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Coronavirus disease 2019 (COVID-19) reportedly affects male reproductive function by causing spermatogenesis dysfunction and suppressing testosterone secretion. However, the relationship between COVID-19 and impaired reproductive function, such as whether these effects on reproductive function are a direct effect of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection in male reproductive organs or an indirect effect of high fever, is not known. Here, we examined whether the cell entry molecules of SARS-CoV-2, namely, ACE2, NRP1, TMPRSS2, and FURIN, are expressed in the male reproductive organs using the testes and accessory gonads of macaques during the breeding season. RT-PCR expression analysis showed that the testes alone expressed all four molecules. Immunohistochemical staining of testis tissue sections revealed that ACE2 is expressed in Leydig cells and the apical region of Sertoli cells, whereas NRP1 is expressed in the cell bodies surrounding the Leydig and Sertoli cell nuclei. FURIN is mainly expressed in Leydig cells, secondary spermatocytes, and spermatids. However, TMPRSS2 immunopositive cells were not observed. Therefore, it was not possible to observe cells expressing all four molecules in the gonads and accessory gonads of male primates. These results suggest that SARS-CoV-2 is unlikely to directly affect spermatogenesis in primates or proliferate in cells of the seminiferous tubules and undergo release into the semen through the previously known ACE2-mediated infection route. However, the expression of three molecules, including ACE2, was observed in Leydig cells, suggesting that testosterone synthesis and secretion may be affected when primates, including humans, are infected with SARS-CoV-2.
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BACKGROUND: Preoperative chemotherapy/chemoradiotherapy has been generally considered for the treatment of esophageal squamous cell carcinoma (ESCC) to improve prognosis. We examined the effects of anticancer drugs on the expression of kallikrein-related peptidase 13 (KLK13), a potential ESCC prognostic marker, and its clinical relevance in patients who received chemotherapy/chemoradiotherapy for ESCC. METHODS: Overall, 105 patients with ESCC who received chemotherapy or chemoradiotherapy before esophagectomy were enrolled. The expression of KLK13 in biopsy samples obtained before chemotherapy/chemoradiotherapy and resected ESCC tumors was assessed by immunohistochemical staining. The effects of 5-fluorouracil (5-FU) and/or cisplatin (CDDP) exposure on the expressions of KLK13 and ten-eleven translocation dioxygenases (TET) in ESCC cells were examined by reverse transcription-polymerase chain reaction. RESULTS: Immunohistochemical staining of paired ESCC specimens before (biopsy samples) and after (resected specimens) chemotherapy/chemoradiotherapy demonstrated a change in KLK13 expression. KLK13 and TET2/3 transcriptions were induced when human ESCC cell lines were treated with 5-FU and/or CDDP. Among patients with KLK13-negative status before chemotherapy/chemoradiotherapy, those with KLK13-positive resected tumors had a significantly poorer prognosis than those with KLK13-negative resected tumors (p = 0.0477). By using tumor cells isolated from ESCC biopsy tissues obtained before chemotherapy/chemoradiotherapy, we established a primary culture system and detected the induction of KLK13 expression by anticancer drugs. CONCLUSIONS: Preoperative treatments alter KLK13 expression in ESCC. The conversion of KLK13 expression from a negative status in biopsy samples to a positive status in resected tumor samples is a predictor of poor prognosis. KLK13 status is a potential marker for decision making to avoid harmful chemotherapy/chemoradiotherapy in patients with ESCC.
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Antineoplásicos , Dioxigenases , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimiorradioterapia , Cisplatino/farmacologia , Proteínas de Ligação a DNA , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas do Esôfago/terapia , Fluoruracila , Calicreínas , Prognóstico , Terapia NeoadjuvanteRESUMO
Lung squamous cell carcinoma (LSCC) is associated with poor prognosis. Molecular targeting drugs have been demonstrated to be effective for lung adenocarcinoma; however, they are often not effective for LSCC. Kallikrein-related peptidase 13 (KLK13) expression enhances the malignancy of lung adenocarcinoma; however, its expression and crucial role in LSCC remain largely unknown. The present study examined the relationship between the KLK13 expression and clinicopathological features of LSCC. A total of 94 patients diagnosed with LSCC who underwent lobectomy, segmentectomy or wedge resection were selected. KLK13 expression was evaluated through immunostaining of formalin-fixed paraffin-embedded sections of surgical specimens. Of the 94 LSCC samples, 70 exhibited no KLK13 expression, while the remaining 24 exhibited ectopic expression. KLK13 expression in tumors was focal and restricted to the cytoplasm of keratinized cells. LSCC cases were classified into KLK13-negative and KLK13-positive groups, and KLK13 expression was positively associated with E-cadherin expression (P=0.0143). Associations between KLK13 expression and keratinization (P=0.0052) or absence of lymphatic vessel invasion (P=0.0603) were observed; however, these trends did not reach statistical significance. The present findings indicated that KLK13 expression in keratinized LSCC may have a protective role in lymphatic vessel invasion of LSCC, which suggests its significance for therapeutic applications against LSCC.
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Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites during the refolding and auto-processing reactions were determined by mass spectrometric analyses and were mutated to suppress the numerous degradation reactions that occur during the reactions. In addition, the Lys8 residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase. Finally, a degradation-suppressed [K8D,K63G,K131G,K133A]-proCCN protein was prepared and was found to refold readily without significant degradation. The results of an enzyme assay using casein or Bz-Arg-OEt suggested that the mutations had no significant effect on either the enzyme activity or the protein conformation. Thus, we, herein, provide the non-degradative cocoonase protein to investigate the propeptide-mediated protein folding of the molecule. We also examined the catalytic residues using the degradation-suppressed cocoonase. The point mutations at the putative catalytic residues in cocoonase resulted in the loss of catalytic activity without any secondary structural changes, indicating that the mutated residues play a role in the catalytic activity of this enzyme.
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Dobramento de Proteína , Precursores de Proteínas , Sequência de Aminoácidos , Mutação Puntual , MutaçãoRESUMO
Cocoonase, a protein that is produced by the silkworm (Bombyx mori), is thought to specifically digest the sericin protein of the cocoon and has a high homology with trypsin. Similar to trypsin, cocoonase is folded as an inactive precursor protein which is activated by releasing the propeptide moiety. However, the mechanism responsible for the activation of its catalytic structure has not yet been determined in detail. Therefore, to investigate the activation and folding mechanism of cocoonase, recombinant cocoonase (CCN) and prococoonase (proCCN) were over-expressed in E. coli cells. Both recombinant proteins (proCCN and CCN) were expressed as inclusion bodies in E. coli cells and their folding was examined under several sets of conditions. After the refolding reactions, both of the recombinant proteins were present as the oxidized soluble forms. The proCCN protein was then auto-processed to release the propeptide region for activation. Interestingly, the CCN (CCN∗) derived from the refolded proCCN showed a much stronger protease activity than the refolded CCN from the reduced CCN in a protease assay using Bz-Arg-OEt as a substrate. In addition, the secondary structure of the refolded CCN protein was similar to that of the CCN∗ protein, as evidenced by CD measurements. These results suggest that the CCN protein becomes trapped in a molten globule-like state without the assistance of the propeptide region during the folding process. We therefore conclude that the propeptide region of CCN kinetically accelerates the folding of CCN to adopt the correct conformation of cocoonase at the final step of the folding pathway.
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Bombyx , Escherichia coli , Animais , Bombyx/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Peptídeo Hidrolases/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tripsina/metabolismoRESUMO
Glutathione S-transferase omega 2 (GSTO2) lacks any appreciable GST activity, but it exhibits thioltransferase activity. The significance of GSTO2 in lung function has been reported; however, the precise expression and molecular function of GSTO2 in the lungs remain unclear. In the present study, we found that GSTO2 is expressed in airway basal cells, non-ciliated, columnar Clara cells, and type II alveolar cells, which have self-renewal capacity in the lungs. Contrastingly, no GSTO2 expression was observed in 94 lung squamous cell carcinoma (LSCC) samples. When human LSCC cell lines were treated with 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant GSTO2 hypermethylation in LSCC is the cause of its downregulation. Forced GSTO2 expression in LSCC cell lines inhibited cell growth and colony formation in vitro. In a subcutaneous xenograft model, GSTO2-transfected cells formed smaller tumors in nude mice than mock-transfected cells. Upon intravenous injection into nude mice, the incidence of liver metastasis was lower in mice injected with GSTO2-transfected cells than in those injected with mock-transfected cells. In addition, GSTO2 induction suppressed the expression of ß-catenin and the oxygen consumption rate, but it did not affect the extracellular acidification rate. Furthermore, GSTO2-transfected cells displayed lower mitochondrial membrane potential than mock-transfected cells. When GSTO2-transfected cells were treated with a p38 inhibitor, ß-catenin expression and mitochondrial membrane potential were recovered. Our study indicated that the loss of GSTO2 via DNA hypermethylation contributes to the growth and progression of LSCC, probably by modulating cancer metabolism via the p38/ß-catenin signaling pathway.
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Carcinoma de Células Escamosas/patologia , Regulação para Baixo , Glutationa Transferase/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/patologia , Animais , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Decitabina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise , Humanos , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fosforilação OxidativaRESUMO
BACKGROUND: A previous study conducted a transcriptome analysis of paired normal and esophageal squamous cell carcinoma (ESCC) tissue samples. The results showed that the expression of serine protease 27 (PRSS27) was perturbed in tumor samples. Hence, this retrospective study aimed to validate the prognostic significance of PRSS27 in patients with preoperative treatment for ESCC. METHODS: We enrolled 86 patients who received preoperative treatment before esophagectomy for ESCC. The expression of PRSS27 in resected ESCC and biopsy tissue samples obtained before preoperative treatment was evaluated via immunostaining, and its relationship with clinicopathological features and prognosis was analyzed. RESULTS: In normal esophageal mucosa tissue samples, PRSS27 was expressed in the cytoplasm of spinous cells in the suprabasal layer and basal cells in the basal layer. Of 64 resected ESCC tissue samples, 35 (54.7%) expressed PRSS27 and 29 (45.3%) did not. Moreover, ectopic nuclear expression of PRSS27 was observed. Based on multivariate analysis, PRSS27 expression in resected tumor samples was a predictor of poor prognosis. In cases in which PRSS27 expression was observed in biopsy samples, patients with PRSS27-negative resected tumors had a better postoperative prognosis than those with PRSS27-positive resected tumors. CONCLUSIONS: PRSS27 expression in resected ESCC tissue samples is a poor prognostic factor in ESCC patients with preoperative treatment. Furthermore, conversion of PRSS27 expression from positive in biopsy samples to negative in resected tumor samples is a predictor of good prognosis in these patients. Hence, PRSS27 status is an effective tool for decision making regarding adjuvant treatment in ESCC patients.
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Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Neoplasias de Cabeça e Pescoço , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/terapia , Quimiorradioterapia , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas do Esôfago/terapia , Humanos , Prognóstico , Estudos Retrospectivos , Serina Endopeptidases , Serina ProteasesRESUMO
BACKGROUND: Although maternal MeHg-exposure causes hydrocephalus in the offspring of mice, its pathogenesis has not been fully explained. In the present study, we examined the issue of how maternal MeHg-exposure in mice affects ependymal ciliary movement in the offspring and whether the lateral ventricles in offspring show dilation. METHODS: Pregnant mice were given drinking water containing 0, 10, or 20 mg/L MeHg, or a single dose of 2 mg/kg MeHg. Brain slices were prepared from the offspring and the ependymal ciliary movement of ependymal cells in the third ventricle were observed by a high-speed digital camera. The dilation of the lateral ventricles in the offspring was assessed by histological examination. RESULTS: The administration of MeHg in the drinking water of pregnant mice at levels of 10 mg/L and 20 mg/L MeHg from GD10 to birth caused a significant decrease of ciliary beating frequency (CBF) in ependymal cells of the third ventricle in the weaned offspring. The ependymal ciliary movement of the weaned offspring was particularly sensitive in the case of the administration of MeHg at GD10. Moreover, there was a significant dilation of cross-sectional areas of lateral ventricles in weaned offspring from the pregnant mice that had been administered MeHg. The CBF and the cross-sectional areas of the lateral ventricles improved with time. CONCLUSIONS: These results suggest that the impairment of ependymal ciliary movement by maternal MeHg-exposure contributes to the development of hydrocephalus in the offspring.
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Compostos de Metilmercúrio , Terceiro Ventrículo , Animais , Cílios , Dilatação , Feminino , Humanos , Ventrículos Laterais , Exposição Materna/efeitos adversos , CamundongosRESUMO
Negative energy balance in domestic animals suppresses their reproductive function. These animals commonly use long-chain fatty acids (LCFAs) from adipocytes as an energy source under states of malnutrition. The G-protein coupled receptor, GPR120, is a specific receptor for LCFAs, but its role in reproductive function remains unknown in domestic animals. The purpose of this study was to examine whether GPR120 is involved in the reproductive system of cattle. GPR120 mRNA expression was evaluated in brain, pituitary, and ovarian tissue samples by RT-PCR. GPR120 gene expression was detected with high intensity only in the anterior pituitary sample, and GPR120-immunoreactive cells were found in the anterior pituitary gland. Double immunohistochemistry of GPR120 in the anterior pituitary hormone-producing cells, such as gonadotropes, thyrotropes, lactotropes, somatotropes, and corticotropes, was performed to clarify the distribution of GPR120 in the anterior pituitary gland of ovariectomized heifers. Luteinizing hormone ß subunit (LHß)- and follicle-stimulating hormone ß subunit (FSHß)-immunoreactive cells demonstrated GPR120 immunoreactivity at 80.7% and 85.9%, respectively. Thyrotropes, lactotropes, somatotropes, and corticotropes coexpressed GPR120 at 21.1%, 5.4%, 13.6%, and 14.5%, respectively. In conclusion, the present study suggests that GPR120 in the anterior pituitary gland might mediate LCFA signaling to regulate gonadotrope functions, such as hormone secretion or production, in cattle.
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Subunidade beta do Hormônio Folículoestimulante/metabolismo , Gonadotrofos/metabolismo , Hormônio Luteinizante Subunidade beta/metabolismo , Adeno-Hipófise/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Bovinos , Feminino , Imuno-HistoquímicaRESUMO
Malnutrition is one of the factors that induces reproductive disorders. However, the underlying biological processes are unclear. AMP-activated protein kinase (AMPK) is an enzyme that plays crucial role as a cellular energy sensor. In the present study, we examined the effects of AMPK activation on the transcription of the murine gonadotropin subunit genes Cga, Lhb, and Fshb, and the gonadotropin-releasing hormone receptor Gnrh-r. Real-time PCR and transcription assay using LßT2 cells demonstrated that 5-amino-imidazole carboxamide riboside (AICAR), a cell-permeable AMP analog, repressed the expression of Lhb. Next, we examined deletion mutants of the upstream region of Lhb and found that the upstream regulatory region of Lhb (-2527 to -2198 b) was responsible for the repression by AICAR. Furthermore, putative transcription factors (SP1, STAT5a, and TEF) that might mediate transcriptional control of the Lhb repression induced by AICAR were identified. In addition, it was confirmed that both AICAR and a competitive inhibitor of glucose metabolism, 2-deoxy-D-glucose, induced AMPK phosphorylation in LßT2 cells. Therefore, the upstream region of Lhb is one of the target sites for glucoprivation inducing AMPK activation. In addition, AMPK plays a role in repressing Lhb expression through the distal -2527 to -2198 b region.
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Proteínas Quinases Ativadas por AMP/metabolismo , Hormônio Luteinizante Subunidade beta/genética , Transcrição Gênica/fisiologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Linhagem Celular , Subunidade beta do Hormônio Folículoestimulante/genética , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Hormônio Luteinizante Subunidade beta/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , Receptores LHRH/genética , Receptores LHRH/metabolismo , Ribonucleotídeos/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Glutathione S-transferase omega 2 (GSTO2), which belongs to the superfamily of GST omega class, lacks any appreciable GST activity. Although GSTO2 exhibits thioltransferase and glutathione dehydrogenase activities, its precise expression and physiological functions are still unclear. In the present study, we found that GSTO2 is exclusively expressed in the basal cell layer in Ki67-negative non-proliferative cells in the human esophageal mucosa. GSTO2 overexpression in esophageal squamous cell carcinoma (ESCC) cell lines inhibited cell growth and colony formation, and GSTO2-transfected cells formed smaller tumors in nude mice compared with mock-transfected cells. Interestingly, GSTO2 induction suppressed the expressions of E-cadherin and ß-catenin at the cell-cell contact site. We quantified the phosphorylation levels of key proteins of MAPK signaling pathway and identified phosphorylation of p38. Additionally, HSP27, a downstream molecule of p38, was accelerated in GSTO2-transfected cells, unlike in mock-transfected cells. When GSTO2-transfected cells were treated with a p38 inhibitor, the expression of ß-catenin and the membrane localization of E-cadherin was recovered. We next examined GSTO2 expression in 61 ESCC tissues using quantitative reverse transcription polymerase chain reaction and immunostaining. The results showed that GSTO2 mRNA and protein were significantly reduced in ESCC compared with normal tissues. When human ESCC cell lines were treated with 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant hypermethylation is the cause of the down-regulated expression. Our results indicate that GSTO2 expression inhibits the membrane localization of E-cadherin, probably by modulation of the p38 signaling pathway. Down-regulation of GSTO2 by DNA hypermethylation contributes to the growth and progression of ESCC.
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Caderinas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Glutationa Transferase/genética , beta Catenina/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Metilação de DNA/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Camundongos , Transdução de Sinais/genéticaRESUMO
In the original publication of the article, Fig. 1 was published incorrectly.
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We previously conducted transcriptome analysis of a paired specimen of normal and esophageal squamous cell carcinoma (ESCC) tissues and found that mRNA expression of cystatin A (CSTA), a member of the cystatin superfamily, was perturbed in tumors compared with that in the background mucosa. However, little is known about the significance of CSTA expression in ESCC.The mRNA expression of CSTA was evaluated by qRT-PCR using 28 paired frozen samples of tumor and nontumor mucosae. The protein expression of CSTA was evaluated by the immunostaining of formalin-fixed, paraffin-embedded sections of ESCC samples from 59 patients who underwent surgery, and its relationship with clinical features was analyzed.The mRNA expression of CSTA was significantly decreased in ESCC compared with that in matched normal mucosa (Pâ<â.0001). The protein expression of CSTA was limited in stratum granulosum and stratum spinosum but not in stratum basal in normal esophageal mucosa. It was reduced in all ESCC tissue samples compared with normal tissues; however, CSTA expression levels in tumors showed considerable variation. Of the 59 samples, 20 did not express CSTA, whereas 39 clearly expressed it. The expression of CSTA in tumors was significantly associated with pT classification (deeper tumor invasions) (Pâ=â.0118) and advanced TNM stages (Pâ=â.0497). In CSTA-positive tumor samples, CSTA-expressing cancer cells often expressed Ki67, a proliferation marker, which was in sharp contrast to normal mucosa, where Ki67-expressing cells were limited to the basal layer and did not express CSTA. Furthermore, CSTA expression was observed in all 22 lymph node metastases analyzed.Relatively high levels of CSTA expression in tumors were correlated with tumor progression and advanced cancer stage, including lymph node metastasis.
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Carcinoma de Células Escamosas , Cistatina A , Mucosa Esofágica , Neoplasias Esofágicas , Idoso , Biomarcadores Tumorais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Cistatina A/genética , Cistatina A/metabolismo , Progressão da Doença , Mucosa Esofágica/metabolismo , Mucosa Esofágica/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Japão , Antígeno Ki-67/análise , Metástase Linfática/genética , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , RNA Mensageiro/análise , Estatística como AssuntoRESUMO
OBJECTIVE: Our previous differential transcriptome analysis between a paired specimen of normal and esophageal squamous cell carcinoma (ESCC) tissues found aberrant expression of kallikrein-related peptidase 13 (KLK13) in tumors. In this study, we evaluated the expression of KLK13 in many ESCC cases in relation with clinical features, and the prognosis. METHODS: Eighty-eight ESCC cases were subjected to immunohistological staining for KLK13 and classified into KLK13-negative and KLK13-positive groups. Difference of clinical features and the prognosis between the groups was analyzed. RESULTS: In normal esophageal mucosa, KLK13 expression was evident but limited in the stratum granulosum in all cases. By contrast, only 27 of 88 ESCC samples showed KLK13 expression, whereas the remaining 61 tumors showed no KLK13 expression. The KLK13-positive group was significantly associated with pT classification (deeper tumor invasions; P = 0.0282), pN classification (lymph node metastasis; P = 0.0163), and advanced TNM stage (P = 0.0198). In KLK13-positive samples, KLK13-expressing cells often expressed Ki67, a proliferation marker, unlike normal mucosa, in which Ki67-expressing cells were limited to the basal layer and did not express KLK13. Compared with patients with KLK13-negative group, KLK13-positive group showed poorer postoperative prognosis. CONCLUSION: Relatively high levels of KLK13 expression in ESCC were associated with cell proliferation and correlated with tumor progression, advanced cancer stage, and poor prognosis.
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Carcinoma de Células Escamosas/genética , DNA de Neoplasias/genética , Mucosa Esofágica/metabolismo , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Calicreínas/genética , Idoso , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Mucosa Esofágica/patologia , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Imuno-Histoquímica , Calicreínas/biossíntese , Masculino , Estadiamento de Neoplasias , Prognóstico , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Esophageal squamous cell carcinoma (ESCC) is associated with the accumulation of genetic and epigenetic changes in the background mucosa. Dysregulated DNA methylation is known to lead to the inactivation of tumor suppressor genes and the activation of oncogenes. To identify the genes whose expression is perturbed by abnormal DNA methylation in ESCC, integrative transcriptomics by serial analysis of gene expression (SAGE) and methylome sequencing by methyl-DNA immunoprecipitation (MeDIP) analysis were performed. We found 159 genes with significantly decreased expression in ESCC compared to that in noncancerous esophageal mucosa. MeDIP-seq analysis identified hypermethylation in the promoter region of 56 of these genes. Using surgically resected tissues of 40 cases, we confirmed that the paired-like homeodomain 1 (PITX1) gene was hypermethylated in ESCC compared to that in normal tissues (P < 0.0001) by pyrosequencing. PITX1 overexpression in ESCC cell lines inhibited cell growth and colony formation, whereas PITX1 knockdown accelerated cell growth. A PITX1-transfected ESCC cell line, KYSE30, formed smaller tumors in nude mice than in mock-transfected cells. Hypermethylation of PITX1 was associated with tumor depth (P = 0.0011) and advanced tumor stage (P = 0.0052) and predicted poor survival in ESCC (hazard ratio, 0.1538; 95% confidence interval, 0.03159-0.7488; P = 0.0169). In this study, we found a novel tumor suppressor gene of ESCC, PITX1, which is silenced by DNA hypermethylation. Downregulation of PITX1 contributes to the growth and progression of ESCC. Hypermethylation of the PITX1 in ESCC correlated with tumor progression and advanced stage cancer, and may predict a poor prognosis.
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Fasting-refeeding in mice induces transient hyperproliferation of colonic epithelial cells, which is dependent on the lactate produced as a metabolite of commensal bacteria. We attempted to manipulate colonic epithelial cell turnover with intermittent fasting to prompt recovery from acute colitis. Acute colitis was induced in C57BL/6 mice by administration of dextran sulfate sodium in the drinking water for 5 days. From day 6, mice were fasted for 36 h and refed normal bait, glucose powder, or lactylated high-amylose starch. On day 9, colon tissues were subjected to analysis of histology and cytokine expression. The effect of lactate on the proliferation of colonocytes was assessed by enema in vivo and primary culture in vitro. Intermittent fasting resulted in restored colonic crypts and less expression of interleukin-1ß and interleukin-17 in the colon than in mice fed ad libitum. Administration of lactate in the colon at refeeding time by enema or by feeding lactylated high-amylose starch increased the number of regenerating crypts. Addition of lactate but not butyrate or acetate supported colony formation of colonocytes in vitro. In conclusion, intermittent fasting in the resolution phase of acute colitis resulted in better recovery of epithelial cells and reduced inflammation.
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PURPOSE: The concentration-sensitive sodium channel (NaC) is expressed in alveolar type II epithelial cells and pulmonary microvascular endothelial cells in mouse lungs. We recently reported that NaC contributes to amiloride-insensitive sodium transport in mouse lungs (Respiratory Physiology & Neurobiology, 2016). However, details regarding its physiological role in the lung remain unknown. To examine whether NaC is involved in alveolar fluid clearance during an acute lung injury (ALI), we analyzed the relationship between NaC gene expression in the lung and the development of pulmonary edema in lipopolysaccharide (LPS)-induced ALI mice. METHODS: LPS-induced ALI mice were prepared by the intratracheal administration of LPS. Bronchoalveolar lavage (BAL) neutrophils and lung water content (LWCs) were used as a marker of ALI and pulmonary edema, respectively. NaC protein production in the lung was detected by immunoblotting and immunofluorescence. The gene expressions of NaC and the epithelial sodium channel (ENaC) of LPS-induced ALI mice were examined by quantitative RT-PCR over a time course of 14 days. RESULTS: The BAL neutrophil count increased until day 2 after LPS administration and had nearly recovered by day 6. LWCs in LPS-induced mice gradually increased until day 8 and had recovered by day 14. The expression of the NaC protein in the lungs of LPS-induced mice dramatically decreased from day 2 to day 6, but recovered by day 8. The mRNA expression of NaC decreased in the lung, as well as those for α-, ß-, and γ-ENaC during ALI. Thus, NaC expression is suppressed during the development stage of pulmonary edema and then recovers in the convalescent phase. CONCLUSION: Our results suggest that suppression of the gene expression of NaC is involved in the development of pulmonary edema in ALI.
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Lesão Pulmonar Aguda/induzido quimicamente , Canais Epiteliais de Sódio/análise , Canais de Sódio/análise , Células Epiteliais Alveolares , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Expressão Gênica , Lipopolissacarídeos/efeitos adversos , Pulmão/metabolismo , Camundongos , Alvéolos Pulmonares , Edema PulmonarRESUMO
AIM: To clarify the roles of TWEAK and its receptor Fn14 in 5-fluorouracil (5-FU)-induced diarrhea. METHODS: Diarrhea was induced in wild-type (WT), Fn14 knockout (KO), and IL-13 receptor (IL-13R)α1 KO BALB/c mice using a single injection of 5-FU. Histological analysis, cytokine analysis, and flow cytometry was performed on ileal tissues and cells. Murine colon carcinoma-bearing mice were co-treated with an anti-TWEAK antibody and 5-FU. Embryonic fibroblast response to cytokines was also analyzed. RESULTS: 5-FU induced high Fn14 expression in epithelial cells. The severity of 5-FU-induced diarrhea was lower in Fn14 KO mice compared with WT mice. Administration of anti-TWEAK antibody reduced 5-FU-induced diarrhea without affecting the antitumor effects of 5-FU in vivo. 5-FU-induced expression of IL-13, IL-17A, TNF-α, and IFN-γ in the ileum was Fn14 dependent. The severity of 5-FU-induced diarrhea was lower in IL-13Rα1 KO mice, indicating major role for IL-13 signaling via IL-13Rα1 in pathogenesis. We found that IL-13Rα2, an IL-13 neutralizing/cell protective receptor, was strongly induced by IL-33 in vitro and in vivo. IL-13Rα2 was upregulated in the ileum of 5-FU-treated Fn14 KO mice. Thus, the deletion of Fn14 upregulated IL-13Rα2 expression, which reduced IL-13 expression and activity. CONCLUSION: Disruption of the TWEAK/Fn14 pathway affects several interconnected pathways, including those associated with IL-13, IL-33, and IL-13Rα2, to attenuate 5-FU-induced intestinal side effects.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Citocina TWEAK/metabolismo , Diarreia/induzido quimicamente , Fluoruracila/efeitos adversos , Receptor de TWEAK/metabolismo , Animais , Linhagem Celular Tumoral , Citocina TWEAK/antagonistas & inibidores , Diarreia/metabolismo , Diarreia/prevenção & controle , Interleucina-13/metabolismo , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Interleucina-33/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptor de TWEAK/antagonistas & inibidoresRESUMO
We recently reported that cerebral sodium-glucose transporter type 1 (SGLT-1) plays a role in exacerbation of cerebral ischemia. However, the mechanism by which cerebral SGLT-1 acts remains unclear. Here we demonstrated that sodium influx through cerebral SGLT-1 exacerbates cerebral ischemic neuronal damage. SGLT-specific sodium ion influx was induced using α-methyl-D-glucopyranoside (α-MG). Intracellular sodium concentrations in primary cortical neurons were estimated using sodium-binding benzofuran isophthalate fluorescence. SGLT-1 knockdown in primary cortical neurons and mice was achieved using SGLT-1 siRNA. The survival rates of primary cultured cortical neurons were assessed using biochemical assays 1 day after treatment. Middle cerebral artery occlusion (MCAO) was used to generate a focal cerebral ischemic model in SGLT-1 knockdown mice. The change in fasting blood glucose levels, infarction development, and behavioral abnormalities were assessed 1 day after MCAO. Treatment with 200mM α-MG induced a continuous increase in the intracellular sodium concentration, and this increase was normalized after α-MG removal. Neuronal SGLT-1 knockdown had no effect on 100µM H2O2-induced neuronal cell death; however, the knockdown prevented the neuronal cell death induced by 17.5mM glucose and the co-treatment of 100µM H2O2/8.75mM glucose. Neuronal SGLT-1 knockdown also suppressed the cell death induced by α-MG alone and the co-treatment of 100µM H2O2/0.01mM α-MG. Our in vivo results showed that the exacerbation of cerebral ischemic neuronal damage induced by the intracerebroventricular administration of 5.0µg α-MG/mouse was ameliorated in cerebral SGLT-1 knockdown mice. Thus, sodium influx through cerebral SGLT-1 may exacerbate cerebral ischemia-induced neuronal damage.
Assuntos
Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Neurônios/patologia , Transportador 1 de Glucose-Sódio/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Hiperglicemia/complicações , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Metilglucosídeos/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , RNA Interferente Pequeno/genética , Proteínas de Transporte de Sódio-Glucose/metabolismo , Transportador 1 de Glucose-Sódio/deficiência , Transportador 1 de Glucose-Sódio/genéticaRESUMO
BACKGROUND: Obesity is associated with risk of adenocarcinoma in the proximal stomach. We aimed to identify the links between dietary fat and gastric premalignant lesions. METHODS: C57BL/6 mice were fed high fat diet (HFD), and gastric mucosa was histologically analysed. Morphological changes were also analysed using an electron microscope. Transcriptome analysis of purified parietal cells was performed, and non-parietal gastric corpus epithelial cells were subjected to single-cell gene-expression profiling. Composition of gastric contents of HFD-fed mice was compared with that of the HFD itself. Lipotoxicity of free fatty acids (FFA) was examined in primary culture and organoid culture of mouse gastric epithelial cells in vitro, as well as in vivo, feeding FFA-rich diets. RESULTS: During ~8-20 weeks of HFD feeding, the parietal cells of the stomach displayed mitochondrial damage, and a total of 23% of the mice developed macroscopically distinct metaplastic lesions in the gastric corpus mucosa. Transcriptome analysis of parietal cells indicated that feeding HFD enhanced pathways related to cell death. Histological analysis and gene-expression profiling indicated that the lesions were similar to previously reported precancerous lesions identified as spasmolytic polypeptide-expressing metaplasia. FFAs, including linoleic acid with refluxed bile acids were detected in the stomachs of the HFD-fed mice. In vitro, FFAs impaired mitochondrial function and decreased the viability of parietal cells. In vivo, linoleic acid-rich diet, but not stearic acid-rich diet induced parietal-cell loss and metaplastic changes in mice. CONCLUSIONS: Dietary lipids induce parietal-cell damage and may lead to the development of precancerous metaplasia.