中草藥對乳癌細胞輻射增敏作用之研究; The radiosensitizing effect of Chinese herbal medicines in cultured breast cancer cells

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Title:	中草藥對乳癌細胞輻射增敏作用之研究;The radiosensitizing effect of Chinese herbal medicines in cultured breast cancer cells
Authors:	吳蓉茹;Jung-Ju Wu
Contributors:	中國醫藥大學中國醫學研究所
Keywords:	人類乳癌細胞;輻射增敏作用;細胞凋亡;微核;radiosensitizing effect;herbal medicines;human breast cancer cells;apoptosis;micronuclei
Date:	2004
Issue Date:	2010-01-20
Abstract:	乳癌發生率及死亡率逐年攀升，臨床上普遍應用乳房保留手術合併放射線來治療。而輻射治療時可用輻射增敏劑(Radiosensitizer)，來加強輻射線對腫瘤的毒殺作用，然而傳統的輻射增敏劑，因有較大的毒副作用，故限制其臨床應用。本論文希望能由中草藥中尋找高效低毒的乳癌輻射增敏劑，以期能成為提高放療效果的新藥。首先以MTS分析法觀察20味清熱解毒、活血化瘀等中草藥(青蒿、蒲公英、敗醬草、紫花地丁、白花蛇舌草、金銀花、牡丹皮、虎杖、大青葉、黃芩、黃柏、丹參、紅花、三稜、莪朮、三七、薑黃、王不留行、甘草、夏枯草)與中藥有效成分茶多酚、Baicalin、Baicalein等對MDA-MB-231細胞(p53-, Rb-, ER-)生長的影響，並以已知對MDA-MB-231乳癌細胞有明顯輻射增敏作用的Roscovitine為陽性對照組；結果發現Roscovitine 5 μg/ml與7.5 μg/ml的濃度下，其相對於陰性對照組之生長抑制率分別為16.6%與37.3%，都有統計上差異(p<0.05)。部分的中草藥，例如薑黃、三稜、蒲公英、大青葉對MDA-MB-231細胞沒有明顯的生長抑制作用(P>0.05)。有些中草藥與其生物活性成分，例如黃芩、黃柏、王不留行、Baicalin、Baicalein等對MDA-MB-231細胞有明顯的生長抑制作用(P<0.05)，即有明顯毒殺乳癌細胞的效果。其50% 生長抑制所需濃度(IC50)的比較如下：Roscovitine為10.3 μg/ml 、Baicalin為13.8 μg/ml、Baicalein為26.0 μg/ml、黃柏為32.3 μg/ml、王不留行為36.9 μg/ml、黃芩為207.7 μg/ml。另以MTS分析法觀察中草藥對MCF-7細胞(p53＋, Rb＋, ER＋)生長的影響，可看出大部分的中草藥(例如黃芩、金銀花、夏枯草、甘草、大青葉)對細胞沒有明顯的生長抑制作用(P>0.05)。其中Roscovitine、黃柏則可明顯抑制MCF-7細胞之生長(P<0.05)。以MTS分析法觀察輻射對MDA-MB-231與MCF-7細胞生長的影響，無論是於輻射後24、48、72、96小時測量，可看出輻射(3?7 Gy)對此兩種乳癌細胞，以MTS分析法觀察並沒有明顯的生長抑制作用。然而，進一步以細胞群落法觀察輻射對MDA-MB-231細胞生長的影響，可看出輻射(2、3、4、5 Gy)後，輻射對MDA-MB-231細胞有顯著的生長抑制作用，且呈現明顯的劑量反應關係，其生長抑制率分別是2 Gy為26%、3 Gy為39%、4 Gy為54%、5 Gy為65%。以細胞群落試驗法觀察中草藥對MDA-MB-231細胞的輻射增敏作用，以相對應於5.0 μM Roscovitine對MDA-MB-231細胞的細胞毒性(MTS法)約60-80%的存活率，來挑選中草藥的濃度。結果顯示部分中草藥(例如黃芩、莪朮、白花蛇舌草、黃柏、三稜、Baicalin、Baicalein)，對MDA-MB-231細胞有明顯增強輻射之生長抑制作用。與單獨輻射組(3 Gy)比較，2.5 μM Roscovitine並無輻射增敏效果(P>0.05)，但輻射(3 Gy)分別併用5.0 μM Roscovitine、12.5 μg/ml黃芩、25 μg/ml黃芩、500 μg/ml 莪朮、250 μg/ml白花蛇舌草、500 μg/ml白花蛇舌草、6 μg/ml黃柏、12.5 μg/ml黃柏、18 μg/ml黃柏、25 μg/ml黃柏、6.25 μg/ml Baicalin、12.5 μg/ml Baicalin、3 μg/ml Baicalein、或6.25 μg/ml Baicalein時，其所增強的生長抑制率分別為26.8%、19.5%、59.3%、32.9%、41.5%、47.4%、27.7%、54.5%、57.0%、74.9%、49.0%、71.8%、32.0%、55.4% (p<0.05，與單獨3 Gy輻射組比較)。顯微鏡下的細胞型態觀察結果發現，輻射併用中草藥其細胞生長受到比輻射更明顯的生長抑制作用，有輻射增敏的效果。進一步研究其可能的輻射增敏作用機制，利用PI染色法分析細胞週期Sub G1期與G2-M期的比例，觀察到輻射(5 Gy)會增長MDA-MB-231細胞之G2-M期(P<0.05)。然而，黃芩與黃柏加輻射(4 Gy)組，與單獨輻射組(4 Gy)比較，其Sub G1期和G2-M期並沒有明顯增加(P>0.05)。另以染色體傷害指標微核試驗法觀察，輻射可明顯增加MDA-MB-231細胞之微核率，與陰性對照組比較，輻射約增加3-4倍；另發現輻射(4 Gy)併用25 μg/ml黃芩或12.5 μg/ml黃柏，與輻射(4 Gy)比較，其微核數沒有明顯增加。綜合以上結果，黃柏、王不留行、黃芩與生物活性成分對MDA-MB-231乳癌細胞有良好的輻射增敏效果，具有研究與開發作為乳癌輻射增敏劑的潛力，然其可能作用機制尚待進一步的研究。; The incidence and mortality of breast cancer in Taiwan are increasing year by year. Breast conserving therapy including conserved mastectomy combined with radiation therapy is the contemporary choice of treatment. Radiosensitizers are often used in radiation therapy program to augment the anti-tumor effect. However, the clinical application of traditional radiosensitizers is limited due to their serious side effects. In this thesis, the radiosensitizing effect of Chinese herbal medicines in cultured breast cancer cells was examined to search for new radiosensitizers with high therapeutic effects but low side effects. First, the effects of 20 Chinese herbal medicines within the categories of clearing away heat and detoxication as well as promoting circulation and dispelling stagnant blood (such as Artemisia apiacea, Taraxacum mangolicum, Patrinia scabiosaefolia, Viola yedoensis, Hedyotis diffusae, Lonicera japonica, Paeonia suffruticosa, Polygonum cuspidatum, Isatis indigotica, Scutellaria baicalensis, Phellodendron amurense, Savia tatarica, Carthamus tinctorius, Sparganium stoloniferum, Curcuma zedoaria, Panxa pseudo-ginseng var.notoginseng, Curcuma longa, Vaccaria pyramidata、Glycyrrhiza uralensis, and Prunella vulgaris) on cell growth of MDA-MB-231 (p53-, Rb-, ER-) human breast cancer cells were examined. The components of some herbal medicines, such as Epigallocatechin gallate, baicalin, and baicalein, were also examined. Roscovitine, a known radiosensitizer in MDA-MB-231 cells, was used as a positive control. The growth inhibition induced by herbal medicines was measured by MTS assay. Some herbal medicines, such as Curcuma longa, Sparganium stoloniferum, Taraxacum mangolicum, and Isatis indigotica, showed no significant growth inhibition in MDA-MB-231 cells (P>0.05). Some herbal medicine and their components, such as Scutellaria baicalensis, Phellodendron a
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