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Objective To investigate the effects of huaier granules on invasion and metastasis of colorectal cancer SW480 cells in vitro, and explore the basic mechanism. Methods The appropriate concentration and duration of huaier granules promoting SW480 cell apoptosis were determined by SubG1 method. Wound healing assay and transwell assay were used to observe the effect of huaier granules on SW480 cell invasion and metastasis. The changes of E-cadherin, twist, snail and vimentin at protein and mRNA levels were examined by Western blotting and Real-Time PCR. Results After treatment with huaier granules at 3. 0 g·L-1 for 36 h, apoptosis of SW480 cells was most significant, and wound healing assay revealed that relative mobility was (31. 36±2. 39)%, compared with (61. 11±1. 09)% in control group (P<0. 01). Number of invaded cells per field of view was (129±12) in treatment group and (354±20) in control group (P<0. 01). After treatment with huaier granules at 3. 0 g·L-1 for 36 h, protein and mRNA levels of E-cadherin were increased, while those of twist, snail and vimentin were decreased. Conclusion Huaier granules can inhibit invasion and metastasis of colorectal cancer SW480 cells in vitro through effectively depressing epithelial-mesenchymal transition.
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The normal range of oral mucosal cell apoptosis and proliferation rate through a larger sample of non-malnourished crowd was investigated, and the nutritional status of clinical patients was assessed. Of 194 clinical patients selected according to "NRS2002" guidance, there were 167 non-malnourished patients and 27 malnourished cases, respectively. Twelve patients with toxic reactions of grade III after postoperative chemotherapy (POC) were chosen. The oral mucosal epithelial apoptosis and proliferation rate were measured by using flow cytometry. The statistical significance was processed by using unpaired t-test. The results showed that there was no significant difference in gender, age and body weight between malnourished and non-malnourished groups. The normal range of oral mucosal epithelial apoptosis and the proliferation rate was (27.50±1.50)% and (15.12±1.68)% in non-malnourished patients, and that was (19.90±4.14)% and (6.66±5.83)% in the malnourished patients, respectively. It is concluded that the normal range of oral mucosa cell apoptosis and proliferation rate is achieved, which can not be influenced by gender, age, weight and other factors, and could be used as a sensitive and accurate index to assess the nutritional status of clinical patients.
ABSTRACT
The normal range of oral mucosal cell apoptosis and proliferation rate through a larger sample of non-malnourished crowd was investigated, and the nutritional status of clinical patients was assessed. Of 194 clinical patients selected according to "NRS2002" guidance, there were 167 non-malnourished patients and 27 malnourished cases, respectively. Twelve patients with toxic reactions of grade III after postoperative chemotherapy (POC) were chosen. The oral mucosal epithelial apoptosis and proliferation rate were measured by using flow cytometry. The statistical significance was processed by using unpaired t-test. The results showed that there was no significant difference in gender, age and body weight between malnourished and non-malnourished groups. The normal range of oral mucosal epithelial apoptosis and the proliferation rate was (27.50±1.50)% and (15.12±1.68)% in non-malnourished patients, and that was (19.90±4.14)% and (6.66±5.83)% in the malnourished patients, respectively. It is concluded that the normal range of oral mucosa cell apoptosis and proliferation rate is achieved, which can not be influenced by gender, age, weight and other factors, and could be used as a sensitive and accurate index to assess the nutritional status of clinical patients.
Subject(s)
Female , Humans , Male , Middle Aged , Apoptosis , Physiology , Cell Proliferation , Mouth Mucosa , Pathology , Physiology , Nutritional Status , PhysiologyABSTRACT
This study examined the role of regulated upon activation normal T cell expressed and secreted (RANTES) and its receptor C-C chemokine receptor type 5 (CCR5) in gastric cancer metastasis and the associated mechanism. The expression of RANTES and CCR5 was detected by using immunohistochemical staining and Western blotting in the gastric cancer tissues obtained from 60 gastric cancer patients with or without lymph node metastasis (n=30 in each). The results showed that the expression levels of RANTES and CCR5 were higher in gastric cancer with lymph node metastasis than in that without metastasis (P<0.05). The expression levels of RANTES in 30 lymph nodes with cancerous invasion were higher than in 30 normal lymph nodes (P<0.05). Chemotactic test revealed that the number of migrating gastric cancer cells (n=295.0±54.6) induced by the protein of cancer-invading lymph nodes was greater than that by the protein mixture from cancer-invading lymph nodes and RANTES antibody (n=42.5±11.6) (P<0.05). RT-PCR showed that the expression levels of the main Th1 cytokines (IL-2, Γ-IFN) were lower in gastric cancer with lymph node metastasis (2.22±0.90, 3.26±1.15 respectively) than in that without metastasis (3.07±1.67, 4.77±1.52 respectively) (P<0.05), but the expression level of the main Th 2 cytokine (IL-10) was higher in gastric cancer with lymph nodes metastasis (6.06±2.04) than in that without metastasis (4.88±1.87) (P<0.05). It was concluded that RANTES and its receptor CCR5 may contribute to gastric cancer metastasis through influencing the balance of Th1/Th2. RANTES and CCR5 may become a marker of gastric cancer metastasis.
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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 (P<0.05). 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.
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In this study, CD133+ subpopulations were isolated from 41 primary colorectal cancer tissues, the proliferation and cell cycle distribution of the cells were examined without in vitro expansion, and then compared to those of cell lines. The detection of CD133 in colorectal cancer tissues, isolation of CD133+ and CD133- epithelial subpopulations, Ki-67/DNA multiparameter assay and cell volume analysis were flow cytometrically conducted. The results showed that Ki-67 expression was correlated with CD133 level in primary cancer tissues, while cell cycle G2/M phase distribution or clinicopathological characteristics was not. In addition, the CD133+ cells showed larger cell volume and higher Ki-67 expression as compared with CD133- cells. But there was no statistically significant difference in G(2)/M phase distribution between the two subpopulations. Our results demonstrated that the CD133+ subpopulation in colorectal cancer tissue contained more actively cycling and proliferating cells, which was not correlated to clinicopathological factors but might contribute to tumor progression and poor clinical outcome.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , AC133 Antigen , Antigens, CD , Metabolism , Cell Cycle , Physiology , Cell Proliferation , Colorectal Neoplasms , Pathology , Glycoproteins , Metabolism , Ki-67 Antigen , Metabolism , Neoplastic Stem Cells , Metabolism , Pathology , Peptides , Metabolism , PrognosisABSTRACT
Objective:To investigate the effect of retinoid-interferon-induced mortality (GRIM-19) gene on the apoptosis of colon cancer. Methods: A GRIM-19 eukaryotic expression vector (pCMV-Flag-GRIM-19) was constructed and transfected into SW480 cells. Expressions of GRIM-19 and apoptosis-related proteins were detected by Western blotting analysis. Apoptosis of SW480 cells was measured by Annexin-V/PI assay and mitochondrial membrane potential JC-1 staining. Results: The GRIM-19 eukaryotic expression vector pCMV-Flag-GRIM-19 was successfully constructed. Expression of GRIM-19 in SW480 cells was up-regulated and that of apoptosis-related protein Bcl-xl was down-regulated after transfection with pCMV-Flag-GRIM-19. Apoptosis rate was (7.7±1.39)% in SW480 cells transfected with pCMV-Flag empty vector and (15.0 ± 2.52)% in pCMV-Flag-GRIM-19 transfected cells (P<0.05). Mitochondrial membrane potential was decreased in (7.5±2.09)% of pCMV-Flag transfected cells and (17.5±3.07)% of pCMV-Flag-GRIM-19 transfected cells (P<0.05). Conclusion: In vitro GRIM-19 transfection can effectively induce apoptosis of colon cancer SW480 cells.
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In this study, CD133+ subpopulations were isolated from 41 primary colorectal cancer tissues, the proliferation and cell cycle distribution of the cells were examined without in vitro expansion, and then compared to those of cell lines. The detection of CD133 in colorectal cancer tissues, isolation of CD133+ and CD133- epithelial subpopulations, Ki-67/DNA multiparameter assay and cell volume analysis were flow cytometrically conducted. The results showed that Ki-67 expression was correlated with CD133 level in primary cancer tissues, while cell cycle G2/M phase distribution or clinicopathological characteristics was not. In addition, the CD133+ cells showed larger cell volume and higher Ki-67 expression as compared with CD133- cells. But there was no statistically significant difference in G(2)/M phase distribution between the two subpopulations. Our results demonstrated that the CD133+ subpopulation in colorectal cancer tissue contained more actively cycling and proliferating cells, which was not correlated to clinicopathological factors but might contribute to tumor progression and poor clinical outcome.
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Objective To study the mechanism of unscheduled Cyclin B1 expression at G_1 phase which is usually at G_2/M phase.Methods Human peripheral blood lymphocytes(PBL) from healthy volunteers were firstly activated by PHA and then went into cell cycle.The cells were collected at 0,36,48 and 60 h after activation and divided into two parts:one for Cyclins/DNA muhiparameter assay,and another for Post-sorting Western blot.Results After activation by PHA,Cyclin B1 and CDK1 of lymphocytes were expressed at G_1 phase.Conclusion Unscheduled Cyclin B1/CDKl probably contributes to lymphocytes in vivo into cell cycle.
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To study the effect of HBx gene on the apoptosis of the cell lines (L02, HepG2) and the interaction between HBx and X-linked inhibitor of apoptosis protein (XIAP), the apoptosis of pcDNA3.1-HBx transiently transfected cell lines (L02, HepG2) was detected by flow cytometry and the mRNA expression of XIAP was assayed by real-time RT-PCR. Our study showed (1) the morphology of L02/pcDNA3.1-HBx was changed and the appearance of the cells mimicked that of HepG2 cells; (2) HBx gene could be detected in L02/pcDNA3.1-HBx and HepG2/ pcDNA3.1-HBx; (3) the apoptosis rate of L02/pcDNA 3.1-HBx was higher than that of L02 cells (P<0.01) and the apoptosis rate of HepG2/pcDNA3.1-HBx was lower than that of HepG2 cells (P<0.05); (4) the XIAP expression in L02 was about 3 times that in L02/pcDNA3.1-HBx cells (P<0.01), and the expression of XIAP in HepG2/pcDNA3.1-HBx was about 4 times that in HepG2 (P<0.01). It is concluded that HBx gene may promote the apoptosis of normal hepatocytes and inhibit the apoptosis of cells of hepatic carcinoma by regulating the expression of XIAP.
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To study the effect of HBx gene on the apoptosis of the cell lines (L02, HepG2) and the interaction between HBx and X-linked inhibitor of apoptosis protein (XIAP), the apoptosis of pcDNA3.1-HBx transiently transfected cell lines (L02, HepG2) was detected by flow cytometry and the mRNA expression of XIAP was assayed by real-time RT-PCR. Our study showed (1) the morphology of L02/pcDNA3. 1-HBx was changed and the appearance of the cells mimicked that of HepG2 cells; (2) HBx gene could be detected in L02/pcDNA3.1-HBx and HepG2/pcDNA3.1-HBx;(3) the apoptosis rate of L02/pcDNA 3.1-HBx was higher than that of L02 cells (P＜0.01) and the apoptosis rate of HepG2/pcDNA3. 1-HBx was lower than that of HepG2 cells (P＜0.05); (4) the XIAP expression in L02 was about 3 times that in L02/pcDNA3.1-HBx cells (P＜0.01), and the expression of XIAP in HepG2/pcDNA3. 1-HBx was about 4 times that in HepG2 (P＜0.01). It is concluded that HBx gene may promote the apoptosis of normal hepatocytes and inhibit the apoptosis of cells of hepatic carcinoma by regulating the expression of XIAP.
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The threshold of cyclin E expression at G1/S boundary is a characteristic feature of cell cycle progressing. In this study, we tried to develop a quantitative approach to analyze cyclin E threshold by multiparameter flow cytometry. The expression of cyclin E in exponentially growing MOLT-4 cells was detected under different photomultiplier tube (PMT) voltages by cyclin E/DNA multiparameter flow cytometry. Additionally, cyclin E was detected in cells which were treated with caffeine and cycloheximide (CHX) under the same PMT voltage. Moreover, the expression of cyclin E in MOLT-4 cells was compared with that in JURKAT cells. Cyclin E threshold was quantified by formula B2/AxC (A, B, C indicates the minimum, threshold, and maximum of cyclin E fluorescence intensity, respectively). Results showed that in MOLT-4 cells, cyclin E threshold calculated by formula B2/AxC was invariable under different PMT settings. It was decreased in cells treated with caffeine and remained changeless in cells treated with cycloheximide. Cyclin E threshold in JURKAT cells was much lower than that in MOLT-4 cells. It was suggested that Formula B2/AxC we firstly set up could be used to analyze cyclin E expression threshold quantitatively.
Subject(s)
Caffeine/pharmacology , Cell Cycle/physiology , Cell Line, Tumor , Cyclin E/analysis , Cycloheximide/pharmacology , DNA, Neoplasm/analysis , Flow Cytometry/methods , Jurkat Cells , Leukemia, Lymphoid/pathologyABSTRACT
The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67antigen were analyzed. The patients were followed-up for about 3 years and the relationship between the above-mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03 %. The S phase cells accounted for 0 to 54 % in the cell cycle and the positive expression of Ki67 ranged from 0 to 52 %.There were 40 cases of LPI (64.5 % ) including 15 negative cases and 22 cases of HPI (35.5 % ) respectively. The DNA anueploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.
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Summary: The threshold of cyclin E expression at G1/S boundary is a characteristic feature of cell cycle progressing. In this study, we tried to develop a quantitative approach to analyze cyclin E threshold by multiparameter flow cytometry. The expression of cyclin E in exponentially growing MOLT-4 cells was detected under different photomultiplier tube (PMT) voltages by cyclin E/DNA multiparameter flow cytometry. Additionally, cyclin E was detected in cells which were treated with caffeine and cycloheximide (CHX) under the same PMT voltage. Moreover, the expression of cyclin E in MOLT-4 cells was compared with that in JURKAT cells. Cyclin E threshold was quantified by formula B2/A×C (A, B, C indicates the minimum, threshold, and maximum of cyclin E fluorescence intensity, respectively). Results showed that in MOLT-4 cells, cyclin E threshold calculated by formula B2/A×C was invariable under different PMT settings. It was decreased in cells treated with caffeine and remained changeless in cells treated with cycloheximide. Cyclin E threshold in JURKAT cells was much lower than that in MOLT-4 cells. It was suggested that Formula B2/A×C we firstly set up could be used to analyze cyclin E expression threshold quantitatively.
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The expression of the costimulatory molecules B7/CD28 in peripheral blood mononuclear cells (PBMC) of the patients with systemic lupus erythematosus (SLE) and its relation to the pathogenesis of SLE were studied. The expression of the costimulatory molecules in PBMC in 30 patients with active SLE and 20 cases of healthy controls was detected by using the techniques of immunofluorescence and flow cytometer. The result showed that the expression percentage of CD28+, CD4+ CD28+ in T cells of PBMC from the patients with SLE decreased significantly as compared with that in healthy control group, while the expression percentage of CD80+, CD19+ CD80+ in B cells was significantly increased than that in healthy control group (P<0.01). It suggested that the abnormal expression of costimulatory molecules B7/CD28 played a role in the pathogenesis of SLE.
Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , B7-1 Antigen , Genetics , CD28 Antigens , Genetics , Flow Cytometry , Leukocytes, Mononuclear , Metabolism , Lupus Erythematosus, Systemic , Blood , Allergy and Immunology , Lymphocyte ActivationABSTRACT
<p><b>OBJECTIVE</b>To confirm the unscheduled in vivo and in vitro expression models of cyclin B1 in cancer cells so as to study the different profiles of cyclin B1 in G(1)-phase immortal cells under different culture states and culture conditions.</p><p><b>METHODS</b>Multiparameter flow cytometry (FCM) was used to correlate the expression of cyclin B1 with the position in cell cycle of immortal cells in vivo and in vitro using the MOLT-4 cell line as control. Cells which belonged to G(1)-phase were sorted by FCM according DNA diploidy, and then the expression of cyclin B1 was examined by confocal microscope to confirm the results. For further analysis, different subgroups in G(1) phase were sorted according to the fluorescent intensity of cyclin E, and then the exact period in G(1) phase when cyclin B1 was expressed, were assayed by Western blot.</p><p><b>RESULTS</b>Unscheduled expression of cyclin B1 expressed in G(1)-phase was found not only in synchronized leukemia cells MOLT-4 and in vivo transformed T-7 cells, but also in vivo tumor cells detached from clinical samples. In the synchronized growing cells, cyclin B1 was mainly detected in the early G(1) phase, while in transformed T7 cells, cyclin B1 was mainly detected in the late G(1) phase.</p><p><b>CONCLUSION</b>The limitation of detecting cyclin B1 is due to its unscheduled expression, rending cyclin B1 being detected at different time-spots in the G(1) phase. This phenomenon may be related to the adjustment between the loss of control in cell proliferation and cell apoptosis, thereby leading to tumorigenesis.</p>
Subject(s)
Humans , Apoptosis , Physiology , Cell Line, Transformed , Cyclin B , Cyclin B1 , Flow Cytometry , G1 Phase , Physiology , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of the spleen on hepatocyte proliferative activity during experimental hepatocarcinogenesis in rats.</p><p><b>METHODS</b>Cell-cycle and expression of proliferating cell nuclear antigen (PCNA) of hepatocytes were studied by immunohistochemical and flow cytometric technique in two groups: splenectomy (45 rats) and sham-operation with laparotomy (45 rats).</p><p><b>RESULTS</b>(1) Hepatocirrhosis was formed in group A earlier than in group B. Marked degenerative changes of the liver parachyma showing vacuolization of hepatocytes were observed in hepatic lobules. (2) The proliferative level of hepatocytes increased with the progression of hepatocarcinogenesis, and topped at the 18 th week. (3) The proliferative level of the splenectomy group was lower than that of the sham-operation group in the mid-stage of carcinogenesis (t = 4.76, P < 0.05). After stopping feeding diethylnitrosamine (DENA), however, no difference was found in the hepatocyte proliferative activity between the two groups at the 18th week.</p><p><b>CONCLUSIONS</b>There is a close relationship between hepatic proliferative activity and spleen, and the spleen may play an important role in facilitating hepatic proliferation.</p>
Subject(s)
Animals , Male , Rats , Cell Cycle , Diethylnitrosamine , Liver , Pathology , Liver Neoplasms, Experimental , Pathology , Proliferating Cell Nuclear Antigen , Rats, Wistar , Spleen , Physiology , SplenectomyABSTRACT
The immunophenotyping expression levels of lymphocyte in the peripheral blood from 21 patients with active systemic lupus erythematosus (SLE) were analyzed by using the immunofluorescence labeling-flow cytometry technique to investigate the immunophenotyping expression of lymphocytes T and B in the peripheral blood of active SLE patients and its clinical value. It was showed that, compared with normal controls, the expression of CD+3, CD+4 and the ratio of CD+4/CD+8 in the peripheral blood of these patients were decreased (P<0.01), while the expression of CD+8, CD+20 was significantly increased (P<0.01). It was suggested that both T and B cells in patients with active SLE involved in immunoregulation, were activated. The abnormal expression of lymphocyte immunophenotyping could influence the immune reaction in SLE patients, which might be one of the important pathogenesis factors in SLE.
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The immunophenotyping expression levels of lymphocyte in the peripheral blood from 21 patients with active systemic lupus erythematosus (SLE) were analyzed by using the immunofluorescence labeling-flow cytometry technique to investigate the immunophenotyping expression of lymphocytes T and B in the peripheral blood of active SLE patients and its clinical value. It was showed that, compared with normal controls, the expression of CD+3, CD+4 and the ratio of CD+4/CD+8 in the peripheral blood of these patients were decreased (P<0.01), while the expression of CD+8, CD+20 was significantly increased (P<0.01). It was suggested that both T and B cells in patients with active SLE involved in immunoregulation, were activated. The abnormal expression of lymphocyte immunophenotyping could influence the immune reaction in SLE patients, which might be one of the important pathogenesis factors in SLE.