氯己定二葡萄糖酸增加五胺基酮戊酸光動力療法對具各治療抗性口腔癌細胞效果影響之研究

The Study on Chlorhexidine Digluconate in Enhancing the Cytotoxic Effect of 5-ALA Photodynamic Therapy on Oral Cancer Cells with Variant Resistance

安明克(Jan Michael Silvosa Antes)

指導教授 ： 陳信銘

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903478

摘要

導論：光動力治療 (Photodynamic Therapy, PDT) 是對口腔癌症前病變和早期口腔癌症的微創療法之一。然而，光動力的缺點之一（其中一個缺點）是由因多次治療而產生具有PDT抗性的癌症細胞，而抗凋亡蛋白Bcl-2的過度表現是造成口腔癌症細胞抵抗光動力療法胞毒性效果的原因之一提供癌症細胞被免受光動力治療的細胞毒性效果 （PDT cytotoxic effect）的保護機制可能來自調控細胞凋亡的Bcl-2 蛋白過度表現。

目標：本研究旨在探討癌症細胞因多次光動力治療而造成Bcl-2蛋白表現增加的抗性結果。利用氯己定二葡萄糖酸鹽(Chlorhexidine digluconate, CHX) 已知的毒性可以抑制Bcl-2蛋白表現，藉此使這些具抗性癌症細胞再次恢復對光動力的敏感效果。因爲在臨床常使用光動力與CHX的組合治療，所以本研究進一步探討CHX對癌症細胞多次使用的影響。

材料與實驗方法：首先將SAS與Ca9-22細胞通過多次暴露半數致死量（LD50）建立具有抗性的細胞，先暴露確立的半數致死量（LD50）CHX 30秒再暴露於光動力治療。接著進行MTT測定細胞的存活率以比較每種處理條件（單獨PDT處理，單獨CHX處理和組合PDT和CHX處理）。利用西方墨點法（Western blot assay）測定CHX暴露前與暴露後（親代與具CHX抗性的細胞）Bcl-2/Bax的表現。在SAS與Ca9-22細胞多次暴露CHX後，使用西方墨點法（Western blot assay）偵測上皮間充質轉換（Epithelial-Mesenchymal Transition, EMT）標記物E-cadherin和Vimentin的蛋白表現，並使用Transwell assay比較親代細胞與多次暴露CHX細胞的遷移能力。

結果：CHX與PDT的組合治療對殺死癌症細胞與具PDT抗性的癌症細胞有增效作用。此機制可能是由於CHX抑制Bcl-2/Bax蛋白交互作用的結果。本實驗也有發現多次暴露CHX 會使SAS產生CHX抗性，而且會誘導細胞產生EMT的現象。然而，雖然SAS由於多次的CHX暴露而發現有EMT的外型變化，細胞的遷移能力卻反而下降。同時儘管經過多次暴露CHX的Ca9-22也有與SAS相同的遷移結果，然而卻沒有觀察到EMT的現象。

結論：本研究結果顯示CHX的優點不僅可以作爲對具有抗性的口腔癌及口腔癌病變的輔助療法，而且還有助於限制病灶和抑制病變轉移的效果。然而，還需要更進一步的研究來闡明CHX誘導的EMT及其對SAS細胞產生的抗性結果。

關鍵字

口腔癌症病變 ； 口腔癌症 ； 光動力治療 ； 氯己定二葡萄糖酸鹽 (CHX) ； Bcl-2 家族蛋白

並列摘要

Introduction: Photodynamic therapy (PDT) is a minimally invasive treatment for oral premalignant lesions and early cancers. However, one of the drawbacks of PDT is the formation of resistant cancer cells from multiple treatments. The mechanism that provides oral cancer cells protection from the cytotoxic effects of PDT could be from the overexpression of the Bcl-2 protein that regulates the apoptosis of the cells.

Objective: This paper demonstrates the acquired resistance of the cells to PDT with subsequent increase of Bcl-2 expression from multiple exposures. The use of Chlorhexidine digluconate (CHX) could alter the Bcl-2 expression resulting in sensitizing these resistant cells again to the effects of PDT. Multiple exposures of CHX to the parental oral cancer cells were also performed to further explore its effects when used in conjunction with multiple treatments of PDT in the clinical setting.

Materials and Methods: Cultured PDT-resistant SAS and Ca9-22 cells were established through multiple LD50 exposures of PDT. These cells were treated with determined LD50 concentration of CHX for 30s and then were treated with PDT. MTT assay was performed to compare the cell viability of each treatment conditions (PDT-alone, CHX-alone, and Combination PDT and CHX). The ratio of Bcl-2/Bax expression was also compared between pre- and post-exposures of CHX on parental and resistant cell lines through Western blot assay. Parental cells of SAS and Ca9-22 were also exposed to multiple CHX treatments. Subsequently, the protein expression of EMT markers E-cadherin and Vimentin were detected through Western blot Assay and migration ability through transwell assay.

Result: It has been shown that the combination of CHX and PDT had a synergistic effect on the killing of cancer cells and their resistant strains. The mechanism involved may be due to the inhibitory interaction of CHX to the Bcl-2 proteins. We also found out that multiple exposures to CHX could also yield resistance to the antiseptic and even induce EMT phenomenon on oral SAS cells. However, although EMT was noted, the migration rate of these post-EMT SAS cells was paradoxically decreased. Likewise, the migration ability was also observed to have decreased in multiple CHX-exposed Ca9-22 cells, though no EMT phenomenon was noted.

Conclusion: The findings show the advantage of CHX that not only it could be used as an adjunct treatment to deal with PDT-resistant oral cancer lesions but also aid in the localization of the lesion and inhibition of any metastatic effect. However, further studies are needed to elucidate the CHX-induced EMT and its consequent resistance on SAS cells.

並列關鍵字

Oral Premalignant Lesions ； Oral Cancer ； Photodynamic Therapy ； Chlorhexidine ； Bcl-2 Family of Proteins

參考文獻

1. Chin D, Boyle GM, Porceddu S, Theile DR, Parsons PG, Coman WB. Head and neck cancer: past, present and future. Expert Rev Anticancer Ther. 2006;6(7):1111-1118.

2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394-424.

3. Vigneswaran N, Williams MD. Epidemiologic trends in head and neck cancer and aids in diagnosis. Oral Maxillofac Surg Clin North Am. 2014;26(2):123-141.

4. Shield KD, Ferlay J, Jemal A, et al. The global incidence of lip, oral cavity, and pharyngeal cancers by subsite in 2012. CA Cancer J Clin. 2017;67(1):51-64.

5. Taipei: Health Promotion Administration, Ministry of Health and Welfare. Health Promotion Administration, Ministry of Health and Welfare Cancer Registry Annual Report, Taiwan Available at: http://health99hpagovtw/educZone/edu_detailaspx?CatId=21937&Type=002&kw=. 2016.