癌症標靶材料作用於細胞與分子機制之探討：1.多光子趨動標靶化奈米鑽石誘發癌細胞死亡之機制；2.anti-CEACAM6與anti-HER-2單一抗原結合功能區抗體於乳癌標靶治療之研究。

Abstract

癌症的標靶治療可標定癌細胞，減少對正常細胞的傷害，故比傳統治療更有效。本研究結合奈米技術以及生物技術，發展出對癌細胞具有靶向性的奈米粒子。 在癌症細胞當中，通常會存在一些特殊表現或調控之因子，這些調控因子即為標靶治療的理想目標。本研究中以肺癌與乳癌作為模式，針對肺癌大量表現之生長激素受體、乳癌特殊表達之癌胚抗原相關細胞黏著分子6 (Carcinoembryonic antigen-related cell adhesion molecule 6, CEACAM6)與人類表皮生長因子受體2 (Human epidermal growth factor receptor 2, HER-2)，分別設計出標靶化之奈米粒子與單一抗原結合功能區抗體作為標靶治療之工具。 許多癌症細胞會內生性分泌生長因子，利用生長激素在腫瘤細胞的特殊表現，使羧酸化奈米鑽石與重組的生長激素受體結合蛋白以及綠色螢光蛋白接合成一複合體，此複合體能夠專一性的結合於具有高度表現生長激素受體的A549人類非小細胞肺腺癌細胞株的細胞膜上。細胞毒性測試結果指出，羧酸化奈米鑽石-生長激素受體結合蛋白-綠螢光蛋白複合體具有低生物毒性，高度生物相容之特性。當細胞羧酸化奈米鑽石-生長激素受體結合蛋白-綠螢光蛋白複合體接合於A549肺腺癌細胞表面，同時經由脈衝雷射的誘導使得奈米鑽石爆炸並造成細胞表面受損，並經由細胞凋亡機制導致細胞死亡。因此，此實驗之設計猶如一奈米手術，或許能夠建立為一種新的癌症治療策略，亦或為生物醫學所應用。 另一方面，癌胚抗原相關細胞黏著分子6與人類表皮生長因子受體2皆高度表現於乳癌細胞當中，且與乳癌之進程相關。藉由免疫駱馬後，分離、純化出單一抗原結合功能區抗體，並以抗體保守區(conserve region, Fc)接合不同的單一抗原結合功能區抗體，測試其對乳癌細胞株MCF-7之影響。同時，我們也以歐洲紫杉醇作為對照組與單一抗原結合功能區抗體之治療效果作比較。由實驗結果顯示，針對乳癌之進程，單一抗原結合功能區抗體可以經由誘導乳癌細胞凋亡進而抑制腫瘤細胞之增生，並且藉由減低基質金屬蛋白酶2 (MMP-2)、基質金屬蛋白酶9 (MMP-9)之活性，以及抑制單核球趨化蛋白1 (MCP-1)、細胞內黏附因子1 (ICAM1)、血管細胞黏附因子1(VCAM-1)等等，以減少巨噬細胞浸潤乳癌細胞之能力、降低乳癌細胞遷移、侵犯、轉移，以及阻礙癌症血管新生、使腫瘤體不再增大。相較於歐洲紫杉醇之療效，單一抗原結合功能區抗體更為一具有臨床潛力之抗體藥物。 總而言之，癌症的標靶化治療對周圍細胞不具傷害性，且可針對不同癌症細胞所表現特定分子，設計擁有較佳治療效果的分子或方法。於本實驗中，以針對生長激素受體所設計的奈米鑽石複合物，經由奈秒雷射誘發導致癌細胞凋亡，為高專一性的光敏療法平台開發。而針對CEACAM6與HER-2的單一抗原結合功能區抗體，更具全面性阻止癌症進程的療效，故此研究之奈米鑽石複合物與單一抗原結合功能區抗體分別為非小細胞肺腺癌與乳癌標靶治療之理想因子。

Targeted cancer therapy is more effective than conventional treatments and less harmful to normal cells. In this study, we have combined nanotechnology and biotechnology to develop a novel cancer cell targeting therapy. Growth factor are autocines, will be overexpressed in varies tumors. We expressed recombinant green fluorescence protein (GFP) and growth hormone receptor binding protein (GHRBP) which could specify recognize growth hormone receptor (GHR) which is located on cell surface of the non-small cell lung cancer cells line, A549. By conjugated with carboxylated nanodiamond (cND) and expressed protein, the cND-GHRBP-GFP complex shows a low toxicity, high biocompatible, specificity, and observable properties. Following recognizing by cND-GHRBP-GFP, A549 were triggered by pulse laser. Results indicate that laser pulsed cND-GHRBP-GFP nano-blast recognized A549 will induces cell surface damage and resulting cell death by apoptosis pathway. According, we have developed a novel strategy for non-small cell lung cancer targeting therapy by combining nanodiamond conjugated hormone receptor binding protein and laser pulse. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) and human epithelial growth factor receptor 2 (HER2) are overexpression in breast cancer cell, but not in normal cells. They are associated with the progression of breast cancer. Therefore, they could be use as cancer markers of antibodies targeting therapy. Anti-CEACAM6 and anti-HER-2 single domain antibodies (sdAb) were cloned from heavy chain only IgG of an immunized llama immune library and fused with Fc fragment. A breast cancer cell line, MCF-7, was used to test the therapy efficiency, and conventional chemotherapy drug, docetaxel, was used as a control of the experiment. Our data indicated that anti-CEACAM6 and anti-HER-2 sdAb could reduce breast cancer cell proliferation and induced tumor cell apoptosis. Western blotting indicated anti-CEACAM6 and anti-HER-2 could induced caspase-9 activation, and PARP cleavage; reduced MCF-7 secreted MMP-2, MMP-9 activity; inhibited MCP-1, ICAM-1, and VCAM-1 expression. In other words, anti-CEACAM6 and anti-HER-2 sdAb could inhibit breast cancer progression by reduce breast cancer cell migration, invasion, monocytes infiltration, and angiogenesis. Therefore, anti-CEACAM6 and anti-HER-2 sdAb are ideal candidates for the breast cancer with CEACAM-6 and HER-2 overexpression. Conclusion, Growth hormone receptor, CEACAM6, and Her-2 are idea target markers for non-small cell lung cancer and breast cancer targeting therapy.