| Abstract: | 近年來，自從三氧化二砷被證實對急性前骨髓性白血病具有療效後，也已經被應用到許多癌症如慢性骨髓白血病、惡性淋巴瘤、皮膚癌及子宮頸癌等的治療試驗上，甚至有部分實驗已進入臨床試驗階段。但是因限於對砷化合物毒性之考量以及較長期低劑量暴露之風險，尚有許多問題亟待解決。此外，有關於腐植酸與慢性疾病如糖尿病與癌症之關係亦有更深入之探討，發現腐植酸和砷單獨或共同暴露皆會增加HIT-T15細胞株之自由基與氧化性壓力傷害並誘發細胞毒性。低劑量(≦100μg/ml)腐植酸在細胞實驗中也被發現會誘導JB6鼷鼠皮膚細胞癌化及促進A549肺癌細胞之惡化(progress)、轉移(migration)與侵襲(invasion)能力。
子宮頸癌症是目前常見的婦女癌症。近年來，醫學上已經證實人類乳突病毒是造成子宮頸癌症的元兇。人類乳突病毒有約200種亞型，其中第16及18亞型和子宮頸細胞癌前病變與子宮頸癌症有著密切關係。三氧化二砷可以顯著的抑制趨化因子CXCR4 的表現，此種抑制作用在不同腫瘤的細胞皆可產生作用，包括HeLa、SiHa（子宮頸癌細胞株），因此三氧化二砷有可能可作為現代抗癌技術的輔助性藥物。
腐植酸和三氧化二砷在不同濃度之藥物會啟動不同之細胞分子作用機制，導致不同之細胞作用。因此，本篇研究將腐植酸與三氧化二砷依高低不同濃度區塊分開探討其對子宮頸癌細胞之生物效應及作用機制。其中，第一部分為三氧化二砷1μM~25μM 以及腐植酸100μg/mL~1000μg/mL之濃度範圍探討其對子宮頸癌細胞之毒性作用與機制探討。結果顯示將腐植酸及三氧化二砷共同暴露，可以明顯增強砷的細胞毒性使砷在較低劑量暴露時即達到較強之細胞毒殺效果。而其作用機制主要是過增加細胞內的過氧化物與自由基，進而誘發細胞凋亡途徑，抑制子宮頸癌細胞之生長。所以，在ㄧ定濃度範圍內，腐植酸與三氧化二砷的組合可以作為開發以三氧化二砷為主體之化療組成藥物之重要參考。這一部分實驗證明了腐植酸可以增強三氧化二砷對子宮頸癌細胞的毒殺作用，同時也降低其使用的劑量，對未來臨床上使用三氧化二砷作為抗癌藥時對的重要參考，不僅可提高藥物之作用效果，也能降低後續殘留毒性對正常組織器官之影響。
第二部份為觀察低於1μM三氧化二砷以及100μg/mL腐植酸對子宮頸癌細胞在體外及體內(NOD/SCID小鼠腫瘤生長模式)之促進生長作用及其機制探討。 建立低劑量腐植酸與三氧化二砷暴露之動物試驗模式，以SCID老鼠為子宮頸癌細胞株-SiHa之生長載體，探討其生長速率?氧化性壓力?細胞轉移?蛋白質表現與酵素活性等。結果顯示低劑量之腐植酸(≦100μg/mL)及三氧化二砷的暴露會促使二株癌細胞的轉移能力增加，值得注意的是單獨暴露的增強效果更明顯，同時加藥暴露組比對照組要明顯或相近。在體內試驗模式下，低濃度之腐植酸及三氧化二砷亦有顯著之促癌作用而且與增加金屬基質蛋白?及血管新生因子之表現以及誘發低強度之氧化性壓力影響等有關。
本研究嘗試將兼具藥物與環境毒性物質之腐植酸與三氧化二砷，依不同劑量範圍分別探討其藥用機制與毒性影響。高劑量範圍之藥物暴露，可經由較強之氧化性壓力之誘發而活化細胞凋亡機制，產生癌細胞之毒殺效應。 較低劑量範圍之藥物暴露，在體外試驗能誘發細胞增生與轉移能力增加，而在體內試驗能促使腫瘤生長，且與VEGF-A、MMPs之蛋白質表現增加有關。
Based on the studies of Chinese clinicians in 1971, As2O3 was recognized as an ideal anticancer drug for treating acute promyelocytic leukemia (APL), and these results renewed interest in this metalloid for treating various forms of cancers. Recent studies have demonstrated As2O3 has therapeutic effects on cervical cancer by promoting apoptosis and inhibiting metastasis in vitro and in vivo.
Exposure to Humic acid (HA) damages tissues and cells, and is involved in goiter and cancer and has been implicated as a causal factor of Blackfoot disease. HA has been shown to generate ROS, such as superoxide anion, and deplete glutathione and several antioxidant enzymes. HA has been utilized in traditional Chinese medicine and possesses various pharmacologic properties, including anti-inflammatory, anti-hypertensive, anti-neoplastic, and hemostatic activities, and HA displays anti-proliferative effects by inducing apoptosis. In addition, low concentration HA could promoted neoplastic transformation of mouse epidermal JB6 cells and enhanced the progression of lung cancer A549 cells in vitro.
Cervical cancer is the second leading cancer affecting women, and recent studies have demonstrated arsenic trioxide (As2O3) has therapeutic effects on cervical cancer by promoting apoptosis and inhibiting metastasis in vitro and in vivo. Humic acid (HA) possesses various pharmacologic properties, including anti-inflammatory, anti-neoplastic, and anti-proliferative effects by inducing apoptosis. We examined the growth inhibition properties and the combined effects of HA and As2O3 in human cervical adenocarcinoma cell lines. Our results shown both higher concentration of As2O3 and HA induced inhibition of cell growth, most likely by ROS-mediated cell damage and activation of the apoptosis pathway, and HA enhanced the antiproliferative action of As2O3 in HeLa and SiHa cells. This study is relevant to the development of chemotherapeutic approaches using As2O3 in treating human cervical cancer. On the other hand, pretreated lower concentrations of HA, As2O3, or both of them could significantly enhance the ability of cell migration on SiHa and HeLa cells, and lower concentrations of HA or As2O3 alone exposure could promote cell proliferation of both cell lines. Pretreatment of HA and As2O3 or both of them could also enhanced the tumor growth and there were significant differences between exposure groups and control, which were correlated with the protein expression of MMPs and VEGF-A in the tumor. |
