All trans-RETINOIC ACID 對於鼻咽癌細胞株之生長控制影響以及合併化療藥物治療之效用

Effect of All trans-RETINOIC ACID on the growth of nasopharyngeal cancer cell lines and treatment potential in combined use with cisplatin

洪士涵(Shih-Han Hung)

指導教授 ： 李飛鵬

共同指導教授 ： 吳瑞裕

臺北醫學大學/醫學院/臨床醫學研究所/碩士(2009年)

https://doi.org/10.6831/TMU.2009.00051

摘要

目的:
Retinoids是一群存在於自然界或是人工合成的維生素A的類似物，其中包括了retinol、 isotretinoin、 tretinoin(ATRA)、 retinylpalmitate、 Fenretinide(4-HPR)、 etretinate， arotinoids等等。 此類化合物在許多的動物實驗上，對於許多上皮組織 (包括口腔， 皮膚， 膀胱， 攝護腺和乳腺) 表現出明顯抑制癌化的效果。 近年來各種維他命A的代謝衍生物更被運用到其他類型的癌症上試圖提供更好的腫瘤治療效果。
將ATRA運用在頭頸部鱗狀細胞癌不論是在細胞株的試驗或是動物實驗上已經得到具體的成果，進一步的人體試驗同時也在不同的國家進行當中。 甚至於在一些國家，對於頭頸部鱗狀細胞癌症的治療已經將ATRA納入了原本的化療配方組合之中。 然而，同屬於源自上皮細胞組織的鼻咽癌，卻仍然缺少對於這類促細胞分化藥物的反應研究。
本實驗計畫希望透過ATRA合併使用現有治療鼻咽癌化療藥物Cisplatin，以鼻咽癌細胞株作為實驗材料，試圖找出ATRA對於鼻咽癌細胞的影響，以及合併使用Cisplatin是否具有加成的治療效果。

方法:
實驗材料為兩株本土鼻咽癌細胞株 NPC-TW01以及 NPC-TW04。 各別於不同濃度之ATRA培養下，在光學顯微鏡下觀察細胞型態學的變化，並測量個別之生長曲線。 最後將細胞株分別培養於混有不同濃度的cisplatin、ATRA、以及兩者藥物合併的培養液中，選定6天之生長期，以MTT assay光學方法測光密度OD值來估算細胞的數目。

結果:
NPC-TW01細胞株在單獨使用Cisplatin 0.5μmol/L 開始產生細胞生長抑制，並且隨著加入藥物的濃度增加而有加強。 單獨使用ATRA 1μmol/L生長開始產生抑制，然而對於細胞生長的抑制作用並未能隨著ATRA濃度的增加而加強。 而NPC-TW04細胞株則對個別使用Cisplatin以及ATRA的反應相較之下明顯變差。
在合併使用的效用上，兩者皆有著顯著的加成效果。 NPC-TW01在ATRA在合併較低濃度的cisplatin之下，對細胞生長抑制的作用與單獨使用ATRA並沒有差異。 然而當cisplatin的濃度達到5μM的時候，合併ATRA 1μM比起對照組可以造成吸光值平均下降至23.7%。 比起單獨使用cisplatin 5μM 以及單獨使用ATRA 1μM，細胞抑制的差異皆可達到統計上的意義。 NPCTW04的反應也有類似的結果，當cisplatin的濃度提升到1 μM以上的時候，加入原本合併使用時反應不甚明顯的最低濃度的ATRA 1μM，就可以達到大幅下降吸光值的效果。

結論:
ATRA對於本實驗中的兩株鼻咽癌細胞株皆有抑制生長的作用，此外當合併使用cisplatin時可以於低濃度就達到良好的加成效果，顯示出ATRA作為鼻咽癌合併治療藥物的潛力。 但是由於在合併cisplatin以及ATRA治療下，腫瘤的抑制加成效果須依賴一定的cisplaitn濃度，而且並未能隨同ATRA的濃度增加而更加明顯。 因此除了同時使用cisplain與ATRA預期可以達到相當好的鼻咽癌合併治療效果之外，若能加入經由不同機轉作用的抗癌藥物，應能進一步再提高療效。

關鍵字

維他命A ； 視黃質 ； 鼻咽癌

並列摘要

Objectives:
Retinoids are a group of naturally existing or synthetic vitamin A derivatives including retinal, isotretinoin, tretinoin(ATRA), retinylpalmitate, Fenretinide (4-HPR), etretinate, arotinoids. In many animal models, these compounds have shown significant anti-neoplastic effect on many epithelial tissues, and more recently they have been applied to other types of cancer in order to obtain better therapeutic effects.
Previous research have already achieve remarkable results in the application of ATRA on human head and neck squamous cell carcinoma using cell line and animal models. In some countries, ATRA was included as a combined chemotherapy regimen. However, related researches about the nasopharyngeal cancer, which are also originated from the epithelial tissue, were still unclear.
The purpose of this study is to understand the effect of ATRA on the nasopharyngeal cancer cell line and evaluate the additional effect when used together with Cisplatin which is currently a standard chemotherapeutic agent for nasopharyngeal carcinoma.

Materials and Methods:
Two NPC cell lines NPC-TW01 and NPC-TW04 were used and observed under optical microscope for morphological changes after treating with different concentrations of ATRA. Growth curves were also determined individually. Finally after six days culturing with various concentration of ATRA alone, cisplatin alone, and combination of both, viable cell numbers were estimated by OD value using MTT assay.

Results:
Cell growth inhibition can be observed on NPC-TW01 starting with Cisplatin 0.5μmol/L, and this effect was further enhanced by increased cisplatin concentration. Growth inhibition also started with ATRA 1μmol/L treated alone, but unlike cisplain, was not enhanced by increased ATRA concentration. Both cisplatin and ATRA showed decreased inhibitory effect on NPC-TW04.
Both cell lines showed remarkable growth inhibition under combined used of cisplatin and ATRA. Under low concentrations of cisplatin, NPC-TW01 cell growth showed no differences between combined use and using ATRA alone. However, when the concentration of cisplatin increased to 5μM, combined use with ATRA 1μM results in decrease in OD value to 23.7% of control group, which is statistically significant comparing to treating with cisplatin and ATRA alone. Similar result was also found on NPC-TW04. When cisplatin level was raised above 1 μM, combined use with low concentration of ATRA showed remarkable decrease in measured OD value.

Conclusion:
Both cell lines used in this study respond to ATRA and results in cell growth inhibition. Low concentration of ATRA is required for synergy effect while combining with cisplatin, implying the potential use for combination therapy regimen. However, the increased cytotoxicity is cisplatin concentration dependent, and also, not enhanced by further increase of ATRA concentration. Therefore, we expect quite effective nasopharyngeal cancer cell control using ATRA and cisplatin together, and possibly even better control effect if other anti-cancer agents using different mechanisms were added together.

並列關鍵字

Vitamin A ； Retinoids ； nasopharyngeal cancer

參考文獻

4. 朱水松，柯政郁，徐茂銘 預防癌症的生力軍-類視黃質(retinoid)

1. Alan G. Kerr, Scott-Brown`s Otolaryngology, 6th edition volume 5 chapter 13.

5. Blomhoff R, Green MH, Berg T & Norum KR. Transport and storage of vitamin A. Science 250: 399-404 (1990).

7. Miller WH, Jr. The emerging role of retinoids and retinoic acid metabolism blocking agents in the treatment of cancer. Cancer 83:1471-1482 (1998).

8. Sporn MB, Roberts AB & DeWitt SG. The retinoids: biology, chemistry and medicine. 2nd edition. Raven Press, New York (1994).