MicroRNA-409-5p Inhibits GIST Tumorigenesis and Improves Imatinib Resistance by Targeting KDM4D Expression.

OBJECTIVE: Gastrointestinal stromal tumors (GISTs) can rapidly proliferate through angiogenesis. Previous studies indicated the potential influence of microRNA on the progression of tumor immature angiogenesis. This study aimed to explore the specific mechanism by which microRNA-409-5p (miR-409-5p) contributes to GIST. METHODS: To identify genes potentially involved in the development and progression of GIST, the differences of miR-409-5p between tumors and adjacent tissues were first analyzed. Following this analysis, target genes were predicted. To further investigate the function of miRNA in GIST cells, two GIST cell lines (GIST-T1 and GIST882) were transfected with lentiviruses that stably expressed miR-409-5p and scrambled miRNA (negative control). Later, the cells were subjected to Western blotting and ELSA to determine any differences in angiogenesis-related genes. RESULTS: In GISTs, there was a decrease in the expression levels of miR-409-5p compared to the adjacent tissues. It was observed that the upregulation of miR-409-5p in GIST cell lines effectively inhibited the proteins hypoxia-inducible transcription factor 1ß (HIF1ß) and vascular endothelial growth factor A (VEGF-A). Further investigations revealed that miR-409-5p acted as an inhibitor of angiogenesis by binding to the 3'-UTR of Lysine-specific demethylase 4D (KDM4D) mRNA. Moreover, the combination of miR-409-5p with imatinib enhanced its inhibitory effect on angiogenesis. CONCLUSION: This study demonstrated that the miRNA-409-5p/KDM4D/HIF1ß/VEGF-A signaling pathway could serve as a novel target for the development of therapeutic strategies for the treatment of imatinib-resistance in GIST patients.

Aseptic abscess in the abdominal wall accompanied by monoclonal gammopathy simulating the local recurrence of rectal cancer: A case report.

BACKGROUND: Infectious abscesses in the abdominal wall can be secondary to retained foreign bodies (e.g., stones, use of artificial mesh, use of silk yarn in surgical suture), inflammatory diseases (e.g., acute appendicitis), and perforated malignancies of the digestive tract (particularly the colon). Aseptic abscesses (AAs) are relatively rare. To the best of our knowledge, this is the first report of an AA in the abdominal wall accompanied by monoclonal gammopathy of undetermined significance (MGUS) at 5 years after laparoscopic proctectomy. CASE SUMMARY: A 72-year-old female patient presented with an enlarged painless mass in the lower abdomen for 1 year. She had a history of obesity, diabetes, and MGUS. Her surgical history was laparoscopic resection for rectal cancer 6 years prior, followed by chemotherapy. She was afebrile. Abdominal examination revealed a smooth abdomen with a clinically palpable solid mass under a laparotomy scar in the left lower quadrant. No obvious tenderness or skin redness was spotted. Laboratory data were not remarkable. Computed tomography scan revealed a low-density mass of 4.8 cm in diameter in the lower abdominal wall, which showed high uptake on positron emission tomography. The preoperative diagnosis was an abscess or tumor, and surgical resection was recommended. The mass was confirmed to be an AA by microbiological and pathological examinations. The patient recovered well after surgery. There was no evidence of recurrence 2 years later. CONCLUSION: It is important to consider underlying conditions (diabetes, chemotherapy, MGUS) which may contribute to AA formation in the surgical wound.

Survey and analysis of the nutritional status in hospitalized patients with malignant gastric tumors and its influence on the quality of life.

BACKGROUND/OBJECTIVES: The assessment of nutritional status and the quality of life in patients with gastric cancer has become one of the important goals of current clinical treatment. The purpose of this study was to assess the nutritional status in hospitalized gastric cancer patients by using patient-generated subjective global assessment (PG-SGA) and to analyze the influence of nutritional status on the patients' quality of life (QOL). METHODS: We reviewed the pathological diagnosis of gastric cancer for 2322 hospitalized patients using PG-SGA to assess their nutritional status and collected data on clinical symptoms, the anthropometric parameters (height, weight, body mass index (BMI), mid-arm circumference (MAC), triceps skin-fold thickness (TSF), and hand-grip strength (HGS). We also collected laboratory data (prealbumin, albumin, hemoglobin) within 48 h after the patient was admitted to the hospital. The 30-item European Organization for Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30) was used for QOL assessment in all patients. RESULTS: By using PG-SGA, we found 80.4% of the patients were malnourished (score ≥ 4) and 45.1% of the patients required urgent nutritional support (score ≥ 9). In univariate analysis, old age (> 65 years, p < 0.001), female (p = 0.007), residence in a village (p = 0.004), a lower level of education (p < 0.001), and self-paying (p < 0.001) were indicated as risk factors of patients with gastric cancer to be suffering from severe malnutrition. There was a negative correlation between PG-SGA and various nutritional parameters (p < 0.05). The quality of life was significantly different in gastric cancer patients with different nutritional status (p < 0.01). CONCLUSION: Malnutrition of hospitalized patients with gastric cancer in China is common and seriously affects the patients' quality of life. The nutritional status should be evaluated in a timely manner and reasonable nutritional intervention should be provided as soon as possible. The PG-SGA was fit for using as a clinical nutrition assessment method, being worthy of clinical application.

[Relationship of apoptotic receptor expression in normal human peripheral blood lymphocytes to cell apoptosis].

The aim of this study was to detect the expression and cell cycle specificity of Fas, TNFRI and TNFRII in human peripheral blood lymphocytes (PBL), and to study the potential role of Fas, TNFRIand TNFRII in cell cycle specific apoptosis. The improved double-parameter flow cytometry was used to detect the expressions of Fas, TNFRI and TNFRII and cell cycle specificity in PBL which were incubated for 24 hours in the presence or absence of phytohaematoagglutinin (PHA) respectively. Apoptosis induced by IgM type anti-Fas and TNF-alpha was detected by API method. The results showed that compared with PBL treated in the absence of PHA in G(0) phase, the ratio of Fas, TNFRI and TNFRII expressions in PHA-stimulated PBL entering cell cycle increased (35.55 +/- 6.63)%, (30.63 +/- 2.66)%, (26.62 +/- 5.14)% respectively (P < 0.01), and mainly appeared at G(1)-phase; no apoptosis was induced by anti-Fas and TNF-alpha in G(0)-phase PBL cultured in the absence of PHA. On the contrary, the apoptosis was induced by anti-Fas and TNF-alpha in PBL which entered cell cycle after stimulation with PHA and mainly initiated at G(1)-Phase. It is concluded that there is evident dose-effect relationship between apoptotic receptor and receptor-mediated apoptosis. Moreover, the cell cycle specificity of receptor-mediated apoptosis is correlated with the cell cycle specific expressions of apoptotic receptor. The induction of apoptosis by apoptotic factors (anti-Fas and TNF-alpha) depends on whether cell entering cell cycle or not.

[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.

[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.

[Cell balance between apoptosis and proliferation in colon cancer and its correlation with prognosis].

BACKGROUND & OBJECTIVE: In normal tissues and organs, cell apoptosis and proliferation maintain a homeostasis. Alterations of this physiologic balance may lead to malignant transformation of cells and tumorigenesis. This study was to investigate cell balance (ratio of apoptosis index to proliferation index, AI/PI) in colon cancer and its correlation with prognosis. METHODS: The apoptotic population and the proliferating population of colon cancer cells were quantitatively analyzed by Sub-G1 method and Ki-67/DNA bivariate analysis of flow cytometry. Cell proliferation was observed under confocal microscope. Kaplan-Meier method and log-rank test were used to analyze patients' survival. RESULTS: AI/PI ratio of cells was significantly higher in normal colon tissue than in colon adenoma tissue, Dukes' A colon cancer, Dukes' B colon cancer, and Dukes' C and D colon cancer (0.45+/-0.19 vs. 0.30+/-0.07, 0.29+/-0.11, 0.28+/-0.10, and 0.26+/-0.07, respectively, P < 0.01). AI/PI ratio showed a down-regulating trend in variables tested including tumor size, pathologic type, differentiation grade, Dukes' stage, and nodal involvement. AI/PI ratio of peripheral lymphocytes was significantly higher in colon cancer patients than in healthy people (0.64+/-0.11 vs. 0.49+/-0.12, P < 0.01). The expression of Ki-67 was observed in normal colon tissue, colon adenoma, and colon cancer under confocal microscope. Survival rate of patients with AI/PI ratio of < 0.285 (using the median value as the cutoff) and >/= 0.285 was not significantly different (P> 0.05). CONCLUSIONS: Dysregulation (down-regulation or up-regulation) of cell balance between apoptosis and proliferation in colon cancer cells and lymphocytes might play an important role in tumorigenesis and tumor progression. AI/PI ratio can' t be used as a prognostic factor of colon cancer.

[Expressions of cyclins A, B1, D3, and E in asynchronous and synchronous MOLT-4 cells of G1 phase].

BACKGROUND & OBJECTIVE: We have testified expression imbalance of Cyclins A, B1, D3, and E in MOLT-4 cells, which were synchronized by "double thymidine blocks", with flow cytometry (FCM). However, comparisons of Cyclins expressions between synchronized cells and asynchronized cells haven't been performed at that time because of technique limitations. This study was to compare expressions of Cyclins A, B1, D3, and E in G(1) phase of asynchronized and synchronized cells by newly established "postsorting Western blot", and to conform the unreasonableness of using double thymidine blocks to synchronize cells to analyze normal cell cycles. METHODS: MOLT-4 cells were synchronized at G(1) phase by double thymidine blocks, asynchronous cells of G(1) phase were sorted by FCM. Western blot and double parameters analysis of DNA/Cyclins were performed to detect Cyclins A, B1, D3, and E expressions in asynchronous and synchronous MOLT-4 cells of G(1) phase. RESULTS: There were almost no expressions of Cyclins A, B1 in asynchronous MOLT-4 cells of G(1) phase, and obvious expressions in synchronized cells of G(1) phase. Expressions of Cyclins D3, E in synchronous MOLT-4 cells of G(1) phase were higher than those in asynchronous cells of G(1) phase. The FCM results were accordant with Western blot results. CONCLUSIONS: The expressions of Cyclins in synchronized cells obtained through "double thymidine blocks" can't represent their expressions in normal cells. Thus, synchronous cells produced by "double thymidine blocks" are not ideal experimental models for analyzing normal cell cycles.

[Promotive effect of decreased cyclin E threshold on cell proliferation].

BACKGROUND & OBJECTIVE: Many studies showed that high expression of Cyclin E promotes cell proliferation, but contrary data was also reported that cell proliferation didn't decrease with low expression of Cyclin E. In addition, we observed that many tumor cells have strong capability of proliferation with low expression of Cyclins, including Cyclin E. This study was to analyze effect of reduced Cyclin E threshold on proliferation of acute lymphocyte leukemia cell line MOLT-4 to explain the above phenomena. METHODS: We have established the model of decreased Cyclin E threshold in MOLT-4 cells by treating cells with low concentration (5 mmol/L) of caffeine for 2, and 4 h. The positive rates of proliferation cell nuclear antigen (PCNA), Ki67, and DNA strand break induction by photolysis (SBIP) were analyzed by flow cytometry. RESULTS: MOLT-4 cells presented sharply decrease of Cyclin E threshold, and increase of positive rates of PCNA, Ki67, and SBIP after treated with caffeine, especially at 2-h point. CONCLUSIONS: Decrease of Cyclin E threshold was accompanied by increase of cell proliferation. MOLT-4 cells may remain high proliferation capability with low level of Cyclin expression.

[Expression of estrogen receptor in different breast cancer cell lines transfected with estrogen receptor plasmid HEGO5 mediated by FuGENE6].

BACKGROUND & OBJECTIVE: Gene transfection is a major approach in studies of estrogen receptor (ER)expression in breast cancer cells,and ER's functional mechanism. This study was designed to detect the transfection efficiency of different human breast cancer cell lines mediated by FuGENE6 reagent, and ER expression in these cells after transfected with ER plasmid HEGO5. METHODS: Breast cancer cell lines were transfected with plasmid pEGFP-N1,and the transfection efficiency was measured by flow cytometry (FCM). The cell lines with high transfection efficiency were transfected with HEGO5,and then the expression of ER was determined by FCM and Western blot. RESULTS: There were obvious differences in transfection efficiencies of breast cancer cell lines MM-231, MM330, MM134 VI, MM175VII, MM157, MM361, MM436, MM453, UaCC812, UaCC893, BT-549, BT-20, HBL-100, Hs578t, MCF-7, T-47d, and ZR-75-1. Repetitious transfection test in HBL-100 cells showed that co-efficient of variation was 5.1. HEGO5 was transfected into the cells with high plasmid transfection efficiency (MCF-7, HBL-100, Hs578t, MM436, MM453, and BT-20), positive rates of ER in these cells ranged from 12.9% to 54.8%, corresponding to the pEGFP-N1 transfection efficiency, and the expression of ER detected by FCM were in accordance with those tested by Western blot. To some extent, the expression of ER in transfected cells was cell cycle specific. CONCLUSIONS: Transfection of breast cancer cell lines mediated by FuGENE6 reagent has a good repetition,and the results were stable and reliable. In the cells transfected with HEGO5,the expression of ER can be qualitatively detected by Western blot, and quantitatively detected by FCM.

[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.

[Western blot analysis and flow cytometric analysis of estrogen and progesterone receptors in fixed breast cancer cells].

BACKGROUND & OBJECTIVE: Quantitative and qualitative detection of estrogen receptor (ER) and progesterone receptor (PR) levels are very important for predicting prognosis and evaluating the outcome of endocrine therapy of breast cancer patients. Western blot analysis and Flow cytometry (FCM) are important means for quantitative and qualitative analysis of proteins, but conventional Western blot analysis need to extract proteins from fresh cells. The present study was designed to establish Western blot analysis of human estrogen receptor (ER) and progesterone receptor (PR) in fixed breast cancer cells, and to explore the possibility of ER and PR analysis in fixed cells by flow cytometry and Western blot analysis simultaneously. METHODS: Proteins extracted from fresh and fixed cells of different exponentially growing breast cancer cell lines were labelled using 1D5 and PgR636, which were monoantibodies to ERalpha and PR, respectively. The expression of ER and PR were determined using Western blot analysis, and the results were compared with that of fixed cells measured with flow cytometry. RESULTS: Clear and correct ERalpha bands were observed with Western blot analysis in cell lines T-47d, MCF-7, and ZR-75-1, and the band density of fixed T-47d and ZR-75-1 was higher than that of fresh cells. The expression of ERalpha in MM231 cells was negative. Clear and correct PR bands were visible in T-47d and ZR-75-1 cells with Western blot analysis, and the band density of fixed cells was higher than that of fresh cells. And the expression of PR in MM231 and MCF-7 cells were negative. In addition, positive expression of ER and PR in different cell lines measured by flow cytometry were the same with that analyzed by Western blot analysis. CONCLUSION: After fixed with 0.25% paraformaldehyde and 75% ethanol, breast cancer cells can be used for not only quantitative measurement of ER and PR, but also Western blot analysis of ER and PR.

[Selective apoptosis in HeLa cells induced by sodium butyrate and its mechanism].

OBJECTIVE: To investigate apoptosis induced by sodium butyrate in cervix cancer cell line HeLa and primary human embryo lung fibroblasts and its mechanism. METHODS: Cell apoptosis was assessed by morphology, cell viability, DNA fragmentation, the percentage of sub-G1 cells and phosphatidylserine (PS) externalization. The effects of sodium butyrate on transcription of Bax and Bcl-2 was analyzed by RT-PCR. RESULTS: Sodium butyrate inhibited proliferation in a time and dose-dependant manner. The inhibition of proliferation in HeLa cells was more significant than that in primary human embryo lung fibroblasts. DNA fragmentation, sub-G1 peak and AnnexinV/PI by flow cytometry showed very high apoptosis rates in HeLa cells 72 hours after treated with sodium butyrate, while pretty low in primary human embryo lung fibroblasts. RT-PCR showed sodium butyrate had little effects on transcription of Bax and Bcl-2 in HeLa cells. CONCLUSION: Sodium butyrate can induce apoptosis in HeLa cells without changing the expression of Bax and Bcl-2. Sodium butyrate comparatively has little effects on fibroblasts.