RESUMO
OBJECTIVE: To elucidate the effect of hSav1 expression on Mst1-mediated apoptosis in HeLa cells. METHODS: Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 micromol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. RESULTS: Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSav1 could be detect from the anti-Mst1 immunoprecipitation complex. The immunofluorescent labeling showed that hSav1 and Mst1 had the same localization in cells. Overexpressed protein hSav1 did not induce a significant cell apoptosis. However, co-expression of hSav1 with Mst1 resulted in a significant increase of apoptosis above the level seen with Mst1 alone (24.5% +/- 2.4% vs. 39.3% +/- 4.0%, P < 0.05). CONCLUSION: Our findings indicate that hSav1 is a newly identified protein that interacts with Mst1 and augments Mst1-mediated apoptosis.
Assuntos
Apoptose , Proteínas de Ciclo Celular/metabolismo , Citoplasma/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ciclo Celular/genética , Células HeLa , Fator de Crescimento de Hepatócito/genética , Humanos , Plasmídeos , Proteínas Proto-Oncogênicas/genética , TransfecçãoRESUMO
AIM: To detect the existence of isolated cancer cells in the mesentery of colorectum (named as Metastasis V), and investigate its clinical significance in colorectal cancer (CRC) patients. METHODS: Sixty-three CRC patients who received radical excision between January 2012 and September 2015 were included. All the patients underwent laparoscopy-assisted radical colorectomy or proctectomy [with complete mesocolic excision (CME) or total mesorectal excision (TME)] with R0 dissections at the Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The location and size of the primary lesions were recorded immediately after the tumor was removed, with the surrounding mesenterium completely separated along the intestinal wall. Each dissected mesentery sample was analyzed for hematoxylin-eosin staining and immunohistochemistry using cytokeratin 19 antibody. Image Pro Plus Software 6.0 (Media Cybernetics, CA, United States) was used to semi-quantitatively measure the concentration of the cytokeratin 19 immunohistochemistry. The correlation between metastasis found in mesentery and clinicopathological characteristics was examined. The prognosis of patients was also evaluated by preoperative serum CEA level. RESULTS: Metastasis V was detected in 14 of 63 (22.2%) CRC patients who underwent laparoscopy-assisted radical colorectomy or proctectomy (with CME or TME) with R0 dissection in our hospital between January 2012 and September 2015. There was no significant difference in age, gender, tumor size, and tumor location in patients with Metastasis V (P > 0.05). Metastasis V was more likely to occur in poorly differentiated tumor (5/11; 45.5%) than moderately (8/46; 17.4%) and well- differentiated one (1/6; 16.7%). The Metastasis V in N2 stage (9/14; 64.3%) was more frequent that in the N0 stage (3/35; 8.6%) or N1 stages (2/14; 14.3%). In addition, Metastasis V was positively related to the tumor invasive depth (T1:0/1, 0%; T2:1/12, 8.3%; T3:7/39, 17.9%; T4:6/11, 54.5%). Furthermore, preoperative serum CEA level in Metastasis V-positive patients was significantly higher than in Metastasis V-negative patients (4.27 ng/mL vs 3.00 ng/mL). CONCLUSION: Metastasis V might be associated with a poor prognosis of CRC patients.
Assuntos
Neoplasias Colorretais/patologia , Queratina-19/análise , Mesentério/patologia , Neoplasias Peritoneais/patologia , Adulto , Antígeno Carcinoembrionário/sangue , Colectomia/métodos , Neoplasias Colorretais/sangue , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Imuno-Histoquímica , Laparoscopia/métodos , Masculino , Mesentério/citologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Peritoneais/sangue , Neoplasias Peritoneais/diagnóstico , Neoplasias Peritoneais/secundário , Período Pré-Operatório , Prognóstico , Estudos Retrospectivos , Fatores de RiscoRESUMO
BACKGROUND & OBJECTIVE: p55 gamma is one of the regulatory subunits of phosphoinositide 3-kinase (PI3K), which plays an important role in the regulation of PI3K activity. This study was to explore the inhibitory effect of the N-terminal 24 amino acids of the p55 gamma on proliferation of colon carcinoma cell line HT29. METHODS: Ad-N24p55-GFP, containing N-terminal 24 amino acids of the p55 gamma, and control vector Ad-GFP were constructed, and used to infect HT29 cells. The cell cycle progression was detected using flow cytometry. DNA synthesis was analyzed using the BrdU/PI method. The nude mice model bearing HT29 xenograft tumors was used to study the effect of Ad-N24p55-GFP against the tumor. RESULTS: Compared with cells infected with Ad-GFP, cells infected with Ad-N24p55-GFP at the G0/G1 phase were increased from 65.11% to 73.39% P < 0.05, and cells at S and G2/M phases were decreased from 17.37% to 15.08% and from 17.51% to 11.13%, respectively; BrdU-positive cells were decreased from 24.82% to 9.27%. In the nude mice model, the tumor growth was inhibited after the administration of Ad-N24p55-GFP. The tumor weight was (0.32+/-0.08)g vs. (10.67+/-0.3)g and (0.72+/-0.28)g in the Ad-N24P55-GFP group,the blank control group and the Ad-GFP group, respectively. CONCLUSION: In HT29 cells, overexpression of the N-terminal 24 amino acids of the p55 gamma can induce cell cycle arrest, inhibit DNA synthesis and inhibit tumor growth in the nude mice model of HT29 xenograft tumors.
Assuntos
Ciclo Celular , DNA de Neoplasias/biossíntese , Vetores Genéticos , Fosfatidilinositol 3-Quinases/metabolismo , Carga Tumoral/efeitos dos fármacos , Adenoviridae/genética , Animais , Feminino , Proteínas de Fluorescência Verde , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
BACKGROUND & OBJECTIVE: GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) gene is a specific protein to inhibit signal transducers and activators of transcription 3 (STAT3). STAT3 and its pathway are involved in modulating cell proliferation, apoptosis, differentiation, and mediating malignant transformation of cells. This study was to investigate the expression of GRIM-19 and its target gene STAT3 in human colorectal carcinoma tissues, and explore their roles in the tumorigenesis of colorectal carcinoma. METHODS: The expression of GRIM-19, STAT3 and its activated form p-STAT3 in 40 specimens of colorectal carcinoma, adjacent tissue, and normal tissue was determined by immunohistochemistry and Western blot. The correlations of the expression of GRIM-19, STAT3, and p-STAT3 to various clinicopathologic characteristics of colorectal carcinoma were analyzed statistically. The mRNA expression and gene mutation of GRIM-19 in colon cancer cell line SW480 and 23 specimens of colorectal carcinoma, adjacent tissue, and normal tissue were detected by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. RESULTS: The expression of both STAT3 and p-STAT3 were up-regulated in colorectal carcinoma. The mRNA and protein expression of GRIM-19 was obviously lower in colorectal carcinoma than in normal tissues. The expression of GRIM-19 was correlated to clinical stage and cell differentiation of colorectal cancer (P< 0.05). GRIM-19 expression in colorectal cancer was negatively correlated to STAT3 and p-STAT3 expression (Chi2 = 9.95, P = 0.00; Chi2 = 5.10, P = 0.02). No mutation of GRIM-19 gene was detected in colorectal carcinoma tissues. CONCLUSIONS: The low expression or absence of GRIM-19 may play an important role in the tumorigenesis of colorectal carcinoma. The high expression of STAT3 and the low expression of GRIM-19 co-exist in colorectal carcinoma, and may be related to malignant transformation and abnormal proliferation of cells.