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Title: | 猴病毒40型T/t-common蛋白質在HER2大量表現癌細胞可抑制新生血管生成並增加化療藥物的毒殺能力 SV40 T/t-common polypeptide inhibits angiogenesis and enhances the cytotoxic activity of chemotherapeutic agents in human HER2-overexpressing tumors |
Authors: | Shu-Ping Hsueh 薛淑萍 |
Advisor: | 王萬波 |
Keyword: | HER2,猴病毒四十型T/t-common,血管新生,腫瘤生長,VEGF-A,TSP-1,cisplatin,doxorubicin,藥物感受性, HER2,SV40 T/t-common,angiogenesis,tumor growth,VEGF-A,TSP-1,cisplatin,doxorubicin,chemosensitivity, |
Publication Year : | 2011 |
Degree: | 博士 |
Abstract: | HER2 致癌基因在許多人類癌細胞 (特別是卵巢癌和乳癌) 中有大量表現的情形。研究發現HER2會促進癌細胞生長、存活、血管新生 (angiogenesis)、癌細胞轉移 (metastasis)、抑制癌細胞對化療藥物的感受性 (chemosensitivity) 和降低生命存活率,若在HER2大量表現的癌細胞中抑制HER2的表現則會降低癌細胞生長、存活、血管新生、增加癌細胞對化療藥物感受性和增加生命存活率。我們先前證明了猴病毒四十型(SV40)的T/t-common polypeptide在HER2大量表現的癌細胞會透過抑制HER2的表現進而抑制癌細胞形成腫瘤的能力,此外也可使HER2大量表現的人類卵巢癌和乳癌細胞進行細胞凋亡(apoptosis)。本論文第一部分探討T/t-common在具有HER2大量表現的人類卵巢癌細胞SK-OV-3中是否會抑制血管新生。利用具有T/t-common 表現的SK-OV-3 細胞培養液(conditioned medium) 發現會抑制內皮細胞的移動 (migration)、血管生成 (tube formation) 和動物體內血管生成 (microvessel formation),證明了T/t-common 可抑制SK-OV-3誘發血管新生的能力。若將HER2基因重新送入可表現T/t-common的SK-OV-3細胞 (T/t-common stable clones)中並使HER2再大量表現;收集細胞培養液進行實驗,發現HER2會降低T/t-common抑制內皮細胞移動和血管生成的能力,因此T/t-common抑制血管新生能力是透過抑制了HER2的表現。T/t-common在SK-OV-3中會抑制一些血管新生因子 (proangiogenic factors) 例如(VEGF-A、IL-8、bFGF、uPA、MMP-2)的表現和促進一些抑制血管新生因子 (antiangiogenic factors) 例如(TSP-1、TIMP-1)的表現。我們也進一步証明T/t-common在SK-OV-3中是透過抑制 ERK1/2活性進而抑制轉錄因子HIF-1α 和血管新生因子VEGF-A的表現,此外T/t-common可促進p38活性進而增加抗血管新生因子TSP-1的表現,因此T/t-common可透過HER2-ERK1/2-HIF-1α-VEGF-A和HER2-p38-TSP-1訊息傳遞路徑來抑制HER2誘發血管新生能力。將HER2大量表現的卵巢癌細胞SK-OV-3種植在免疫缺陷老鼠NOD/SCID並以T/t-common治療,發現T/t-common在NOD/SCID老鼠可抑制HER2大量表現腫瘤的血管新生和腫瘤生長。因此利用T/t-common做基因治療可能是一種治療HER2大量表現癌症的新方法。
本論文第二部份則探討T/t-common在具抗藥性的HER2大量表現的癌細胞中,是否能增強癌細胞對化療藥物cisplatin (CDDP)及doxorubicin (DXR)的感受性,並進一步探討其增強化療藥物療效的機制。結果顯示T/t-common可特異性地加強CDDP或DXR 對HER2大量表現癌細胞的毒殺能力,並經由增加Caspase 3的活性誘發癌細胞進行細胞凋亡而增加化療藥物療效。當HER2大量表現的癌細胞同時以T/t-common 和CDDP或DXR作用下,發現T/t-common可抑制ERK1/2的活性和促進JNK的活性,並藉此抑制Bcl-2和Bcl-XL的表現,因此T/t-common可能是經由調控上述細胞凋亡因子進而增強CDDP或DXR誘發HER2大量表現癌細胞細胞凋亡。最後將HER2大量表現的卵巢癌細胞SK-OV-3.ip1植入免疫缺陷老鼠NOD/SCID,並以T/t-common和化療藥物DXR一同治療,發現腫瘤體積比任一單獨試劑治療還小。因為T/t-common可增強CDDP或DXR毒殺HER2大量表現癌細胞的能力,所以T/t-common與CDDP或DXR的合同療法也許可克服HER2大量表現癌細胞對化療藥物的抗藥性,因此是一種具有潛力的治療HER2大量表現癌細胞的新方法。 Overexpression of HER2 has been frequently detected in many types of human cancer, most notably breast and ovarian cancers. HER2 overexpression is associated with increased angiogenesis, increased metastasis, increased chemoresistance and reduced survival. Inhibition of HER2 in HER2-overexpressing cancers can lead to reduced angiogenesis, reduced chemoresistance and improved survival. Previously, we reported that Simian Virus 40 T/t-common polypeptide can inhibit HER2, suppress the tumorigenic potential of HER2-overexpressing cancer cells and specifically induce apoptosis in HER2-overexpressing human cancer cell lines. In the part I of this thesis, we further explored the effect of T/t-common on tumor angiogenesis. We found that T/t-common could inhibit the ability of HER2-overexpressing cancer cells, but not low HER2-expressing cancer cells, to induce the migration and tube formation of endothelial cells. Reexpression of HER2 could block T/t-common’s activity to inhibit the ability of HER2-overexpressing cancer cells to induce the migration and tube formation of endothelial cells. We further tested whether T/t-common could affect HER2-overexpressing cancer cells to express and secrete angiogenic and anti-angiogenic factors. We found that T/t-common could inhibit the expression of proangiogenic factor VEGF-A, IL-8, bFGF, uPA and MMP-2, and induce the expression of anti-angiogenic factor TSP-1and TIMP-1 in HER2-overexpressing human SK-OV-3 ovarian cancer cells. T/t-common was found to be able to repress the expression of HIF-1α, a transcription factor required for VEGF-A expression, through inhibiting HER2-mediated ERK1/2 activity. Moreover, T/t-common was found to be able to activate p38 pathway, leading to up-regulation of TSP-1. Through regulating HER2-ERK1/2-HIF-1α-VEGF-A and HER2-p38-TSP-1 signaling pathways in HER2-overexpressing cancer cells, T/t-common could inhibit these cancer cells to recruit endothelial cells, leading to inhibition of angiogenesis in HER2-overexpressing tumors. Two experiments described below demonstrated that T/t-common indeed can inhibit tumor angiogenesis. First, in Matrigel plug assays, we demonstrated that conditioned medium from T/t-common-transduced SK-OV-3 cancer cells had lower ability to induce blood vessel formation in Matrigel plugs than that from control SK-OV-3 cells. Second, infection of adenovirus carrying T/t-common gene could lead to inhibition of the growth and microvessel formation of SK-OV-3 tumors in NOD/SCID mice model. Taken together, the above suggest that T/t-common had the potential to be developed as a new antiangiogenic agent specific for treating HER2-overexpressing ovarian cancers. In the part II of this thesis, we investigated whether T/t-common could enhance the sensitivity of HER2-overexpressing human cancer cells to chemotherapeutic agents such as cisplatin (CDDP) and doxorubicin (DXR). We found that T/t-common could specifically enhance the sensitivity of HER2-overexpressing human cancer cells, but not that of HER2 low-expressing human cancer cells, to CDDP and DXR. T/t-common could specifically enhance CDDP- or DXR-induced apoptosis by activating caspase-3 activity in HER2-overexpressing cancer cells. T/t-common expression led to down-regulation of Bcl-2 and Bcl-XL, inhibition of ERK activity, and activation of JNK activity in CDDP- or DXR-treated HER2-overexpressing BT-474 cancer cells. This modulation of apoptosis regulatory molecules by T/t-common may contribute to its ability to enhance CDDP- or DXR-induced apoptosis in HER2-overexpressing cancer cells. Finally, we showed that T/t-common could enhance the antitumor activity of DXR on HER2-overexpressing SK-OV-3.ip1 tumor in NOD/SCID mice. Together, these data demonstrated that T/t-common could sensitize HER2-overexpressing cancer cells to chemotherapeutic agents CDDP or DXR, and suggest that combination therapy using T/t-common gene and chemotherapeutic agents (such as CDDP or DXR) may overcome tumor chemoresistance and thus provide a new approach for treatment of HER2-overexpressing cancers. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66684 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 微生物學科所 |
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