Monitoring the functional expression of multidrug resistance gene (MDR1) and study on potentiation of antitumor agent by MDR1 inhibitors in brain tumors.

本研究的目的是探讨~(99)m；~(99)m&GT；~(99)Tc-MIBI是否适用于肿瘤多药耐药的研究和MDR1抑制剂对抗肿瘤药物增强作用的预测。为了诱导和维持肿瘤细胞(RG2和C6胶质瘤，Walker 256肿瘤：W256)的多药耐药，用小剂量长春新碱(VCR，1 ng/ml)连续孵育肿瘤细胞。不同浓度的长春新碱(0.001~10μg/ml)作用3d后，用四甲基偶氮唑盐比色法检测细胞的多药耐药情况。在1μg/mlVCR作用下，所有耐药细胞株(RG2R、C6R、W256R)的存活分数均显著高于耐药细胞株(RG2、C6、W256)(P<0.01)。逆转录聚合酶链式反应显示，在所有长春新碱耐药细胞系中，mdr1mRNA的表达均高于未用药的细胞系。高表达mdr1 m RNA的长春花碱耐药细胞株…对~(99m)Tc-MIBI的Vd值显著低于未用药细胞株Mdr1基因表达水平较低的细胞株较多(p&lt；0.01)。在药物初治细胞系和长春新碱耐药亚系中，99m&GT；TcMIBI的Vd与mdr1基因表达呈负相关(r=0.98，p&lt；0.01)。经5μM浓度的mdr1抑制剂(维帕米、环孢素、FK506)处理3d后，所有耐药亚系的存活率均显著低于未处理的耐药亚系。四甲基偶氮唑盐比色法显示MDR-1抑制剂预处理对VCR细胞毒性有增强作用。在所有长春新碱耐药亚系中，mdr1抑制剂5μM处理1h后，所有耐药亚系的Vd均显著增加(p&lt；0.01)。这些结果表明，MIBI依赖的允许VCR排出的机制可被MDR-1抑制剂抑制，这可能具有重要的临床意义，如~(99)m；~(99)m&gt；Tc-MIBI可有效地监测主要作用于MDR_1的系统的治疗。~(99)m；99m&gt；Tc-MIBI SPECT有望为监测恶性脑肿瘤患者化疗前后的多药耐药提供更明确的标准。较少

The aim of this study is to explore whether ^<99m>Tc-MIBI is suitable to elucidate multidrug resistance and prediction of potentiation of antitumor agents by MDR1 inhibitors in malignant tumors.In order to induce and maintain multidrug resistance, tumor cells (RG2 and C6 gliomas, Walker 256 carcinoma : W256) were continuously incubated with low dose vincristine (VCR,1 ng/ml). MTT assay was performed following a 3-day exposure of various concentration of VCR (0.001-10 μg/ml) to assess the status of multidrug resistance. At a dose of 1 μg/ml of VCR, MTT assay demonstrated significant increase of surviving fractions in all VCR-resistant sublines (RG2R, C6R, W256R) as compared with those of drug-naive cell lines (RG2, C6, W256)(p<0.01). In all VCR-resistant sublines, RT-PCR revealed higher expression of MDR1 mRNA as compared with drug-naive cell lines. Vds of ^<99m>Tc-MIBI in VCR-resistant sublines expressing higher level of MDR1 mRNA was significantly lower than those of drug-naive cell l … More ines expressing lower levels of MDR1 mRNA (p<0.01). Vd of ^<99m>Tc-MIBI is negatively correlated with MDR1 mRNA expression (r=0.98, p<0.01) among drug-naive cell lines and VCR-resistant sublines. These findings indicated that the development of drug resistance was associated with enhanced ^<99m>Tc-MIBI extrusion.After 3-day pretreatment with MDR 1 inhibitors (verpamil, cyclosporin, FK506) at a concentration of 5 μM, surviving fractions of all VCR-resistant sublines significantly decreased as compared with those of non-treated VCR-resistant sublines. MTT assay revealed the enhancing effects on VCR cytotoxity following pretreatment with MDR 1 inhibitors. Vd of ^<99m>Tc-MIBI significantly increased after one-hour pretreatment with MDR 1 inhibitors at a concentration of 5 μM in all VCR-resistant sublines (p<0.01). These findings indicated that MIBI-dependent mechanisms allowing VCR extrusion were inhibited by MDR 1 inhibitors, which might have significant clinical implications such as ^<99m>Tc MIBI could be effective in monitoring therapies which act primarily on systems effected by MDR 1. ^<99m>Tc-MIBI SPECT is expected to provide more definitive criteria in monitoring multidrug resistance before and after chemotherapy in patients with malignant brain tumors. Less