氯黴素對癌症細胞株之缺氧誘導性因子1α表現影響和機轉探討

Effects and Mechanism of Chloramphenicol on the Hypoxia-inducible Factor 1α Expression in Cancer Cells

吳芊樺(Chien-Hua Wu)

指導教授 ： 康照洲

國立臺灣大學/醫學院/毒理學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02514

摘要

氯黴素（Chloramphenicol，CAP）是一種廣效、抑菌性抗生素，它於長期以來被懷疑具有致癌性，但是證據仍相當有限。缺氧誘導性因子1α（Hypoxia-inducible factor 1α，HIF-1α）是一種缺氧敏感性轉錄因子，因可調控許多癌症相關的基因表現，被認為在癌症發展上扮演關鍵角色，若其表現增加則癌症病患的預後較差。而它的表現受到PI3K（phosphatidylinositol 3-kinase）和MAPK（mitogen-activated protein kinase）路徑調控。由於過去在本實驗室的研究中發現，氯黴素會活化PI3K和MAPK路徑，以及增加癌細胞浸襲力和抗凋亡，因此想進一步來了解氯黴素是否會影響癌細胞之缺氧誘導性因子1α的蛋白表現。
　　本研究以SDS-PAGE（Sodium dodecyl sulfate polyacrylamide gel electrophoresis）和西方墨點法分析，在正常氧壓下對H1299和A549細胞處理氯黴素（1∼100 µg/ml, 1∼12小時），並不影響缺氧誘導性因子1α的蛋白表現。但當對H1299、A549和MCF-7細胞前處理氯黴素(1-100 µg/ml，1小時)，再使用氯化亞鈷（Ⅱ）（cobalt chloride，CoCl2）作為缺氧模擬劑，則短時間內可有效抑制缺氧誘導性因子1α蛋白表現增加的現象，而此抑制作用在缺氧狀況（0.5% O2）下也會產生。此外，MTT試驗的結果顯示，於本研究投予氯黴素之劑量和處理時間之下，並無產生明顯的細胞毒性。
　　本研究以H1299進行氯黴素抑制缺氧誘導性因子1α蛋白表現作用的機轉研究。以cycloheximide（蛋白轉譯抑制劑）研究缺氧誘導性因子1α蛋白降解，發現氯黴素會加速缺氧誘導性因子1α蛋白的降解；至於蛋白生成的相關訊息傳遞方面，則發現氯化亞鈷（Ⅱ）和缺氧所增加的缺氧誘導性因子1α蛋白表現現象，和PI3K及ERK1/2（Extracellular signal-regulated kinase 1/2）路徑活化有關，而前處理氯黴素會抑制由氯化亞鈷（Ⅱ）刺激之AKT、mTOR磷酸化，但不會抑制ERK1/2的磷酸化。此外，氯化亞鈷（Ⅱ）和氯黴素皆不會影響細胞缺氧誘導性因子1α的mRNA表現。
　　本研究以半定量RT-PCR觀察缺氧誘導性因子1α所調控的下游基因p21和血管內皮生長因子（vescular endothelial growth factor，VEGF）的mRNA表現；以酵素免疫法（ELISA，Enzyme-linked immunosorbent assay）觀察血管內皮生長因子的蛋白表現，結果它們的表現量皆會因氯化亞鈷（Ⅱ）的處理而增加、因前處理氯黴素而減少。
　　總而言之，本研究首度提出了短時間處理氯黴素於癌症治療上可能具有正面的影響，而以低劑量效果尤佳。它的作用主要是透過轉錄後的影響，會加速缺氧誘導性因子1α蛋白降解；此外它可經抑制PI3K / AKT / mTOR路徑之活化，來減低缺氧誘導性因子1α的蛋白表現。而氯黴素也影響了缺氧誘導性因子1α對下游基因的調控。儘管如此，就整體而言是否確實為正面影響，仍需要進行更多研究才能確認。

關鍵字

氯黴素 ； 缺氧誘導性因子1α ； 氯化亞鈷 ； 缺氧 ； 癌症

並列摘要

Chloramphenicol (CAP) is a potent, broad-spectrum, bacteriostatic antibiotic agent. It is suggested that CAP is reasonably anticipated to be a human carcinogen. Although it has been doubted to be a carcinogen for decades, there still have very limited evidence to prove the carcinogenic hypothesis. Hypoxia-inducible factor 1α(HIF-1α) is a hypoxia-sensitive transcription factor, regulating many genes that code for proteins involved in cancer progression. Overexpression of HIF-1α was associated with poor prognosis of cancer patients. Its expression was regulated via PI3K (phosphatidylinositol 3-kinase), and MAPK (mitogen-activated protein kinase) pathways. Because previous studies of our labortory showed that CAP activated PI3K and MAPK pathway, and enhanced invasion and antiapoptosis abilities of cancer cells, we further investigated whether CAP could affect HIF-1αprotein level.
We used SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and western blotting analysis for the investigation. In normoxia, the HIF-1α protein level in CAP-treated (1-100 µg/ml, 1-12hours) H1299 and A549 cells were unaffected. Interestingly, when we pretreated H1299, A549 and MCF-7 cells with CAP (1-100 µg/ml, 1 hour) and used CoCl2（cobalt chloride） as a hypoxia-mimetic agent, CAP inhibited the CoCl2-induced HIF-1α accumulation in cells. Besides, the inhibitory effect
was also happened in hypoxia (0.5% O2). Short term treatment of CAP had no significant cytotoxicity on H1299 cells. The inhibitory effect of CoCl2-induced HIF-1α expression by CAP might be due to acceleration of HIF-1α degradation rate, and interfering with activation of PI3K / AKT / mTOR signaling induced by CoCl2, but not ERK1/2 signaling in H1299 cells. Both CAP and CoCl2 treatment had no obvious effect on HIF-1α mRNA expression in H1299 cells. In addition, CAP could also reduced the CoCl2-induced mRNA level elevation of VEGF (vescular endothelial growth factor) and p21, both were downstream genes regulated via HIF-1α. And it could also inhibited the CoCl2-induced VEGF165 secretion.
As a whole, although CAP has been thought to be a reasonable carcinogen in the past, we proposed a possible good effect of short term treatment of CAP in cancer therapy, especially with low dose. However, we need more investigations to further confirm the benefits and toxicities of CAP.

並列關鍵字

chloramphenicol ； hypoxia-inducible factor 1α ； cobalt chloride ； hypoxia, cancer

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