探討epirubicin用於光動力治療 對三陰性乳癌細胞的影響及作用機制

Photodynamic Effects of Epirubicin on Triple Negative Breast Cancer Cells

林弘婷(Hung-Ting Lin)

指導教授 ： 呂鋒洲

中山醫學大學/醫學院/醫學研究所/碩士(2014年)

https://doi.org/10.6834/CSMU.2014.00002

摘要

研究背景：常用於三陰性乳癌（triple-negative breast cancer；TNBC）化學治療的泛艾黴素（epirubicin），同時也是一種對光敏感的藥物。但是，泛艾黴素所累積的治療劑量易導致心臟毒性，使用時需特別注意監控其劑量及心臟功能。光動力療法（photodynamic theraphy；PDT）目前已應用在皮膚癌、口腔癌、大腸癌、膀胱癌等癌症，其原理為利用光敏感藥物受到光刺激而產生的單線態氧分子（1O2）或自由基，會產生對細胞本體或細胞的重要結構造成損傷而使癌細胞死亡。本研究希望利用光動力療法合併化療藥物泛艾黴素可以藉著降低泛艾黴素劑量（減低心臟毒性）而又能同時達到毒殺癌細胞的目的。
材料與方法：分別以不同濃度的泛艾黴素處理三陰性乳癌BT20細胞株後再給予光照治療（Epi-PDT），以細胞存活率分析（MTT Assay）評估細胞毒性，並以雙染法（annexin V-FITC and PI）、乳酸脫氫酶測定法（lactate dehydrogenase；LDH assay）、硫代巴比妥酸反應產物分析法（thiobarbituric acid reactive substances；TBARS assay）、西方點墨法（Western blot），進而探討Epi-PDT所引起細胞死亡的機制。
研究結果：BT-20細胞無論是經過高劑量短時間或低劑量長時間的Epi-PDT處理，細胞存活率皆比只有單純泛艾黴素處理組低。Epi-PDT處理也會增加細胞產生細胞壞死和細胞凋亡的現象，此現象與細胞壞死性凋亡相關的PARP cleavage及細胞核內的凋亡誘導因子（apoptosis-inducing factor；AIF）增加有關。Caspase 8、caspase 9及caspase 3於Epi-PDT處理後也有表現量增加的情形，細胞內ATP含量則有下降的趨勢。TBARS分析顯示於光照後2小時細胞內脂質過氧化（lipid peroxidation）程度明顯上升；beclin-1及LC3BII表現量的增加則證明Epi-PDT也啟動細胞自噬（autophagy）的機制。
結論：實驗結果證實Epi-PDT可以降低泛艾黴素使用的劑量，而Epi-PDT引起乳癌細胞死亡的機制包括細胞凋亡（apoptosis）、細胞自噬及壞死後細胞凋亡（necroptosis）等。目前文獻仍無以光動力療法合併泛艾黴素治療三陰性乳癌的相關研究，希望本研究結果能夠提供臨床上治療三陰性乳癌的參考。

關鍵字

光動力療法 ； 乳癌 ； 泛艾黴素

並列摘要

Background: Epirubicin is a common chemotherapy medicine for treating Triple-negative breast cancer (TNBC). In addition to its photosensitivity, epirubicin can be accumulated to a harmful dosage that may cause serious heart problems because of its cardiotoxicity. Therefore, the dosage and the cardiac function need to be closely monitored during and after the treatment. Photodynamic therapy (PDT) has been used to treat various types of cancer, including skin cancer, oral cancer, colorectal cancer, bladder cancer, etc. PDT is a treatment that uses a photosensitizer and a particular type of light. When photosensitizers are exposed to the light, they produce 1O2 or free radical that destroy the cell body or important structures of the cancer cells which will die as a result .The conducting of the experiment is that epirubicin injection is used in combination with photodynamic therapy in hopes of reducing the dosage of epirubicin and thus cardiotoxicity while killing the cancer cells.
Materials and Methods: On TNBC cell lines BT-20, apply different concentration of epirubicin solution, and then apply photodynamic therapy (Epi-PDT). Next, determine how Epi-PDT can lead to cell death by MTT assay (cell viability analysis), annexin V-FITC and PI staining, lactate dehydrogenase (LDH assay), thiobarbituric acid reactive substances (TBARS) assay, and Western blot.
Results: As for cytotoxicity, whether it’s high doses of epirubicin with short exposure to the light or vice versa, Epi-PDT can lead to much less cell viability comparing to solely using of epirubicin. As a result, the experiment shows Epi-PDT does help to increase epirubicin’s curing of cancer: Annexin V-FITC and PI staining shows that Epi-PDT can result in necrosis and apoptosis of the breast cancer cells. Moreover, the increase of necroptosis-related PARP cleavage and the apoptosis-inducing factor (AIF) in nucleus further proves that Epi-PDT can help activate the necrptoosis of the cells. The increasing activation of caspase 8, 9, and 3 during Epi-PDT also serves as a proof that Epi-PDT help induces apoptosis in cells. In Epi-PDT group, the amount of adenosine triphosphate (ATP) molecules is generally decreasing. The result of lipid peroxidation assay also shows that not only TBARS has significantly increased after two hours of light exposure but also lipid peroxidation happens in cells. Lastly, the increase of beclin-1 and LC3BII protein also indicates that Epi-PDT can help activate autophagy of the cells.
Conclusions: According to the findings from the experiment, the combination of photo-sensitive chemotherapy medicine epirubicin with PDT can help reduce the dosage of epirubicin. Epi-PDT can also help lead to physiological mechanism of breast cancer cell death, such as apoptosis, autophagy, and necroptosis. There is currently no other research on the combination of epirubicin with photodynamic therapy in treating triple-negative breast cancer. The findings in this research could serve as future clinical reference for the treatment of triple-negative breast cancer.

並列關鍵字

Photodynamic therapy ； Breast cancer ； Epirubicin

參考文獻

7.Chang K-J, Kuo W-H and Wang M-Y: The epidemiology of breast cancer in Taiwan. Journal of Oncology Society 24: 85-93, 2008.

36.張金堅: 計畫性的細胞死亡通道－細胞凋亡（apoptosis）、細胞自噬（autophage）與細胞壞死性凋亡（necroptosis）與癌症治療. 當代醫學 666-673, 2013.

1.Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin 61: 69-90, 2011.

2.McCarthy N, Mitchell G, Bilous M, Wilcken N and Lindeman GJ: Triple-negative breast cancer: making the most of a misnomer. Asia Pac J Clin Oncol 8: 145-155, 2012.

3.Simunek T, Sterba M, Popelova O, Adamcova M, Hrdina R and Gersl V: Anthracycline-induced cardiotoxicity: overview of studies examining the roles of oxidative stress and free cellular iron. Pharmacol Rep 61: 156-173, 2009.