one-humped camel is similar to that of other mammals. Finally, the distribution of these bioactive agents may give clues as to how these agents aid in the function of the intestinal tract of this desert animal.


Subject(s)
Intestine, Large/physiology , Intestine, Small/physiology , Serotonin/analysis , Animals , Camelus , Cholecystokinin/analysis , Gastric Inhibitory Polypeptide/analysis , Gastrin-Releasing Peptide/analysis , Immunohistochemistry , Intestine, Large/cytology , Intestine, Small/cytology , Neurokinin A/analysis , Nitric Oxide Synthase Type III/analysis , Sincalide/analysis , Somatostatin/analysis , Substance P/analysis
17.
Obes Surg ; 17(3): 406-13, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17546851

ABSTRACT

BACKGROUND: Gastric/intestinal electrical stimulation (GIES) has been used to suppress appetite in the treatment of obesity with promising results. However, the mechanisms by which GIES benefits obese patients are not completely understood. This study investigated the acute effects of GIES on gastric and intestinal tissue levels of peptide hormones related to satiety and appetite in rats. METHODS: 32 rats were divided into 4 groups: 1) sham stimulation, 2) gastric electrical stimulation (GES) with pulse trains, 3) GES with long pulse, and 4) duodenal electrical stimulation (DES) with pulse trains. After 2 hours of GIES, the rats were sacrificed immediately, and gastric fundus, duodenum and distal colon were harvested and extracted. Hormone levels of ghrelin, obestatin, cholecystokinin-8 (CCK-8) and peptide YY (PYY) were measured by radioimmunoassay (RIA). RESULTS: 1) The mean gastric fundus ghrelin level was 1789.04+/-362.81 pg/mg in the sham stimulation and significantly decreased with GES of pulse trains (597.85+/-195.33 pg/mg, P=0.012), GES of long pulse (754.6+/-282.6 pg/mg, P=0.039) and DES (731.69+/-110.84 pg/mg, P=0.037). 2) The mean duodenal CCK-8 concentration was 413.27+/-42.14 pg/mg in the sham stimulation and significantly increased by DES (762.6+/-98.75 pg/mg, P=0.013) but not in others. 3) Neither gastric obestatin nor distal colonic PYY was altered by any of GES or DES. CONCLUSIONS: GIES significantly impacts appetite-related peptide hormones in gastric and duodenal tissues. Acute GIES-induced manipulation of gut peptide hormones related to appetite and satiety is a nonpharmacologic, well-tolerated clinical procedure that could substantially contribute to the successful treatment and long-term management of obesity.


Subject(s)
Appetite/physiology , Duodenum/physiology , Peptide Hormones/analysis , Peptide YY/analysis , Sincalide/analysis , Stomach/physiology , Animals , Electric Stimulation , Ghrelin , Male , Rats , Rats, Sprague-Dawley , Satiety Response/physiology
18.
Fish Shellfish Immunol ; 22(3): 252-63, 2007 Mar.
Article in English | MEDLINE | ID: mdl-16844386

ABSTRACT

In recent years a new parasite, causing severe losses, has been detected in farmed turbot, Scophthalmus maximus (L.), in Northwestern Spain. Dead fish showed emaciation and cachexia caused by severe necrotizing enteritis, which affected all areas of the digestive tract. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi. This study was designed to assess the response of the turbot neuroendocrine system against E. scophthalmi infection. Immunohistochemical tests were applied to sections of the gastrointestinal tract of uninfected and E. scophthalmi-infected turbot, and the presence of cholecystokinin (CCK-8), serotonin (5-HT), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) were documented. A higher abundance of both endocrine epithelial cells (ECs) and nerve cell bodies and fibres for CCK-8, 5-HT and SP were recorded in the gastrointestinal tract of infected turbot, whereas VIP-like substance decreased. The results indicate that E. scophthalmi infection in turbot induced changes in the neuroendocrine system, which may cause alterations in gut motility, electrolyte and fluid secretion, and vascular and immune functions.


Subject(s)
Fish Diseases/parasitology , Flatfishes/parasitology , Gastrointestinal Tract/immunology , Neurosecretory Systems/immunology , Protozoan Infections, Animal/immunology , Animals , Calcitonin Gene-Related Peptide/analysis , Calcitonin Gene-Related Peptide/metabolism , Enteroendocrine Cells/immunology , Fish Diseases/immunology , Fish Diseases/pathology , Flatfishes/immunology , Gastrointestinal Tract/innervation , Gastrointestinal Tract/pathology , Immune Sera/immunology , Immunohistochemistry/veterinary , Neurosecretory Systems/metabolism , Protozoan Infections, Animal/parasitology , Protozoan Infections, Animal/pathology , Serotonin/analysis , Serotonin/metabolism , Sincalide/analysis , Sincalide/metabolism , Substance P/analysis , Substance P/metabolism , Vasoactive Intestinal Peptide/analysis , Vasoactive Intestinal Peptide/metabolism
19.
Drug Dev Ind Pharm ; 32(8): 947-53, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16954107

ABSTRACT

The purpose of this research was to study the thermal stability of cholecystokinin octapeptide (CCK-8) in aqueous solution at pH 12 and ionic strength 0.01 M, which were kept as constants, by using isothermal and nonisothermal methods. The isothermal decomposition of CCK-8 was investigated as a function of temperature (40 degrees C to 70 degrees C). Nonisothermal stability studies were performed using a linear increasing temperature program. Two different nonisothermal studies were carried out at 0.25 degrees K and 0.5 degrees K per hour, and the temperature interval varied from 40 degrees C to 82 degrees C. The degradation of CCK-8 followed first-order kinetics, obeying the Arrhenius equation in the experimental temperature range. This indicated that the degradation mechanism of CCK-8 could be the equal within the temperature range studied. The nonisothermal approach resulted in activation energy (Ea) and shelf-life (t90%) values that agree well with those obtained by the isothermal method. The level of uncertainty in the estimates of t90% and Ea values is determined mainly by the extent of drug degradation and temperature change during the experiment. Therefore, nonisothermal experiments save time, labor and materials (i.e. the amount of drugs necessary to conduct the experiment) compared to the classic isothermal experiments, if they are performed using a suitable experimental design and a precise analytical method.


Subject(s)
Radiopharmaceuticals/chemistry , Sincalide/chemistry , Chromatography, High Pressure Liquid , Drug Stability , Sincalide/analysis , Temperature
20.
Mol Nutr Food Res ; 50(4-5): 406-12, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16598808

ABSTRACT

Recently the first Monascus metabolites with a pyridine ring were detected, the monascopyridines A and B. They are formally dehydrogenated derivatives of the red rice pigments rubropunctamine and monascorubramine. Because of their structural similarity, the toxicological effects of these secondary metabolites were studied using immortalized human kidney epithelial cells. The cytotoxicity was determined with the following different endpoint detection methods: metabolic activity, trypan blue exclusion, and electronic cell counting. The compounds led to EC(50) values between 11 and 31 micromol/L but the pigments caused a stronger reduction of the cell viability. Also, the apoptotic potential was examined by measuring caspase 3 activity and detecting apoptotic bodies, but none of the tested compounds induced apoptosis. All four substances caused a rise of the mitotic index to about 9% (100 micromol/L monascopyridine A and B) and 20% (25 micromol/L rubropunctamine and monascorubramine). The significant decrease of the ratio of cells in the ana- and telophase to cells in the prometa- and metaphase proved a stop of the mitosis at the meta- to anaphase control point. The compounds caused mitotic arrest and the formation of structural damages like c-mitosis through interaction with the mitotic spindle. These effects point to an aneuploidy inducing potential, which is linked to cancer formation.


Subject(s)
Apoptosis/drug effects , Mitosis/drug effects , Monascus/metabolism , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/metabolism , 4-Butyrolactone/toxicity , Caspase 3 , Caspases/metabolism , Cell Count , Cell Line, Transformed , Chromatography, High Pressure Liquid , Epithelial Cells , Fermentation , Humans , Isoquinolines/metabolism , Isoquinolines/toxicity , Kidney , Magnetic Resonance Spectroscopy , Mitotic Index , Oryza/microbiology , Sincalide/analysis
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