| Background Esophageal carcinomas rank as the eighth most common cancer in the world and the sixth highest cause of cancer-related mortality. The most common histological type of esophageal carcinoma in high incidence areas in the world is oesophageal squamous cell carcinoma(ESCC). Paclitaxel has a broad spectrum of antitumor activity and is widely used in the treatment of various malignant tumors. However, intrinsic or acquired resistance to paclitaxel severely limits the therapeutic potential of this drug.The molecular mechanisms that underlie this chemoresistance are still largely unknown. A more detailed understanding of the molecular mechanisms by which tumor cells survive chemotherapy is likely to lead to novel therapeutic targets with more successful outcomes. A variety of cancer stem-like cells(CSCs) have been shown to be responsible for cancer tumorigenicity, relapse and metastasis. Despite several reports demonstrating the presence of CSCs in human esophageal carcinoma,their identities are still under debate. There were few studies have examined whether paclitaxel resistance human oesophageal squamous cell carcinoma cell line have the biological characteristics of cancer stem cell.Objective Established of paclitaxel resistant of human oesophageal squamous cell carcinoma cell line, and studies whether the cell line have the biological characteristics of cancer stem cell.Object of study Human oesophageal squamous cell carcinoma cells EC109 were bought from Shanghai cell bank of Chinese academy of sciences.Part one The establishment of paclitaxel resistance human oesophageal squamous cell carcinoma cell lineMethods The resistant cells was established by intermittent exposure human oesophageal squamous cell carcinoma cells EC109 to a high concentration of paclitaxel with time-stepwise increment over a period of 7 months. The multidrug resistance of R-ECl09 to anticancer agents was evaluated by MTT assay.Results Over 7 months, the paclitaxel resistant cell line of human oesophageal squamous cell carcinoma(R-EC109) was established. The half inhibitory concentration(IC50) of EC109 was 0.068±0.003μg/m L, and the IC50 of R-EC109 was 3.578±0.089μg/m L(P<0.001). R-ECl09 cells were of 67.258 fold resistance to paclitaxel. After testing 30 d,60d, 90 d, 120 d were determined by MTT methods, however, there show the R-EC109 cells resistant stability was good.Summary The study successfully established the stability highly paclitaxel resistance human oesophageal squamous cell carcinoma cell line R-ECl09.Part two The identification of biological characteristics of cancer stem cell between human oesophageal squamous cell carcinoma cells EC109 and paclitaxel re sistance human oesophageal squamous cell carcinoma cells R-EC109Methods To cultivate the R-EC109 cells and EC109 cells, after the third generation use d in the experiment.1. Sphere formation experiment 5000 R-EC109 cells or EC109 cells were vacc inated in low adhesion 6 orifice per hole. Replaced half of the culture every 2d. After 10 d, observed and counted the sphere formation of R-EC109 cells and EC109 cells, and counted number of > 20 cloned cells.2. Clone formation experiment 600 R-EC109 cells or EC109 cells were inocul ated to the cultivation of bottle(25cm2), changing culture every 2-3d. After 10 d,left to broth, fixed cells, stained cells, and counted number of > 50 cloned cells.3. Expression of stem cell gene mRNA extracted the total stem cell gene RN A of R-EC109 cells and EC109 cells, then stem cell gene m RNA reverse tran scribed. Quantitatively detected the expression of m RNA by the PCR reaction.4. Side population separating R-EC109 cells and EC109 cells were divided int o two groups, experimental group: add fluid of verapamil and Hoechst33342; control group: add Hoechst33342. Using flow cytometry analyzed the content of SP cells in each group, and analyzed Hoechst33342 dual parameter.5. Expression of tumor metastasis gene m RNA Extracted the total tumor metastasis gene RNA of R-EC109 cells and EC109 cells, then tumor metastasis gen e m RNA reverse transcribed. Quantitatively detected the expression of mRNA by the PCR reaction.Results1.The self-renewal ability of R-EC109 cells(13.2±2.1%) was higher than EC-109 cells(7.6±1.7%)(P<0.01).2.The clones proliferation ability of R-EC109 cells(65.2±4.1%) was higher than EC-109 cells(36.8±3.9%)(P<0.05).3.The expression of stem cell gene m RNA of R-EC109 cells[Bmi-1(3.3544±0.4570), Nanog(2.5657±0.2677), Oct4(3.5767±0.3754), Sox2(1.6788±0.1688), ABCG2(2.8655±0.1700), Nestin(3.1567±0.5879) and Ki-67(4.1678±0.5967)] was higher than EC-109 cells[Bmi-1(1±0.1468), Nanog(1±0.0875), Oct4(1±0.0976), Sox2(1±0.1132), ABCG2(1±0.0645), Nestin(1±0.1670) and Ki-67(1±0.0746)](P<0.05).4.The content of SP cell of R-EC109 cells(5.8%) was higher than EC109 cells(2.3%)(P<0.05).5. The expression of tumor metastasis gene m RNA between R-EC109 cells[E-c adherin(0.6245±0.0598), N-cadherin(2.5882±0.1330), Snail(2.4767±0.0798) and Twist(2.8563±0.0797)] and EC109 cells[E-cadherin(1±0.0479), N-cadherin(1±0.0743), Snail(1±0.0579) and Twist(1±0.0780)] was difference(P<0.05). The EMT process of R-EC109 cells was more active.SummaryR-EC109 cells have the multiple characteristics of cancer stem cells. The self-r enewal ability, clones proliferation ability, expression of stem cell gene mRNA,and content of SP cell of R-EC109 cells were higher than EC-109 cells. R-E C109 cells have stronger ability of metastasis and invasion, and its EMT process was more active than EC-109 cells.Conclusions1. Human oesophageal squamous cell carcinoma paclitaxel resistant cells R-EC109 have the multiple characteristics of cancer stem cells.2. Cancer stem cells may lead to the root causes of paclitaxel resistance in R-EC109 cells. |
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