類胡蘿蔔素對倉鼠誘發性口腔腫瘤之預防與作用機制之探討

類胡蘿蔔素對倉鼠誘發性口腔腫瘤之預防與作用機制之探討

 中文摘要

 類胡蘿蔔素是廣泛存在於自然界的一群亮黃色至暗紅色天然脂溶性色素，近年來在許多癌症的預防已被廣泛的研究及探討，但對於口腔癌症預防的角色尚 未廣泛的研究及明瞭，國內有許多病患因嚼食檳榔而導致口腔癌症的發生且逐年增加，因此本論文主要以類胡蘿蔔素為口腔癌症預防之主要對象，並以倉 鼠口腔頰囊（ hamster cheek pouch ）病變為實驗模式，分成四部分探討類胡蘿蔔素對誘發性口腔癌症預防作用與機制之研究。

 實驗一係探討單獨塗抹維生素 E （ α-tocopherol ）和類胡蘿蔔素（ β- 胡蘿蔔素、蕃茄紅素、木質黃素）或混合使用時對口腔癌症之預防效果。結果顯示混 合使用和蕃茄紅素的局部塗抹能降低 7,12-dimethylben[a]-anthracene （ DMBA ）致癌物所誘導出原位癌和鱗狀細胞癌的發生率；若以個體產生之腫瘤負荷

（ tumor burden ）的數量及平均體積（ mm3 ）而言，混合組的預防效果最好，其餘依序為維生素 E 、蕃茄紅素、 β- 胡蘿蔔素、木質黃素等組。

 實驗二進而探討單獨塗抹維生素 E 和類胡蘿蔔素（ β- 胡蘿蔔素、蕃茄紅素、木質黃素）或四種抗氧化劑混合使用時對檳榔嚼塊萃取物誘發之口腔癌症預防 效果。實驗前期 4 週內以 DMBA 為誘癌物，在實驗後期 12 週內則以檳榔嚼塊萃取物（ Betel quid extract, BQE ）和類胡蘿蔔素每日交互塗抹於倉鼠右側口 頰。實驗結果發現病理變化在口腔或食道黏膜上皮病灶皆以混合組最輕微而以對照組較嚴重。經由致癌物 DMBA 的誘導，檳榔嚼塊萃取物的介入確實有 促癌作用的產生，而類胡蘿蔔素的局部塗抹具有抑制檳榔嚼塊萃取物誘發口腔黏膜上皮腫瘤的產生，尤其是蕃茄紅素組和混合組較為顯著。不論在食道及 口腔黏膜上皮的腫瘤數目和腫瘤負荷，皆是以對照組最為嚴重，而類胡蘿蔔素局部塗抹能顯著抑制食道及口腔黏膜上皮腫瘤數目和腫瘤負荷的產生，其中 以混合組抑制效果最為顯著。

 實驗三主要以類胡蘿蔔素的攝取對檳榔萃取物誘發倉鼠口腔癌症預防效果為主要目的。結果顯示實驗組血漿及肝臟各組類胡蘿蔔素濃度皆顯著上升。蕃茄 紅素組和混合組紅血球超氧化物歧化酉每（ SOD ）活性顯著低於對照組（ p<0.05 ），木質黃素組、 4,4’- 二酮 -β- 胡蘿蔔素組和混合組肝臟麩胱甘月太過 氧化酉每（ GSH-Px ）活性顯著低於對照組；在脂質過氧化產物（ malondialdehyde, MDA ）方面，混合組血漿 MDA 濃度顯著小於對照組（ p<0.05 ），而 肝臟 MDA 濃度則為類胡蘿蔔素各組皆小於對照組（ p<0.05 ）。在口腔癌症發生率方面，混合組和蕃茄紅素組乳突瘤發生率顯著低於對照組，其次為木質 黃素組、 β- 胡蘿蔔素組和 4,4’- 二酮 -β- 胡蘿蔔素組，而在鱗狀細胞癌的發生率方面，類胡蘿蔔素各組發生率低於對照組，混合組、蕃茄紅素組、木質黃素 組和 4,4’- 二酮 -β- 胡蘿蔔素組沒有鱗狀細胞癌的產生，而只有 β- 胡蘿蔔素組有鱗狀細胞癌的產生。以口腔腫瘤數目及腫瘤負荷（ tumor burden ）之觀點評 估類胡蘿蔔素的預防效果，蕃茄紅素、 4,4’- 二酮 -β- 胡蘿蔔素和混合類胡蘿蔔素有較佳的抑制效果。

 實驗四探討藉由分子細胞層級中增殖細胞核抗原（ proliferating cell nuclear antigen, PCNA ）和角蛋白 cytokeratin （ Pan, AE1 and CK14 ）的表現來探討檳 榔嚼塊萃取物誘導口腔癌症過程中類胡蘿蔔素預防的效力。結果顯示實驗各組細胞增殖、細胞異常化生、乳突瘤和惡性腫瘤病灶 Pan 及 AE1 染色方面皆很 顯著，實驗各組並沒有顯著差異；在 PCNA 方面，在口腔黏膜上皮增生方面，木質黃素組、蕃茄紅素組、混合組和維生素 E 組具有抑制 PCNA 的表現；在 黏膜上皮異常化生方面，類胡蘿蔔素和維生素 E 各組均具有抑制 PCNA 的表現；在乳突瘤方面， β- 胡蘿蔔素組、混合組和維生素 E 組具有抑制 PCNA 的 表現；在惡性腫瘤方面，蕃茄紅素和混合組並無惡性腫瘤的產生，就其他各組而言，維生素 E 和 β- 胡蘿蔔素組也具有抑制 PCNA 的表現。在 CK14 方面

，在口腔黏膜上皮增生方面，木質黃素組、蕃茄紅素組和維生素 E 組與對照組比較具有抑制 CK14 的表現；在乳突瘤方面，類胡蘿蔔素和維生素 E 各組具 有抑制 CK14 的表現；在黏膜上皮異常化生方面，木質黃素組、蕃茄紅素組、混合組和維生素 E 組均具有抑制 CK14 的表現；在惡性腫瘤方面，蕃茄紅素 和混合組則無惡性腫瘤的產生，就其他各組而言，維生素 E 和 β- 胡蘿蔔素組也具有抑制 CK14 的表現。

 綜觀本論文研究結果得知，（ 1 ）類胡蘿蔔素對致癌物 DMBA 口腔癌症的發生皆有明顯的抑制效果，混合投與則有協同抑癌作用；而不同類胡蘿蔔對抑 制誘發腫瘤負荷的數量和平均體積（ mm3 ）之能力略有差異。（ 2 ）在檳榔嚼塊萃取物介入誘發癌症的過程中類胡蘿蔔素能顯著抑制口腔癌症的發生，

而混合投與也具有協同抑癌的功效。（ 3 ）攝取類胡蘿蔔素能降低誘發性口腔癌症倉鼠體內脂質過氧化產物 MDA 的濃度，特別是蕃茄紅素、 4,4’- 二酮 - β- 胡蘿蔔素和混合類胡蘿蔔素，而蕃茄紅素、 4,4’- 二酮 -β- 胡蘿蔔素和混合類胡蘿蔔素有較佳的抑制口腔腫瘤數目及腫瘤負荷效果。（ 4 ）類胡蘿蔔素能 預防口腔癌症的發生，在細胞增殖、細胞異常化生、乳突瘤和惡性腫瘤病灶中 PCNA 和 CK14 的表現降低，而不同的類胡蘿蔔素之間預防癌症的機制可能 不盡相同。

The Preventive Effect and Mechanism of Carotenoids on Induced Oral Tumorgenesis in Hamster

 英文摘要

 The incidence of oral cancer is highly associated with betel quid chewing in Taiwan. Previous studies have shown that betel quid represents a promoter role during the carcinogenesis of h amster buccal pouch carcinoma. The thesis was divided with four studies for evaluate the preventive effect and mechanism of carotenoids on induced hamster oral cancer.

 This first study was designed to evaluate the DMBA （ 9,10-Dimethyl- 1,2-benz[a]anthracene ） induced oral cancer prevention ability and safety of single or combined carotenoids, incl uding β-carotene, lycopene, lutein, and a-tocopherol. Over a 14-week experimental period, the mixture group presented the most effective oral cancer prevention. The other carotenoid gr oups presented inconsistent results for oral carcinoma prevention. According to the inhibitory number and average volume （ mm3 ） of tumors, the results indicated that the mixture gro up represented the most effective prevention, followed by a-tocopherol, lycopene, β-carotene, and lutein. The four different antioxidants （ carotenoids & vitamin E ） significantly inhib ited the incidence of oral cancer when administrated alone as well in mixtures of antioxidants.

 The second study was to investigate the preventive effects of carotenoids on betel quid extracts （ BQE ） induced hamster oral cancer. The results indicated the buccal or esophageal his tological symptom of the mixture group is the less severe compared with the controlled group. Carotenoids present effectively inhibition on the development of oral mucosal malignant tu mor, especially in the lycopene and mixture group. From the carotenoid preventive viewpoint, the tumor number and burden of the esophagus and the buccal mucosa are the most severe i n the controlled group, the application of carotenoids could significantly inhibit the development of both esophageal and buccal tumor number and burden, especially in the mixture grou p.

 The third study was to investigate the effects of various carotenoids consumption on oral carcinogenesis and activity of antioxidative enzymes in male hamsters. The results indicated the plasma and liver carotenoid level of experimental groups were significantly higher than the control group （ p<0.05 ） . The red blood cell （ RBC ） superoxide dismutase （ SOD ） activity of the lycopene and mixture group were significantly lower than the control group, so did the liver SOD activity of the canthaxanthin and mixture group. The liver glutathione per oxidase （ GPx ） activity of the lutein, canthaxanthin and mixture group were significantly lower than the control group, however there was no difference between the activity of RBC GPx in each group. The plasma malnodialdehyde （ MDA ） level in the mixture group was significantly lower than the control group （ p<0.05 ） , furthermore, the hepatic MDA level of the carotenoid treated groups were all significantly lower than the control group （ p<0.05 ） . In the aspect of oral cancer incidence, the papilloma incidence of the mixture group and lycopene group were significantly lower than the control group, and the others were as follow: the lutein group, theβ-carotene group and the canthaxanthin group. The squamous cell carc inoma （ SCC ） incidence of each carotenoid groups were significantly lower than the control group, but there were no SCC incidence production in the mixture group, the lycopene gr oup, the lutein group and the canthaxanthin group. The number and volume of tumor burden of the experimental groups were significantly lower than the control group. The consumption of carotenoids could provide the inhibitory capability on the BQE induced hamster oral carcinogenesis.

 The forth study investigates the expression of different CK （ Pan, AE1, CK14 ） and PCNA during the process of carotenoids chemopreventive effect on betel quid extract （ BQE ） induced hamster oral cancer. The results indicated all groups present significantly expression of Pan and AE1 and there were no differences between each group, but not in the CK14 and PCNA expression. In the hyperplasia lesions, the application of lutein, lycopene, mixture and vitamin E could significantly inhibit the PCNA expression, and the same as the CK14 expre ssion of the lycopene and vitamin E. In the papilloma lesions, the application of the β-carotene, mixture and vitamin E could significantly inhibit the PCNA expression ， and the same as the CK14 expression of all the carotenoid and vitamin E. In the dysplasia lesions, the application of the carotenoid and vitamin E could significantly inhibit the PCNA and CK14 expressi on, but in the carcinoma lesion there were the β-carotene and vitamin E application expressing the inhibiting effect of the PCNA expression and only the vitamin E application expressing the inhibiting effect of the CK14 expression. There were no carcinoma production in the lycopene and mixture groups. These findings suggest that carotenoids may prevent the carcinoge nesis of BQE indued hamster oral cancer by act as suppressor inhibiting the expression of PCNA and CK14.

 The results of the thesis presents as follow: （ 1 ） The ability of different carotenoids to inhibit the amount and average volume of the induced tumors was different. This may be becaus e the carotenoids and a-tocopherol inhibited different developmental stages of cancer. （ 2 ） The carotenoids significantly inhibit the incidence of oral cancer while administrating alone as well as the mixture of antioxidants produced a significant synergistic chemoprevention of oral cancer. （ 3 ） The consumption of carotenoids could significantly decline the plasma a nd liver MDA levels and decreased the tumor burden on BQE induced hamster, especially in lycopene, canthaxanthin and mixture carotenoids. （ 4 ） The carotenoids may prevent the c arcinogenesis of BQE indued hamster oral cancer by act as suppressor inhibiting the expression of PCNA and CK14. By the way, the anti-cancer mechanism of different carotenoids may be not the same pathway.