含氟修飾之順鉑藥物於抗腫瘤作用機制之探討與臨床試驗前之評估

The characterizing and assessments of the modified fluorinated cisplatin analogues on antitumor effects

甘慈珺(Tzu-Chun Kan)

指導教授 ： 張榮善

臺北醫學大學/醫學院/醫學科學研究所/碩士(2013年)

摘要

順鉑(順一雙氨雙氯鉑)，英文商品名為Cisplatin，是常見的化療藥物，適用於多種癌症，包含乳癌、大腸直腸癌和卵巢癌。然而在長期使用下，臨床許多病例出現對順鉑的抗藥性，除此之外，順鉑原本的副作用，例如腎毒性、耳毒性等，也深深影響病患的治療效果與預後，因此，開發新式、具抗癌性的順鉑修飾化合物是目前重要並且迫切的研究議題。
在本研究中所檢測的順鉑修飾化合物(4,4'4F PtCl2)是以順鉑為基底，使用含氟聯吡啶(fluorinated bipyridine)取代原有的胺基(amine group) 結構，此新式藥物針對多種癌症均展現強效細胞毒殺性，甚於傳統的順鉑藥物，尤其以乳癌與大腸癌。實驗裡以順鉑為對照組，比較順鉑修飾化合物的抗癌效果，所測試的腫瘤細胞有MCF-7 (人類乳腺癌)、MDA-MB-231 (人類三重陰性乳腺癌) 和HCT-15 (人類大腸腺癌)三種癌細胞。實驗以流氏細胞儀偵測凋亡試驗與細胞週期，佐以DNA片段電泳，細胞存活不僅以MTT試驗檢視，細胞經由吉姆薩染液染色再次確認，最後動物實驗則以順鉑或順鉑修飾化合物經由靜脈注射或腫瘤內注射，測量腫瘤的生長情況。
結果可知此新合成的順鉑修飾化合物表現強而有力的毒殺特性， MDA-MB-231細胞的IC50為24μΜ，在80μΜ作用下低於10%的細胞存活，約90%的細胞進入凋亡階段。而HCT-15細胞的IC50則是48μM，同樣給予80μΜ藥物後僅有22%細胞存活，接近74%處於晚期凋亡，更重要的是此合成物克服了兩種癌症對順鉑的抗藥性。細胞週期方面，可見週期停頓於subG1和G1階段，不同於已往的順鉑效果會停留在G1/S。In vivo實驗以異體移植小鼠模式建立，順鉑修飾化合物經由靜脈注射不具有治療效果，但是更改給藥途徑為腫瘤內注射後，化合物成功抑制大腸腫瘤的生長，由此一連串實驗證實了化合物對兩種癌症皆擁有抗癌的能力。
本論文所描述的順鉑修飾化合物表現高度潛力於取代傳統順鉑作為臨床治療的替代藥品。未來的實驗以進一步研究此化合物的抗癌分子機制，並與傳統順鉑相互比較，希望能開發新的化療藥物以改善患者的病情結果與預後情況。

關鍵字

順鉑 ； 乳癌 ； 大腸直腸癌 ； 抗癌藥物 ； 細胞凋亡 ； 細胞週期

並列摘要

Cisplatin is a common therapeutic anti-cancer drug for patients with several forms of cancers including breast cancer, colorectal cancer, and ovarian cancer. Currently, however it has been revealed that tumors can develop the resistance to cisplatin in clinical. Moreover, the considerable severe side effects also affect the drug efficacy and prognostic outcomes. Therefore, to develop new and tumor-targeted analogues of cisplatin is important and urgent.
In this study, we presented the cisplatin containing chemotherapeutic candidates with fluorinated bipyridine instead of amine group displaying more powerful cytotoxicity for various forms of cancers. It demonstrated that one of these candidates (4, 4' 4F PtCl2) is more effective especially for breast cancers and colorectal cancers, comparing to commercial cisplatin. Commercial cisplatin has been used as control therapeutic agent to verify and compare the anti-tumor effects with new synthesized cisplatin analogues. Several forms of cancer cell lines have been investigated including MCF-7 (Human breast adenocarcinoma cells), MDA-MB-231 (Human breast adenocarcinoma cells) and HCT-15 (Human colorectal adenocarcinoma cells). The analogue shows the most profound cytotoxic effects on the MDA-MB-231 cells with IC50 at 24μΜ, less than 10% were survived and 90% of cells entered apoptotic phase under the concentration of 80μM. In HCT-15 cells, the IC50 of these cells was 48μM, 22% cells were alive and 74% of cells located at late apoptosis after 80μM of analogue was administrated. Moreover, cisplatin-resistance was observed in both cell lines. Besides, cell cycle arrested at subG1 and G1 stage rather than previous results of cisplatin-treated. Finally, the in vivo experiment show inhibition of colorectal tumor growth followed by intratumoral injection of modified cisplatin analogue. It demonstrated that our modified cisplatin analogue possess much stronger cytotoxic effects on all investigated cancer cell lines, comparing to commercial cisplatin. The cell survival rate was determined by MTT assay, rechecked and imaged through Giemsa staining, and the cell death program would be evaluated by apoptotic assay and DNA fragmentation. The improvement of cisplatin analogues on the anti-cancer effects had also been evaluated in xenograft murine (nude mice). This result demonstrated that no improved therapeutic effects in vivo xenogafts, when putative anti-cancer candidates were intravenously administrated. However, there is increased therapeutic efficacy observed in xenograft when intratumoral injection of drug were applied.
Our cisplatin analogue displays powerful cytotoxicity in both breast and colorectal cancer cells and potentially can replace cisplatin to turn as a good therapeutic candidate for patients. We will further investigate the molecular mechanism of cisplatin analogues on the anti-cancer effects and then unbraid its improved therapeutic effects by comparing to cisplatin and hope this study can generate a new chemotherapeutic drug for cancer patients with an improved outcome and prognosis.

並列關鍵字

Cisplatin ； Breast cancer ； Colorectal cancer ； Anti-cancer drug ； Apoptosis ； Cell cycle

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