NAD(P)H-Quinone Oxidoreductase 1參與人類乳癌細胞對放射線的反應

The involvement of NAD(P)H-Quinone Oxidoreductase 1 in radiation response of human breast cancer cells

張慕亞(Mu-Ya Chang)

指導教授 ： 張文瑋

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2018年)

摘要

乳癌在全球婦女當中為常見的癌症之一，而放射治療常作為乳癌的療法，但臨床上常見放射線治療後復發的乳癌患者，因此了解乳癌產生放射線抗性的原因有助於發展放射線治療的輔助療法。NAD(P)H-Quinone Oxidoreductase 1 (NQO1)是一種含有黃素腺嘌呤二核苷酸(FAD)的醌還原酶，能夠催化大部分的醌進行雙電子還原，在正常組織中具有提供細胞保護和預防化學物毒害的作用。在許多發炎的環境和腫瘤組織中NQO1也會有大量活性的表現，例如乳癌、肺癌和胃癌。在本研究中，我們利用MDA-MB-231人類乳癌細胞株(簡稱231-P)來建立放射線抗性的亞細胞株(簡稱231-RR)，並利用這兩株細胞株分析NQO1在乳癌細胞產生放射線抗性的角色。我們首先發現NQO1的蛋白質表現在231-RR中較231-P細胞高40倍，並且NQO1的活性同樣在231-RR中較231-P細胞呈現增加的現象。我們進一步的利用NQO1生物活性藥物，beta-Lapachone，比較231-P和231-RR細胞對於beta-Lapachone的敏感性，證明231-RR細胞對beta-Lapachone生長抑制的敏感性較231-P細胞高。而以NQO1抑制劑-Dicoumarol處理231-RR細胞，則能抑制其生長能力。接著我們將細胞處理抑制NQO1或NQO1生物活性化合物的處理並照射放射線，我們發現透過抑制NQO1或NQO1生物活性化合物的處理，231-RR細胞對放射線的敏感性明顯增加，這表明抑制NQO1或NQO1生物活性化合物的處理能夠作為上這治療的輔佐療法。然而我們在探討其上游調控路徑時，發現Notch1訊號路徑與AP-1訊號路徑並不會影響NQO1的表現。這些結果說明NQO1的蛋白表現及活性的增加，可能是乳癌細胞產生放射線抗性的原因之一，並且NQO1的表現可能作為乳癌患者是否在放射線治療後產生復發的潛力預測指標。但乳癌細胞中NQO1如何受到放射線調控的分子訊號途逕進而使細胞產生放射線抗性的方法仍不清楚，是未來必須繼續探討的。

關鍵字

NQO1 ； Nrf2 ； Notch1 ； AP-1 ； 氧化壓力 ； Dicoumarol ； beta-Lapachone ； 放射治療

並列摘要

Breast cancer is one of the common cancer in women worldwide. In recent years, the radiation therapy was used as a common therapy in breast cancer and often combined with other treatments. The NAD(P)H-Quinone Oxidoreductase 1 (NQO1) is an FAD containing quinone reductase that catalyzes the 2-electron reduction of a broad range of quinones to provide cytoprotection and chemoprevention in normal tissue. NQO1 also presents high-level activity in many inflammatory conditions and in the tumorous tissues, such as breast, lung, and gastric cancers. In the present study, our laboratory used the MDA-MB-231 human breast cancer cell line (called 231-P) to establish a radioresistant subline (called 231-RR) and to examine the NQO1 expression in breast cancer cells after requiring radioresistant feature. We first confirmed that the NQO1 protein expression was elevated in 231-RR then that in 231-P cells to 40 fold. In addition, the NQO1 activity was also increased in 231-RR cells then 231-P cells. We further used beta-Lapachone, which is bioactivated by NQO1 to become cytotoxic, to compare the sensitivity between 231-P and 231-RR cells and results indicated that 231-RR was more sensitive to growth inhibition by beta-Lapachone then 231-P cells. With the treatment of dicoumarol, an NQO1 inhibitor, the inhibition of NQO1 activity suppressed cell growth of 231-RR cells. We using dicoumarol or beta-Lapachone to treated cells followed by radiation exposure and found that the radiosensitivity of 231-RR were obviously elevated by pre-treatment these two compounds. These results indicated that NQO1 inhibitors or NQO1 bioactive compounds could serve as an adjuvant therapy for cancer radiation therapy. When investigated the underlying molecular pathway of NQO1 upregulation in radioresisant breast cancer cells, we found that inhibition of Notch or JNK activity by specific inhibitors did not influence the expression of NQO1 protein. Taken together, our results indicate that NQO1 is a potential target for treatment of breast cancer patients who encounter with radiation therapy failure. In the future, it is necessary to examine the underlying molecular mechanism in the upregulation of NQO1 in radioresistant breast cancer cells.

並列關鍵字

NQO1 ； Nrf2 ； Notch1 ； AP-1 ； Oxidative stress ； Dicoumarol ； beta-lapachone ； Radio Therapy

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