探討藥物YC-1在視網膜細胞存活與泡沫細胞形成的作用

Study the effects of YC-1 on retinal cell survival and foam cell formation

崔立有(Leo Tsui)

指導教授 ： 馮琮涵

共同指導教授 ： 王一中(I-Jong Wang)

臺北醫學大學/醫學院/醫學科學研究所/博士(2014年)

摘要

生物體內由於缺乏氧氣供應會造成缺氧的發生，並且引起許多細胞層級的反應去因應氧氣濃度的改變，例如缺氧會導致視網膜神經節細胞的死亡與誘發巨噬細胞形成充滿脂質堆積的泡沫細胞。在缺氧環境中，HIF-1alpha是其中一個主要調節者去調控與細胞存活或是代謝相關的特定基因表現。已知抗癌藥物YC-1可以增加細胞內cGMP的量並抑制HIF-1alpha蛋白質的表現。本篇研究的目的即是想探討YC-1這種藥物對於視網膜細胞存活以及泡沫細胞形成的作用。
由於視網膜感光細胞株661W(又被稱為RGC-5)受到HIF-1alpha的刺激劑CoCl2處理後會誘發HIF-1alpha表現並造成細胞死亡，我們因此假設藥物YC-1會透過抑制HIF-1alpha而影響正常661W的細胞存活。根據免疫轉漬的結果顯示，濃度20 microM的YC-1處理4小時後確實會降低正常情況下661W中HIF-1alpha蛋白質表現。MTT細胞存活率分析的結果顯示，在正常環境中濃度20 microM的YC-1處理24小時後可以降低約25%細胞存活率。但是LIVE/DEAD細胞存活率分析的結果顯示在正常環境下YC-1並不會造成661W細胞死亡，而我們透過高內涵篩選技術去偵測細胞核中細胞增生指標物Ki-67的表現，發現YC-1是透過抑制細胞增生來降低細胞存活率。上述實驗結果證實在一般正常氧氣供應的情況下，YC-1確實會抑制HIF-alpha並且阻礙661W的細胞增生。
我們根據免疫轉漬的結果更進一步發現，濃度20 microM的YC-1處理2小時可以抑制200 microM CoCl2刺激下661W細胞中的HIF-1alpha蛋白質表現。MTT細胞存活率分析的結果顯示，在CoCl2模擬的缺氧情況與0.5%低氧環境中，濃度20 microM的YC-1處理24小時各降低約50%與25%的細胞存活率。高內涵篩選技術的數據則顯示，在CoCl2模擬的缺氧環境下，YC-1可以降低661W細胞核中細胞增生指標物Ki-67的表現。然而，流式細胞儀的結果顯示YC-1會在CoCl2模擬的缺氧環境下造成細胞凋亡與死亡，而透過定量反轉錄聚合酶連鎖反應可以發現腫瘤抑制蛋白p53的基因轉錄增加。我們的發現指出在缺氧環境下，YC-1會抑制CoCl2所刺激HIF-1alpha的表現，除了造成細胞增生停滯，還會誘發視網膜細胞凋亡與死亡，並且增加p53基因的表現。
此外，在總共640種美國藥物食品管理局核可的藥物中，我們利用高效能篩選系統與LIVE/DEAD細胞存活率分析試劑，評估何種藥物可以降低缺氧環境造成的細胞死亡。我們的實驗結果顯示在CoCl2模擬的缺氧環境中，statin類藥物應是所有藥物中保護效果最佳的藥物，而且還會導致661W轉變成類似神經細胞的型態。利用高內涵篩選技術去偵測神經細胞指標物MAP2的免疫細胞化學染色，所得到的數據更進一步顯示出在0.5%氧氣的缺氧環境中，fluvastatin、mevastatin和simvastatin可以誘發661W分化成神經細胞，而且與單獨接受缺氧處理的細胞相比可增加約2倍的神經突起總長。我們的發現證實在缺氧環境下statins不但具有神經保護作用，還可以誘導661W分化成神經細胞。
另一方面，我們想要探討YC-1在小鼠的巨噬細胞株RAW 264.7中對於泡沫細胞形成的影響。Oil red O染色的結果顯示濃度20 microM的YC-1處理24小時可以增加巨噬細胞中的脂肪滴形成。利用高內涵篩選技術結合AdipoRedTM染色的結果顯示，與對照組相比YC-1會增加巨噬細胞內約30%的總脂質含量。我們的結果也指出YC-1可以增強氧化型低密度脂蛋白所誘發的泡沫細胞形成。透過oil red O染色，我們的初步結果顯示cGMP類似物8-br-cGMP可以增促進巨噬細胞形成脂肪滴和泡沫細胞，而PKG活化的抑制劑KT5823則可以降低YC-1所誘發的脂肪堆積。根據上述結果，我們推測YC-1可能是透過cGMP相關的途徑促進泡沫細胞形成。
總結我們的研究發現，YC-1可以抑制HIF-1alpha的表現繼而造成正常或缺氧情況下視網膜細胞的凋亡或是增生被抑制，而只有在缺氧狀態下YC-1才會促進細胞死亡。我們也發現statin可以保護缺氧所誘發的視網膜細胞死亡。在巨噬細胞中，我們的結果證實YC-1會誘發細胞形態轉變形成富含脂肪滴的泡沫細胞。

關鍵字

YC-1 ； 缺氧誘導因子-1alpha ； 環磷酸鳥苷視網膜感光細胞 ； 巨噬細胞 ； 泡沫細胞

並列摘要

Hypoxia is a condition with low oxygen supply and results in many cellular responses to adapt the change of oxygen concentration, such as induction of retinal cell death and macrophage differentiation to lipid-laden foam cells. Under hypoxia, hypoxia inducible factor (HIF)-1alpha is one of the major modulator to regulate specific genes which involved in cell survival or metabolism. 3-(5'-Hydroxymethyl-2'-furyl)- 1-benzyl indazole (YC-1) is a potential anticancer drug which could increase the level of cyclic guanosine monophosphate (cGMP) and inhibit HIF-1alpha protein expression. The aim of our study is to explore the effects of YC-1 on retinal cell survival and foam cell formation.
The HIF-1alpha inducer cobalt chloride (CoCl2) could induce HIF-1alpha expression and lead to cell death in a retinal photoreceptor cell line 661W (also known as RGC-5). We hypothesized that YC-1 may affect 661W cell survival under normoxia through inhibition of HIF-1alpha. Our immunoblotting results showed the inhibition of normoxic HIF-1alpha expression in response to 20 microM YC-1 for 4 h. Treatment with 20 microM YC-1 for 24 h decreased ~25% of survival rate (compared to control group) using 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay. However, the LIVE/DEAD cell viability assay indicated that YC-1 had no effect on cell death of 661W under normoxia, and we found that YC-1 could reduce normoxic cell viability through inhibition of cell proliferation by high content screening (HCS) and antigen identified by monoclonal antibody Ki-67 (Ki-67) immunocytochemistry. Our findings demonstrate that YC-1 could inhibit HIF-1alpha and consequently reduce cell proliferation in normoxic 661W.
We further found that treatment with 20 microM YC-1 for 2 h could inhibit CoCl2 (200 microM)-stimulated HIF-1alpha protein expression in 661W using immunoblotting. The MTT cell viability assay indicated that treatment with 20 microM YC-1 for 24 h could respectively reduce ~50% and ~25% of cell viability (compared to control group) under CoCl2-mimicked hypoxia and environmental hypoxia (0.5% O2). The HCS data showed that YC-1 could decrease cell proliferation marker Ki-67 expression in the nuclei of 661W under CoCl2-mimicked hypoxia. However, we found that YC-1 increased cell apoptosis and cell death by flow cytometry and tumor suppressor gene p53 transcription by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) under CoCl2-mimicked hypoxia. Our findings indicate that YC-1 could inhibit HIF-1alpha and subsequently increase p53 transcription and induce of cell apoptosis, death and cell cycle arrest in 661W under hypoxia.
Moreover, in a commercial library with total 640 US FDA approved drugs, HCS and LIVE/DEAD cell viability assay were used to evaluate which one could rescue 661W loss under hypoxia. We found that statins could improve 661W cell survival and induce cell transformation to neuron-like cells under CoCl2-mimicked hypoxia. HCS and microtubule-associated protein (MAP) 2 immunocytochemistry indicated that fluvastatin, mevastatin, and simvastatin could induce neurite outgrowth and increase ~200% of total neurite length (compared to hypoxia group) in 661W under environmental hypoxia (0.5% O2). Our findings establish that statins not only prevented 661W cell death but also induced neural differentiation under hypoxia.
On the other hand, we wanted to explore the impact of YC-1 on foam cell formation in a murine macrophage cell line RAW 264.7. The oil red O staining indicated that treatment with 20 microM YC-1 for 24 h increased the intracellular lipid droplets in macrophages, and HCS with AdipoRed assay revealed that YC-1 could increase ~30% of total lipid content compared to control group. Our finding also revealed that YC-1 enhanced oxidized low density lipoproteins (ox-LDL)-induced foam cell formation. We further found that cGMP analogue 8-bromo-cGMP (8-br-cGMP) could promote lipid droplet and foam cell formation, and the cGMP-dependent protein kinase (PKG) inhibitor KT5823 could reduce YC-1-induced lipid accumulation. Our findings suggest that YC-1 could increase foam cell formation through a cGMP-related pathway.
Taken together, we found that YC-1 could inhibit HIF-1alpha expression and subsequently induce cell apoptosis and cell cycle arrest in retinal cells under normoxia and hypoxia both, but it only increased cell death under hypoxia. We also found that statins could protect hypoxia-induced retinal cell death. In macrophages, our finding demonstrated that YC-1 could induce cell transformation to lipid-laden foam cells.

並列關鍵字

YC-1 ； hypoxia inducible factor-1alpha ； cyclic guanosine monophosphate ； retinal photoreceptor cells ； macrophages ； foam cells

參考文獻

Ak, P., and Levine, A.J. (2010). p53 and NF-kappaB: different strategies for responding to stress lead to a functional antagonism. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 24, 3643-3652.

An, W.G., Kanekal, M., Simon, M.C., Maltepe, E., Blagosklonny, M.V., and Neckers, L.M. (1998). Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha. Nature 392, 405-408.

Aranha, A.M., Zhang, Z., Neiva, K.G., Costa, C.A., Hebling, J., and Nor, J.E. (2010). Hypoxia enhances the angiogenic potential of human dental pulp cells. Journal of endodontics 36, 1633-1637.

Araya, J., Maruyama, M., Inoue, A., Fujita, T., Kawahara, J., Sassa, K., Hayashi, R., Kawagishi, Y., Yamashita, N., Sugiyama, E., et al. (2002). Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages. American journal of physiology Lung cellular and molecular physiology 283, L849-858.

Arjamaa, O., and Nikinmaa, M. (2006). Oxygen-dependent diseases in the retina: role of hypoxia-inducible factors. Experimental eye research 83, 473-483.