Ding Lei, Chen Xiao-ping, Zhang Zhi-wei, Zhang Wan-guang, Cao Bin, Wang Zhi-hui, Li Chun-lei
Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Gan Zang Bing Za Zhi. 2006 May;14(5):353-7.
To investigate the mechanism of reversing multidrug resistance of hepatocarcinoma by bromocriptine (BCT) in vitro and in vivo.
Three groups of cultured HepG2 cells were used: HepG2 (group A), the multidrug resistance HepG2/ADM cells (group B), and the HepG2/ADM cells treated with BCT (group C). Rhodamine 123 test was used to detect the function of P-gp protein. MTT assay was performed to examine the IC50. Multidrug resistance index to common five anticancer drugs of different concentrations of BCT and immunocytochemistry was performed to detect the expression of PKC-a protein. P-gp protein levels of the cells of each group were determined by Western blot. In addition, HepG2 and HepG2/ADM were injected into the livers of the nude mice (NM) (named NM HepG2, NM ADM, NM BCT groups respectively). The BCT group mice were treated with bromocriptine through gastric feedings. The sizes of the tumor growths in the livers were measured using B ultrasound. The MDR1mRNA levels in these tumor tissues were determined by reverse transcription polymerase chain reaction (RT-PCR) and the apoptosis rates of them were measured with TUNEL assay. 99mTc-MIBI SPECT was performed to detect the tumor 99mTc-MIBI accumulation index before and after the BCT treatment.
The rate of reversing resistance to ADM by BCT was 45.68% shown by using MTT assay, and the intracellular Rho123 accumulation increased more than two times compared with the control group shown by flow cytometric assay at the concentration of 10 micromol/L BCT and the effect was time-dependent. Between group B and group C there was a significant difference in the expression of PKC-alpha protein by immunocytochemistry detection (q = 5.37, P < 0.01), but there was no significant difference in the expression of P-gp protein between the two groups (q = 1.86, P > 0.05) . There was no notable difference of growth rates of the transplanted liver tumors among the three NM groups (F = 6.39, P > 0.05), and the inhibition rate of tumor volume and weight in NM BCT was higher than that in NM ADM (q1 = 5.89, q2 = 4.92, P < 0.01), but similar to that in NM HepG2 (q1 = 2.47, q2 = 3.02, P > 0.05). No difference was detected in MDR1mRNA between NM ADM and NM BCT using RT-PCR (q = 3.71, P > 0.05). The average number of apoptotic cells per high-power field (25.7+/-1.8) in NM BCT tumor tissues was higher than that in group ADM (2.7+/-0.2) (q = 3.72, P < 0.01), but similar to that of NM HepG2 (23.9+/-1.6) (q = 1.43, P > 0.05). The uptake of 99mTc-MIBI in all the NM after BCT therapy was significantly higher than that before the BCT treatment (t = 3.58, P < 0.01).
BCT can reverse multidrug resistance of hepatocarcinomas by inhibiting the function of P-gp protein and can enhance the susceptibility of HepG2/ADM cells to cytotoxic drugs.
探讨溴隐亭（BCT）在体内外逆转肝癌多药耐药的机制。
使用三组培养的HepG2细胞：HepG2（A组）、多药耐药的HepG2/ADM细胞（B组）和用BCT处理的HepG2/ADM细胞（C组）。采用罗丹明123试验检测P-糖蛋白的功能。进行MTT法检测半数抑制浓度（IC50）。对不同浓度BCT对五种常用抗癌药物的多药耐药指数及免疫细胞化学法检测蛋白激酶C-α（PKC-α）的表达。采用蛋白质印迹法测定各组细胞P-糖蛋白水平。此外，将HepG2和HepG2/ADM细胞接种到裸鼠肝脏（分别命名为裸鼠HepG2组、裸鼠ADM组、裸鼠BCT组）。BCT组小鼠通过灌胃给予溴隐亭。用B超测量肝脏肿瘤生长大小。采用逆转录聚合酶链反应（RT-PCR）测定这些肿瘤组织中多药耐药基因1（MDR1）mRNA水平，并用末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）检测其凋亡率。在BCT治疗前后进行99m锝-甲氧基异丁基异腈（99mTc-MIBI）单光子发射计算机断层扫描（SPECT）检测肿瘤99mTc-MIBI摄取指数。
MTT法显示BCT对阿霉素（ADM）的耐药逆转率为45.68%，在10μmol/L BCT浓度下，流式细胞术检测显示细胞内罗丹明123蓄积较对照组增加两倍以上，且呈时间依赖性。免疫细胞化学检测显示B组和C组PKC-α蛋白表达有显著差异（q = 5.37，P < 0.01），但两组P-糖蛋白表达无显著差异（q = 1.86，P > 0.05）。三个裸鼠组移植性肝肿瘤生长速率无显著差异（F = 6.39，P > 0.05），裸鼠BCT组肿瘤体积和重量抑制率高于裸鼠ADM组（q1 = 5.89，q = 4. q2 = 92，P < 0.01），但与裸鼠HepG2组相似（q1 = 2.47，q2 = 3.02，P > 0.05）。RT-PCR检测显示裸鼠ADM组和裸鼠BCT组MDR1mRNA无差异（q = 3.71，P > 0.05）。裸鼠BCT组肿瘤组织中每高倍视野平均凋亡细胞数（25.7±1.8）高于ADM组（2.7±0.2）（q = 3.72，P < 0.01），但与裸鼠HepG2组相似（23.9±1.6）（q = 1.43，P > 0.05）。BCT治疗后所有裸鼠99mTc-MIBI摄取均显著高于BCT治疗前（t = 3.58，P < 0.01）。
BCT可通过抑制P-糖蛋白功能逆转肝癌多药耐药，并可增强HepG2/ADM细胞对细胞毒性药物的敏感性。
