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...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 laparoscopyassisted 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 usedto 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 welldifferentiated 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/m L vs 3.00 ng/m L).CONCLUSION Metastasis V might be associated with a poor prognosis of CRC patients.展开更多
This study examined the synergetic effect of class ⅠA Phosphoinositide 3-kinases catalytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA ...This study examined the synergetic effect of class ⅠA Phosphoinositide 3-kinases catalytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29, SW620 and HCT116. MTT assay was used to measure the inhibitory effect of p110β knockdown on the proliferation of colon cancer cell lines. SubG1 assay and Annexin-Ⅴ FITC/PI double-labeling cytometry were applied to detect cell apoptosis. And cell cycle was evaluated by using PI staining and flow cytometry. The expression of caspase 3, cleaved PARP, p-Akt, T-Akt and p110β was determined by western blotting. The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G0-G1 phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in HCT116). Moreover, inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29, HCT116 and SW620 cell lines. In addition, increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by immunoblotting in p110β knockdown group compared with normal control group and wildtype group. It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.展开更多
Little is reported about the role of PTEN gene in the progression and prognosis of GISTs. This study examined the clinical implications of the tumor suppressor gene PTEN as a prognostic factor in the GISTs. Immunohist...Little is reported about the role of PTEN gene in the progression and prognosis of GISTs. This study examined the clinical implications of the tumor suppressor gene PTEN as a prognostic factor in the GISTs. Immunohistological staining and immunoblotting were employed to examine the PTEN protein expression, and its association with clinical measures. Clinicopathological features were reviewed by a retrospective examination of medical records. Reduced PTEN expression was significantly associated with tumor diameter, mitotic figure count, metastasis and pathological stage of tumor （P〈0.05）, and was not correlated with age, gender and tumor location （P〉0.05）. The 3-year survival rate of the patients with reduced PTEN expression was significantly lower than those with high PTEN expression （P〈0.01）. These results suggest that the expression of PTEN gene was significantly linked with the progression and metastasis of GISTs and it is an independent prognostic factor.展开更多
DNA damage response （DDR） in different cell cycle status of human peripheral blood lymphocytes （PBLs） and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin...DNA damage response （DDR） in different cell cycle status of human peripheral blood lymphocytes （PBLs） and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin （PHA）. The apoptotic ratio and the phosphorylation H2AX （S139） were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin （CPT） or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks （DSBs） were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes （P0.05）. The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.展开更多
Objective： To analyze and discuss cell cycle＇s correlation of y-H2AX, so as to accumulate the data for the further studies of y-H2AX. Methods： MOLT-4 cells, and peripheral blood lymphocytes （PBLs）, with or withou...Objective： To analyze and discuss cell cycle＇s correlation of y-H2AX, so as to accumulate the data for the further studies of y-H2AX. Methods： MOLT-4 cells, and peripheral blood lymphocytes （PBLs）, with or without 48 h stimulation of phytohemagglutinin （PHA）, were irradiated by ultraviolet rays （UV rays）. Fluorescence-labeled y-H2AX antibody was used to detect γ-H2AX foci at the DNA double-strand breaks （DSBs） in chromatin, DNA damage was analyzed by flow cytometry, cell cycle and cell apoptosis were detected by sub-G1 peak method, the expression of γ-H2AX was detected by Western blot. Results： With the progression of time, sub-G1 peak emerged apparently in the DNA histograms, and the cells of apoptosis increased gradually; with the progression of time, the increase of γ-H2AX emerged and firstly raised, then decreased; PBLs with 48 h stimulation of PHA entered apparently cell cycle, cells of S and G2/M phase emerged, and PBLs without stimulation of PHA did not enter cell cycle; Western blot showed the increase of the expression of γ-H2AX, and the increase also firstly raised, then decreased. Conclusion： γ-H2AX expressed in the cells of stationary phase and proliferative phase, and with the progression of time, the increase of γ-H2AX firstly raised, and then decreased.展开更多
Objective： To establish a system in detecting the cell cycle specificity induced by recombinant human Fas ligand in vitro, so as to provide a reliable platform for further exploring the mechanism of cell cycle contro...Objective： To establish a system in detecting the cell cycle specificity induced by recombinant human Fas ligand in vitro, so as to provide a reliable platform for further exploring the mechanism of cell cycle control and regulation in Fas-mediated apoptosis. Methods： The target cells-leukaemia cell lines and activated peripheral blood lymphocytes stimulated by phytohemagglutinin were incubated with recombinant human Fas ligand for 6 to 36 h, apoptosis was detected by sub-G1, common annexin-Ⅴ/PI and modified annexin Ⅴ and propidium iodide （API） methods and analysed by flow cytometry. Results： The modified API method demonstrated that Fas-mediated apoptosis was cell cycle specific and initiated at G1 phase. The common annexinⅤ/PI method showed the most appropriate condition for the detection of typical cell cycle-specific apoptosis. The sub-G1 method could only illuminate late apoptosis and DNA histogram. Conclusion： Fas-mediated apoptosis was cell cycle-specific and initiated at G 1 phase. Based on the modified API and common AnnexinⅤ/PI methods, the establishment of stable and typical cell cycle-specific model in Fas-mediated apoptosis in vitro was feasible.展开更多
基金Supported by the National Natural Science Foundation of China,No.81572861,No.81302309 and No.81372324
文摘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 laparoscopyassisted 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 usedto 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 welldifferentiated 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/m L vs 3.00 ng/m L).CONCLUSION Metastasis V might be associated with a poor prognosis of CRC patients.
基金supported by grants from National Natural Science Foundation of China(No.81072431,30872472,30973496,and30800569)a grant from Innovation Founda-tion of Huazhong University of Science and Technology(No.2010MS027)+1 种基金grants of Foundation of Program973(No.2009CB521802)Special Fund for Central University Basic Scientific Research(No.2011JC062,and2011JC063)
文摘This study examined the synergetic effect of class ⅠA Phosphoinositide 3-kinases catalytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29, SW620 and HCT116. MTT assay was used to measure the inhibitory effect of p110β knockdown on the proliferation of colon cancer cell lines. SubG1 assay and Annexin-Ⅴ FITC/PI double-labeling cytometry were applied to detect cell apoptosis. And cell cycle was evaluated by using PI staining and flow cytometry. The expression of caspase 3, cleaved PARP, p-Akt, T-Akt and p110β was determined by western blotting. The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G0-G1 phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in HCT116). Moreover, inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29, HCT116 and SW620 cell lines. In addition, increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by immunoblotting in p110β knockdown group compared with normal control group and wildtype group. It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.
基金supported by a grant from 973 program from Ministry of Science and Technology of China (No. 2004CB518705,2009CB521802)the National Natural Science Foundation of China (No. 30872472, 30800569)New Century Eminent Talents (No. NCET-04-0699)
文摘Little is reported about the role of PTEN gene in the progression and prognosis of GISTs. This study examined the clinical implications of the tumor suppressor gene PTEN as a prognostic factor in the GISTs. Immunohistological staining and immunoblotting were employed to examine the PTEN protein expression, and its association with clinical measures. Clinicopathological features were reviewed by a retrospective examination of medical records. Reduced PTEN expression was significantly associated with tumor diameter, mitotic figure count, metastasis and pathological stage of tumor （P〈0.05）, and was not correlated with age, gender and tumor location （P〉0.05）. The 3-year survival rate of the patients with reduced PTEN expression was significantly lower than those with high PTEN expression （P〈0.01）. These results suggest that the expression of PTEN gene was significantly linked with the progression and metastasis of GISTs and it is an independent prognostic factor.
基金supported by grants from Program 973 from Ministry of ScienceTechnology of China (Nos. 2004CB518705, 2009CB5218702)the National Natural Sciences Foundation of China (Nos. 30872472, 30800569)
文摘DNA damage response （DDR） in different cell cycle status of human peripheral blood lymphocytes （PBLs） and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin （PHA）. The apoptotic ratio and the phosphorylation H2AX （S139） were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin （CPT） or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks （DSBs） were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes （P0.05）. The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.
基金grants from the National Natural Sciences Foundation of China(No.30570908)Clinical Key Subject Foundation of Health Ministry of China"Cell Cycle Diagnosis and Analysisin ClinicalTumor(Ⅲ)"
文摘Objective： To analyze and discuss cell cycle＇s correlation of y-H2AX, so as to accumulate the data for the further studies of y-H2AX. Methods： MOLT-4 cells, and peripheral blood lymphocytes （PBLs）, with or without 48 h stimulation of phytohemagglutinin （PHA）, were irradiated by ultraviolet rays （UV rays）. Fluorescence-labeled y-H2AX antibody was used to detect γ-H2AX foci at the DNA double-strand breaks （DSBs） in chromatin, DNA damage was analyzed by flow cytometry, cell cycle and cell apoptosis were detected by sub-G1 peak method, the expression of γ-H2AX was detected by Western blot. Results： With the progression of time, sub-G1 peak emerged apparently in the DNA histograms, and the cells of apoptosis increased gradually; with the progression of time, the increase of γ-H2AX emerged and firstly raised, then decreased; PBLs with 48 h stimulation of PHA entered apparently cell cycle, cells of S and G2/M phase emerged, and PBLs without stimulation of PHA did not enter cell cycle; Western blot showed the increase of the expression of γ-H2AX, and the increase also firstly raised, then decreased. Conclusion： γ-H2AX expressed in the cells of stationary phase and proliferative phase, and with the progression of time, the increase of γ-H2AX firstly raised, and then decreased.
基金Supported by the Major State Basic Research Development Program of China (973 program) (No. 2004CB518705, 2002CB513100-2) and Clinical Key Subject Foundation from Ministry of Health of China "Cell Cycle Diag-nosis and Analysis in Clinical Tumor (III)".
文摘Objective： To establish a system in detecting the cell cycle specificity induced by recombinant human Fas ligand in vitro, so as to provide a reliable platform for further exploring the mechanism of cell cycle control and regulation in Fas-mediated apoptosis. Methods： The target cells-leukaemia cell lines and activated peripheral blood lymphocytes stimulated by phytohemagglutinin were incubated with recombinant human Fas ligand for 6 to 36 h, apoptosis was detected by sub-G1, common annexin-Ⅴ/PI and modified annexin Ⅴ and propidium iodide （API） methods and analysed by flow cytometry. Results： The modified API method demonstrated that Fas-mediated apoptosis was cell cycle specific and initiated at G1 phase. The common annexinⅤ/PI method showed the most appropriate condition for the detection of typical cell cycle-specific apoptosis. The sub-G1 method could only illuminate late apoptosis and DNA histogram. Conclusion： Fas-mediated apoptosis was cell cycle-specific and initiated at G 1 phase. Based on the modified API and common AnnexinⅤ/PI methods, the establishment of stable and typical cell cycle-specific model in Fas-mediated apoptosis in vitro was feasible.
