ABSTRACT
PURPOSE: Lapatinib is a candidate drug for treatment of trastuzumab-resistant, human epidermal growth factor receptor 2 (HER2)–positive gastric cancer (GC). Unfortunately, lapatinib resistance renders this drug ineffective. The present study investigated the implication of forkhead box O1 (FOXO1) signaling in the acquired lapatinib resistance in HER2-positive GC cells. MATERIALS AND METHODS: Lapatinib-resistant GC cell lines (SNU-216 LR2-8) were generated in vitro by chronic exposure of lapatinib-sensitive, HER2-positive SNU-216 cells to lapatinib. SNU-216 LR cells with FOXO1 overexpression were generated by stable transfection of a constitutively active FOXO1 mutant (FOXO1A3). HER2 and MET in SNU-216 LR cells were downregulated using RNA interference. The sensitivity of GC cells to lapatinib and/or cisplatin was determined by crystal violet assay. In addition, Western blot analysis, luciferase reporter assay and reverse transcription–polymerase chain reaction were performed. RESULTS: SNU-216 LR cells showed upregulations of HER2 and MET, but downregulation of FOXO1 compared to parental SNU-216 cells. FOXO1 overexpression in SNU-216 LR cells significantly suppressed resistance to lapatinib and/or cisplatin. In addition, FOXO1 negatively controlled HER2 and MET at the transcriptional level and was negatively controlled by these molecules at the post-transcriptional level. A positive crosstalk was shown between HER2 and MET, each of which increased resistance to lapatinib and/or cisplatin. CONCLUSION: FOXO1 serves as an important linker between HER2 and MET signaling pathways through negative crosstalks and is a key regulator of the acquired lapatinib resistance in HER2-positive GC cells. These findings provide a rationale for establishing a novel treatment strategy to overcome lapatinib resistance in a subtype of GC patients.
Subject(s)
Humans , Blotting, Western , Cell Line , Cisplatin , Down-Regulation , Drug Resistance , Gentian Violet , In Vitro Techniques , Luciferases , Parents , ErbB Receptors , Receptor, ErbB-2 , RNA Interference , Stomach Neoplasms , Transfection , Up-RegulationABSTRACT
PURPOSE: We previously reported that forkhead transcription factors of the O class 1 (FOXO1) expression in gastric cancer (GC) was associated with angiogenesis-related molecules. However, there is little experimental evidence for the direct role of FOXO1 in GC. In the present study, we investigated the effect of FOXO1 on the tumorigenesis and angiogenesis in GC and its relationship with SIRT1. MATERIALS AND METHODS: Stable GC cell lines (SNU-638 and SNU-601) infected with a lentivirus containing FOXO1 shRNA were established for animal studies as well as cell culture experiments. We used xenograft tumors in nude mice to evaluate the effect of FOXO1 silencing on tumor growth and angiogenesis. In addition, we examined the association between FOXO1 and SIRT1 by immunohistochemical tissue array analysis of 471 human GC specimens and Western blot analysis of xenografted tumor tissues. RESULTS: In cell culture, FOXO1 silencing enhanced hypoxia inducible factor-1alpha (HIF-1alpha) expression and GC cell growth under hypoxic conditions, but not under normoxic conditions. The xenograft study showed that FOXO1 downregulation enhanced tumor growth, microvessel areas, HIF-1alpha activation and vascular endothelial growth factor (VEGF) expression. In addition, inactivated FOXO1 expression was associated with SIRT1 expression in human GC tissues and xenograft tumor tissues. CONCLUSION: Our results indicate that FOXO1 inhibits GC growth and angiogenesis under hypoxic conditions via inactivation of the HIF-1alpha-VEGF pathway, possibly in association with SIRT1. Thus, development of treatment modalities aiming at this pathway might be useful for treating GC.
Subject(s)
Animals , Humans , Mice , Angiogenesis Modulating Agents , Hypoxia , Blotting, Western , Carcinogenesis , Cell Culture Techniques , Cell Line , Down-Regulation , Forkhead Transcription Factors , Heterografts , Lentivirus , Mice, Nude , Microvessels , RNA, Small Interfering , Stomach Neoplasms , Tissue Array Analysis , Transcription Factors , Vascular Endothelial Growth Factor AABSTRACT
Although protein kinase B (PKB, AKT) has been investigated extensively for its roles in oncogenic transformation and apoptotic prevention, controversial results are also reported. Here we assessed the role of AKT in the cell growth and expression of a key set of cell cycle regulators in the human normal uroepithelial cell line, SVHUC-1. AKT activity was suppressed by permanent transfection of dominent negative (DN)-AKT with Lipofectamine Plus. Cell viability was measured by the crystal violet assay. DNA contents stained by propidium iodide were measured by flow-cytometry for cell cycle analysis. Cell growth curve showed that overexpression of DN-AKT which suppressed the AKT activity decreased the cell growth. In the cell cycle analysis, overexpression of DN-AKT resulted in a 6% increase in the proportion of cells in G1 phase. Furthermore, DN-AKT overexpression increased the p27(Kip1) protein expression and the activation of a transcription factor FKHR, which induces p27(Kip1) transcription. Our results suggest that, in normal uroepithelial cells, AKT activation increases the cell growth through the influence on p27(Kip1) expression and FKHR activation. Thus, AKT may be used as a biomarker for tumor transformation of bladder uroepithelial cells.
Subject(s)
Humans , Cell Cycle , Cell Line , Cell Survival , DNA , G1 Phase , Gentian Violet , Propidium , Proto-Oncogene Proteins c-akt , Transcription Factors , Transfection , Urinary BladderABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)induces apoptosis in some cancer cells such as breast,prostate,lung,and colon cancer cells,but not normal cells.However,because the effects of TRAIL in gastric cancer cells is unclear,we undertook this study to clarify the effects of TRAIL and its mechanism. To assess the cytotoxicity of TRAIL,two human gastric cancer cell lines,SNU-484 and SNU601,were treated with TRAIL (0-200 ng/mL)in the presence or absence of cycloheximide (1 microgram/mL)for 24 h.Both SNU-484 and SNU-601 were sensitive to TRAIL-induced cell death in a dose-dependent manner.The combination of TRAIL (100 ng/mL)and cycloheximide (1 microgram/mL)for 24 h enhanced cell death and PARP cleavage by promoting activations of caspase-8, caspase-9,and caspase-3,relative to that of TRAIL alone.We further examined the expressions of death receptor 4 (DR4),death receptor 5 (DR5),and FLICE inhibitory protein (FLIP).Although DR4 and DR5 were expressed in both cell lines,the expression of long form (FLIPL )and short form (FLIPS )of FLIP were detected at the low levels. Overexpression of FLIPL or FLIPS in both cell lines rendered the cells resistant to TRAIL.Taken together,our results suggest that FLIP promotes human gastric cancer cell survival against TRAIL-induced apoptosis and is important modulator for TRAIL-induced cell death in human gastric cancer cells.
Subject(s)
Humans , Apoptosis , CASP8 and FADD-Like Apoptosis Regulating Protein , Caspase 8 , Cell Death , Cell Line , Cell Survival , Colonic Neoplasms , Cycloheximide , Necrosis , Receptors, TNF-Related Apoptosis-Inducing Ligand , Stomach NeoplasmsABSTRACT
PURPOSE: The human CD24 antigen is a small heavily glycosylated cell surface protein, which is expressed in hematological malignancies, as well as in a large variety of solid tumors. Its expression is now known to be related to the prognosis of several kinds of tumors. This study is designed to examine the prognostic significance of CD24 in Korean gastric cancer patients. MATERIALS AND METHODS: In the present study, we examined CD24 expression in 300 consecutive cases of gastric carcinoma by immunohistochemical staining using the tissue-array method. We also investigated the clinicopathological profiles related to CD24 expression. RESULTS: One hundred and three cases out of 300 (34.3%) showed the positive expression of CD24. The altered expression of CD24 was significantly associated with differentiated cancer (p=0.003), the intestinal subtype according to the Lauren classification (p<0.001), the advanced stage cancer (p=0.027), with lymphatic invasion (p=0.038) and with vascular invasion (p=0.006). The survival analysis revealed that the patients with CD24 positive expression showed significantly poorer survival than those without CD24 expression. Moreover, a combined evaluation revealed that PTEN /CD24 cases showed the best survival compared to other groups (p=0.01). CONCLUSION: Positive CD24 expression occurs in a subset of gastric carcinomas and it correlates significantly with lymphatic invasion, blood vessel invasion and poor survival.
Subject(s)
Humans , CD24 Antigen , Blood Vessels , Classification , Hematologic Neoplasms , Immunohistochemistry , Prognosis , Stomach Neoplasms , Survival AnalysisABSTRACT
Apoptosis is a genetically programmed cell death that is required for morphogenesis during embryogenic development and for tissue homeostasis in adult organisms. Although apoptosis is important in the development, expression of apoptosis-related genes has been studied mostly in the cancers and neurodegenerative diseases. We intended to obtain the basic data in order to understand the role of the apoptosis-related genes including bcl-2 in the apoptosis in the human development. Immunohiostochemistry for Bcl-2 was performed using Korean fetal lung, kidney, thymus, placenta, testis, small intestine, pancrease, skin, urinary bladder tissues in the 14~30 weeks of the human development. Our results showed that Bcl-2 appeared in early stages of human development in the lung, kidney, thymus, placenta, small intestine, pancreas. As differentiation grew, expression of Bcl-2 decreased and had the tendency of localizing in the bronchial epitheliums, tubular epitheliums, Bowman's epithelium, lymphocytes, synchytial trophoblasts, intestinal epitheliums, ganglionic cells, ductal epitheliums of pancreas. We suggested that in the early stages when differentiation didn't occur cell death was suppressed, in the late stages when differentiation was achieving cell death increased to remove the innecessary portions of the organs to protect the specific cells of the organs having functions. For the efficiency of the experiment, a high-throughput technique, a tissue-array method was applied which contributed to save time, money and labor without performance errors. Tissue-array technique will be useful to fasten the developmental studies.
Subject(s)
Adult , Male , Female , HumansABSTRACT
BACKGROUND: Mutations in the human Cu, Zn-superoxide dismutase(SOD1) gene have been identified in some cases of familial amyotrophic lateral sclerosis(ALS). The aim of this study is to delineate the effect of the SOD1 mutation on neural differentiation, and to investigate the mechanism of neuronal death. METHODS: We studied motorneuron-neurob-lastoma hybrid cells(VSC 4.1) expressing wild type or mutant SOD1(G93A, A4V) during differentiation by dibutyryl cAMP and aphidicolin. RESULTS: Mutant cells(G93A) revealed a decreased viability compared with the control cells, mainly in the early stage ofdifferentiation. The release of cytochrome c and increased nuclear fragmentation were observed. However, cell death was not protected by nonselective caspase inhibitor(z-VAD-fmk), but by the antioxi-dant( Trolox). CONCLUSIONS: The results suggest that oxidative stress may be the main mechanism of neuronal death, particularly in the early stage of differentiation.
Subject(s)
Humans , Aphidicolin , Cell Death , Cytochromes c , Motor Neurons , Neurons , Oxidative StressABSTRACT
Some of the pituitary prolactinomas were reported that they don't have active dopamine receptors and do not respond to bromocriptine which is a dopamine agonist. GH3 cell line which is derived from the rat pituitary tumor cells lacks affinity of dopamine receptors and secrete prolactin as well as small amount of growth hormone. Although it has been reported that epidermal growth factor (EGF) induces functional expression of dopamine receptors on GH3 cells in vitro, there has been a contradictory result. In the present study, EGF effect on the GH3 cell response to the bromocriptine was observed in order to investigate whether EGF induces dopamine receptor expression on dopamine resistant tumors in the absence of serum. GH3 cells were cultured for 4 days in the serum-supplemented medium (SSM) followed by culture in serum-free medium (SFM) with or without EGF. Additionally, effect of tamoxifen was also observed. EGF decreased the cell number and the ratio of cell division of GH3 cells while the ratio of prolactin-immunoreac-tive cells was increased. However, EGF did not show any significant effect on the GH3 cell response to bromocriptine treatment. Although tamoxifen decreased the GH3 cell number by increasing apoptosis, it did not influence GH3 cell response to bromocriptine. Our results indicate that EGF does not increase the affinity of dopamine receptors on GH3 cells and is not useful for the treatment of the dopamine-resistant prolactinoma.
Subject(s)
Animals , Rats , Apoptosis , Bromocriptine , Cell Count , Cell Division , Cell Line , Dopamine , Dopamine Agonists , Epidermal Growth Factor , Growth Hormone , Pituitary Neoplasms , Prolactin , Prolactinoma , Receptors, Dopamine , TamoxifenABSTRACT
This study was performed in order to establish the culture system optimal for the study on pituitary prolactin cells using growth factor and extra cellular matrix components as the culture substrate. The effect of epidermal growth factor (EGF) alone or along with extracellular marix components on GH3 cell growth and PRL expression was assessed using cell count, BrdU-immunocytochemistry and PRL-immunocytochemistry in in vitro cultures on plastic, laminin and Matrigel. EGF decreased the cell growth, BrdU-labeling and increased the PRL-immunoreactive cells regardless of the culture substrate by day 3 of the culture. Matrigel was the best culture substrate to decrease the cell growth and to increase the PRL expression. EGF treatment in the Matrigel culture showed about 80.5% of PRL-immunoreactive cells by day 6 of the culture. These results indicated that Matrigel is the better culture substrate than plastic or laminin to inhibit the overgrowth and to increase the prolactin expression of the GH3 cell and that EGF and Matrigel causes very effective culture environment for the long-term culture of the GH3 cell by synergistic mechanism.
Subject(s)
Cell Count , Epidermal Growth Factor , Extracellular Matrix , Lactotrophs , Laminin , Pituitary Neoplasms , Plastics , ProlactinABSTRACT
Although somatotropes of the pituitary gland which secret growth hormone occupy the highest percentage among pituitary hormone cells, the regulatory mechanism of the somatotropes has not been investigated as much as that of the mammotropes. The present study was performed to investigate the direct effects of estrogen on the somatotropes after estrogen treatment in culture of rat pituiary cells. Quantification of bromodeoxyuridine (BrdU)-labeled cells after double immunocytochemistry for BrdU and growth hormone (GH) or prolactin (PRL) demonstrated that estrogen treatment did not bring about the difference in either BrdU labeling index (BLI), apoptosis or cell number of somatotropes. In comparison, estrogen increased the BLI and relative cell number of mammotropes. It was suggested that the changes in the relative cell number of the somatotropes or mammotropes do not attribute to cell death, but to cell proliferation.
Subject(s)
Animals , Rats , Apoptosis , Bromodeoxyuridine , Cell Count , Cell Death , Cell Proliferation , Estrogens , Growth Hormone , Immunohistochemistry , Pituitary Gland , ProlactinABSTRACT
This study was performed to investigate the effect of Matrigel, a reconstituted basement membrane, on the expression of the anterior pituitary hormones in culture. Rat pituitary cells cultured for 6 days on Matrigel showed 3-dimensional, lobular structures with connecting cells while those on plastic showed flat, polygonal cells forming a monolayer. Western blot analysis showed that prolactin (PRL) content in the anterior pituitary cells was higher compared to those cultured on plastic. In comparison, TSH expression was not increased in cultures on Matrigel. The total cell number and the proportion of fibroblasts was decreased. These results suggested that Matrigel is a useful culture substrate for the enhanced expression of PRL but not for TSH. Further studies are needed in order to find a useful culture substrate for TSH cells.
Subject(s)
Animals , Rats , Basement Membrane , Blotting, Western , Cell Count , Fibroblasts , Pituitary Hormones, Anterior , Plastics , Prolactin , ThyrotrophsABSTRACT
Nitric oxide (NO) involvement has been demonstrated in mechanisms of synaptic plasticity, particularly in hippocampal long-term potentiation, a mechanism that underlies certain forms of learning and memory. Further, NO has been shown to regulate various neurotransmitters which play an important role in learning and memory. Several findings suggest that NO production may be decreased in the aged rat. Changes in the nNOS-containing neurons with aging were demonstrated by immunocytochemistry and in situ hybridization. NOS-immunoreactive cells in aged rats were present in all cortical areas and the hippocampus, and the pattern of distribution was similar to that of the control group. The number of NOS-immunoreactive cells in the cerebral cortex was significantly decreased in the aged rats, but the extent of changes was variable in each area, and ranged from mild decrease (50%). Severely decreased areas were the cingulate cortex, parietal cortex area 1, temporal cortex area 1, 2, 3, medial part of occipital cortex area 2, monocular and binocular part of occipital cortex area 1, entorhinal cortex, hippocampus proper, dentate gyrus and subiculum. Moderately decreased areas (30~50%) were frontal cortex area 1, 2, 3, parietal cortex area 2, forelimb, hindlimb, lateral part of occipital cortex area 2. Slightly decreased area was insular cortex. Morphologically, the number of dendritic branches seemed to be decreased in aged group and the length of dendrites of NOS-IR neurons showed a tendency to shorten. These results indicate the involvement of neuronal system containing NOS in the aging brain, and provide the first morphological evidence for the loss of NOS neurons in the cerebral cortex of the aged rats by immunocytochemistry. Further multidisciplinary investigations involving normal aging and neurodegenerative disease such as Alzheimer's disease are needed to clarify the importance of nitric oxide changes in the cerebral cortex with aging.
Subject(s)
Animals , Rats , Aging , Alzheimer Disease , Brain , Cerebral Cortex , Dendrites , Dentate Gyrus , Entorhinal Cortex , Forelimb , Gyrus Cinguli , Hindlimb , Hippocampus , Immunohistochemistry , In Situ Hybridization , Learning , Long-Term Potentiation , Memory , Neurodegenerative Diseases , Neurons , Neurotransmitter Agents , Nitric Oxide , Plastics , Rabeprazole , TelescopesABSTRACT
To elucidate the endocrine mechanism of human parturition, the expression of c-Jun and c-Fos mRNA were examined in relation to estrogen receptor (ER) and progesterone receptor (PR) in human myometrium. c-Jun mRNA was detected in all myometrial tissues (n=5) during labor but not before labor (n=5) and in oxytocin-resistant postterm pregnancy (n=3). c-Fos mRNA was detected in only one myometrial tissue from a woman in labor. The distribution and intensity of immunostaining for ER and PR were semiquantitatively scored. During the late pregnancies, no significant difference was seen in the receptor scores for myometrial ER and PR between the patients who experienced labor and those who did not. Receptor scores for ER and PR were significantly lower in postterm pregnancy than in late pregnancy, regardless of the labor status. These data suggest that there are no changes in ER and PR in human myometrium during parturition. On the other hand, postterm pregnancy is associated with low ER and PR. c-Jun, induced during labor without changes in ER and PR, may play a role as a signaling mechanism in human myometrium.
Subject(s)
Adult , Female , Humans , Pregnancy , Blotting, Northern , Genes, jun/genetics , Immunohistochemistry , Labor, Obstetric/metabolism , Myometrium/metabolism , Myometrium/cytology , RNA, Messenger/analysis , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Reference ValuesABSTRACT
This study was performed to investigate the effect of progesterone (P) on the estrogen-induced proliferation of mammotropes in cultures of normal rat pituitary cells. Dispersed pituitary cells from male rats were cultured in Dulbecco's Modified Eagle's medium containing striped 10% fetal bovine serum for 6 days. Part of cells were treated with 10(-9) M 17beta-estradiol with or without P (10(-6) M). For the simultaneous detection of mitotic cells and mam-motropes, bromodeoxyuridine (BrdU) labeling and double immunocytochemical staining for BrdU and prolactin were performed. The ratio of BrdU-labeled cells among mammotropes (BLI) was increased by 6.3-fold after 6 day treatment with estradiol, which was partly suppressed by 2.3-fold by additional P treatment. The percentage of mammotropes among pituitary cells was increased by 1.5-fold by estradiol treatment, which was suppressed by 1.1-fold after coadminis-tration of progesterone. Thus, coadmistration of progesterone could suppress the estradiol-induced effect on the BLI and the proportion of mammotropes by 75% and 80%, respectively. In addition, apoptotic ratio of mammotropes was not changed by hormone treatment. The present data demonstrated that progesterone inhibits estradiol-induced proliferation of mammotropes, which may contribute to the decrease in the proportion of mammotrope. The indirect modulatory effect of progesterone on the estradiol-induced increase in the prolactin secretion in the rat pituitary was suggested.
Subject(s)
Animals , Humans , Male , Rats , Bromodeoxyuridine , Estradiol , Estrogens , Progesterone , ProlactinABSTRACT
GH3 cells are derived from rat pituitary tumor cells that secrete prolactin and growth hormone, and important in studying prolactin-secreting pitutary tumors. This study was performed to examine the effects of polylysine on growth and differentiation of GH3 cells by means of (a) cell attachment assay (b) cell count and bromodeoxyuridine labeling and (c) immunohistochemistry for prolactin in the absence or presence of epidermal growth factor (EGF). Cell shape, attachment to the culture surface and growth of GH3 cells were not affected by polylysine coating. The percentages of prolactin-immunoreactive cells were higher in the cells cultured on the polylysine-coated surface compared to those on the plastic surface. Cell number and BrdU incorporation were lower in the EGF-treated cells on both culture surfaces. The results provided basic information on the effects of polylysine coating on GH3 cells in culture and suggested that polylysine coating was useful for the study on GH3 cells because it enhanced cell differentiation as well as it provided stronger attachment than plastic surfaces.
Subject(s)
Animals , Rats , Bromodeoxyuridine , Cell Count , Cell Differentiation , Cell Shape , Epidermal Growth Factor , Growth Hormone , Immunohistochemistry , Pituitary Neoplasms , Plastics , Polylysine , ProlactinABSTRACT
The technique of in situ hybridization using synthetic oligonucleotides labelled by non-radioactive method was developed to localize vasoactive intestinal polypeptide, arginine-vasopressin and oxytocin mRNAs in the rat brain. Also double in situ hybridization technique where combination of non-radioactive and radioactive probes were applied was developed to localize 2 neuropeptide mRNAs in single tissue section. The results were as follows; In non-radioactive in situ hybridization methods using digoxigenin-labelled oligonucleotide probe, alkaline-phosphates method using NBT and BCIP as substrates gave the best result that specific hybridization signals were observed. In radioactive in situ hybridization methods using 35S-labelled oligonucleotide probe, specific hybridization signals were observed in both nuclear track emulsion and X-ray film autoradiography. In double in situ hybridization methods using combination of 35S-labelled and digoxigenin-labelled oligonucleotide probes, specific hybridization signals were observed in the group where K5 emulsion was applied as nuclear track emulsion. The technique of in situ hybridization using digoxigenin-labelled oligonucleotide applied in this study will be useful as alternative for radioactive in situ hybridization technique. Moreover, combination of non-radioactive and radioactive labelled probes in double in situ hybridization technique will be a useful tool for the simultaneous localization of various mRNAs in single section for the study of various neurotransmitters, neuropeptides, receptors and signal transduction molecules.