乳癌細胞快速生長以及快速轉移至其他器官是造成致死率居高不下的主因。因此，研究以及開發相關抑制癌症藥物及治療乳癌的方式一直都是相當重要的課題之一。GCIP(Grap2 cyclin-D interacting protein)蛋白包含360 個氨基酸，其分子量約為42kDa。GCIP 含有五個區域，分別為：N 端區域、HLH 區域(helix-loop-helix
domain) 、acidic 區域(acidic domain)、leucine zipper 區域(leucine zipper domain)以及C 端區域。文獻指出GCIP 在許多高度分化的細胞均有大量表現，例如心臟、肌肉、周邊血液白血球以及腦部。而我們在先前的研究發現，GCIP 蛋白其表現量與乳癌細胞癌化程度呈現負相關，所以我們推測，GCIP 蛋白在細胞癌化的過
程當中為一重要調節因子。為了探討GCIP 在癌細胞中所扮演的角色，我們利用暫時性轉染(transient transfection)以及穩定表現細胞系統(stable clone)將GCIP 送入第三期乳癌細胞MDA-MB-231 中。我們藉由觀察細胞型態(morphology)，生長(proliferation)，細胞週期(cell cycle)，移行(migration)以及侵入(invasion)的影響。利用免疫螢光染色，我們發現擁有GCIP 的細胞其細胞突處(lame-podia)以及肌動蛋白應力纖維(stress fiber)較少，代表細胞其較缺乏移動及生長能力。利用MTT方式我們也發現，擁有GCIP 的細胞其生長明顯比控制組細胞緩慢，且利用滴流細胞儀分析發現，擁有GCIP 的細胞其細胞週期大部分停留於G0/G1 時期。另外，我們利用boyden chamber 觀察細胞移行能力。實驗發現GCIP 的存在使得癌細胞移行能力有約60~75%的下降情形。除此之外，我們利用特製的invasion chamber探討癌細胞其侵入能力是否有何改變。實驗發現，擁有GCIP 的細胞其侵入能力有40~70%下降。我們更進一步探討GCIP 是否會調控癌化相關蛋白表現或訊號傳遞路徑。在癌細胞移轉的過程當中MMP(基質的金屬蛋白酵素)扮演重要的角色。所以我們利用zymography 分析癌細胞其MMP-2,-9 表現狀況。實驗結果發現，GCIP 的存在會抑制MMP-2 以及MMP-9 的表現，代表細胞其侵入能力有減弱的現象。文獻指出GCIP 存在的細胞中細胞週期調控蛋白cyclin D1 表現量也
會下降，綜合以上的實驗以及參考文獻，我們認為GCIP 的存在有可能會藉著調控細胞訊號傳遞，達到抑制蛋白表現的結果。我們首先利用西方墨點法分析MAPK(Mitogen-Activated Protein Kinase)路徑當中ERK1/2, p38 以及JNK 被活化程度。實驗結果發現，擁有GCIP 的細胞其ERK1/2, p38 以及JNK 之磷酸化程度
均被明顯的抑制，並且與GCIP 蛋白表現情形呈現負相關情況；我們認為GCIP擁有直接或間接調控細胞週期達到抑制細胞癌化之能力。

Breast cancer is the most common malignant disease in Western women. In these patients, it is not the primary tumor, but its metastasis at distant sites that are the main cause of death. Hence, it is important to discover new diagnoses approach and therapeutic target that against the malignancy of breast cancer cells. Grap2 cyclin-D
interacting protein (GCIP) is ubiquitously expressed in human tissues with a relatively
higher level in the heart, skeletal muscle, kidney, and peripheral blood leukocytes. The
full length of GCIP contains 360 amino acids with predicts molecular mass of 40 kDa.
Previous evidences show that GCIP is a negative regulator of transcription factors. In our previous study we found that the expression level was related to breast cancer cells’ malignancy. We suggest that GCIP was an important key regulator in cancer progression. GCIP was transient transfected and stable expressed in MDA-MB-231. Cell morphology, proliferation, cell cycle progression, migration and invasion were performed in observation. In fluorescent microscopic observation we found that GCIP transfected cells had less lame-podia and stress fibers. This data indicated that GCIP
expressed cells have lower migrating ability. In MTT assay, we demonstrated that GCIP stable clone had lower proliferating rate than control cells. Moreover, we found that GCIP also inhibit cell progression which is consistent to previous research. Furthermore, we also indicated that GCIP dramatically reduced breast cancer cell
migration and invasion ability. Evidenced showed that metalloprotease (MMPs) play important role in breast cancer cells migration and invasion, we performed zymography assay to investigate whether GCIP inhibits MMP secretion. Our results
indicated that GCIP down-regulate MMP-2 and MMP-9 expression in mRNA and protein level. The data indicated that GCIP reduced cell invasion ability. However,recent study claimed that GCIP inhibits cyclin D1 expression in tumor cells. Hence, we suggested that upstream MAPK pathway play major role in regulating MMP and
cyclin D1 expression. We found that the activation of ERK1/2, p38 and JNK were much lower in GCIP stable clone than in control cells. Here we proposed a novel regulatory mechanism of GCIP in breast cancer cells. In conclusion, we suggested that GCIP plays an important role in regulation of breast cancer metastasis furthermore GCIP will be a potential therapeutic target in discovering new anti-cancer
agents.

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