耐他莫昔芬人乳腺癌细胞株的建立及其^18F-FDG摄取的降低机制

Establishment of an acquired tamoxifen-resistant human breast cancer cell line and its mechanism of ^18F-FDG uptake reduction

目的建立耐他莫昔芬（TAM）人乳腺癌细胞耐药细胞株T47D－TamR，比较其与亲本细胞株T47D在^18F－FDG细胞摄取率上的差异，并进行机制研究。方法采用长时间逐步增加药物浓度冲击法，诱导建立耐TAM人乳腺癌细胞耐药细胞株T47D－TamR。利用MTT法测定耐药株的耐药指数（RI）及增殖能力。分别在不同细胞数、反应时间、^18F－FDG用量及葡萄糖浓度的实验条件下，测定T47D－TamR和T47D细胞的^18F－FDG细胞摄取率。利用试剂盒检测2种细胞内LDH活性、ATP水平及上清液乳酸含量。通过Western blot法检测2组细胞中ERα和Glut－1的表达量及AMPK和mTOR的磷酸化水平。采用两样本t检验或双因素方差分析处理数据。结果成功构建T47D－TamR细胞株，其RI为1.97±0.08，且增殖能力明显低于亲本细胞（F＝230.10，P〈0.05）。分别改变细胞数、反应时间及^18F－FDG用量时，T47D－TamR细胞的^18F－FDG细胞摄取率均低于T47D细胞，2组间差异均有统计学意义（F值：419.00～1 116.00，均P〈0.05）。T47D和T47D－TamR细胞内LDH活性分别为（0.42±0.04）和（0.32±0.02） U/mg蛋白质，ATP水平分别为（19.99±0.32） 和（14.01±0.70） nmol/mg蛋白质，上清液乳酸浓度分别为（2.95±0.05） 和（2.02±0.07） mmol/L，2组间差异有统计学意义（t值：4.39～18.80，均P〈0.05）。T47D和T47D－TamR细胞中ERα、磷酸化AMPK（p－AMPK）、磷酸化mTOR （p－mTOR）、Glut－1的相对表达量分别为0.26±0.03、0.36±0.06、0.75±0.11、0.35±0.07和0.17±0.02、0.61±0.09、0.52±0.08、0.21±0.04，2组间差异有统计学意义（t值：12.20～16.45，均P〈0.05）。结论T47D－TamR细胞的^18F－FDG摄取率、胞内LDH活性、ATP水平及上清液乳酸含量均低于T47D细胞，且其p－AMPK的表达量升高，ERα、p－mTOR、Glut－1的表达量降低，提示耐TAM乳腺癌细胞糖酵解水平降低 。

ObjectiveTo establish the acquired tamoxifen （TAM）-resistant human breast cancer cell line T47D-TamR, compare the ^18F-FDG uptake rate between T47D-TamR and its parental cell line T47D, and study the mechanism.MethodsLong-term step-wise drug stimulation was used for cell line T47D-TamR establishment and then the cell proliferation and resistance index （RI） were determined by MTT assay. The ^18F-FDG uptake rates of T47D-TamR and T47D cells were measured in the setting of different cell counts, reaction time, ^18F-FDG dosages and glucose concentrations. The LDH activity, cellular ATP level and lactic acid concentration in cell supernatant of T47D-TamR and T47D cells were detected. Western blot was used to examine the expression of ERα, Glut-1, phosphorylated AMPK （p-AMPK） and phosphorylated mTOR （p-mTOR）. Two-sample t test and two-way analysis of variance were used to analyze the data.ResultsT47D-TamR cell line was successfully established and its drug RI was 1.97±0.08, with a significantly decreased cell proliferation efficacy （F=230.10, P〈0.05）. Significant differences of ^18F-FDG uptake rates were found between T47D-TamR cell and T47D cell when changing the cell count, reaction time, and ^18F-FDG dosage （F values： 419.00-1 116.00, all P〈0.05）. The LDH activities of T47D cell and T47D-TamR cell were （0.42±0.04） and （0.32±0.02） U/mg protein, cellular ATP levels were （19.99±0.32） and （14.01±0.70） nmol/mg protein, lactic acid concentrations in cell supernatant were （2.95±0.05） and （2.02±0.07） mmol/L, respectively. The differences of above parameters between the two groups were significant （t values： 4.39-18.80, all P〈0.05）. The relative expressions of ERα, p-AMPK, p-mTOR, Glut-1 were 0.26±0.03, 0.36±0.06, 0.75±0.11, 0.35±0.07 in T47D cell, and 0.17±0.02, 0.61±0.09, 0.52±0.08, 0.21±0.04 in T47D-TamR cell, and there were significant differences （t values： 12.20-16.45, all P〈0.05） between the two groups.ConclusionsCompared with parental cells, T47D-TamR cells have lower ^18F-FDG uptake rate, LDH activity, cellular ATP level and lactic acid concentration, increased p-AMPK expression and decreased ERα, p-mTOR, Glut-1 expression, indicating the decreased glycolysis ability in TAM-resistant breast cancer cells.