Detection of metastatic cancer cells in mesentery of colorectal cancer 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 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.

[Down-regulation of Chk1/Chk2 gene expression increases apoptosis in irradiated HeLa cells and its mechanism].

OBJECTIVE: To explore the increasing effect of blocking Chk1 and /or Chk2 gene by Chk1 or Chk2-specific antisense oligodeoxynucleotides (AsODN) on apoptosis in HeLa cell line after irradiation and its mechanism of action. METHODS: Asynchronized HeLa cells were exposed to (60)Co-irradiation at different dosage to activate G(2)/M checkpoint arrest. The cell cycle profiles were observed in HeLa cells after irradiation at a range of various doses and different time points by flow cytometry. In the experimental groups, Chk1/2 sODN and AsODN alone or in combination were transfected into HeLa cells, and the cells were exposed to (60)Co-irradiation at 24 h after transfection. The changes of Chk1/2 protein expression were assayed by Western blot and confocal laser scanning microscopy (Confocal), and the cell cycles, apoptosis rates and cell cycle specific apoptosis were detected by annexin V-PI labeling and flow cytometry. RESULTS: Apoptotic response was significantly increased in the Hela cells after G(2)/M arrest and was inversed to activation of G(2)/M checkpoint. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 90% approximately 120%, compared to corresponding sODN control (P < 0.05). Unexpectedly, combined use of Chk1- and Chk2-specific AsODN did not produce synergistic effect as compared to treatment with Chk1- or Chk2-specific AsODN alone (P > 0.05). While irradiated HeLa cells underwent apoptosis preferentially in G(1)-phase, apoptosis occurred in either of G(1)-, S- or G(2)/M -phase in the presence of Chk1 and/or Chk2 AsODN. CONCLUSION: The radioresistance is mainly induced by activating the cell cycle checkpoint signal transduction pathway after irradiation, and abrogating of the key effector Chk1 and Chk2 may increase the apoptotic sensitivity to irradiation due to changes of the pattern of cell cycle specific apoptosis.

[Silencing of cell cycle checkpoint kinase gene enhances cisplatin-induced apoptosis of lung cancer cells].

OBJECTIVE: To investigate the changes in cell cycle induced by cisplatin (DDP) and the effect of antisense oligonucleotide (AsODN) targeting Chk1/2 on DDP-induced apoptosis in lung cancer cell line A549 cells. METHODS: The characteristics of cell cycle and apoptosis induced by DDP were detected by flow cytometry using SubG1 method. Chk1/2 mRNA and protein expression were assayed by RT-PCR and Western blot under best condition of transfection of AsODN targeting Chk1/2 by lipofection. Apoptosis of A549 cells induced by DDP was determined by flow cytometry using AnnexinV-FITC staining after transfection of Chk1/2 AsODN. RESULTS: Asynchronized A549 cells were treated with 10 micromol/L DDP, and significant S-phase arrest was observed at 12 h later. Transfection with antisense oligonucleotide targeting Chk1/2 inhibited the Chk1/2 expression at both mRNA and protein levels. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 100% - 200%, compared with that in the sODN control (P < 0.05), but combined use of Chk1- and Chk2-specific AsODN did not show synergistic effects as compared with that induced by treatment with Chk1- or Chk2-specific AsODN alone (P > 0.05). CONCLUSION: Chk1 and Chk2 may be regarded as effective targets of chemotherapy for lung cancer. Silencing the key effector Chk1 and Chk2 genes may significantly increase the chemosensitivity of lung cancer cells.

[Paclitaxel induces apoptosis of acute leukemia cells in S phase].

BACKGROUND & OBJECTIVE: Cell cycle specificity, an important feature of anticancer drugs, is commonly used in the design of combined chemotherapy. Paclitaxel is widely accepted as an G2/M phase-specific agent. However, cumulative evidences revealed that the cell cycle specificity of anticancer drugs in vitro is not always consistent as in vivo. This study was to observe the effect of paclitaxel on the cell cycle specificity in different cell models. METHODS: Effects of paclitaxel on cell apoptosis of human lymphocyte leukemia cell line Molt-4 and 17 clinical specimens of acute leukemia were investigated using flow cytometry. RESULTS: Paclitaxel induced G2/M phase-specific apoptosis in exponentially growing Molt-4 cells, G0/G1 phase-specific apoptosis in high-density cultured Molt-4 cells, and S phase-specific apoptosis in acute leukemia specimens. CONCLUSIONS: Paclitaxel exhibits different cell-cycle specificity in different models. Different from other studies, paclitaxel induces S phase-specific apoptosis in clinical leukemia specimens. This difference is most likely related to the growing status of the target cells.

[Correlation of receptor-mediated apoptosis to cell cycle].

BACKGROUND & OBJECTIVE: Chondriosome-mediated apoptosis is closely related to cell cycle, however, the correlation of receptor-mediated apoptosis to cell cycle progression is unclear yet. This study was to observe the receptor-mediated apoptosis and cell cycle specificity in cultured normal and tumor lymphocytes, and investigate their correlation. METHODS: Exponentially growing human leukemia cell lines Molt-4 and Jurkat were treated with tumor necrosis factor-alpha (TNF-alpha) or Anti-Fas. Peripheral blood lymphocytes (PBLs) from healthy donors were stimulated by phytohemagglutinin (PHA), and further incubated with the presence of TNF-alpha or anti-Fas. Annexin V/PI was used to detect the apoptosis, and API method was used to illustrate the cell cycle specificity of apoptotic cells. RESULTS: Unstimulated PBLs kept blunt to stimulation with TNF-alpha or anti-Fas, and the apoptotic rate was 6%-8%. Molt-4 cells, Jurkat cells, and stimulated PBLs which were treated with TNF-alpha or anti-Fas went to apoptosis, and the apoptosis rates were 15%-28%. Most receptor-mediated apoptosis happened in early G1 phase. CONCLUSION: Receptor-mediated apoptosis is closely related to cell cycle and presents cell cycle specificity.

[Effect of caffeine on camptothecin-induced apoptosis of Molt-4 cells].

BACKGROUND & OBJECTIVE: Caffeine could act on cell cycle checkpoints and affect the progression of cell cycle, but its impact on apoptosis of tumor cells is in debate. This study was carried out to investigate the effects of caffeine on camptothecin-induced apoptosis and cell cycle checkpoints of leukemia cell line Molt-4. METHODS: The cell apoptosis was induced by camptothecin, and caffeine was used to interfere with cell cycle checkpoints. The apoptosis rate and cell cycle during apoptosis were analyzed using sub-G1 method and Annexin V-propidium iodide (Annexin V/PI) staining. RESULTS: Caffeine (2.0-20.0 mmon/L) had no effect on proliferation of Molt-4 cells in exponentially growth phase. Camptothecin selectively induced apoptosis of Molt-4 cells in S phase; when induced with camptothecin (0.15 micromol/L) for 4 or 6 h, the apoptosis rates were (23.69+/-2.26)% and (36.99+/-1.42)%. This cell cycle-specific apoptosis were inhibited obviously by caffeine with the apoptosis rates of (4.79+/-0.64)% and (2.69+/-0.56)%. When caffeine was removed, the apoptosis rates increased obviously to (46.23+/-0.21)% and (55.81+/-0.41)%, and still mainly happened in S phase. CONCLUSIONS: Caffeine could inhibit camptothecin-induced apoptosis of Molt-4 cells. As a drug acting on cell cycle checkpoints, caffeine could transiently shield the surveillance of checkpoints to damaged cells and inhibit cell apoptosis. The effect may be reversed when caffeine is removed away.

[Analysis of cell cycle checkpoints by cyclins/DNA flow cytometry].

BACKGROUND & OBJECTIVE: Eukaryotic cell cycle events progress strictly in order which is controlled by the mechanism of checkpoint. At present, most analyses of checkpoint use the flow cytometry (FCM) based on DNA histogram to detect cell cycle distribution. This study was designed to set up and evaluate a new method, Cyclins/DNA multiparameter FCM based on the model of late G1 phase (G1L) checkpoint, for analyzing cell cycle checkpoint. METHODS: After irradiation by ultraviolet (UV), human acute lymphatic leukemia MOLT-4 cells were gathered and fixed at different time points, and divided into 2 groups. In one group, the total G0/G1 phase cells were calculated by Modifit software using DNA histogram method; in the other group, fluorescence intensity and threshold of Cyclin E in G1L cells and G0/G1 phase cells were quantitatively analyzed by Cyclins/DNA multiparameter method. RESULTS: When analyzed by DNA histogram method, the percentage of G0/G1 phase cells was unchanged after irradiated for 0-4 h, but increased to 12.6% after irradiated for 6 h. When analyzed by Cyclins/DNA multiparameter method, the Cyclin E fluorescence intensity of G1L cells was increased from 295.1 (control) to 341.2 (15.6%) after irradiated for 1 h, and increased to 577.6 (95.7%) with the threshold increased from 2.0 (control) to 5.4 after irradiated for 6 h; G1L cells was slightly decreased after irradiated for 6 h when the apoptosis rate was 5.61%, and early G1 phase (G1E) cells was increased slowly. CONCLUSION: Cyclin E/DNA multiparameter FCM could quantitatively detect fluorescence intensity and threshold of Cyclin E, and is more sensitive and precise than DNA histogram FCM in detecting G1L checkpoint.

[PTEN induces anoikis through its phosphatase activity in hepatocellular carcinoma cells].

OBJECTIVE: To investigate the effect and mechanisms of tumor suppressor gene PTEN on the induction of anoikis of hepatocellular carcinoma SMMC-7721 cells. METHODS: SMMC-7721 cells were transfected with GFP plasmids containing wild-type PTEN or phosphatase inactivating mutant PTEN (C124A-PTEN) in vitro; The PTEN expression and the phosphorylation levels of focal adhesion kinase (FAK) and protein kinase B (PKB/Akt) were detected by Western blotting; Flow cytometry assay and laser scanning confocal microscopy were used to analyze apoptosis in adherent and non-adherent cells. RESULTS: Compared with the control, PTEN expression in the cells transfected with wild-type PTEN increased to 248%, while the phosphorylation level of FAK and Akt decreased 65.2% and 89.1%, respectively; and the anoikis percentage increased from 9.5% to 31.3%. In the cells transfected with C124A-PTEN, neither the phosphorylation of FAK and Akt nor the anoikis percentage had obviously changed, although the PTEN expression enhanced dramatically in comparison with the control. CONCLUSION: Through its phosphatase activity, tumor suppressor gene PTEN can suppress the phosphorylation of FAK and Akt, and induce anoikis in hepatocellular carcinoma cells.

Peplomycin induces G1-phase specific apoptosis in liver carcinoma cell line Bel-7402 involving G2-phase arrest.

AIM: To investigate the mechanism of peplomycin (PEP)-induced apoptosis in liver carcinoma cell line (Bel-7402). METHODS: Growth inhibition by PEP was analyzed using 3- 4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic cells were detected using Hoechest 33258 staining, and confirmed by flow cytometric analysis and DNA fragmentation analysis. The expression of cyclin A and B1 were determined by flow cytometry and Western blot. Annexin V assay was measured by flow cytometric analysis. RESULTS: PEP induced apoptosis and then inhibited cell proliferation in liver carcinoma cell line Bel-7402. Cells treated with PEP 50 mumol/L for 15 h were arrested in G2-phase with dramatical expression of cyclin A and a little change in cyclin B1. Almost all the apoptosis occurred in cells undergoing the G1-phase after treatment for 24 h. CONCLUSION: Peplomycin induced G1-phase specific apoptosis in Bel-7402 involving G2-phase arrest.

[PTEN induces apoptosis and up-regulates p53 expression in HepG2 cells].

OBJECTIVE: To investigate the effects of tumor suppressor gene PTEN on apoptosis and protein expression of p53 in HepG2 cells, as well as to explore its mechanisms. METHODS: HepG2 cells were transfected with GFP plasmids containing wild-type PTEN or G129E-PTEN and C124A-PTEN in vitro. Both the expression of wild-type p53 and the phosphorylation of protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) were detected by Western blotting. Flow cytometry and confocal microscopy were used to analyze apoptosis of the transfected cells. RESULTS: Compared with the control, the expression of phosphorylated FAK and phosphoylated Akt were down-regulated in HepG2 cells transfected with wild-type PTEN (-65%, -93%) and G129E-PTEN (-65%, -35%), whereas the apoptosis percentage increased to (19.8+/-1.2)% and (9.2+/-0.6)%, and p53 expression was up-regulated by 120% and 50%, respectively. However, in the cells transfected with C124A-PTEN, neither the phosphorylation of FAK and Akt nor the apoptosis percentage and p53 expression had changed. CONCLUSION: PTEN can dephosphrylate FAK through its protein phosphatase activity, and suppress phosphorylation of Akt mainly through its lipid phosphatase activity. Consequently, it can induce apoptosis of HepG2 cells and up-regulate p53 expression.

[Correlation of intra-cellular localization and variations of active caspase-3 to morphology changes in apoptotic MOLT-4 cells induced by X-ray].

BACKGROUND & OBJECTIVE: How pro-caspase-3 activation lead to serial morphology changes during progress of cell apoptosis is unclear. This study was to investigate the variations and intra-localization of active Caspase-3, determine cell morphology changes in apoptotic MOLT-4 cells induced by X-ray, and evaluate their relationship. METHODS: MOLT-4 cells were irradiated by 10 Gy X-ray. Sub G(1)peak method, and DNA fragmentation assay were used to detect variations of DNA in apoptotic cells. Annexin V/PI method was used to determine the cell membrane reversion, and fluorescence labeled inhibitor of Caspases (FLICA) was used to detect the active Caspase-3 in apoptotic cells. Cell morphology and Caspase-3 intra-localization were determined by confocal microscopy. RESULTS: MOLT-4 cells irradiated by 10 Gy X-ray presented classical apoptotic morphology changes such as membrane reversion, and apoptotic body. Caspase-3 was activated after irradiation, and increased remarkably after irradiated for 4 hours. Activated Caspase-3 moved from sub-membrane toward cytoplasm and nucleus. Caspase-3 activity was detected 2 hours earlier than membrane reversion. CONCLUSIONS: Caspase-3 was activated in MOLT-4 cells induced by X-ray, and its intra-localization correlated with the apoptotic morphology changes. The spatial shift of active Caspase-3 in MOLT-4 cells induced by X-ray is one of the mechanisms of apoptosis.

[Quantitative analysis of cell cyclin E expression threshold].

BACKGROUND & OBJECTIVES: It is important to analyze the threshold of cyclins when we research the mechanism of cell cycle progressing. However, there was no effective way to caculate quantitatively. This study was designed to analyze cyclin E expression threshold quantitatively. METHODS: MOLT-4 cells were detected at different photomultiplier tube (PMT) voltage by cyclin E/DNA multiparameter flow cytometry. Using this method, MOLT-4 cells were detected at the same voltage after being treated with caffeine and cycloheximide (CHX), and then MOLT-4 cells and JURKAT cells were detected at the same voltage. Threshold of cyclin E was counted by using formula B2/A x C (A, B, C indicates the minimum, threshold, and maximum of cyclin E fluorescence intensity respectively). RESULTS: Cyclin E threshold of MOLT-4 cells calculated by formula B2/A x C was invariable at different voltage. It decreased when cells treaded with caffeine and unchanged when treated with cycloheximide. At the same time, cyclin E threshold of JURKAT cells calculated by formula was much lower than that of Molt-4 cells. CONCLUSIONS: Formula B2/A x C can be used to analyze cyclin E expression threshold quantitatively.