當歸黃耆對非小細胞肺癌的腫瘤增生抑制作用及免疫調控效應

Effectiveness of Angelica and Astragliin proliferation inhibition and immunoregulation on non-small cell lung cancer

顏慈怡(Chi-Yi Yen)

指導教授 ： 梁有志

共同指導教授 ： 朱彥

臺北醫學大學/醫學院/醫學科學研究所/碩士(2010年)

摘要

中文摘要

背景
肺癌是世界上常見的惡性腫瘤。在臺灣，肺癌更是男性排行第一大與女性第二大癌症死亡病因。早期非小細胞肺癌的五年存活率有60%; 但是在臺灣，有高達70%的肺癌病患為不可開刀之局部擴散 (IIIB期) 或遠處轉移病患 (IV期)，而這些已擴散或遠處轉移的肺癌病患的平均存活期約只有六至九個月，其5年存活率也只有15%。雖然外科手術與輔助性療法在肺癌的各分期都有詳盡的研究，但治療的結果仍然明顯令人失望。
針對肺癌研發之新一代抗癌化學藥物或是標靶治療藥物雖可有意義地延長平均存活期，但其臨床上所增加的平均存活時間不過是有限的一至三個月。大部分肺癌病患於治療過程中多有可能盲目投醫，在醫療花費、生活品質及病人心情方面，都造成負面的影響。
近年來，我們發現許多肺癌病人在接受外科手術與輔助性治療時，多曾嘗試使用中藥相關的另類療法，但相關的研究報告卻非常少。所以，我們開始對中草藥於肺癌病人的潛在效應感到興趣。
當歸與黃耆在台灣是非常普遍使用的補藥。臨床上常見抗癌的補中益氣湯也是以當歸黃耆為主成分的方劑。許多研究已指出當歸與黃耆在一些癌症上具有抗腫瘤效應並可提升免疫力的功用，但在肺癌相關的研究，結果卻非常有限。

目的
本研究目的是以 (1)人類肺癌細胞模式(human lung cancer cell model)，(2)人類肺腺癌細胞免疫化裸鼠之轉殖動物模式(xenotransplantation model)及(3)黃耆複方-補中益氣湯之人體試驗模式(clinical study)探討當歸黃耆的可能抗癌效應。

材料和方法
我們以人類肺癌細胞模式和人類肺腺癌細胞免疫化裸鼠之轉殖動物模式，利用MTT assay、Western blot、IHC、ELISA等實驗方法來評估當歸和黃耆是否會對人類肺癌細胞有腫瘤毒殺作用，並檢測腫瘤血管新生相關分子及凋亡蛋白的表現量。同時，我們也收集非小細胞肺癌病患的血液檢體，評估黃耆複方-補中益氣湯對肺癌病患的Th1及Th2細胞所分泌的IL-10和IFN-gamma與免疫調控功能之間的相關性。

結果
當歸與黃耆對肺癌細胞株A431，A549，及實驗室從肺癌病人身上所培養的細胞株(Luca)均具有抑制腫瘤細胞增生效應。在人類肺腺癌細胞免疫化裸鼠之轉殖動物模式結果也發現，合併餵飲當歸和黃耆則可顯著增加肺癌腫瘤組織的凋亡現象，同時並不會增生與腫瘤轉移相關的血管新生。在臨床實驗也發現非小細胞肺癌病患在手術後服用黃耆複方－補中益氣湯有調理或復原免疫機能(Th1/Th2 switching)的可能趨勢。同時服用黃耆複方－補中益氣湯後的第24個月，肺癌病患的血清中sIL-2R值會顯著下降。

重要性
我們希望藉此研究來評估當歸與黃耆在肺癌細胞模式和肺癌動物模式下可能的細胞毒殺效應，並進一步評估黃耆複方─補中益氣湯對肺癌病患的免疫調控作用，以奠定未來系統性研究中草藥複方的研究基礎，增加臨床醫師及肺癌病患對使用中草藥的信心。

關鍵字

肺癌 ； 當歸和黃耆

並列摘要

Abstract

Backgroud
Lung cancer is the commonest malignant tumor in the world. The five-year survival rate of early stage, operable non-small cell lung cancer (NSCLC) patients was 60%, but there were approximately 70% of patients with NSCLC presenting with advanced disease (stage IIIB with pleural effusion or stage IV) at the time of diagnosis in Taiwan. However, the prognosis for patients with advanced NSCLC remains extremely poor with a median survival time of 6-9 months and a 5-year survival rate of 15%. Although surgical and neoadjuvant therapies in each stages of lung cancer are under investigation and some progress has been achieved in the management of local and advanced lung cancer, treatment outcomes for lung cancer are still to be considered disappoint. New generations of chemotherapy or newly developed target therapy can significantly prolong the medium survival in statistics but the range is only 1 to 3 months, which is of limited significance in clinical practice, suggesting a limitation of current modalities of treatment.
The majority of lung cancer patients experienced metastasis and optimization of futile therapies impact negatively on cost, quality of life, and patient morale. In recent years, we found that many lung cancer patients receiving surgery and adjuvant therapy have tried to use Traditional Chinese Medicine (TCM). However, the uncertain effectiveness of TCM in cancer treatment still hesitating both physicians and patients and the related reports of TCM in lung cancer research are extremely limited.
Angelica and Astragali (A & A) is the most common tonic used in Taiwanese, especially in patients with cancer. Astragalus-based herb formula composed of A & A commonly in clinical use. A & A has been reported to possess immunostimulation and antitumor effects for cancer subjects, however, these results in lung neoplasm are extremely limited.

Purpose
We aimed to evaluate the potential proliferation inhibition and immunological effectiveness of A & A on 1) human lung tumor cell lines, 2) human lung adenocarcinoma-immunized nude mice and 3) clinical studies of Astragali-based herb formula in patients with NSCLC.

Materials and Methods
The methodologies of MTT assay, Western blot, IHC, and ELISA were to evaluate the potential effectiveness of A & A on 1) proliferation inhibition, 2) tumor apoptosis, 3) tumor neo-angiogenesis, and 4) cytokine expressions, respectively. In the period of 2-year clinical studies, patients with NSCLC continuously administrated with Astragali-based herb formula and the levels of Th1- and Th2- related cytokines were carefully evaluated in every 6 months.

Results
In vitro studies showed A & A could induce significant proliferation inhibition in two different lung tumor cell lines (i.e., A431 and A549), including lab-established patient derived lung adenocarcinoma cell line (Luca). In xenotransplantation mice model, increased apoptosis in tumor tissue was found when administrated A & A for 10 weeks, and neo-angiogenesis was not seen. In clinical studies, the levels of IL-10 were not significantly increased in patients with NSCLC who continuously received Astragli-based herb formula for two years. Furthermore, the levels of INF-gamma were significantly increased after 2-year administration. In addition, the levels of serum soluble IL-2 receptor were decreased.

Significance
This cross sectional study of angelica and astragali could high light the effectiveness of potential herb medicine in anti-tumor treatment. Explorations of the Astragalus-based herb formula in modulating tumor cell proliferation and reversing immunosuppression in patients with NSCLC is undergoing. Further studies warrant the prognostic effectiveness of Astragli-based herb formula in patient with NSCLC. We sincerely hope the basis of our comprehensive research in herb medicines could benefit not only clinical physicians but cancer patients as well.

並列關鍵字

lung cancer ； Angelica and Astragli

參考文獻

Abul KA, Kenneth MM, Alan S. Functional diversity of helper T lymphocytes. Nature 1996; 383: 787-93.

Aoki Y, Tsuneki I, Sasaki M, Watanabe M, Sato T, Aida H, Tanaka K. Analysis of Th1 and Th2 Cells by Intracellular Cytokine Detection with Flow Cytometry in Patients with Ovarian Cancer. Gynecol Obstet Invest. 2000; 50: 207-11.

Bremnes RM, Camps C, Sirera R. Angiogenesis in non-small cell lung cancer: The prognostic impact of neoangiogenesis and the cytokines VEGF and bFGF in tumours and blood. Lung Cancer 2006; 51: 143-58.

Cao W, Li XQ, Wang X, Fan HT, Zhang XN, Hou Y, Liu SB, Mei QB. A novel polysaccharide, isolated from Angelica sinensis (Oliv.) Diels induces the apoptosis of cervical cancer HeLa cells through an intrinsic apoptotic pathway, Phytomedicine 2010; 17: 598-605.

Chan RW, Lai FM, Li EK, Tam LS, Chow KM, Li PK, Szeto CC. Imbalance of Th1/Th2 transcription factors in patients with lupus nephritis. Rheumatology (Oxford) 2006; 45: 951-7.