Angiostrongylus cantonensis ; isolation & purification ; Animals ; China ; Snails ; parasitology ; Strongylida Infections ; epidemiology

OBJECTIVETo study the interaction between transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust.

METHODS(1) The immortalized human bronchial epithelial cell line BEAS-2B and human embryo lung fibroblast cell line WI-38 were grown in MEM medium containing 5% and 10% FBS, respectively, at 37 degrees C and 5% CO2 with saturated humidity. The cells were subcultured every 6 days. BEAS-2B cells and WI-38 cells were induced with Yunnan tin mine dust on every other generation at the concentration of 100 microg/ml. From the 11th generation, the cells were co-cultured. Epithelial cell transformation was tested using concanavalin A (ConA) agglutination and anchorage-independent growth assays. The cell cycles were analyzed through flow cytometry. The expressions of alpha-SMA in fibroblasts were determined with immunocytochemistry.

RESULTS(1) Cell morphology of mine dust-exposed epithelial cells began to transform at the 28th generation. Similar transformations were observed with mine dust-induced epithelial cells co-cultured with fibroblasts from the 20th generation and mine dust-induce epithelial cells co-cultured with mine dust-induced fibroblasts from the 16th generation. ConA agglutination assay and anchorage-independent growth assays were negative in normal BEAS-2B cells. At the 26 th generation, the agglutination test result of the mine dust-exposed epithelial cells was positive. Co-cultured with fibroblasts and mine dust-exposed fibroblasts, the agglutination time of the mine dust-exposed epithelial cells became short. Epithelial cell anchorage-independent growth assay was positive for mine dust-exposed epithelial cells co-cultured with fibroblasts at the 36th generations and for mine dust-exposed epithelial cells co-cultured with mine dust-exposed fibroblasts at the 26th generations. The clone formation rate of the 26th generation was 6.00 per thousand +/- 1.00 per thousand and 15.33 per thousand +/- 2.52 per thousand respectively, with the significant differences (P < 0.05). With generation adding, the portion of S phase increased for mine dust-exposed epithelial cells. (2) At the 26th generations, fibroblasts expressed alpha-SMA. Co-cultured with epithelial cell, the alpha-SMA expression of fibroblasts increased. Especially, positive cell numbers and intensity of staining dramatically increased with generation adding.

CONCLUSIONS(1) The tin mine dust can induce malignant transformation of human pulmonary epithelial cells BEAS-2B and activation of fibroblasts WI-38. (2) The epithelial cells are major target in carcinogenesis induced by Yunnan tin mine dust. (3) Transformation of epithelia and activation of fibroblasts co-evolve in the developing process of induced lung cancer by Yunnan tin mine dust.

Cell Cycle ; drug effects ; Cell Line ; Cell Transformation, Neoplastic ; drug effects ; Coculture Techniques ; Dust ; Epithelial Cells ; pathology ; Fibroblasts ; metabolism ; pathology ; Humans ; Tin ; toxicity

Objce tive To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells.Methods Breast cancer cell line MDA-MB-231 cells were used in this study.Breast cancer stem cells were isolated and enriched by serum-free culture.The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice.RT-PCR, real-time PCR (qPCR) and Western blot were used to detect Oct4 expression.RNA interference was applied to induce Oct4 down-regulation.The interference experiment set up a control group ( no siRNA transfection) , negative control group ( negative siRNA group,transfection of siRNA sequences without any interfering effect on the cells) and Oct4 siRNA group ( transfection of siRNA with interfering effect on the Oct4 gene) .Methyl thiazolyl tetrazolium ( MTT ) and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. Resulst MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension.MDA-MB-231 breast cancer stem cells showed a higher percentage of CD44+C/D24 -/low cells (97.2%) than that in MDA-MB-231 breast cancer cells ( 76.6%) ( P<0.05) .The tumor size in mice inoculated with MDA-MB-231 breast cancer stem cells was (124.60±13.65)mm3, significantly larger than that of mice inoculated with breast cancer cells (68.20±9.99 mm3) (P=0.0007).MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration (IC50) [(4.40±0.48) μg/ml vs.(8.20±0.34) μg/ml, P<0.05].However, the expression of transcriptional factors Oct4 was higher in MDA-MB-231 breast cancer stem cells than that in breast cancer cells (P<0.05).The proliferation potential of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was significantly lower than that in the negative siRNA and control groups ( P<0.05) from the third day.The invasion ability of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was obviously reduced than that in the control and negative siRNA groups shown by number of penetrated cells [(46.52±2.58) vs.(79.67±3.85) and (77.29±2.13), P<0.05 for both].As for resistance to paclitaxel, IC50 of MDA-MB-231 breast cancer stem cells with Oct4siRNA interference was significantly decreased [(4.48±0.22) μg/ml] compared with that in the control [(7.99±0.59) μg/ml] and negative siRNA group [(8.10±0.68) μg/ml] (P<0.05 for both).Conclusions MDA-MB-231 breast cancer cells are successfully obtained by serum-free culture. The proliferation potential, invasion ability and drug resistance of breast cancer stem cells were down-regulated by Oct4 gene knock-down.

Objce tive To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells.Methods Breast cancer cell line MDA-MB-231 cells were used in this study.Breast cancer stem cells were isolated and enriched by serum-free culture.The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice.RT-PCR, real-time PCR (qPCR) and Western blot were used to detect Oct4 expression.RNA interference was applied to induce Oct4 down-regulation.The interference experiment set up a control group ( no siRNA transfection) , negative control group ( negative siRNA group,transfection of siRNA sequences without any interfering effect on the cells) and Oct4 siRNA group ( transfection of siRNA with interfering effect on the Oct4 gene) .Methyl thiazolyl tetrazolium ( MTT ) and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. Resulst MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension.MDA-MB-231 breast cancer stem cells showed a higher percentage of CD44+C/D24 -/low cells (97.2%) than that in MDA-MB-231 breast cancer cells ( 76.6%) ( P<0.05) .The tumor size in mice inoculated with MDA-MB-231 breast cancer stem cells was (124.60±13.65)mm3, significantly larger than that of mice inoculated with breast cancer cells (68.20±9.99 mm3) (P=0.0007).MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration (IC50) [(4.40±0.48) μg/ml vs.(8.20±0.34) μg/ml, P<0.05].However, the expression of transcriptional factors Oct4 was higher in MDA-MB-231 breast cancer stem cells than that in breast cancer cells (P<0.05).The proliferation potential of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was significantly lower than that in the negative siRNA and control groups ( P<0.05) from the third day.The invasion ability of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was obviously reduced than that in the control and negative siRNA groups shown by number of penetrated cells [(46.52±2.58) vs.(79.67±3.85) and (77.29±2.13), P<0.05 for both].As for resistance to paclitaxel, IC50 of MDA-MB-231 breast cancer stem cells with Oct4siRNA interference was significantly decreased [(4.48±0.22) μg/ml] compared with that in the control [(7.99±0.59) μg/ml] and negative siRNA group [(8.10±0.68) μg/ml] (P<0.05 for both).Conclusions MDA-MB-231 breast cancer cells are successfully obtained by serum-free culture. The proliferation potential, invasion ability and drug resistance of breast cancer stem cells were down-regulated by Oct4 gene knock-down.