探討ABCB1 在多型性神經膠母細胞瘤中循環性缺氧所誘導化療抗性之角色與機轉

背景：腫瘤細胞循環性缺氧（tumor cycling hypoxia）在動物和人類腫瘤是一個眾所皆知的現象。不過，目前還不清楚腫瘤循環性的缺氧如何影響化療（chemotherapy）。在本研究中，我們探討了細胞循環性缺氧的機制和影響在腫瘤環境下化療抗性（chemoresistance）的調節。方法：將人類多型性神經膠母細胞瘤（glioblastoma multiforme, GBM）異體移植（xenograft）細胞，利用免疫螢光影像技術和螢光激活細胞搜尋法將腫瘤切片用Hoechst 33342染色和缺氧誘發因子1（hypoxia- inducible factor-1, HIF-1）激活做標記，找尋出來源於人類多型性神經膠母細胞瘤的缺氧腫瘤細胞亞群。而人類多型性神經膠母細胞瘤的缺氧腫瘤異體移植細胞或經由體外（in vitro）循環缺氧處理過（cycling hypoxic stress-treated）的多型性神經膠母細胞瘤細胞之ABCB 1表達、P-糖蛋白(P-glycoprotein)功能和化療敏感度則是藉由西方墨點法 (Western blot analysis) 藥物聚集及排外法 (drug accumulation and efflux assay)及MTT assay分別呈現。結果：在多型性神經膠母細胞瘤的細胞循環性缺氧下，正調控ABCB1的表達和P-糖蛋白的功能伴著多型性神經膠母細胞瘤細胞對doxorubicin和BCNU反應下降。然而，把ABCB1基因 knockdown，就會抑制這些現象。此外，免疫螢光影像和流式細胞儀分析ABCB1、HIF-1的活性及在多型性神經膠母細胞瘤內的Hoechst3342染色，顯示局部性高度ABCB1的表達主要是伴隨HIF-1活化和血流灌注之實體腫瘤微環境(microenvironment) 的潛在細胞循環性缺氧區域內。從人類多型性神經膠母細胞瘤異體移植取得細胞循環性缺氧的腫瘤細胞，比慢性持續性缺氧細胞和常氧細胞(normoxic cells)表現出更高的ABCB1與P-糖蛋白以及化療抗性。而在荷瘤小鼠(bearing mice)給與一種HIF-1α的抑制劑YC-1，結果顯示抑制腫瘤微環境誘導ABCB1 誘發及增加BCNU化療的存活率。結論：細胞循環性缺氧在腫瘤微環境藉由HIF-1誘發ABCB1所引起的化療抗性過程中扮演著重要的角色。如果 HIF-1阻斷劑在化療之前和化療中同時使用，可以抑制細胞循環性缺氧誘發的化療抗性。

Background: Tumor cycling hypoxia is now a well-recognized phenomenon in animal and human solid tumors. However, how tumor cycling hypoxia impacts chemotherapy is unclear. In the present study, we explored the impact and the mechanism of cycling hypoxia on tumor microenvironment-mediated chemoresistance. Methods: Hoechst 33342 staining and hypoxia-inducible factor-1 (HIF-1) activation labeling together with immunofluorescence imaging and fluorescence-activated cell sorting were utilized to isolate hypoxic tumor subpopulations from human glioblastoma xenografts. ABCB1 expression, P-glycoprotein function, and chemosensitivity in tumor cells derived from human glioblastoma xenografts or in vitro cycling hypoxic stress-treated glioblastoma cells were determined by western blot analysis, drug accumulation and efflux assays, and MTT assay, respectively. Results: ABCB1 expression and P-glycoprotein function were upregulated under cycling hypoxia in glioblastoma cells concomitant with decreased responses to doxorubicin and BCNU. However, ABCB1 knockdown inhibited these effects. Moreover, immunofluorescence imaging and flow cytometric analysis for ABCB1, HIF-1 activation, and Hoechst 3342 in glioblastoma revealed highly localized ABCB1 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion within the solid tumor microenvironment. The cycling hypoxic tumor cells derived from glioblastoma xenografts exhibited higher ABCB1 expression, P-glycoprotein function, and chemoresistance than chronic hypoxic and normoxic cells. Tumor-bearing mice that received YC-1, an HIF-1α inhibitor, exhibited suppressed tumor microenvironment-induced ABCB1 expression and enhanced survival rate in BCNU chemotherapy. Conclusions: Cycling hypoxia plays a vital role in tumor microenvironment-mediated chemoresistance via the HIF-1-dependent induction of ABCB1. HIF-1 blockade before and concurrent with chemotherapy could suppress cycling hypoxia-induced chemoresistance.