201242593 六、發明說明： 【發明所屬之技術領域】 本發明係關於抑制微衛星不穩定之醫藥組成物及包含 其之癌症治療用醫藥組成物，尤指可抑制因慢性發炎或抗 癌藥物所導致之微衛星不穩定之醫藥組成物，其分別可降 低癌症發生率及死亡率。 【先前技術】 癌症疋一個複雜的基因疾病，其中2 5 %是來自慢性發 炎病人中。以癌症病人之臨床治療而言，除了手術治療外， 更須搭配化療以達到最佳治療功效。目前，用於癌症治療 之化療藥物相當多，如：順鉑（cispUtin)、氟脲嘧啶 (5-flu〇rouracn，5_FU)、曱氨蝶呤（meth〇trexate)、洛莫司汀 (lomustine)、草酸鉑（0)^丨丨13丨31丨11)等。其中，順鉑及洛莫司 >丁係為一種DNA損傷試劑，其會對鹼基進行修飾、股間交 聯、股外交聯、單股斷裂、或雙股斷裂；而5 FU及曱氨蝶 呤則會對DNA合成造成干擾。 目前已有研究指出，在慢性發炎病人、癌症病人、化 療導致之第二種癌症病人中，常因癌症化療及氧化壓力， 而谷易τιτ另微紀星不穩定性（micr〇sate丨丨㈣❶丨丨丨^， MSI)。其中，微衛星不穩定性之發生，主要起因為錯 配修復系統（DNA mismatch repair，MMR)的功能無法正常 運作，而無法修復基因複製時之錯誤。慢性發炎與癌症病 患者，常因MMR功能的喪失，而無法修復微衛星序列長度 201242593 上在DNA複製時所產生之錯誤，如：在編碼區（c〇d ing region) 之仅t作了生的重複單元缺失，則造成讀框序列位移丨⑴ 大k產生。同時，MMR也辨識並修復活性氧化物質（rad丨ca| oxygen species, R〇S)所造成之8_氧-鳥嘌呤 DNA(8-〇XO-guanine DNA)。除了 h2〇2為容易產生 r〇s 的主 要因子之一外，已知5_FU&順鉑亦會產生R〇s，因而也能 導致讀框序列位移突變及微衛星不穩定性。 除此之外，臨床上更發現，當病人呈現微衛星不穩定 性時，往往容易對化療藥物產生抗藥性。藥物產生的基因 毒化（genotoxic)壓力，能導致DNA損傷，因而導致癌症細胞 的凋亡。但若細胞的MMR#能喪失，則無法走向細胞凋亡， 因而產生抗藥性。 口此若發展出可抑制微衛星不穩定之藥物，則可 用於杈性發炎病人，進而降低癌症之發生率。同時，若能 將可抑制微衛星不穩定之藥物與抗癌藥物搭配使用，勢必 可提升癌症治療之成功率。 【發明内容】 本發明之主要目的係在提供抑制因活性氧化物質所導 致的微衛星不穩定之醫藥組成物，俾能降低人體内之因慢 ^生發乂所導致之微衛星不穩定性’而可降低癌症之發生率。 本發明之另一目的係在提供抑制化療所導致的微衛星 不％定之醫藥組成物’藉由將癌症藥物與可抑制微衛星不 穩定之化合物一同使用，而可降低癌症之死亡率。 201242593 為達成上述目的’本發明提供種抑制微衛星不穩定之 醫藥組成物，包括：有效劑量之至少一種抑制微衛星不穩 定之化合物，其中，抑制微衛星不穩定之化合物係為乙醯 半脱胺酸（N- acetylcysteine)、麩胱甘肽（giutathi〇ne)、其衍 生物、或其鹽類；以及醫藥上可接受之載體。更詳細而言， 抑制微衛星不穩定之化合物係為乙醯半胱胺酸、乙醯半胱 胺酸之衍生物或鹽類、麩胱甘肽 '或麩胱甘肽之衍生物或 鹽類。 藉由使用本發明所提供之抑制微衛星不穩定之醫藥組 成物時，可降低病患中之微衛星不穩定性。特別是，當本 發明之抑制微衛星不穩定之醫藥組成物用於慢性發炎病 患，可降低微衛星不穩定的發生。目前已知慢性發炎病患 中所產生之微術星不穩定會導致癌症發生，故使用本發明 所提供之抑制微衛星不穩定之醫藥組成物，可降低因發炎 所導致之癌症發生率。 另一方面，本發明更發現當此抑制微衛星不穩定之化 合物與抗癌藥物一同使用時，可達到抑制因抗癌藥物所導 致微衛星不穩定之功效。據此，本發明亦提供一種癌症治 療之醫藥組成物，包括：有效劑量之至少一種上述抑制微 衛星不穩定之化合物；治療癌症之化合物；以及醫藥上可 接受之載體。 由於本發明之癌症治療之醫藥組成物，除了包含可治 療癌症之化合物外，更包含抑制微衛星不穩定之化合物。 因此，於施予藥物的同時，除了可達到癌症治療之效果外， 201242593 更可達到抑制因抗癌藥物所導致微衛星不穩定之目的，進 而降低抗藥性的產生。同時，因本發明之癌症治療之醫藥 組成物可抑制微衛星不穩定之發生，故可降低因基因突變 所造成之癌症惡化，進而降低癌症之死亡率。 於本發明之癌症治療之醫藥組成物中’較佳係包括有 效劑S之治療癌症之化合物，且此治療癌症之化合物即所 謂之抗癌藥物。更佳為，於本發明之癌症治療之醫藥組成 物中’治療癌症之化合物係為化療用之抗癌藥物。 此外’於本發明之醫藥組成物中，抑制微衛星不穩定 之化合物較佳為乙醯半胱胺酸、麩胱甘肽、或其衍生物。 已知癌症病患於化學藥物治療的過程中，出現微衛星 不穩疋性時’往往容易對化療藥物產生抗藥性。由於本發 明之癌症治療之醫藥組成物除了包括化療用之抗癌藥物 外’更包括抑制微衛星不穩定之化合物，故於化療的過程 中可降低微衛星不穩定性之發生率，進而降低病患對化療 藥物產生抗藥性’以提升化療之治療效果及癌症病患存活 率。 其中’治療癌症之化合物較佳為抗癌藥物，如順麵 (cisplatin)、氟腺。密咬（5-fiuorouracn)、曱氨蝶吟 (methotrexate)、洛莫司汀（lomustine)、草酸鉑（〇xa|ipUtin)。 於本發明之醫藥組成物中，「醫藥上可接受之載體」 意指載體須能與該組成物之活性成分相容（較佳係能穩定 活性成分），且不可在治療過程中危害個體。其中，載體可 為至少一選自：活性劑、輔劑、分散劑 '潤濕劑、及懸浮 201242593 劑所組成之群組，如微晶質纖維素（micr〇crysuUine cellu丨ose)、甘露糖醇（mannitol)、葡萄糖、脫脂奶粉、聚乙 烯、聚乙烯吡咯烷酮^。卜^丨”如⑺丨丨如时广澱粉或其組 合物。 此外，於本發明之醫藥組成物中，「有效劑量」音指： 每一活性劑的量為為了達到個體治療之目的而所需之劑 I，其劑量可單獨使用或和其他一種或一種以上之活性劑 组合使用。本領域中具有通常知識者均瞭解，其有效劑量 會依據投藥方式、輔劑之選擇、及其他活性劑的合併使用 而有所變化。 【實施方式】 以下係藉由特定的具體實施例說明本發明之實施方 式’熟習此技藝之人士可由本說明書所揭示之内容輕易地 了解本發明之其他優點與功效。本發明亦可藉由其他不同 的具體實施例加以施行或應用，本說明書中的各項細節亦 可針對不同觀點與應用，在不悖離本創作之精神下進行各 種修飾與變更。 建構雙螢光微衛星報導質體 首先’先從 pDsRedl-Nl 質體（Clontech)取出 DsRed cDNA(可表現rfp紅光螢光蛋白），並以Sacn&NotI之酶切 位點，將 中’以製得pRFP-IRES-GFP載體。 201242593 而後，將一微衛星序列ATG-(CA)I3(SEQ ID NO:丨）或 一控制組序列 ATG-(N)I6(SEQ ID NO: 2)，以 SacI 及 Agel 之 酶切位點，插入至上述pRFP-丨RES-GFP載體之DsRed起始子 之上游區域，並將RFP(即，DsRed)之讀框序列（reading frame) 位移至-1位置。接著，透過Cre介導重組反應（Cre-mediated recombination)，則可插入 pExchange Module EC-Hyg (Stratagene)之抗潮霉素基因（hygromycin resistance gene)， 以建構一雙螢光微衛星報導質體 p(CA)l3RFP-IRES-GFP-Hyg 、以及一控制組質體 p(N)l3RFP-IRES-GFP-Hyg。 接著，使用將上述所建構之雙螢光微衛星報導質體、 及控制組質體，轉染至人類結腸癌細胞株HCT11 6、或 HCT116+chr3 (American Type Culture Collection)中，以表 現上述之表現質體。其中，HCTΠ6細胞株因其/zA/L///基因 有同型接合子突變（homozygous mutation)，故為一 DN A錯配 修復缺失細胞（MMR-deficient cells);而 HCT116+chr3 細胞 株因其具有帶正常/zMI///基因之染色體3，故為一DNA錯配 修復徤全細胞（MMR-proficient cells)。此外，HCT116細胞 株之培養，係於5°/。C02及3 7°C下，以含有10%胎牛血清及2 mM之L-麵醢胺酸（L-glutamine)之DMEM/F -12培養基進行 培養；而HCT116+chr3細胞株之培養係與HCT116細胞株相 似，除了更於培養基中添加400 pg/ml之G4 1 8。 在此，將說明如何將上述所建構之雙螢光微衛星報導 質體 '及控制組質體，轉染至HCT11 6細胞株。首先，於六 201242593 孔盤中，每孔種植6><丨05個細胞；待種植細胞16小時後’使 用脂質體轉染試劑（Lipofectamine 2000ΊΜ)’將雙螢光微衛 星報導質體、及控制組質體轉染至HCT1 1 6細胞株。在此， 轉染有雙螢光微衛星報導質體之HCT1 16細胞株係稱為 HCT1 16-(CA)I3，而轉染有控制組質體之HCT116細胞株係 稱為HCT116-(N)I6。於轉染反應兩天後，以200 gg/ml之抗 潮霉素篩選HCT〗 16-(CA)13及HCT116-(N)I6轉染細胞株》 此外’更以上述相同方法，將雙螢光微衛星報導質體 轉染至HCT 1 1 6+chr3細胞株，而所得之轉染細胞株則稱為 HCT1 16+chr3-(CA)i3。 以高通量螢光顯微系統判斷讀框序列位移 將細胞以4%之二聚曱酸 (paraforma丨dehyde)固定，並以 1 pg/ml之Hochest 33258試劑染細胞核。在此，係使用 ImageXpressMicro 系統（Molecular Devices)以 10x 放大倍率201242593 VI. OBJECTS OF THE INVENTION: TECHNICAL FIELD The present invention relates to a pharmaceutical composition for inhibiting microsatellite instability and a pharmaceutical composition for cancer treatment comprising the same, and particularly to inhibiting chronic inflammation or anticancer drugs Microsatellite unstable pharmaceutical compositions that reduce cancer incidence and mortality, respectively. [Prior Art] Cancer is a complex genetic disease in which 25 percent is from chronic inflammatory patients. In the clinical treatment of cancer patients, in addition to surgery, it is necessary to use chemotherapy to achieve the best therapeutic effect. At present, there are quite a few chemotherapeutic drugs for cancer treatment, such as: cispUtin, fluorouracil (5-FU), meth〇trexate, lomustine , oxalic acid platinum (0) ^ 丨丨 13 丨 31 丨 11) and so on. Among them, cisplatin and lovastatin are a DNA damage reagent, which will modify bases, inter-strand cross-linking, femoral diplomatic union, single-strand break, or double-strand break; and 5 FU and sulphate呤 will interfere with DNA synthesis. At present, it has been pointed out that in patients with chronic inflammation, cancer patients, and cancer patients, the second cancer patients often suffer from cancer chemotherapy and oxidative stress, while Gu Yi τιτ is also micro-star instability (micr〇sate丨丨 (4)❶丨丨丨^, MSI). Among them, the occurrence of microsatellite instability mainly occurs because the function of DNA mismatch repair (MMR) cannot function normally, and it is impossible to repair errors in gene duplication. Chronic inflammation and cancer patients, often due to the loss of MMR function, can not repair the errors generated by DNA replication in the microsatellite sequence length 201242593, such as: only in the coding region (c〇d ing region) The absence of a repeating unit results in a shift in the reading frame sequence 丨(1) large k. At the same time, MMR also recognizes and repairs 8_oxo-guanine DNA (8-〇XO-guanine DNA) caused by active oxidizing substances (rad丨ca| oxygen species, R〇S). In addition to h2〇2 being one of the major factors that are prone to r〇s, it is known that 5_FU& cisplatin also produces R〇s, which can also cause shifts in the reading sequence and microsatellite instability. In addition, it has been found clinically that when patients exhibit microsatellite instability, they are often resistant to chemotherapy drugs. Drug-generating genotoxic stress can lead to DNA damage, which leads to apoptosis in cancer cells. However, if the MMR# of the cell can be lost, it is unable to progress to apoptosis, and thus develops drug resistance. If a drug that inhibits microsatellite instability is developed, it can be used in patients with spastic inflammatory disease, thereby reducing the incidence of cancer. At the same time, if a drug that can inhibit microsatellite instability can be used in combination with an anticancer drug, it will certainly increase the success rate of cancer treatment. SUMMARY OF THE INVENTION The main object of the present invention is to provide a pharmaceutical composition for inhibiting microsatellite instability caused by active oxidizing substances, which can reduce microsatellite instability caused by slow hair growth in the human body. Can reduce the incidence of cancer. Another object of the present invention is to provide a pharmaceutical composition which inhibits microsatellite caused by chemotherapy, which can reduce cancer mortality by using a cancer drug together with a compound which can inhibit microsatellite instability. 201242593 To achieve the above object, the present invention provides a pharmaceutical composition for inhibiting microsatellite instability, comprising: an effective dose of at least one compound which inhibits microsatellite instability, wherein the compound which inhibits microsatellite instability is acetaminophen N-acetylcysteine, giutathi〇ne, a derivative thereof, or a salt thereof; and a pharmaceutically acceptable carrier. In more detail, the compound which inhibits microsatellite instability is acetaminosamine, a derivative or salt of acetaminosamine, a derivative or a salt of glutathione or glutathione. . Microsatellite instability in patients can be reduced by using the pharmaceutical composition of the present invention which inhibits microsatellite instability. In particular, when the pharmaceutical composition for inhibiting microsatellite instability of the present invention is used for chronic inflammatory diseases, the occurrence of microsatellite instability can be reduced. It is known that the instability of microstrips generated in chronic inflammatory patients causes cancer, and the use of the pharmaceutical composition for suppressing microsatellite instability provided by the present invention can reduce the incidence of cancer caused by inflammation. On the other hand, the present invention has further found that when the compound which inhibits microsatellite instability is used together with an anticancer drug, the effect of suppressing microsatellite instability caused by the anticancer drug can be achieved. Accordingly, the present invention also provides a pharmaceutical composition for treating cancer comprising: an effective amount of at least one of the above-mentioned compounds which inhibit microsatellite instability; a compound for treating cancer; and a pharmaceutically acceptable carrier. Since the pharmaceutical composition for cancer treatment of the present invention contains a compound which can treat cancer, it further contains a compound which inhibits microsatellite instability. Therefore, in addition to the effect of cancer treatment, 201242593 can achieve the purpose of inhibiting microsatellite instability caused by anticancer drugs, thereby reducing the emergence of drug resistance. At the same time, the pharmaceutical composition for cancer treatment of the present invention can inhibit the occurrence of microsatellite instability, thereby reducing the cancer deterioration caused by genetic mutations, thereby reducing the mortality rate of cancer. The pharmaceutical composition for cancer treatment of the present invention is preferably a compound for treating cancer comprising an effective agent S, and the compound for treating cancer is a so-called anticancer drug. More preferably, the compound for treating cancer in the pharmaceutical composition for cancer treatment of the present invention is an anticancer drug for chemotherapy. Further, in the pharmaceutical composition of the present invention, the compound which inhibits microsatellite instability is preferably acetylcysteine, glutathione, or a derivative thereof. It is known that cancer patients are often susceptible to chemotherapeutic drugs when microsatellite instability occurs during the course of chemical drug treatment. Since the pharmaceutical composition for cancer treatment of the present invention includes, in addition to the anticancer drug for chemotherapy, a compound which inhibits microsatellite instability, the incidence of microsatellite instability can be lowered during the course of chemotherapy, thereby reducing the disease. Suffering from resistance to chemotherapy drugs to improve the therapeutic effect of chemotherapy and the survival rate of cancer patients. Among them, the compound for treating cancer is preferably an anticancer drug such as cisplatin or fluorogland. 5-fiuorouracn, methotrexate, lomustine, oxalic acid platinum (〇xa|ipUtin). In the pharmaceutical composition of the present invention, "pharmaceutically acceptable carrier" means that the carrier must be compatible with the active ingredient of the composition (preferably to stabilize the active ingredient) and not to harm the individual during the treatment. Wherein, the carrier may be at least one selected from the group consisting of: an active agent, an adjuvant, a dispersing agent, a wetting agent, and a suspension of 201242593, such as microcrystalline cellulose (micr〇crysuUine cellu丨ose), mannose Alcohol (mannitol), glucose, skimmed milk powder, polyethylene, polyvinylpyrrolidone. Further, in the pharmaceutical composition of the present invention, the "effective dose" means that the amount of each active agent is for the purpose of individual treatment. The agent I may be used alone or in combination with one or more other active agents. It is well known to those of ordinary skill in the art that the effective dosage will vary depending on the mode of administration, the choice of adjuvant, and the combined use of other active agents. [Embodiment] The following embodiments of the present invention are described by way of specific embodiments. Those skilled in the art can readily appreciate the advantages and advantages of the present invention from the disclosure herein. The present invention may be embodied or applied in various other specific embodiments. The details of the present invention can be applied to various aspects and applications, and various modifications and changes can be made without departing from the spirit of the invention. Construction of a dual-fluorescence microsatellite reporter plastid firstly extracts DsRed cDNA (which can express rfp red fluorescent protein) from pDsRedl-Nl plastid (Clontech), and cleaves the site with Sacn& NotI, The pRFP-IRES-GFP vector was prepared. 201242593 Then, a microsatellite sequence ATG-(CA)I3 (SEQ ID NO: 丨) or a control group sequence ATG-(N)I6 (SEQ ID NO: 2), with SacI and Agel cleavage sites, The upstream region of the DsRed initiator of the above pRFP-丨RES-GFP vector was inserted, and the reading frame of the RFP (i.e., DsRed) was shifted to the -1 position. Next, by Cre-mediated recombination, the hygromycin resistance gene of pExchange Module EC-Hyg (Stratagene) can be inserted to construct a pair of fluorescent microsatellite reporter plastids. (CA) l3RFP-IRES-GFP-Hyg, and a control group plastid p(N)l3RFP-IRES-GFP-Hyg. Next, the above-described constructed dual-fluorescence microsatellite reporter plastid and control group plastid are transfected into human colon cancer cell line HCT11 6 or HCT116+chr3 (American Type Culture Collection) to express the above. Express plastids. Among them, the HCTΠ6 cell line has a homozygous mutation due to its /zA/L/// gene, so it is a DN A mismatch repairing cell (MMR-deficient cells); and the HCT116+chr3 cell line is It has a chromosome 3 with a normal/zMI/// gene, so it is a DNA mismatch repairing MMR-proficient cells. In addition, the culture of the HCT116 cell line was at 5 °/. C02 and 3 7 ° C, cultured with DMEM/F -12 medium containing 10% fetal bovine serum and 2 mM L-glutamine; and the culture line of HCT116+chr3 cell line The HCT116 cell line was similar except that 400 pg/ml of G4 18 was added to the medium. Here, it will be explained how the above-described constructed dual-fluorescence microsatellite reporter plastid 'and control group plastids are transfected into HCT11 6 cell line. First, in the 201242593 well plate, 6><丨05 cells were planted per well; after 16 hours of planting, 'Lipofectamine 2000ΊΜ' was used to report the plastids with dual-fluorescence microsatellite, and The control group was transfected into HCT1 16 cell line. Here, the HCT1 16 cell line transfected with the dual-fluorescence microsatellite reporter plastid is called HCT1 16-(CA)I3, and the HCT116 cell line transfected with the control plastid is called HCT116-(N). I6. Two days after the transfection reaction, HCT 16-(CA)13 and HCT116-(N)I6 transfected cell lines were screened with 200 gg/ml of antihygromycin. In addition, the same method was used. The light microsatellite reported that the plastid was transfected into the HCT 1 16 + chr3 cell line, and the resulting transfected cell line was called HCT1 16+chr3-(CA)i3. The reading frame sequence was judged by high-throughput fluorescence microscopy. The cells were fixed with 4% diformic acid (paraforma丨dehyde) and the nuclei were stained with 1 pg/ml of Hochest 33258 reagent. Here, using the ImageXpressMicro system (Molecular Devices) at 10x magnification
進行檢測。使用DAPI濾光片組時，係曝光35 ms ;使用FITC 濾光片組時，係曝光丨*75 ms ;而使用TexRed濾光片組時， 則曝光150〇1^’以取得螢光影像。而後’使用1^^)(}31^蛐 V3.1軟體（Molecular Devices)分析所得之螢光影像。 當偵測到GFP螢光蛋白所發出之綠光時，表示可偵測 細胞；且當同時偵測到GFP螢光蛋白所發出之綠光及RFp螢 光蛋白所發出之红光時，表示原先雙螢光微衛星報導質體 或控制組質體中有讀框序列位移的情形產生，而可再次表 現出RFP螢光蛋白。 實驗例1·評估H202對細胞存活率及讀框序列位移之影響 201242593 將HCTl 16及HCTl 16 + chr3細胞株以每孔1 χΙΟ4細胞種 植於96孔盤中。於種植一天後，以Η202處理細胞1小時，且 反應培養基中係含有最終濃度為0、0.25、0.5、0.75、1.0 mM 之H202、以及lx之PBS緩衝溶液。 而後，採習知之MTT分析法分析細胞存活率。於H202 處理細胞後，於每一孔内加入25 μΐ之5 mg/ml的MTT於100 μΐ含細胞的培養基中，反應4小時後於每一孔内加入100 μΐ 的裂解液（lysis buffer)，過夜後終止反應。最後，以酵素免 疫分析儀在595 nm吸光波長下測定其吸光值，藉以計算細 胞的存活率。其中，相對存活率係以未處理之細胞株吸光 值定為100%，計算經處理之細胞株相對於未處理之細胞株 之相對吸光值，而結果係如圖1A所示（圖中亦顯示平均值土 標準差）。 如圖1A結果所示，上述劑量之H202為細胞非致死劑 量’且使用上述劑量評估H202是否產生讀框序列位移而造 成微衛星不穩定的現象。 讀框序列位移之評估，係先將上述HCT116-(CA)I3、 HCTl 16-(N)I6、及 HCT116+chr3-(CA)丨3細胞株以每孔 1 X 1〇5 細胞種植於12孔盤中。於種植一天後，以上述濃度之1〇2 溶液處理細胞i小時；而後去除h2〇2並清洗細胞，且再置於 培養基中繼續成長三天。 接著’使用流式細胞儀（flow cytometry)分析細胞中讀 樞序列位移的情形。首先’將細胞以胰蛋白水解酵素（trypSin) 處理’再懸浮於含1 mM EDTA之PBS緩衝溶液中；而後以 201242593 4〇 μιτι過濾器過渡，再通入流式細胞儀（Quanta1 lV1 SC-MPL Beckman Coulter)進行分析。同時，係使用含有 plRES-hrGFP-la (GFP單一螢光細胞）、pDsRedl-Nl (RFp單 一螢光細胞）、及PRFP_IRES-GFP (GFP/RFP雙螢光細胞）載 體之HCT1 1 6細胞做為分析判斷標準（calibration) ’而未轉植 任何載體或質體之HCT116細胞則做為背景值。在此，讀框 序列位移率則為同一細胞樣品中，同時表現GFP/RFP雙螢 光之細胞數量對表現GFP單一螢光之細胞數量之相對值。讀 框序列位移率之結果係如圖1B所示（圖中亦顯示平均值士標 準差），其中*表示學生t-測試之p<0_02。 如圖1 B所示，隨著H2〇2劑量增加，讀框序列位移率亦 隨之增加；且含有微衛星序列之HCT116-(CA)I：!，其讀框序 列位移率較控制組HCT1 1 6-(N)l6要高。證明了雙螢光系統 即使在DN A錯配修復缺失細胞中仍忠實的報導微衛星不穩 定。此外，於HCT116+chr3-(CA)丨3細胞株中，幾乎偵測不 到讀框序列位移的情形產生，原因在於HCT 1丨6+chr3細胞為 DN A錯配修復徤全細胞，故可修復H2〇2所導致之讀框序列 位移（即’微衛星不穩定性）。因此證明了雙螢光微衛星不穩 定報導系統在DN A錯配修復缺失細胞比在DN A錯配修復健 全細胞中較敏銳（sensitivity)。 此外’若使用高通量螢光顯微系統分析細胞之螢光影 像時，亦可觀察到隨著H2〇2劑量增加，RFP紅色螢光蛋白表 現增加，故讀框序列位移率的情形亦隨之增加。此結果係 201242593 與圖2B之流式細胞儀分析結果一致。也證明此雙螢光微衛 星不穩定報導系統適用於快速與大規模之藥物篩選。 實驗例2-評估MTX抗癌藥物對細胞存活率及讀框序列位移 之影響 將上述HCT1 16-(0八）13及HCT1 16-(N)I6細胞株以每孔 1 X 1 04細胞種植於96孔盤中。於種植一天後，以甲氨蝶呤 (methotrexate, MTX，購自 Sigma- Adrich)處理細胞三天，且Test. When using the DAPI filter set, the exposure is 35 ms; when using the FITC filter set, the exposure is 75*75 ms; and when using the TexRed filter set, the exposure is 150〇1^' to obtain the fluorescent image. Then use '1^^) (}31^蛐V3.1 software (Molecular Devices) to analyze the resulting fluorescent image. When the green light emitted by GFP fluorescent protein is detected, it means that the cells can be detected; When both the green light emitted by the GFP fluorescent protein and the red light emitted by the RFp fluorescent protein are detected, it indicates that the original double-fluorescent microsatellite reports that the reading frame sequence is displaced in the plastid or the control group. The RFP fluorescent protein can be expressed again. Experimental Example 1·Evaluate the effect of H202 on cell viability and reading frame sequence shift 201242593 HCT16 and HCT16+chr3 cell lines were seeded in 96-well plates at 1 χΙΟ4 cells per well. One day after planting, the cells were treated with Η202 for 1 hour, and the reaction medium contained H202 with a final concentration of 0, 0.25, 0.5, 0.75, 1.0 mM, and a PBS buffer solution of lx. Then, the known MTT assay was used. Cell viability was analyzed. After treatment with H202, 25 μM of 5 mg/ml MTT was added to 100 μM of cell-containing medium in each well. After 4 hours of reaction, 100 μM of lysate was added to each well. (lysis buffer), terminate the reverse after overnight Finally, the absorbance of the cells was measured by an enzyme immunoassay at 595 nm absorbance to calculate the cell viability. The relative survival rate was determined by taking the absorbance of the untreated cell line to 100%. The relative absorbance of the cell line relative to the untreated cell line, and the results are shown in Figure 1A (the mean soil standard deviation is also shown in the figure). As shown in the results of Figure 1A, the above dose of H202 is the non-lethal dose of cells. 'And use the above dose to evaluate whether H202 produces a frame-sequence shift and cause microsatellite instability. The evaluation of the reading sequence shift is based on the above HCT116-(CA)I3, HCTl 16-(N)I6, and HCT116. The +chr3-(CA)丨3 cell line was planted in a 12-well plate at 1×1〇5 cells per well. After one day of planting, the cells were treated with the above-mentioned concentration of 1〇2 solution for 1 hour; then h2〇2 was removed and The cells were washed and then placed in the medium for further growth for three days. Then 'flow cytometry was used to analyze the displacement of the reading sequence in the cells. First, 'the cells were treated with trypSin'. Suspension 1 mM EDTA in PBS buffer solution; then with 201242593 4〇μιτι filter transition, and then flow cytometry (Quanta1 lV1 SC-MPL Beckman Coulter) for analysis. Also, use plRES-hrGFP-la (GFP) HCT1 16 cells of single fluorescent cells), pDsRedl-Nl (RFp single fluorescent cells), and PRFP_IRES-GFP (GFP/RFP dual fluorescent cells) were used as analytical calibrations without any retransplantation The vector or plastid HCT116 cells were used as background values. Here, the reading sequence displacement rate is the relative value of the number of cells expressing GFP/RFP double fluorescence to the number of cells expressing GFP single fluorescence in the same cell sample. The result of the displacement rate of the frame sequence is shown in Fig. 1B (the average value of the standard is also shown), where * represents the p<0_02 of the student t-test. As shown in Figure 1B, as the dose of H2〇2 increases, the displacement rate of the reading frame sequence also increases; and the HCT116-(CA)I:! containing the microsatellite sequence has a higher reading rate than the control group HCT1. 1 6-(N)l6 should be high. It has been demonstrated that the dual-fluorescence system faithfully reports microsatellite instability even in the absence of DN A mismatch repair. In addition, in the HCT116+chr3-(CA)丨3 cell line, almost no reading frame sequence shift was detected, because HCT 1丨6+chr3 cells were DN A mismatched to repair 徤 whole cells, so Fix the displacement of the reading frame caused by H2〇2 (ie 'microsatellite instability'). Thus, it was demonstrated that the dual-fluorescence microsatellite instability reporting system is more sensitive in repairing missing cells in DN A mismatches than in DN A mismatch repairing healthy cells. In addition, if high-throughput fluorescence microscopy system is used to analyze the fluorescence images of cells, it can be observed that with the increase of H2〇2 dose, the expression of RFP red fluorescent protein increases, so the displacement rate of the reading frame sequence also Increase. This result is consistent with the flow cytometry analysis of Figure 2B in 201242593. This dual-fluorescence micro-satellite instability reporting system has also proven to be suitable for rapid and large-scale drug screening. Experimental Example 2 - Evaluation of the effect of MTX anticancer drugs on cell viability and reading frame sequence shifts The above HCT1 16-(0-8)13 and HCT1 16-(N)I6 cell lines were planted at 1 X 1 04 cells per well. 96-well plate. One day after planting, cells were treated with methotrexate (MTX, purchased from Sigma-Adrich) for three days, and
反應培養基中係含有最終濃度為〇、5、10、25、50、100 nM 之MTX、以及2.5 nM之NaOH。而後，使用與實驗例1相同 之MTT分析法分析細胞存活率，結果係如圖2 A所示（圖中亦 顯示平均值土標準差）’其顯示MTX細胞非致死劑量約為25 nM。 讀框序列位移之評估，係先將上述HCT1i6_(CA)|3& HCT1 1 6-(Ν)|ό細胞株以每孔1 x 1〇5細胞種植於12孔盤中。於 種植一天後，以最終濃度為〇、6 3、12 5、25 ηΜ2ΜΤχ處 理細胞三天；而後去除1^丁乂並清洗細胞，且再置於培養基 中繼續成長三天。 接著’使用與實施例1相同之流式細胞儀分析細胞中讀 忙序列位移的情形，結果係如圖2B所示（圖中亦顯示平均值 土標準差），其中“*表示學生^測試之p<〇 〇〇〇1。 如圖2B所示，含有微衛星序列之HCT1 16-(CA)n，其讀 ，序列位移率較控制組HCT116-(N),6要高；A尤以MTX劑 1為-5 nM aT，讀框序列位移產生的頻率更加顯著。 201242593 此外，更將HCTlW-fA),3細胞株以最終濃度為25 nM 之MTX處理細胞三天，而後去除ΜΊΓχ並清洗細胞’且再置 於培養基中繼續成長三天。將上述步驟循環處理共四次， 並以PCR放大HCT1 16-(CA)丨3中之DNA，再進行DNA基因定 序（定序引子如SEQ ID NO: 3、SEQ ID NO: 4所示）。 DNA基因定序結果顯示，經MTX處理後之細胞株，微 衛星序列中少了一組CA重複單元，即定序結果為（Ca)|2。 由於一組CA重複單元缺失，則可使RFp讀框序列產生位 移’攸sf框外位移至讀框内。據此，可再次表現R F p蛋白， 故可觀測到RFP螢光蛋白所發出之红色螢光u此證明Μτχ 抗癌藥物會導致微衛星不穩定。 實驗例3-評估CCNU抗癌藥物對細胞存活率及讀框序列位 移之影響 本實驗例之細胞存活率實驗及分析方法係與實驗例2 相同’除了使用洛莫司汀（丨omustine) (1-(2-氣乙基）_3-環己 基-1-亞石肖基尿素（l_(2_Chloroethyl)-3-cyclohexy丨-1· nitrosourea), CCNU,購自 Sigma-Adrich)取代 MTX，且反應 培養基中係含有最終濃度為〇、10、25 ' 50、75、丨〇〇 μΜ之 CCNU、以及1%之乙醇。如圖3Α結果戶斤示（圖中亦顯示平均 值土標準差），CCNU之細胞非致死劑量約為50 μΜ。 此外’本實驗例之讀框序列位移實驗及分析方法亦與 實驗例2相同，除了使用CCNU取代ΜΤΧ，且反應培養基中 係含有最終濃度為0 ' 1 2.5、25 ' 50 μΜ之CCNU、以及1 % 之乙醇。實驗結果係如圖3 Β所示（圖中亦顯示平均值±標準 201242593 差）’其中*表示學生t-測試之p<〇.〇5，"表示p<〇 〇〇5，而*** 表示 p<0.000 1。 如圖3B結果所示，含有微衛星序列之HCTll6-(CA)n， 其讀框序列位移率較控制組HCT 1 1 6-(N)l6要高；且隨著 C C N U劑篁增加，讀框序列位移率亦隨之增加。此證明匸c n u 抗癌藥物會導致微衛星不穩定。 實驗例4-評估CCNU抗癌藥物對細胞存活率及讀框序列位 移之影響 本實驗例之細胞存活率實驗及分析方法係與實驗例2 相同’除了使用順鉑（cisp丨atin)(順式-二胺-二氣鉑⑴） (c/5-diammine-dichloroplatinum (1_I)), CDDP 購 自 Sigma-Adrich)取代MTX，且反應培養基中係含有最終濃度 為 0、2.5、5、10、20、40 μΜ之 CDDP、以及 0.77 mM之 NaCl。 如圖4A結果所示（圖中亦顯示平均值土標準差），cddp之細 胞非致死劑量約為20 μΜ。 此外’本實驗例之讀框序列位移實驗及分析方法亦與 實驗例2相同，除了使用CDDP取代ΜΤΧ，且反應培養基中 係含有最終濃度為0、6.3、12_5、25 μΜ之CDDP、以及0.77 mM之NaCl。實驗結果係如圖4Β所示（圖中亦顯示平均值土 心準差）’其中*表示學生t-測試之p<〇.〇3，* *表示p<〇.〇〇3， 而 *** 表示 p<〇.〇〇〇2。 如圖4B結果所示，含有微衛星序列之hcti16-(CA)I3 隨著CDDP劑量增加，讀框序列位移率亦隨之增加。此證明 CDDP抗癌藥物會導致微衛星不穩定。 201242593 實驗例5-評估5-FU抗癌藥物對細胞存活率及讀框序列位移 之影響 本實驗例之細胞存活率實驗及分析方法係與實驗例2 相同，除了使用氟脲嘧啶（5-fluorouracil)(5-FU，購自 Sigma-Adrich)取代MTX，且反應培養基中係含有最終濃度 為 0、6.3、12.5 ' 25、50、100 μΜ之5-FU、以及0·5。/。之DMSO。 如圖4Α結果所示，5-FU之細胞非致死劑量約為20 μΜ。 此外，本實驗例之讀框序列位移實驗及分析方法亦與 實驗例2相同，除了使用5-FU取代ΜΤΧ，且反應培養基中係 含有最終濃度為0 ' 2.5、5、10 μΜ之5-FU、以及0.5%之 DMSO。如圖4Β結果所示在含有微衛星序列之 HCT1 16-(CA)I3之細胞中，5-FU增高了其讀框序列位移率。 因此證明CDDP抗癌藥物也會導致微衛星不穩定。 實驗例6-評估草酸鉑抗癌藥物對細胞存活率及讀框序列位 移之影響 本實驗例之細胞存活率實驗及分析方法係與實驗例2 相同，除了使用草酸鉑（oxaliplatin)取代MTX，且反應培養 基中係含有最終濃度為〇、0.125 ' 0.25、0.5、〗μΜ之草酸 鉑。如圖5 Α結果所示（圖中亦顯示平均值士標準差），草酸鉑 之細胞非致死劑量約為1 μΜ。 此外，本實驗例之讀框序列位移實驗及分析方法亦與 實驗例2相同，除了使用草酸鉑取rMtx，且反應培養基中 係含有最終濃度為0、0.25、0.5、1 μΜ之草酸鉑。如圖5β 結果所示（圖中亦顯示平均值±標準差），隨著草酸鉑劑量增 201242593 加，讀框序列位移率亦隨之增加。因此證明草酸鉑抗癌藥 物也導致微衛星不穩定。 實驗例7-評估NAC對細胞存活率及讀框序列位移之影響 本實驗例之細胞存活率實驗及分析方法係與實驗例2 相同，單獨加入乙醯半胱胺酸（N-acety lcysteine, N AC)至培 養基中係含有最終濃度為0、1.25、2.5、5、10 mM之NAC。 如圖6A結果所示（圖中亦顯示平均值±標準差），NAC之細胞 非致死劑量之上限約為5 mM。此外NAC也不會影響H202或 % 抗癌藥物對細胞的殺傷率。 除此之外，由上述實驗例1及2之實驗結果證實，H202、 MTX、CDDP、及CCNU確實會增加讀框序列位移率，進而 造成微衛星不穩定的情形增加。為檢測NAC是否可抑制 H202、MTX、CDDP、CCNU所造成之讀框序列位移，係同 時將細胞以NAC及H202、MTX、CDDP、或CCNU處理，並 以與實驗例2相同之讀框序列位移實驗及分析方法，以流式 細胞儀進行分析。 於本實施例中，讀框序列位移實驗及分析方法亦與實 驗例2相同，除了同時加入不同濃度之MAC及H2〇2、MTX、 CDDP、或 CCNU 處理 HCT116-(CA)I3 細胞。 如圖6B所示（圖中亦顯示平均值±標準差），僅以NAC處 理細胞株時，並未觀察到明顯的讀框序列位移現象。當使 用0.5 mM之H202誘發讀框序列位移時，隨著NAC劑量增 加，使用NAC處理之細胞株之讀框序列位移現象也隨之減 201242593 虽同時加入不同濃度之NAC及25 nM之MTX處理 HCTl 16-(CA)n細胞株，以流式細胞儀分析結果係如圖6C 所不（圖中亦顯示平均值士標準差）。當使用25 nm之MTX誘 發忙序列位移時，隨著Nac劑量增加，讀框序列位移現 象也隨之減少（如圖6C所示）。 此外’當同時加入不同濃度之NAC及25 μΜ之CDDP處 理HCT1 16-(CA)l:s細胞株’以流式細胞儀分析結果係如圖 所不（圖中亦顯示平均值土標準差）。當使用25 μΜ之cddP誘 發讀框序列位移時，隨著NAC劑量增加，讀框序列位移現 象也隨之減少（如圖6D所示）。 再者’當同時加入不同濃度之NAC及50 μΜ之CCNU處 理HCT1 1 6-(CΑ)|3細胞株，以流式細胞儀分析結果係如圖6Ε 所示（圖中亦顯示平均值±標準差）。當使用5〇 μ]νι之CCNU誘 發sf框序列位移時，隨著n a C劑量增加，讀框序列位移現 象也隨之減少（如圖6E所示）。 由圖6B至6E的結果顯示，NAC確實可抑制H202或抗癌 藥物所誘發之讀框序列位移現象，故可減少微衛星不穩定 的情形發生3 實驗例8-評估麩胱甘肽對讀框序列位移之影響 於本實施例中’讀框序列位移實驗及分析方法亦與實 驗例2相同，係使用流式細胞儀進行分析，除了同時加入不 同濃度之麩胱甘肽（3丨1^3111丨〇1^)及H2〇2處理HCT1 16-(CA)n 細胞。 201242593 如圖7(圖中亦顯示平均值土標準差）所示，當使用〇 5 πιΜ之4〇2誘發讀框序列位移時，隨著麵胱甘肽劑量增加， 使用麵胱甘肽處理之細胞株之讀框序列位移現象也隨之減 少。 —因此，由實驗例7及8之結果顯示’ NAC或麵胱甘肽確 實可抑制Ηβ2所引發之微衛星不穩定情形，同時nac亦可 抑制抗癌藥物所引發之微衛星不穩定情形。因此，若將NAC 及麩胱甘肽與抗癌藥物一同使用，除了不會影響抗癌藥物 對細胞的殺傷率，更可降低抗癌藥物所導致之微衛星不穩 疋性’進而降低癌症發生率及死亡率。 上述實施例僅係為了方便說明而舉例而已，本發明所 主張之權利範圍自應以申請專利範圍所述為準，而非僅限 於上述貫施例。 【圖式簡單說明】 圖1A係本發明之實驗例1經H202處理之細胞存活率結果 圖。 圖1B係本發明之實驗例1經H2〇2處理之細胞以流式細胞儀 分析讀框序列位移率之結果圖。 圖2A係本發明之實驗例2經MTX處理之細胞存活率結果 圖。 圖2B係本發明之實驗例2經MTX處理之細胞以流式細胞儀 分析讀樞序列位移率之結果圖。 201242593 圖3 A係本發明之實驗例3經CCNU處理之細胞存活率結果 圖。 圖3 B係本發明之實驗例3經CCNU處理之細胞以流式細胞 儀分析讀框序列位移率之結果圖。 圖4八係本發明之實驗例4及5分別經0〇〇卩及5-卩1]處理之細 胞存活率結果圖。 圖4B係本發明之實驗例4及5分別經CDDP及5-FU處理之細 胞以流式細胞儀分析讀框序列位移率之結果圖。 圖5 A係本發明之實驗例6經草酸鉑處理之細胞存活率結果 圖。 圖5B係本發明之實驗例6經草酸鉑處理之細胞以流式細胞 儀分析讀框序列位移率之結果圖。 圖6 A係本發明之實驗例7經NAC處理之細胞存活率結果 圖。 圖6B係本發明之實驗例7經NAC及H202處理之細胞以流式 細胞儀分析讀框序列位移率之結果圖。 圖6C係本發明之實驗例7經NAC及MTX處理之細胞以流式 細胞儀分析讀框序列位移率之結果圖。 圖6D係本發明之實驗例7經NAC及CDDP處理之細胞以流 式細胞儀分析讀框序列位移率之結果圖。 圖6E係本發明之實驗例7經NAC及CCNU處理之細胞以流 式細胞儀分析讀框序列位移率之結果圖。 圖7係本發明之實驗例8經麩胱甘肽及H202處理之細胞以 流式細胞儀分析讀框序列位移率之結果圖。 201242593 【主要元件符號說明】 201242593 序列表 <110〉國立成功大學/ National Cheng Kung University < 120>抑制微衛星不穩定之醫藥組成物及包含其之癌症治療用醫藥組成物 /Pharmaceutical compositions for inhibiting microsatel1ite instability, and pharmaceutical compositions for treating cancer <130> 100-053BP_S4254 <160〉 4 <170〉 Patent In version 3.3 <210〉 1 <21 i> 65 <212> dna <213> An i ficial <220> <223> Art i ficai1 primer for cloning <400> 1 ctggagctca tgcacacaca cacacacaca cacacacagt acgcgtaccg gtcgccacca 60 tggts 65 <210〉 2 <211> 47 <212> DNA <2!3> Artificial <220> <223> Articifial primer for cloning <4(X)> 2 ctggagctca tggatatcat taciagtaac cggtcgccac catggtg 47 <210> 3 201242593 <211> 21 <212> DNA <213> Artificial <22Q> <223> PCR primer for sequencing <400〉 3 gt11 tggcag tacatcaatg g 21 <210〉 4 <211> 20 <212> DNA <213> Artificial <220> <223> PCR primer for sequencing <400〉 4 gtccttatca tcgtcgtctt 20The reaction medium contained MTX at a final concentration of 〇, 5, 10, 25, 50, 100 nM, and 2.5 nM NaOH. Then, the cell viability was analyzed using the same MTT assay as in Experimental Example 1, and the results are shown in Fig. 2A (the average soil standard deviation is also shown in the figure), which showed that the non-lethal dose of MTX cells was about 25 nM. The evaluation of the displacement of the reading frame sequence was carried out by first planting the above HCT1i6_(CA)|3&HCT1 16-(Ν)|ό cell line in a 12-well plate at 1 x 1〇5 cells per well. After one day of planting, the cells were treated at a final concentration of 〇, 63, 12 5, 25 ηΜ2ΜΤχ for three days; then the cells were removed and the cells were washed and placed in the medium for further three days. Then, using the same flow cytometer as in Example 1, the situation of reading the busy sequence displacement in the cells was analyzed, and the results are shown in Fig. 2B (the average soil standard deviation is also shown in the figure), wherein "* indicates the student ^ test p<〇〇〇〇1. As shown in Fig. 2B, the HCT1 16-(CA)n containing the microsatellite sequence has a read sequence shift rate higher than that of the control group HCT116-(N),6; A especially MTX Agent 1 was -5 nM aT, and the frequency of displacement of the reading frame sequence was more pronounced. 201242593 In addition, HCT1W-fA), 3 cell lines were treated with MTX at a final concentration of 25 nM for three days, then the sputum was removed and the cells were washed. 'And then placed in the medium and continued to grow for three days. The above steps were cycled a total of four times, and the DNA in HCT1 16-(CA)丨3 was amplified by PCR, followed by DNA gene sequencing (sequence primer such as SEQ ID) NO: 3, SEQ ID NO: 4) The DNA gene sequencing results showed that after MTX treatment, there was one group of CA repeating units in the microsatellite sequence, that is, the sequencing result was (Ca)|2 Due to the absence of a set of CA repeat units, the RFp reading frame sequence can be shifted to the '攸sf frame outside the frame to the reading frame. According to this, the RF p protein can be expressed again, so the red fluorescence emitted by the RFP fluorescent protein can be observed. This proves that the Μτχ anticancer drug causes microsatellite instability. Experimental Example 3 - Evaluation of CCNU Anticancer Drug Pair Effect of cell viability and reading frame sequence shift The cell viability assay and analytical method of this experimental example was the same as in Experimental Example 2 except for the use of lomustine (1-(2-gasethyl)_3- Cyclohexyl-1- succinyl urea (l_(2_Chloroethyl)-3-cyclohexy丨-1· nitrosourea), CCNU, purchased from Sigma-Adrich) replaces MTX, and the reaction medium contains final concentrations of 〇, 10, 25 ' 50, 75, NUμΜ of CCNU, and 1% of ethanol. As shown in Figure 3, the results are shown in the figure (the average soil standard deviation is also shown in the figure), and the CCNU cell non-lethal dose is about 50 μΜ. The reading sequence shift test and analysis method of the experimental example were also the same as in Experimental Example 2 except that CCNU was used instead of hydrazine, and the reaction medium contained CCNU with a final concentration of 0 '1 2.5, 25 ' 50 μΜ, and 1% ethanol. The experimental results are shown in Figure 3 (also in the figure) Show mean ± standard 201242593 difference) 'where * indicates student t-test p<〇.〇5," indicates p<〇〇〇5, and *** indicates p<0.000 1. As shown in the result of Figure 3B The HCTll6-(CA)n containing the microsatellite sequence has a higher displacement rate of the reading frame than the control group HCT 1 16-(N)l6; and with the increase of the CCNU agent, the reading rate of the reading frame sequence is also followed. increase. This proves that 匸c n u anticancer drugs can cause microsatellite instability. Experimental Example 4 - Evaluation of the effect of CCNU anticancer drugs on cell viability and reading frame sequence shift The cell viability assay and analysis method of this experimental example is the same as in Experimental Example 2 except that cisp丨atin (cis is used) -diamine-dichloroplatinum (1)) (c/5-diammine-dichloroplatinum (1_I)), CDDP purchased from Sigma-Adrich) replaces MTX, and the reaction medium contains final concentrations of 0, 2.5, 5, 10, 20 40 μΜ CDDP, and 0.77 mM NaCl. As shown in the results of Figure 4A (the mean standard deviation is also shown), the non-lethal dose of cddp cells is approximately 20 μΜ. In addition, the reading sequence shift test and analysis method of this experimental example is the same as that of Experimental Example 2 except that CDDP is used instead of hydrazine, and the reaction medium contains CDDP with final concentration of 0, 6.3, 12_5, 25 μΜ, and 0.77 mM. NaCl. The experimental results are shown in Fig. 4Β (the average value of the soil core is also shown in the figure), where * represents the student's t-test p<〇.〇3,** indicates p<〇.〇〇3, and ** * means p<〇.〇〇〇2. As shown in the results of Figure 4B, the hcti16-(CA)I3 containing the microsatellite sequence increased as the CDDP dose increased. This demonstrates that CDDP anticancer drugs can cause microsatellite instability. 201242593 Experimental Example 5 - Evaluation of the effect of 5-FU anticancer drugs on cell viability and reading frame sequence shift The cell viability assay and analytical method of this experimental example was the same as in Experimental Example 2 except that fluorouracil (5-fluorouracil) was used. (5-FU, purchased from Sigma-Adrich) was substituted for MTX, and the reaction medium contained 5-FU at a final concentration of 0, 6.3, 12.5' 25, 50, 100 μΜ, and 0.5. /. DMSO. As shown in the results of Figure 4, the non-lethal dose of 5-FU cells was approximately 20 μΜ. In addition, the reading sequence shift experiment and analysis method of this experimental example are the same as in Experimental Example 2 except that 5-FU is used instead of hydrazine, and the reaction medium contains 5-FU with a final concentration of 0 '2.5, 5, 10 μΜ. And 0.5% DMSO. As shown in the results of Figure 4, in the cells of HCT1 16-(CA)I3 containing the microsatellite sequence, 5-FU increased the rate of displacement of the reading frame sequence. Therefore, it is proved that CDDP anticancer drugs can also cause microsatellite instability. Experimental Example 6 - Evaluation of the effect of oxaliplatin anticancer drugs on cell viability and reading frame sequence shift The cell viability assay and analysis method of this experimental example was the same as in Experimental Example 2 except that oxaliplatin was used instead of MTX. The reaction medium contains platinum oxalate having a final concentration of 〇, 0.125 '0.25, 0.5, and μΜ. As shown in the results in Figure 5 (the mean standard deviation is also shown), the non-lethal dose of oxaliplatin is about 1 μΜ. In addition, the reading sequence shift test and analysis method of this experimental example were also the same as in Experimental Example 2 except that rMtx was taken using oxalic acid platinum, and the reaction medium contained platinum oxalate having a final concentration of 0, 0.25, 0.5, and 1 μM. As shown in Fig. 5β (the mean value ± standard deviation is also shown in the figure), as the dose of oxalic acid platinum increases by 201242593, the displacement rate of the reading frame sequence also increases. Therefore, it has been proved that oxaliplatin anticancer drugs also cause microsatellite instability. Experimental Example 7 - Evaluation of the effect of NAC on cell viability and reading sequence shift The cell viability assay and analysis method of this experimental example was the same as in Experimental Example 2, and N-acety lcysteine (N was added separately). AC) to the medium contains NAC at a final concentration of 0, 1.25, 2.5, 5, 10 mM. As shown by the results in Figure 6A (mean ± standard deviation is also shown), the upper limit of the non-lethal dose of NAC is approximately 5 mM. In addition, NAC will not affect the killing rate of H202 or % anticancer drugs on cells. In addition, it was confirmed by the experimental results of the above Experimental Examples 1 and 2 that H202, MTX, CDDP, and CCNU did increase the displacement rate of the reading frame sequence, which in turn caused an increase in the instability of the microsatellite. To detect whether NAC can inhibit the reading sequence shift caused by H202, MTX, CDDP, and CCNU, the cells were simultaneously treated with NAC and H202, MTX, CDDP, or CCNU, and displaced in the same reading frame sequence as Experimental Example 2. The experimental and analytical methods were analyzed by flow cytometry. In the present example, the reading sequence shift experiment and analysis method were also the same as in Experimental Example 2 except that different concentrations of MAC and H2〇2, MTX, CDDP, or CCNU were added to treat HCT116-(CA)I3 cells. As shown in Fig. 6B (the mean value ± standard deviation is also shown in the figure), when the cell strain was treated only by NAC, no significant reading sequence shift phenomenon was observed. When 0.5 mM H202 was used to induce the reading frame sequence shift, as the NAC dose increased, the reading sequence shift of the cell line treated with NAC was also reduced by 201242593. Although different concentrations of NAC and 25 nM of MTX were added to treat HCT1. The 16-(CA)n cell line was analyzed by flow cytometry as shown in Figure 6C (the mean standard deviation is also shown in the figure). When the 25 nm MTX is used to induce a busy sequence shift, as the Nac dose increases, the displacement of the reading frame sequence is also reduced (as shown in Figure 6C). In addition, 'HCT1 16-(CA) l:s cell line treated with different concentrations of NAC and 25 μΜ of CDDP was analyzed by flow cytometry. The results are shown in the figure (the mean soil standard deviation is also shown in the figure) . When 25 μΜ of cddP is used to induce the reading frame sequence shift, as the NAC dose increases, the reading sequence shift is also reduced (as shown in Figure 6D). Furthermore, 'HCT1 16-(CΑ)|3 cell line was treated with CCNU of different concentrations of NAC and 50 μΜ at the same time, and the results of flow cytometry analysis are shown in Fig. 6Ε (the mean value ± standard is also shown in the figure) difference). When using the CCNU of 5〇 μ]νι to induce the sequence shift of the sf box, as the dose of n a C increases, the displacement of the reading frame sequence also decreases (as shown in Fig. 6E). From the results of Figs. 6B to 6E, it is shown that NAC can inhibit the displacement of the reading frame sequence induced by H202 or anticancer drugs, thereby reducing the occurrence of microsatellite instability. 3 Experimental Example 8 - Evaluation of glutathione reading frame The influence of sequence shift in the present example is the same as that of Experimental Example 2, and the flow cytometry is used for analysis, except that different concentrations of glutathione are added simultaneously (3丨1^3111). HCT1 16-(CA)n cells were treated with 丨〇1^) and H2〇2. 201242593 As shown in Figure 7 (the average soil standard deviation is also shown in the figure), when the reading frame sequence displacement is induced by 4〇2 of 〇5 πιΜ, with the increase of the dose of glutathione, the treatment with glutathione is used. The displacement of the reading sequence of the cell line is also reduced. - Therefore, the results of Experimental Examples 7 and 8 show that 'NAC or glutathione can indeed inhibit microsatellite instability caused by Ηβ2, and nac can also inhibit microsatellite instability caused by anticancer drugs. Therefore, if NAC and glutathione are used together with anticancer drugs, it will not affect the killing rate of anticancer drugs on cells, but also reduce the microsatellite instability caused by anticancer drugs, thereby reducing cancer incidence. Rate and mortality. The above-described embodiments are merely examples for the convenience of the description, and the scope of the claims is intended to be limited to the scope of the claims. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a graph showing the results of cell survival rate of H202 treated in Experimental Example 1 of the present invention. Fig. 1B is a graph showing the results of analyzing the displacement ratio of the reading frame sequence by flow cytometry of the cells treated with H2〇2 in Experimental Example 1 of the present invention. Fig. 2A is a graph showing the results of cell survival of MTX-treated cells of Experimental Example 2 of the present invention. Fig. 2B is a graph showing the results of analyzing the displacement rate of the read-trap sequence by flow cytometry of the MTX-treated cells of Experimental Example 2 of the present invention. 201242593 Fig. 3 A is a graph showing the results of cell survival rate of CCNU-treated Example 3 of the present invention. Fig. 3 B is a graph showing the results of analyzing the displacement ratio of the reading frame sequence by flow cytometry of the CCNU-treated cells of Experimental Example 3 of the present invention. Fig. 4 is a graph showing the results of cell survival of the experimental examples 4 and 5 of the present invention treated with 0 〇〇卩 and 5 卩 1], respectively. Fig. 4B is a graph showing the results of analyzing the displacement ratio of the reading frame sequence by flow cytometry of the cells treated with CDDP and 5-FU in Experimental Examples 4 and 5, respectively. Fig. 5 A is a graph showing the results of cell survival rate of oxalic acid platinum treated in Experimental Example 6 of the present invention. Fig. 5B is a graph showing the results of analyzing the displacement ratio of the reading frame sequence by flow cytometry of the chloroic acid platinum-treated cells of Experimental Example 6 of the present invention. Fig. 6 A is a graph showing the results of cell survival of NAC-treated Example 7 of the present invention. Fig. 6B is a graph showing the results of analyzing the displacement ratio of the reading frame sequence by flow cytometry of the cells treated with NAC and H202 of Experimental Example 7 of the present invention. Fig. 6C is a graph showing the results of analyzing the displacement ratio of the reading frame sequence by flow cytometry of the cells of Example 7 of the present invention treated with NAC and MTX. Fig. 6D is a graph showing the results of analyzing the displacement rate of the reading frame sequence by flow cytometry of the cells of Example 7 of the present invention treated with NAC and CDDP. Fig. 6E is a graph showing the results of analyzing the displacement ratio of the reading frame sequence by flow cytometry of the cells of Example 7 of the present invention treated with NAC and CCNU. Fig. 7 is a graph showing the results of analyzing the displacement rate of the reading frame sequence by flow cytometry of the cells treated with glutathione and H202 in Experimental Example 8 of the present invention. 201242593 [Explanation of main component symbols] 201242593 Sequence Listing <110〉National Success University/National Cheng Kung University <120> Pharmaceutical composition for inhibiting microsatellite instability and pharmaceutical composition for inhibiting it Microsatel1ite instability, and pharmaceutical compositions for treating cancer <130> 100-053BP_S4254 <160> 4 <170> Patent In version 3.3 <210> 1 <21 i> 65 <212> dna <213> An i ficial <220><223> Art i ficai1 primer for cloning <400> 1 ctggagctca tgcacacaca cacacacaca cacacacagt acgcgtaccg gtcgccacca 60 tggts 65 <210> 2 <211> 47 <212> DNA <2!3> Artificial <220><223> Articifial primer for cloning <4(X)> 2 ctggagctca tggatatcat taciagtaac cggtcgccac catggtg 47 <210> 3 201242593 <211> 21 <212> DNA <213> Artificial <22Q><223> PCR primer for sequencing <400> 3 gt11 tggcag Tacatcaatg g 21 <210> 4 <211> 20 <212> DNA <213> Artificial <220><223> PCR primer for sequencing <400> 4 gtccttatca tcgtcgtctt 20