二甲双胍对2型糖尿病合并结直肠癌患者预后的影响及其机制
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摘要
[背景]
目前,我国糖尿病的发病率已经达到9.7%,究其原因,主要是由于人民生活水平的提高,生活方式的迅速改变,进而导致糖尿病的发病率也快速增长。糖尿病除了引起血管及神经系统并发症外,与恶性肿瘤的发生、发展也密切相关。研究显示,2型糖尿病患者发生结直肠癌、肝癌、乳腺癌等恶性肿瘤的风险显著高于一般人群。糖尿病已经成为严重威胁人类健康的疾病之一。
二甲双胍作为一种胰岛素增敏剂,已经广泛应用于2型糖尿病患者的治疗。临床研究显示二甲双胍对肿瘤患者具有一定的保护作用。使用二甲双胍降糖治疗的2型糖尿病患者恶性肿瘤发生的风险性与使用磺脲类降糖药物的患者相比下降了23%,并且肿瘤相关死亡率也显著降低。Diabetologia上的一项队列研究显示,胰岛素或胰岛素促泌剂的治疗与恶性肿瘤风险的增加相关,联用二甲双胍能使其增加的风险显著降低。
二甲双胍的应用与糖尿病患者结直肠癌的发生、发展密切相关。Zhang ZL等通过对108161名2型糖尿病患者的荟萃分析发现,使用二甲双胍治疗可以显著降低结直肠癌的发病率。Lee JH等经过41个月的随访观察发现,患有结直肠癌的糖尿病患者,在结肠癌术后口服二甲双胍与其它降糖方案相比,总的死亡率及结直肠癌相关的死亡率均显著降低。Lee MS等通过队列研究发现2型糖尿病患者结肠癌的发病率是非糖尿病患者的2倍,而口服二甲双胍的糖尿病患者结肠癌的发病率与非糖尿病患者一致。Hosono K等通过临床研究发现,结肠镜检有结肠变性隐窝病灶(癌前病变)的患者口服二甲双胍(250mg/d)1个月后,病灶数目显著减少,同时肠上皮细胞的增殖能力也显著降低。
关于二甲双胍抗癌作用的机制尚不完全清楚,目前的研究多认为与其发挥降糖作用的机制相关。一系列的研究显示,2型糖尿病患者的高血糖及高胰岛素血症状态导致其发生恶性肿瘤的风险性显著高于一般人群。口服二甲双胍可以降低2型糖尿病患者机体内的胰岛素抵抗、改善高胰岛素血症,从而达到抑制肿瘤发生和发展的目的。近年来,体外实验证实二甲双胍可以抑制肿瘤细胞的增殖、促进细胞凋亡,其本身存在着直接的抗肿瘤活性。二甲双胍可以有效激活AMP活化的蛋白激酶(AMPK)及其下游通路,使肝脏糖异生减少,同时增加外周组织对糖的摄取和利用,进而降低血糖。AMP与人体内的丝裂原活化蛋白激酶(MAPK)通路、p53/p21途径、mTOR通路、脂肪酸代谢相关通路、蛋白质代谢相关通路等多种信号传导通路相关。AMPK的活化可以阻断细胞内蛋白质的合成,抑制细胞增殖,促进细胞凋亡。
近来,许多研究显示,二甲双胍具有杀伤肿瘤干细胞的作用。肿瘤干细胞是存在于肿瘤组织中的一小部分具有干细胞性质的细胞群体,均具有自我更新能力和不定向分化潜能,可以导致肿瘤的发生。现认为肿瘤干细胞是肿瘤形成及其不断生长的根源。Vazquez等研究发现,二甲双胍通过降低主要驱动因子(转录因子ZEB1、TWIST1、SNA12)的表达,抑制上皮间质转化(EMT),从而减少乳腺癌肿瘤干细胞的产生。Soritau等通过对III和IV期多形性胶质母细胞瘤患者的手术标本进行组织培养,从中提取的肿瘤细胞具有干细胞的特征,该细胞对常规的化疗药物替莫唑胺(TMZ)耐药,而替莫唑胺合用二甲双胍较单独使用替莫唑胺对肿瘤细胞的抑制率明显增加。Lliopoulos等通过动物实验发现,一定剂量的阿霉素可以抑制裸鼠体内移植的肿瘤组织(包括乳腺癌、前列腺癌和肺癌)的生长和复发,而合用二甲双胍后原剂量1/4的阿霉素对肿瘤组织的抑制效果与之前的剂量相比,无明显差异,考虑是由于二甲双胍能够特异性杀伤对化疗药物阿霉素耐药的肿瘤干细胞。Oliveras等发现二甲双胍可以使乳腺癌细胞MCF-7中抑癌基因let-7a的表达增加18倍,阻碍TGFβ引起的原癌基因181a表达的增加,从而阻滞EMT相关的乳腺癌细胞的自我更新。二甲双胍降低乳腺癌组织内肿瘤干细胞标志CD24的表达,而CD24可以促进肿瘤细胞的转移。卵巢癌及肺癌的研究提示：二甲双胍在抑制肿瘤组织生长、血管生成及转移方面与化疗药物顺铂具有协同作用。
5-氟尿嘧啶(5-FU)是结直肠癌的常规化疗药物,由于它疗效确切,一直处于其他化疗药无法替代的地位,但一些结直肠癌病例对5-FU为主的化疗并不敏感,其对5-FU单药治疗的有效率仅为20%。研究发现,对5-FU耐药的细胞多属于肿瘤干细胞群。二甲双胍对结直肠癌干细胞是否具有特异性杀伤作用,与结直肠癌的常规化疗药物5-FU之间是否具有协同作用,能否改善5-FU耐药,降低5-FU的使用剂量,尚未见相关文献报道。
CD133是结直肠癌干细胞标志之一。2007年,O'Brien CA等在裸鼠体内运用肾被膜移植实验鉴定了人类直肠癌的起始细胞(CC-IC)。纯化实验证实所有的CC-ICs均为CD133+细胞,CD133-细胞组成肿瘤的主要部分但不能促进肿瘤生长。通过有限稀释法发现：CD133+细胞中起始细胞的含量是整个肿瘤细胞中起始细胞含量的200倍。在CD133+细胞群中的CC-ICs在连续的移植中能维持生长、分化以及重建肿瘤的异质性。Wnt/β-catenin信号通路与多种恶性肿瘤的发生发展以及肿瘤细胞耐药性的产生密切相关。肿瘤干细胞内,Wnt/β-catenin通路活化,细胞内β-catenin蛋白表达增加。Wnt/β-catenin通路主要由细胞外因子(Wnt)、跨膜受体(frizzled)、胞质蛋白(β-catenin)及核内转录因子(TCF)等一系列蛋白组成。细胞外因子与受体结合后,通过一系列胞质蛋白间的相互作用,使得细胞浆内β-catenin蛋白含量增加,进入细胞核,与细胞核内的转录因子TCF结合,两者共同作用激活一系列的下游靶基因,包括cylinD1、c-myc等的转录,这些靶基因多数是与细胞增殖及凋亡相关的基因。结肠癌组织中,CD133阳性表达和Wnt/β-catenin通路激活提示肿瘤细胞耐药,预后不良,可以作为肿瘤干细胞的标记。在成骨细胞的研究中发现,二甲双胍能够促进(3-catenin蛋白的磷酸化降解,降低细胞内β-catenin蛋白的水平,进而抑制Wnt/β-catenin通路活性。
患有结直肠癌的糖尿病患者,在结直肠癌术后口服二甲双胍与其它降糖方案相比,总的死亡率及结直肠癌相关的死亡率均显著降低,我们推测,可能与二甲双胍能够特异性的抑制结直肠癌干细胞生长,从而抑制了结直肠癌的复发和转移相关。
本实验通过对南方医院2010年1月-2012年6月期间住院的2型糖尿病合并结直肠癌患者的临床资料进行回顾性分析,探讨了2型糖尿病与结直肠癌的关系以及不同降糖方案对结直肠癌分化程度以及转移的影响；免疫组化检测了使用二甲双胍降糖的2型糖尿病合并结直肠癌患者与未使用二甲双胍的患者相比,手术切除的结直肠癌肿瘤组织内CD133及β-catenin蛋白表达的差异,探讨了二甲双胍是否对结直肠癌干细胞也具有一定的杀伤作用；细胞学实验检测了二甲双胍预处理能否够增强常规化疗药物5-FU对结肠癌细胞SW620的抑制作用及其可能机制,从而为二甲双胍应用于结直肠癌的预防和治疗提供一定的理论依据。
具体研究内容分为以下三部分：
第一章2型糖尿病合并结直肠癌患者的临床特点
[目的]
探讨2型糖尿病合并结直肠癌患者的临床病理特征及降糖药物二甲双胍对肿瘤分化程度及转移的影响。
[方法]
1.采用统一的调查表,记录南方医院2010-1-1至2012-6-30期间住院治疗的病案资料齐全的2型糖尿病合并结直肠癌患者的临床资料,病例共计187例,其中男性患者107例,女性患者80例。
2.调查表的主要内容包括：性别、年龄、2型糖尿病病程、体重指数(BMI)、收缩压(SBP)、舒张压(DBP)、空腹血糖(FPG)、糖化血红蛋白(HbA1c)、尿素氮(BUN)、肌酐(CR)、白蛋白(ALB)、甘油三酯(TG)、总胆固醇(CHOL)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、降糖药的使用情况,结直肠癌病理分级、结直肠癌淋巴结及远处转移情况等。
3.采用SPSS16.0统计软件对实验结果及临床资料进行统计分析,总结结肠癌合并糖尿病患者的临床特点,计量资料用x±sd表示,两组间均数比较采用t检验；计数资料用百分数(%)表示,采用X2检验；P<0.05为差异具有统计学意义。
[结果]
1.187例2型糖尿病合并结直肠癌患者的平均年龄为63.24±11.29岁,平均糖尿病病程为5.25±5.36年,发现结肠癌时才诊断糖尿病的患者占47.06%,患者临床计量资料的单样本t检验显示,除外FBG和HbA1c外,患者的其它资料基本在正常范围内(95%可信区间)。
2.患者结直肠癌的病理类型以中分化腺癌为主,占51.34%,其次为高分腺癌(26.20%),低分化腺癌(7.9%),粘液腺癌(10.70%)。其它病理类型分别为：印戒细胞癌1例；管状绒毛状腺瘤,伴局部癌变1例；病理类型不详伴广泛转移4例。
3.患者淋巴结转移率为25.67%；肝转移16例(8.56%)；肺转移5例(2.67%)；腹腔转移5例(2.67%)；盆腔转移2例(1.07%)；膀胱侵犯2例(1.07%)。
4.按照降糖药物的使用情况,将患者分为二甲双胍组和非二甲双胍组,二甲双胍组患者低分化腺癌比例及远处转移率均显著低于非二甲双胍组,提示二甲双胍组患者结直肠癌的预后较好。
[结论]
1.2型糖尿病合并结直肠癌患者,占同期南方医院住院的结直肠癌患者的12.74%,其中,高达47.06%的患者是在已经诊断结直肠癌的基础上,入院常规检查时才诊断了2型糖尿病,与我国糖尿病患病率高、检出率低的现状是相符合的。患者空腹血糖的平均水平为(9.27±2.58)mmol/L,糖化血红蛋白的平均水平为(7.25±1.65)%,血糖控制差,不利于肿瘤的治疗。
2.研究中的187例2型糖尿病合并结直肠癌患者,初诊结直肠癌时已经合并远处转移者为29例,占15.51%,远处转移率高,预后差；
3.二甲双胍组与非二甲双胍组患者相比,二甲双胍组患者结直肠癌中低分化腺癌比例、远处转移率均低于非二甲双胍组,提示二甲双胍组患者结直肠癌的预后较好。
第二章二甲双胍对结直肠癌组织中CD133及β-catenin蛋白表达的影响
[目的]
初步探讨二甲双胍对结肠癌组织中复发和转移相关的蛋白质CD133及β-catenin表达的影响。
[方法]
1.以2010年1月至2012年6月南方医院普外科住院的2型糖尿病合并结肠癌患者手术切除的肿瘤组织为研究对象,其中二甲双胍组19例(组织病理分级为：高分化者3例,中分化者15例,低分化者1例),非二甲双胍组18例(组织病理分级为：高分化者4例,中分化者11例,低分化者3例)。
2.免疫组织化学方法观察两组标本中CD133及β-catenin蛋白表达的差异。
3.采用SPSS16.0统计软件包进行χ2检验,P<0.05为差异具有统计学意义。
[结果]
1.二甲双胍组患者结直肠癌组织中CD133蛋白阳性表达率为21.1%(4/19),非二甲双胍组阳性表达率为50%(9/18)。非二甲双胍组CD133阳性表达率高于二甲双胍组,但是差异无统计学意义(P=0.065)。
2.二甲双胍组患者结直肠癌组织中β-catenin蛋白阳性表达率为36.8%(7/19),非二甲双胍组阳性表达率为72.2%(13/18),两组间差异具有统计学意义(P=0.031)。
[结论]
1.二甲双胍组与非二甲双胍组患者相比,二甲双胍组患者结直肠癌组织中CD133及β-catenin蛋白的阳性表达率均低于非二甲双胍组,可能是二甲双胍改善结直肠癌患者预后的机制之一。
2.术前未进行放化疗的2型糖尿病合并结直肠癌患者手术标本数量有限,本研究具有一定的局限性,有待进一步扩大样本量深入探讨。
第三章二甲双胍预处理对5-氟尿嘧啶抑制人结肠癌细胞SW620增殖的影响及其机制
[目的]
初步探讨二甲双胍预处理能否增强化疗药物5-FU对人结肠癌细胞SW620的抑制作用及其可能机制。
[方法]
1.以人结肠癌株SW620为实验对象。采用含10%胎牛血清的RPMI1640完全培养基培养,置于37℃、5%CO2的培养箱内。
2.实验分组：
(1)正常对照组(未加药物处理的人结肠癌细胞SW620)；
(2)5-FU干预SW620细胞组,以下简称5-FU组：用含有25mg/ml5-FU的RPMI1640完全培养基培养细胞48小时；
(3)二甲双胍预处理SW620细胞组,以下简称MET+5-FU组：用二甲双胍(5mmol/L)预处理24h后,再用含有25mg/ml5-FU的RPMI1640完全培养基培养细胞48小时。
3.MTT法测定各组细胞的增殖能力。
4.流式细胞仪检测细胞周期、细胞凋亡以及CD133+细胞比例。
5.免疫印迹法(Western Blot)检测各组细胞总蛋白及细胞核蛋白内β-catenin蛋白的表达水平。
6.采用SPSS16.0统计软件进行处理,各指标数据采用均数±标准差(x±sd)表示,各组均数比较采用方差分析,方差齐时,多重比较采用LSD法(Least-significant difference test)检测,方差不齐时,多重比较采用Tambane'sT2法检测。P<0.05被认为差异具有统计学意义。
[结果]
1.二甲双胍预处理增强5-FU对细胞增殖的抑制作用MTT法观察三种细胞的增殖情况：24h,5-FU组细胞的OD值与MET+5-FU组相比差异没有统计学意义；48h和72h,5-FU组细胞的OD值低于MET+5-FU组,两组间的差异具有统计学意义(P<0.05)。
2.二甲双胍预处理降低细胞周期S期细胞比例
正常对照组、5-FU组、MET+5-FU组细胞周期S期细胞比例分别为(43.06±2.75)%；(39.62±0.88)%；(24.14±6.01)%,三组细胞间的差异具有统计学意义(F=20.562,P=0.002),MET+5-FU组S期细胞比例显著低于5-FU组(P=0.003)。
3.二甲双胍预处理增强5-FU促进细胞凋亡的作用
(1)正常对照组、5-FU组、MET+5-FU组细胞早期凋亡率分别为(1.333±0.351)%,(1.767±0.611)%,(3.500±1.082)%,三组细胞早期凋亡率的差异具有统计学意义(F=7.098,P=0.026),SW620组与5-FU组相比差异不具有统计学意义(P=0.503),5-FU组与MET+5-FU组相比具有显著差异(P=0.029)；
(2)正常对照组、5-FU组、MET+5-FU组细胞晚期凋亡率分别为(3.667±0.252)%,(9.467±2.003)%,(18.233±1.159)%,三组细胞晚期凋亡率的差异具有统计学意义(F=81.728,P=0.006),各组间的差异均具有统计学意义(P<0.05)。
4.二甲双胍预处理降低细胞内CD133+细胞比例
正常对照组、5-FU组、MET+5-FU组细胞内CD133+细胞比例分别为(27.63±2.58)%,(21.30±1.40)%,(15.13±3.43)%,三组细胞间CD133+细胞比例的差异具有统计学意义(F=17.254,P=0.003),各组间的差异均具有统计学意义(P<0.05)。
5.二甲双胍预处理降低细胞总蛋白及细胞核蛋白内β-catenin蛋白含量
(1)正常对照组、5-FU组、MET+5-FU组细胞总蛋白内β-catenin蛋白的相对表达量分别为0.936±0.005,0.884±0.017,0.463±0.061,三组细胞总蛋白中β-catenin蛋白表达量的差异具有统计学意义(F=82.531,P=0.000)；
(2)正常对照组、5-FU组、MET+5-FU组细胞核蛋白内β-catenin蛋白的相对表达量分别为1.890±0.242,1.760±0.342,1.163±0.242,三组细胞核蛋白中β-catenin蛋白表达量的差异具有统计学意义(F=5.774,P=0.040)；
(3) MET+5-FU组细胞总蛋白及细胞核蛋白内β-catenin蛋白的表达均显著低于5-FU组(P<0.05),5-FU组与SW620组之间的差异均不具有统计学意义(P>0.05)。
[结论]
1.二甲双胍预处理增强了5-FU对结肠癌细胞SW620增殖的抑制作用,细胞周期S期细胞比例显著降低,细胞凋亡率增加。
2.二甲双胍预处理增强了5-FU对结肠癌细胞SW620中CD133+细胞的杀伤作用,抑制了细胞内Wnt/β-catenin通路活性。
3.二甲双胍与结直肠癌常规化疗药物5-FU具有协同作用,这可能是术后口服二甲双胍降糖的2型糖尿病合并结直肠癌患者肿瘤相关死亡率降低的原因之一,二甲双胍有可能作为一种新的辅助治疗药物,提高结直肠癌患者对5-FU的敏感性,改善化疗效果,降低5-FU的使用剂量,减少对其对正常细胞的损害。
[Background]
With the improvement of people's living standard and the rapid changes of lifestyle, the incidence of diabetes mellitus grows rapidly, which has reached9.7%. In addition to cause damage to macrovascular, microvascular and neuropathy, diabetes mellitus is also closely related to the occurrence and development of malignant tuomours. The risk of malignant tumors of T2DM patients, such as colorectal cancer, liver cancer, breast cancer and so on, was significantly higher than that of the general people. Diabetes mellitus is looming as one of the greatest threats to public health in the21st century.
As an insulin sensitizer, metformin has been widely used in the treatment of patients with T2DM. A series of clinical studies had shown protective effects of metformin on cancer patients with T2DM. Compared to T2DM patients treated with sulfonylurea drugs, the incidence of malignant tumors of patients treated with metformin decreased by23%. The tumor related mortality of T2DM patients treated with metformin was significantly lower than that of the patients treated with sulfonylureas or insulin. A cohort study showed in Diabetologia indicated that insulin or insulin secretagogues treatment is associated with increased cancer risk, and the combination of metformin significantly reduced the cancer risk.
Epidemiologic evidence suggests that diabetes treated with metformin have reduced colorectal cancer risk. A meta-analysis included108,161patients with type2diabetes indicated that metformin treatment was associated with a signicantly lower risk of colorectal neoplasm. A41-month follow-up study indicated that metformin use in CRC patients with diabetes is associated with lower risk of CRC-specific and overall mortality. A representative population prospective cohort study of800,000individuals showed that Type2diabetes increases and metformin reduces colorectal cancer incidences in Taiwanese. A randomized clinical trial among nondiabetic patients with a follow-up of1-month demonstrated that the number of aberrant crypt foci decreased significantly in the metformin group but not in the non-metformin group. However, the inhibitory effect of metformin on the tumor cells cultured in vitro, indicating a directed anti-tumor activity of this drug. Studies have shown that metformin can effectly activate AMPK and its downstream pathways, decrease hepatic gluconeogenesis, increase the glucose uptake and utilization of the peripheral tissue, thus reducing the blood glucose. AMPK is closely related to a variety of signal transduction pathways in vivo, such as mTOR pathway, p53/p21pathway, mitogen-activated protein kinase (MAPK) pathway, and the metabolism pathway of fatty acids and protein. The activation of AMPK can block the protein synthesis within the cell, causing the cell growth stagnation, promoting apoptosis.
However, the mechanisms underlying this antineoplastic potential of metformin for cancer are not yet completely understood. Current studies suggest that it is related to its hypoglycemic effect. Hyperinsulinemia status and insulin metabolism increased the incidence of malignant tumor of T2DM patients. As a insulin sensitizer, metformin can reduce the body's insulin resistance, ameliorate hyperinsulinemia, which can suppress the occurrence and development of the tumor.
Resently, many studies indicate that metformin can selectively kills cancer stem cells of several types of cancers, such as those of breast, prostate, ovarian, thyroid, and liver. The cancer stem cell (CSC) theory posits that both primary and metastatic tumors develop from a small population of cancer cells possessing the characteristics of self-renewal and pluripotency and are responsible for initiation and maintenance of tumors. CSCs have the capacities of selfrenewal, differentiation, and inducing cell proliferation, invasion and metastasis. Chemoresistance of CSCs is one of the reasons for tumor recurrence. Vazquez found that metformin can inhibit epithelial mesenchymal transition (EMT) by reducing the expression of the main drving factors (transcriotion factor ZEB1, TWIS1, SNA12), thereby reducing the generation of breast cancer stem cells. Soritau extracted the tumor cell that have the characteristics of stem cell for the tumor tissue of pleomorphic glioblastoma. They found those cells are resistant to the conventional chemotherapy drug, temozolomide (TMZ). The proliferation capacity of the cells treated with metformin and TMZ was significantly reduced than those of treated with TMZ alone. In animal experiments, metformin reduced the dose of doxorubicin by3/4. The researchers considered that is due to capability of metformin can specifically killing cancer stem cells, which are resistant to chemotherapy drugs. Metformin reduced the cancer stem cell marker CD24expression in breast cancer tissues. CD24can promote the tumor cell metastasis. Researches of ovarian and lung cancer indicated that metformin has synergistic effects with chemotherapy drug cisplatin in inhibiting of tumor growth, angiogenesis and metastasis.
Globally, Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females.5-Fluorouracil (5-FU) is a chemotherapeutic drug used worldwide in the treatment of metastatic colorectal cancer. Over the past50years, despite its many advantages, clinical applications have been greatly limited due to drug resistance. The overall response rate for advanced colorectal cancer of5-FU alone is still only10-15%, and the combination of5-FU with other anti-tumor drugs has merely improved the response rates to40-50%. A series of studies indicated that the cells resistant to5-FU are belonged to cancer stem cell population. It has not been reported that whether metformin can selectively kill cancer stem cells of colorectal cancer, and potentiate the anti-cancer effect of5-FU, reduce the dose of5-FU.
CD133is one of the molecular landmarks of colorectal cancer stem cells. O'Brien use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments made it clear that all CC-ICs were CD133+cells, and the CD133-cells that comprised the majority of the tumour were unable to initiate tumour growth. Wnt signaling pathway regulates self renewal of cells and the formation of multiple organs. The disorder of this pathway induces tumor formation. Classical Wnt signaling pathway is mediated by β-catenin, β-catenin regulates the transcription of Wnt targeting genes and promotes cell proliferation. Activation of endogenous WNT/β-catenin signaling pathway results in a gross disruption of crypt architecture and a disproportionate expansion of CD133expression in the crypt base, and then induces the formation of high grade intraepithelial neoplasia and crypt adenoma, indicating the potential relation between CD133+colorectal CSCs and Wnt/β-catenin signaling pathway.
Metformin treatment significantly reduced the total mortality and CRC related mortality of the diabetic CRC patients. So we speculate it is associated with that metformin can specifically inhibiting the growth of CRC stem cells, thereby inhibiting tumor recurrence and metastasis.
In this study a retrospective review of the medical records of187colorectal cancer patients with type2diabetes was performed. We have investigated the relationship between metformin and clinicopathologic characteristics of diabetic colorectal cancer. Meanwhile we detected the relationship between metformin therapy and the positive expression rate of CD133and β-catenin proteins by immunohistochemistry, to find that whether metformin can be able to kill the cancer stem cells of colorectal cancer, and reduce chemotherapy drug resistance. Cytological experiments were conducted to detect that whether metformin pretreatment can potentiate the anti-cancer effects of5-FU on human colorectal cancer cell line sw620. We hope that our study can provide some theoretical basis for metformin used in the prevention and treatment of colorectal cancer. Three Specific contents are as follows:
Chapter1Clinical characteristics of T2DM with colorectal cancer patients
[Objective]
To analyze the clinicopathologic characteristics of the T2DM with colorectal cancer patients, and the influence of metformin on the degree of tumor differentiation and metastasis.
[Methods]
1. There were187cases of hospitalized T2DM with colorectal cancer patients from January2010to June2012, then we recorded their datas by unified questionnaire.
2. The content of questionnaire is as follows:age, gender, blood pressure, body mass index (BMI), duration of diabetes, fasting plasma glucose (FPG), hemoglobin Alc levels(HbAlc), blood urea nitrogen (BUN), creatinine (CR), albumin (ALB), trilyceride (TG), cholesterol (CHOL), high-density lipoprotein (HDL), very-low-density lipoprotein (VLDL) and the pathological data (tumour histology, lymph node condition, distant metastasis rate).
3. Statistical analysis:Measurement datas are measured by mean±standard deviation. Statistical analysis were performed using the SPSS16.0software Package. Independent sample t test is used to analyze the measurement datas,X2test is used to analyze the enumeration datas, P<0.05is considered statistically significant.
[Results]
1. The average age and average DM duration of the187T2DM with colorectal cancer patients are63.24±11.29years old and5.25+5.36years respectively.47.06%of these patients were diagnosed with diabetes mellitus after their colorectal cancer diagnosis. One-sample t-test indicated that the patients'clinical measurement data were basically within the normal range(95%confidence interval), except for FBG and HbA1c.
2. Moderately differentiated adenocarcinoma is the main pathological type of the colorectal cancer, accounting for51.34%, followed by well differentiated adenocarcinoma (26.10%), poorly differentiated adenocarcinoma (7.9%), mucinous adenocarcinoma (10.70%). Other types:signet ring cell carcinoma; tubular villous adenoma with focal carcinogenesis.
3. The lymph node metastasis rate was25.67%;16cases with liver metastasis (8.56%);5cases with lung metastasis (2.67%);5cases with peritoneal metastasis (2.67%);2cases with pelvic metastasis (1.07%);2cases with bladder assault (1.07%).
4. The patients were divided into two groups metformin group and non-metformin group, according to whether or not they were taking metformin when colorectal cancer was diagnosised. The proportion of patients with poorly differentiated adenocarcinoma and distant metastasis rate were significantly lower in metformin group than those in non-metformin group. Metformin therapy was associated with a better prognosis of colorectal cancer, it should be the first choice of anti-hyperglycaemic drug of T2DM patients.
[Conclusions]
1. T2DM with colorectal cancer patients accounting for12.74of the colorectal cancer patients hospitalized in Nanfang Hospital during the same period, and47.06%of these patients were diagnosed with diabetes mellitus after their colorectal cancer diagnosis. This is consistent with the status of the high prevalence and low detection of diabetes mellitus in our country. The average FPG and HbA1c are (9.27±2.58) mmol/L and (7.25±1.65)%respectively, and the poor glycemic control is not conductive to the treatment of the tumor.
2.29cases of the T2DM with colorectal cancer patients had already merged distant metastasis when the cancer was diagnosed, indicating a high distant metastasis rate and poor prognosis.
3. The proportion of patients with poorly differentiated adenocarcinoma and distant metastasis rate were significantly lower in metformin group than those in non-metformin group. Metformin therapy was associated with a better prognosis of colorectal cancer.
Chapter2The influence of metformin on the expression of CD133and β-catenin proteins in the colorectal cancer tissues
[Objective]
To explore the influence of metformin on the expression of the proteins associated with tumor recurrence and metastasis, CD133and β-catenin, in the colorectal cancer tissues.
[Methods]
1. Take the tumor tissues of the T2DM with colorectal cancer patients as the research object.19of the cases from metformin group (histological grade:well differentiated,3cases; moderately differentiated,15cases; poorly differentiated,1case), and18of the cases from non-metformin group (histological grade:well differentiated,4cases; moderately differentiated,11cases; poorly differentiated,3case).
2. The positive expression rate of CD133and β-catenin proteins of the two groups was detected by immunohistochemistry.
3. Using SPSS16.0as statistical analysis. X2test is used to analyze the enumeration datas, P<0.05is considered statistically significant.
[Results]
1. In metformin group of patients,4cases of19(21.1%) contained more than10%CD133-positive tumor cells. In non-metformin-group of patients,9cases of18(50.0%) specimens contained more than10%CD133-positive tumor cells. However, there was no statistically significant difference of the positive expression rate of CD133protein between the two groups(P=0.065).
2. A significant difference of the positive expression rate of β-catenin protein was found between the two groups (β=0.031). In metformin group of patients,7cases of19(36.8%) specimens nuclear accumulation ofβ-catenin was seen in more than10%of the tumor cells. In non-metformin-group of patients,13cases of18(72.2%) specimens nuclear accumulation of β-catenin was seen in more than10%of the tumor cells.
[Conclusions]
1. Metformin therapy was associated a reduced positive expression rate of CD133and β-catenin proteins, indicating that metformin might be able to kill the cancer stem cells of colorectal cancer, and reduce chemotherapy drug resistance. This maybe one of the mechanisms by which metformin improve the prognosis of T2DM with colorectal cancer patients.
2. The number of cases in this study is limited, further researches are warranted.
Chapter3Metformin pre-treatment potentiate the anti-cancer effect of5-fluorouracil on human colon cancer cell line SW620
[Objective]
To investigate whether the metformin pre-treatment can potentiate the anti-cancer effect of the chemotherapy drug5-fluorouracil (5-FU) on human colon cancer cell line SW620, and to explore the possible mechanisms for this effect.
[Methods]
1. The experiment object was human colorectal cancer cell line SW620. SW620cells were cultured in RPMI1640medium supplemented with10%fetal bovine serum (FBS),100ug/ml penicillin and100ug/ml streptomycin and incubated in a5%CO2incubator at37℃.
2. The cells were divided into3groups according to different intervention:
(1) The normal control group (Control group);
(2)5-FU intervene SW620cells for48h (5-FU group);
(3) Pretreat with metformin for24h before cells were intervened with5-FU for48h (MET+5-FU group).
3. The proliferation of the cells was assessed by MTT assay
4. The cell cycle, apoptosis rate and the sensitivity of CD133+cells changes were analyzed by flow cytometry.
5. The expression of β-catenin protein was detected by Western blot.
6. Statistical analysis:All data are expressed as means±sd. Statistical analysis were performed using the SPSS16.0software Package. Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, P<0.05was considered as significant.
[Results]
1. Effect of MET pre-treatment on5-FU-induced cell growth inhibition
The cells with MET pre-treated, grew significantly more slowly than the cells treated with5-FU alone (5-FU alone vs MET+5-FU, P<0.05).
2. Flow Cytometry Analysis for Cell Cycle
Cell cycle analysis by flow cytometry reveals that the proportion of cells in the G0/G1, S, and G2/M phases. Pre-treatment of SW620cells with MET reduced the proportion of cells in S phase, compared with cells treated with5-FU alone (percentage of cells in S-phase:39.62%vs24.14%for5-FU alone vs MET+5-FU, respectively, P<0.05).
3. MET potentiated the apoptosis of SW620cells induced by5-FU
The percentage of apoptotic cells were significantly increased by pretreatment with MET followed by5-FU (Early apoptosis rate:1.77%vs3.50%; Late apoptosis rate:9.47%vs18.23%;5-FU alone vs MET+5-FU, respectively,P<0.05).
4. Flow Cytometry Analysis for expression of CD133
We examined the expression of CD133in SW620cell line by flow cytometry.27.63%of SW620cells expressed the membrane antigen CD133. Then, the SW620cells were treated for48h with5-FU with MET pre-treatment for24h or not. Flow cytometric analysis revealed that after the treatment, the expression of CD133was reduced (21.3%vs15.1%;5-FU alone vs MET+5-FU; P<0.05).
5. Western Blot Analysis for expression of β-catenin
Treatment of SW620cells with5-FU for48h alone resulted in decreased expression of β-catenin. Compared with control group, this decrease was not significant (5-FU alone vs control:P>0.05). Pre-treatment of cells with MET potentiated the down-regulation of β-catenin expression induced by5-FU (5-FU alone vs MET+5-FU; P<0.05).
[Conclusions]
1.5-FU inhibited the growth of SW620cells, and the inhibition was potentiated by MET pre-treatment, via the reduction of the proportion of cells in S phase and apoptosis induction.
2. The potentiation of the inhibitory effect of5-FU by MET was dependent on the increase of the sensitivity of CD133+cells to5-FU and the reduction of β-catenin protein levels in SW620cells, which is related with the attenuating of Wnt/β-catenin signaling pathway activity.
3. We conclude that the combination therapy with MET might be a novel therapeutic modality to improve efficacy of5-FU based chemotherapy.
目前,我国糖尿病的发病率已经达到9.7%,究其原因,主要是由于人民生活水平的提高,生活方式的迅速改变,进而导致糖尿病的发病率也快速增长。糖尿病除了引起血管及神经系统并发症外,与恶性肿瘤的发生、发展也密切相关。研究显示,2型糖尿病患者发生结直肠癌、肝癌、乳腺癌等恶性肿瘤的风险显著高于一般人群。糖尿病已经成为严重威胁人类健康的疾病之一。
二甲双胍作为一种胰岛素增敏剂,已经广泛应用于2型糖尿病患者的治疗。临床研究显示二甲双胍对肿瘤患者具有一定的保护作用。使用二甲双胍降糖治疗的2型糖尿病患者恶性肿瘤发生的风险性与使用磺脲类降糖药物的患者相比下降了23%,并且肿瘤相关死亡率也显著降低。Diabetologia上的一项队列研究显示,胰岛素或胰岛素促泌剂的治疗与恶性肿瘤风险的增加相关,联用二甲双胍能使其增加的风险显著降低。
二甲双胍的应用与糖尿病患者结直肠癌的发生、发展密切相关。Zhang ZL等通过对108161名2型糖尿病患者的荟萃分析发现,使用二甲双胍治疗可以显著降低结直肠癌的发病率。Lee JH等经过41个月的随访观察发现,患有结直肠癌的糖尿病患者,在结肠癌术后口服二甲双胍与其它降糖方案相比,总的死亡率及结直肠癌相关的死亡率均显著降低。Lee MS等通过队列研究发现2型糖尿病患者结肠癌的发病率是非糖尿病患者的2倍,而口服二甲双胍的糖尿病患者结肠癌的发病率与非糖尿病患者一致。Hosono K等通过临床研究发现,结肠镜检有结肠变性隐窝病灶(癌前病变)的患者口服二甲双胍(250mg/d)1个月后,病灶数目显著减少,同时肠上皮细胞的增殖能力也显著降低。
关于二甲双胍抗癌作用的机制尚不完全清楚,目前的研究多认为与其发挥降糖作用的机制相关。一系列的研究显示,2型糖尿病患者的高血糖及高胰岛素血症状态导致其发生恶性肿瘤的风险性显著高于一般人群。口服二甲双胍可以降低2型糖尿病患者机体内的胰岛素抵抗、改善高胰岛素血症,从而达到抑制肿瘤发生和发展的目的。近年来,体外实验证实二甲双胍可以抑制肿瘤细胞的增殖、促进细胞凋亡,其本身存在着直接的抗肿瘤活性。二甲双胍可以有效激活AMP活化的蛋白激酶(AMPK)及其下游通路,使肝脏糖异生减少,同时增加外周组织对糖的摄取和利用,进而降低血糖。AMP与人体内的丝裂原活化蛋白激酶(MAPK)通路、p53/p21途径、mTOR通路、脂肪酸代谢相关通路、蛋白质代谢相关通路等多种信号传导通路相关。AMPK的活化可以阻断细胞内蛋白质的合成,抑制细胞增殖,促进细胞凋亡。
近来,许多研究显示,二甲双胍具有杀伤肿瘤干细胞的作用。肿瘤干细胞是存在于肿瘤组织中的一小部分具有干细胞性质的细胞群体,均具有自我更新能力和不定向分化潜能,可以导致肿瘤的发生。现认为肿瘤干细胞是肿瘤形成及其不断生长的根源。Vazquez等研究发现,二甲双胍通过降低主要驱动因子(转录因子ZEB1、TWIST1、SNA12)的表达,抑制上皮间质转化(EMT),从而减少乳腺癌肿瘤干细胞的产生。Soritau等通过对III和IV期多形性胶质母细胞瘤患者的手术标本进行组织培养,从中提取的肿瘤细胞具有干细胞的特征,该细胞对常规的化疗药物替莫唑胺(TMZ)耐药,而替莫唑胺合用二甲双胍较单独使用替莫唑胺对肿瘤细胞的抑制率明显增加。Lliopoulos等通过动物实验发现,一定剂量的阿霉素可以抑制裸鼠体内移植的肿瘤组织(包括乳腺癌、前列腺癌和肺癌)的生长和复发,而合用二甲双胍后原剂量1/4的阿霉素对肿瘤组织的抑制效果与之前的剂量相比,无明显差异,考虑是由于二甲双胍能够特异性杀伤对化疗药物阿霉素耐药的肿瘤干细胞。Oliveras等发现二甲双胍可以使乳腺癌细胞MCF-7中抑癌基因let-7a的表达增加18倍,阻碍TGFβ引起的原癌基因181a表达的增加,从而阻滞EMT相关的乳腺癌细胞的自我更新。二甲双胍降低乳腺癌组织内肿瘤干细胞标志CD24的表达,而CD24可以促进肿瘤细胞的转移。卵巢癌及肺癌的研究提示：二甲双胍在抑制肿瘤组织生长、血管生成及转移方面与化疗药物顺铂具有协同作用。
5-氟尿嘧啶(5-FU)是结直肠癌的常规化疗药物,由于它疗效确切,一直处于其他化疗药无法替代的地位,但一些结直肠癌病例对5-FU为主的化疗并不敏感,其对5-FU单药治疗的有效率仅为20%。研究发现,对5-FU耐药的细胞多属于肿瘤干细胞群。二甲双胍对结直肠癌干细胞是否具有特异性杀伤作用,与结直肠癌的常规化疗药物5-FU之间是否具有协同作用,能否改善5-FU耐药,降低5-FU的使用剂量,尚未见相关文献报道。
CD133是结直肠癌干细胞标志之一。2007年,O'Brien CA等在裸鼠体内运用肾被膜移植实验鉴定了人类直肠癌的起始细胞(CC-IC)。纯化实验证实所有的CC-ICs均为CD133+细胞,CD133-细胞组成肿瘤的主要部分但不能促进肿瘤生长。通过有限稀释法发现：CD133+细胞中起始细胞的含量是整个肿瘤细胞中起始细胞含量的200倍。在CD133+细胞群中的CC-ICs在连续的移植中能维持生长、分化以及重建肿瘤的异质性。Wnt/β-catenin信号通路与多种恶性肿瘤的发生发展以及肿瘤细胞耐药性的产生密切相关。肿瘤干细胞内,Wnt/β-catenin通路活化,细胞内β-catenin蛋白表达增加。Wnt/β-catenin通路主要由细胞外因子(Wnt)、跨膜受体(frizzled)、胞质蛋白(β-catenin)及核内转录因子(TCF)等一系列蛋白组成。细胞外因子与受体结合后,通过一系列胞质蛋白间的相互作用,使得细胞浆内β-catenin蛋白含量增加,进入细胞核,与细胞核内的转录因子TCF结合,两者共同作用激活一系列的下游靶基因,包括cylinD1、c-myc等的转录,这些靶基因多数是与细胞增殖及凋亡相关的基因。结肠癌组织中,CD133阳性表达和Wnt/β-catenin通路激活提示肿瘤细胞耐药,预后不良,可以作为肿瘤干细胞的标记。在成骨细胞的研究中发现,二甲双胍能够促进(3-catenin蛋白的磷酸化降解,降低细胞内β-catenin蛋白的水平,进而抑制Wnt/β-catenin通路活性。
患有结直肠癌的糖尿病患者,在结直肠癌术后口服二甲双胍与其它降糖方案相比,总的死亡率及结直肠癌相关的死亡率均显著降低,我们推测,可能与二甲双胍能够特异性的抑制结直肠癌干细胞生长,从而抑制了结直肠癌的复发和转移相关。
本实验通过对南方医院2010年1月-2012年6月期间住院的2型糖尿病合并结直肠癌患者的临床资料进行回顾性分析,探讨了2型糖尿病与结直肠癌的关系以及不同降糖方案对结直肠癌分化程度以及转移的影响；免疫组化检测了使用二甲双胍降糖的2型糖尿病合并结直肠癌患者与未使用二甲双胍的患者相比,手术切除的结直肠癌肿瘤组织内CD133及β-catenin蛋白表达的差异,探讨了二甲双胍是否对结直肠癌干细胞也具有一定的杀伤作用；细胞学实验检测了二甲双胍预处理能否够增强常规化疗药物5-FU对结肠癌细胞SW620的抑制作用及其可能机制,从而为二甲双胍应用于结直肠癌的预防和治疗提供一定的理论依据。
具体研究内容分为以下三部分：
第一章2型糖尿病合并结直肠癌患者的临床特点
[目的]
探讨2型糖尿病合并结直肠癌患者的临床病理特征及降糖药物二甲双胍对肿瘤分化程度及转移的影响。
[方法]
1.采用统一的调查表,记录南方医院2010-1-1至2012-6-30期间住院治疗的病案资料齐全的2型糖尿病合并结直肠癌患者的临床资料,病例共计187例,其中男性患者107例,女性患者80例。
2.调查表的主要内容包括：性别、年龄、2型糖尿病病程、体重指数(BMI)、收缩压(SBP)、舒张压(DBP)、空腹血糖(FPG)、糖化血红蛋白(HbA1c)、尿素氮(BUN)、肌酐(CR)、白蛋白(ALB)、甘油三酯(TG)、总胆固醇(CHOL)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、降糖药的使用情况,结直肠癌病理分级、结直肠癌淋巴结及远处转移情况等。
3.采用SPSS16.0统计软件对实验结果及临床资料进行统计分析,总结结肠癌合并糖尿病患者的临床特点,计量资料用x±sd表示,两组间均数比较采用t检验；计数资料用百分数(%)表示,采用X2检验；P<0.05为差异具有统计学意义。
[结果]
1.187例2型糖尿病合并结直肠癌患者的平均年龄为63.24±11.29岁,平均糖尿病病程为5.25±5.36年,发现结肠癌时才诊断糖尿病的患者占47.06%,患者临床计量资料的单样本t检验显示,除外FBG和HbA1c外,患者的其它资料基本在正常范围内(95%可信区间)。
2.患者结直肠癌的病理类型以中分化腺癌为主,占51.34%,其次为高分腺癌(26.20%),低分化腺癌(7.9%),粘液腺癌(10.70%)。其它病理类型分别为：印戒细胞癌1例；管状绒毛状腺瘤,伴局部癌变1例；病理类型不详伴广泛转移4例。
3.患者淋巴结转移率为25.67%；肝转移16例(8.56%)；肺转移5例(2.67%)；腹腔转移5例(2.67%)；盆腔转移2例(1.07%)；膀胱侵犯2例(1.07%)。
4.按照降糖药物的使用情况,将患者分为二甲双胍组和非二甲双胍组,二甲双胍组患者低分化腺癌比例及远处转移率均显著低于非二甲双胍组,提示二甲双胍组患者结直肠癌的预后较好。
[结论]
1.2型糖尿病合并结直肠癌患者,占同期南方医院住院的结直肠癌患者的12.74%,其中,高达47.06%的患者是在已经诊断结直肠癌的基础上,入院常规检查时才诊断了2型糖尿病,与我国糖尿病患病率高、检出率低的现状是相符合的。患者空腹血糖的平均水平为(9.27±2.58)mmol/L,糖化血红蛋白的平均水平为(7.25±1.65)%,血糖控制差,不利于肿瘤的治疗。
2.研究中的187例2型糖尿病合并结直肠癌患者,初诊结直肠癌时已经合并远处转移者为29例,占15.51%,远处转移率高,预后差；
3.二甲双胍组与非二甲双胍组患者相比,二甲双胍组患者结直肠癌中低分化腺癌比例、远处转移率均低于非二甲双胍组,提示二甲双胍组患者结直肠癌的预后较好。
第二章二甲双胍对结直肠癌组织中CD133及β-catenin蛋白表达的影响
[目的]
初步探讨二甲双胍对结肠癌组织中复发和转移相关的蛋白质CD133及β-catenin表达的影响。
[方法]
1.以2010年1月至2012年6月南方医院普外科住院的2型糖尿病合并结肠癌患者手术切除的肿瘤组织为研究对象,其中二甲双胍组19例(组织病理分级为：高分化者3例,中分化者15例,低分化者1例),非二甲双胍组18例(组织病理分级为：高分化者4例,中分化者11例,低分化者3例)。
2.免疫组织化学方法观察两组标本中CD133及β-catenin蛋白表达的差异。
3.采用SPSS16.0统计软件包进行χ2检验,P<0.05为差异具有统计学意义。
[结果]
1.二甲双胍组患者结直肠癌组织中CD133蛋白阳性表达率为21.1%(4/19),非二甲双胍组阳性表达率为50%(9/18)。非二甲双胍组CD133阳性表达率高于二甲双胍组,但是差异无统计学意义(P=0.065)。
2.二甲双胍组患者结直肠癌组织中β-catenin蛋白阳性表达率为36.8%(7/19),非二甲双胍组阳性表达率为72.2%(13/18),两组间差异具有统计学意义(P=0.031)。
[结论]
1.二甲双胍组与非二甲双胍组患者相比,二甲双胍组患者结直肠癌组织中CD133及β-catenin蛋白的阳性表达率均低于非二甲双胍组,可能是二甲双胍改善结直肠癌患者预后的机制之一。
2.术前未进行放化疗的2型糖尿病合并结直肠癌患者手术标本数量有限,本研究具有一定的局限性,有待进一步扩大样本量深入探讨。
第三章二甲双胍预处理对5-氟尿嘧啶抑制人结肠癌细胞SW620增殖的影响及其机制
[目的]
初步探讨二甲双胍预处理能否增强化疗药物5-FU对人结肠癌细胞SW620的抑制作用及其可能机制。
[方法]
1.以人结肠癌株SW620为实验对象。采用含10%胎牛血清的RPMI1640完全培养基培养,置于37℃、5%CO2的培养箱内。
2.实验分组：
(1)正常对照组(未加药物处理的人结肠癌细胞SW620)；
(2)5-FU干预SW620细胞组,以下简称5-FU组：用含有25mg/ml5-FU的RPMI1640完全培养基培养细胞48小时；
(3)二甲双胍预处理SW620细胞组,以下简称MET+5-FU组：用二甲双胍(5mmol/L)预处理24h后,再用含有25mg/ml5-FU的RPMI1640完全培养基培养细胞48小时。
3.MTT法测定各组细胞的增殖能力。
4.流式细胞仪检测细胞周期、细胞凋亡以及CD133+细胞比例。
5.免疫印迹法(Western Blot)检测各组细胞总蛋白及细胞核蛋白内β-catenin蛋白的表达水平。
6.采用SPSS16.0统计软件进行处理,各指标数据采用均数±标准差(x±sd)表示,各组均数比较采用方差分析,方差齐时,多重比较采用LSD法(Least-significant difference test)检测,方差不齐时,多重比较采用Tambane'sT2法检测。P<0.05被认为差异具有统计学意义。
[结果]
1.二甲双胍预处理增强5-FU对细胞增殖的抑制作用MTT法观察三种细胞的增殖情况：24h,5-FU组细胞的OD值与MET+5-FU组相比差异没有统计学意义；48h和72h,5-FU组细胞的OD值低于MET+5-FU组,两组间的差异具有统计学意义(P<0.05)。
2.二甲双胍预处理降低细胞周期S期细胞比例
正常对照组、5-FU组、MET+5-FU组细胞周期S期细胞比例分别为(43.06±2.75)%；(39.62±0.88)%；(24.14±6.01)%,三组细胞间的差异具有统计学意义(F=20.562,P=0.002),MET+5-FU组S期细胞比例显著低于5-FU组(P=0.003)。
3.二甲双胍预处理增强5-FU促进细胞凋亡的作用
(1)正常对照组、5-FU组、MET+5-FU组细胞早期凋亡率分别为(1.333±0.351)%,(1.767±0.611)%,(3.500±1.082)%,三组细胞早期凋亡率的差异具有统计学意义(F=7.098,P=0.026),SW620组与5-FU组相比差异不具有统计学意义(P=0.503),5-FU组与MET+5-FU组相比具有显著差异(P=0.029)；
(2)正常对照组、5-FU组、MET+5-FU组细胞晚期凋亡率分别为(3.667±0.252)%,(9.467±2.003)%,(18.233±1.159)%,三组细胞晚期凋亡率的差异具有统计学意义(F=81.728,P=0.006),各组间的差异均具有统计学意义(P<0.05)。
4.二甲双胍预处理降低细胞内CD133+细胞比例
正常对照组、5-FU组、MET+5-FU组细胞内CD133+细胞比例分别为(27.63±2.58)%,(21.30±1.40)%,(15.13±3.43)%,三组细胞间CD133+细胞比例的差异具有统计学意义(F=17.254,P=0.003),各组间的差异均具有统计学意义(P<0.05)。
5.二甲双胍预处理降低细胞总蛋白及细胞核蛋白内β-catenin蛋白含量
(1)正常对照组、5-FU组、MET+5-FU组细胞总蛋白内β-catenin蛋白的相对表达量分别为0.936±0.005,0.884±0.017,0.463±0.061,三组细胞总蛋白中β-catenin蛋白表达量的差异具有统计学意义(F=82.531,P=0.000)；
(2)正常对照组、5-FU组、MET+5-FU组细胞核蛋白内β-catenin蛋白的相对表达量分别为1.890±0.242,1.760±0.342,1.163±0.242,三组细胞核蛋白中β-catenin蛋白表达量的差异具有统计学意义(F=5.774,P=0.040)；
(3) MET+5-FU组细胞总蛋白及细胞核蛋白内β-catenin蛋白的表达均显著低于5-FU组(P<0.05),5-FU组与SW620组之间的差异均不具有统计学意义(P>0.05)。
[结论]
1.二甲双胍预处理增强了5-FU对结肠癌细胞SW620增殖的抑制作用,细胞周期S期细胞比例显著降低,细胞凋亡率增加。
2.二甲双胍预处理增强了5-FU对结肠癌细胞SW620中CD133+细胞的杀伤作用,抑制了细胞内Wnt/β-catenin通路活性。
3.二甲双胍与结直肠癌常规化疗药物5-FU具有协同作用,这可能是术后口服二甲双胍降糖的2型糖尿病合并结直肠癌患者肿瘤相关死亡率降低的原因之一,二甲双胍有可能作为一种新的辅助治疗药物,提高结直肠癌患者对5-FU的敏感性,改善化疗效果,降低5-FU的使用剂量,减少对其对正常细胞的损害。
[Background]
With the improvement of people's living standard and the rapid changes of lifestyle, the incidence of diabetes mellitus grows rapidly, which has reached9.7%. In addition to cause damage to macrovascular, microvascular and neuropathy, diabetes mellitus is also closely related to the occurrence and development of malignant tuomours. The risk of malignant tumors of T2DM patients, such as colorectal cancer, liver cancer, breast cancer and so on, was significantly higher than that of the general people. Diabetes mellitus is looming as one of the greatest threats to public health in the21st century.
As an insulin sensitizer, metformin has been widely used in the treatment of patients with T2DM. A series of clinical studies had shown protective effects of metformin on cancer patients with T2DM. Compared to T2DM patients treated with sulfonylurea drugs, the incidence of malignant tumors of patients treated with metformin decreased by23%. The tumor related mortality of T2DM patients treated with metformin was significantly lower than that of the patients treated with sulfonylureas or insulin. A cohort study showed in Diabetologia indicated that insulin or insulin secretagogues treatment is associated with increased cancer risk, and the combination of metformin significantly reduced the cancer risk.
Epidemiologic evidence suggests that diabetes treated with metformin have reduced colorectal cancer risk. A meta-analysis included108,161patients with type2diabetes indicated that metformin treatment was associated with a signicantly lower risk of colorectal neoplasm. A41-month follow-up study indicated that metformin use in CRC patients with diabetes is associated with lower risk of CRC-specific and overall mortality. A representative population prospective cohort study of800,000individuals showed that Type2diabetes increases and metformin reduces colorectal cancer incidences in Taiwanese. A randomized clinical trial among nondiabetic patients with a follow-up of1-month demonstrated that the number of aberrant crypt foci decreased significantly in the metformin group but not in the non-metformin group. However, the inhibitory effect of metformin on the tumor cells cultured in vitro, indicating a directed anti-tumor activity of this drug. Studies have shown that metformin can effectly activate AMPK and its downstream pathways, decrease hepatic gluconeogenesis, increase the glucose uptake and utilization of the peripheral tissue, thus reducing the blood glucose. AMPK is closely related to a variety of signal transduction pathways in vivo, such as mTOR pathway, p53/p21pathway, mitogen-activated protein kinase (MAPK) pathway, and the metabolism pathway of fatty acids and protein. The activation of AMPK can block the protein synthesis within the cell, causing the cell growth stagnation, promoting apoptosis.
However, the mechanisms underlying this antineoplastic potential of metformin for cancer are not yet completely understood. Current studies suggest that it is related to its hypoglycemic effect. Hyperinsulinemia status and insulin metabolism increased the incidence of malignant tumor of T2DM patients. As a insulin sensitizer, metformin can reduce the body's insulin resistance, ameliorate hyperinsulinemia, which can suppress the occurrence and development of the tumor.
Resently, many studies indicate that metformin can selectively kills cancer stem cells of several types of cancers, such as those of breast, prostate, ovarian, thyroid, and liver. The cancer stem cell (CSC) theory posits that both primary and metastatic tumors develop from a small population of cancer cells possessing the characteristics of self-renewal and pluripotency and are responsible for initiation and maintenance of tumors. CSCs have the capacities of selfrenewal, differentiation, and inducing cell proliferation, invasion and metastasis. Chemoresistance of CSCs is one of the reasons for tumor recurrence. Vazquez found that metformin can inhibit epithelial mesenchymal transition (EMT) by reducing the expression of the main drving factors (transcriotion factor ZEB1, TWIS1, SNA12), thereby reducing the generation of breast cancer stem cells. Soritau extracted the tumor cell that have the characteristics of stem cell for the tumor tissue of pleomorphic glioblastoma. They found those cells are resistant to the conventional chemotherapy drug, temozolomide (TMZ). The proliferation capacity of the cells treated with metformin and TMZ was significantly reduced than those of treated with TMZ alone. In animal experiments, metformin reduced the dose of doxorubicin by3/4. The researchers considered that is due to capability of metformin can specifically killing cancer stem cells, which are resistant to chemotherapy drugs. Metformin reduced the cancer stem cell marker CD24expression in breast cancer tissues. CD24can promote the tumor cell metastasis. Researches of ovarian and lung cancer indicated that metformin has synergistic effects with chemotherapy drug cisplatin in inhibiting of tumor growth, angiogenesis and metastasis.
Globally, Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females.5-Fluorouracil (5-FU) is a chemotherapeutic drug used worldwide in the treatment of metastatic colorectal cancer. Over the past50years, despite its many advantages, clinical applications have been greatly limited due to drug resistance. The overall response rate for advanced colorectal cancer of5-FU alone is still only10-15%, and the combination of5-FU with other anti-tumor drugs has merely improved the response rates to40-50%. A series of studies indicated that the cells resistant to5-FU are belonged to cancer stem cell population. It has not been reported that whether metformin can selectively kill cancer stem cells of colorectal cancer, and potentiate the anti-cancer effect of5-FU, reduce the dose of5-FU.
CD133is one of the molecular landmarks of colorectal cancer stem cells. O'Brien use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments made it clear that all CC-ICs were CD133+cells, and the CD133-cells that comprised the majority of the tumour were unable to initiate tumour growth. Wnt signaling pathway regulates self renewal of cells and the formation of multiple organs. The disorder of this pathway induces tumor formation. Classical Wnt signaling pathway is mediated by β-catenin, β-catenin regulates the transcription of Wnt targeting genes and promotes cell proliferation. Activation of endogenous WNT/β-catenin signaling pathway results in a gross disruption of crypt architecture and a disproportionate expansion of CD133expression in the crypt base, and then induces the formation of high grade intraepithelial neoplasia and crypt adenoma, indicating the potential relation between CD133+colorectal CSCs and Wnt/β-catenin signaling pathway.
Metformin treatment significantly reduced the total mortality and CRC related mortality of the diabetic CRC patients. So we speculate it is associated with that metformin can specifically inhibiting the growth of CRC stem cells, thereby inhibiting tumor recurrence and metastasis.
In this study a retrospective review of the medical records of187colorectal cancer patients with type2diabetes was performed. We have investigated the relationship between metformin and clinicopathologic characteristics of diabetic colorectal cancer. Meanwhile we detected the relationship between metformin therapy and the positive expression rate of CD133and β-catenin proteins by immunohistochemistry, to find that whether metformin can be able to kill the cancer stem cells of colorectal cancer, and reduce chemotherapy drug resistance. Cytological experiments were conducted to detect that whether metformin pretreatment can potentiate the anti-cancer effects of5-FU on human colorectal cancer cell line sw620. We hope that our study can provide some theoretical basis for metformin used in the prevention and treatment of colorectal cancer. Three Specific contents are as follows:
Chapter1Clinical characteristics of T2DM with colorectal cancer patients
[Objective]
To analyze the clinicopathologic characteristics of the T2DM with colorectal cancer patients, and the influence of metformin on the degree of tumor differentiation and metastasis.
[Methods]
1. There were187cases of hospitalized T2DM with colorectal cancer patients from January2010to June2012, then we recorded their datas by unified questionnaire.
2. The content of questionnaire is as follows:age, gender, blood pressure, body mass index (BMI), duration of diabetes, fasting plasma glucose (FPG), hemoglobin Alc levels(HbAlc), blood urea nitrogen (BUN), creatinine (CR), albumin (ALB), trilyceride (TG), cholesterol (CHOL), high-density lipoprotein (HDL), very-low-density lipoprotein (VLDL) and the pathological data (tumour histology, lymph node condition, distant metastasis rate).
3. Statistical analysis:Measurement datas are measured by mean±standard deviation. Statistical analysis were performed using the SPSS16.0software Package. Independent sample t test is used to analyze the measurement datas,X2test is used to analyze the enumeration datas, P<0.05is considered statistically significant.
[Results]
1. The average age and average DM duration of the187T2DM with colorectal cancer patients are63.24±11.29years old and5.25+5.36years respectively.47.06%of these patients were diagnosed with diabetes mellitus after their colorectal cancer diagnosis. One-sample t-test indicated that the patients'clinical measurement data were basically within the normal range(95%confidence interval), except for FBG and HbA1c.
2. Moderately differentiated adenocarcinoma is the main pathological type of the colorectal cancer, accounting for51.34%, followed by well differentiated adenocarcinoma (26.10%), poorly differentiated adenocarcinoma (7.9%), mucinous adenocarcinoma (10.70%). Other types:signet ring cell carcinoma; tubular villous adenoma with focal carcinogenesis.
3. The lymph node metastasis rate was25.67%;16cases with liver metastasis (8.56%);5cases with lung metastasis (2.67%);5cases with peritoneal metastasis (2.67%);2cases with pelvic metastasis (1.07%);2cases with bladder assault (1.07%).
4. The patients were divided into two groups metformin group and non-metformin group, according to whether or not they were taking metformin when colorectal cancer was diagnosised. The proportion of patients with poorly differentiated adenocarcinoma and distant metastasis rate were significantly lower in metformin group than those in non-metformin group. Metformin therapy was associated with a better prognosis of colorectal cancer, it should be the first choice of anti-hyperglycaemic drug of T2DM patients.
[Conclusions]
1. T2DM with colorectal cancer patients accounting for12.74of the colorectal cancer patients hospitalized in Nanfang Hospital during the same period, and47.06%of these patients were diagnosed with diabetes mellitus after their colorectal cancer diagnosis. This is consistent with the status of the high prevalence and low detection of diabetes mellitus in our country. The average FPG and HbA1c are (9.27±2.58) mmol/L and (7.25±1.65)%respectively, and the poor glycemic control is not conductive to the treatment of the tumor.
2.29cases of the T2DM with colorectal cancer patients had already merged distant metastasis when the cancer was diagnosed, indicating a high distant metastasis rate and poor prognosis.
3. The proportion of patients with poorly differentiated adenocarcinoma and distant metastasis rate were significantly lower in metformin group than those in non-metformin group. Metformin therapy was associated with a better prognosis of colorectal cancer.
Chapter2The influence of metformin on the expression of CD133and β-catenin proteins in the colorectal cancer tissues
[Objective]
To explore the influence of metformin on the expression of the proteins associated with tumor recurrence and metastasis, CD133and β-catenin, in the colorectal cancer tissues.
[Methods]
1. Take the tumor tissues of the T2DM with colorectal cancer patients as the research object.19of the cases from metformin group (histological grade:well differentiated,3cases; moderately differentiated,15cases; poorly differentiated,1case), and18of the cases from non-metformin group (histological grade:well differentiated,4cases; moderately differentiated,11cases; poorly differentiated,3case).
2. The positive expression rate of CD133and β-catenin proteins of the two groups was detected by immunohistochemistry.
3. Using SPSS16.0as statistical analysis. X2test is used to analyze the enumeration datas, P<0.05is considered statistically significant.
[Results]
1. In metformin group of patients,4cases of19(21.1%) contained more than10%CD133-positive tumor cells. In non-metformin-group of patients,9cases of18(50.0%) specimens contained more than10%CD133-positive tumor cells. However, there was no statistically significant difference of the positive expression rate of CD133protein between the two groups(P=0.065).
2. A significant difference of the positive expression rate of β-catenin protein was found between the two groups (β=0.031). In metformin group of patients,7cases of19(36.8%) specimens nuclear accumulation ofβ-catenin was seen in more than10%of the tumor cells. In non-metformin-group of patients,13cases of18(72.2%) specimens nuclear accumulation of β-catenin was seen in more than10%of the tumor cells.
[Conclusions]
1. Metformin therapy was associated a reduced positive expression rate of CD133and β-catenin proteins, indicating that metformin might be able to kill the cancer stem cells of colorectal cancer, and reduce chemotherapy drug resistance. This maybe one of the mechanisms by which metformin improve the prognosis of T2DM with colorectal cancer patients.
2. The number of cases in this study is limited, further researches are warranted.
Chapter3Metformin pre-treatment potentiate the anti-cancer effect of5-fluorouracil on human colon cancer cell line SW620
[Objective]
To investigate whether the metformin pre-treatment can potentiate the anti-cancer effect of the chemotherapy drug5-fluorouracil (5-FU) on human colon cancer cell line SW620, and to explore the possible mechanisms for this effect.
[Methods]
1. The experiment object was human colorectal cancer cell line SW620. SW620cells were cultured in RPMI1640medium supplemented with10%fetal bovine serum (FBS),100ug/ml penicillin and100ug/ml streptomycin and incubated in a5%CO2incubator at37℃.
2. The cells were divided into3groups according to different intervention:
(1) The normal control group (Control group);
(2)5-FU intervene SW620cells for48h (5-FU group);
(3) Pretreat with metformin for24h before cells were intervened with5-FU for48h (MET+5-FU group).
3. The proliferation of the cells was assessed by MTT assay
4. The cell cycle, apoptosis rate and the sensitivity of CD133+cells changes were analyzed by flow cytometry.
5. The expression of β-catenin protein was detected by Western blot.
6. Statistical analysis:All data are expressed as means±sd. Statistical analysis were performed using the SPSS16.0software Package. Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, P<0.05was considered as significant.
[Results]
1. Effect of MET pre-treatment on5-FU-induced cell growth inhibition
The cells with MET pre-treated, grew significantly more slowly than the cells treated with5-FU alone (5-FU alone vs MET+5-FU, P<0.05).
2. Flow Cytometry Analysis for Cell Cycle
Cell cycle analysis by flow cytometry reveals that the proportion of cells in the G0/G1, S, and G2/M phases. Pre-treatment of SW620cells with MET reduced the proportion of cells in S phase, compared with cells treated with5-FU alone (percentage of cells in S-phase:39.62%vs24.14%for5-FU alone vs MET+5-FU, respectively, P<0.05).
3. MET potentiated the apoptosis of SW620cells induced by5-FU
The percentage of apoptotic cells were significantly increased by pretreatment with MET followed by5-FU (Early apoptosis rate:1.77%vs3.50%; Late apoptosis rate:9.47%vs18.23%;5-FU alone vs MET+5-FU, respectively,P<0.05).
4. Flow Cytometry Analysis for expression of CD133
We examined the expression of CD133in SW620cell line by flow cytometry.27.63%of SW620cells expressed the membrane antigen CD133. Then, the SW620cells were treated for48h with5-FU with MET pre-treatment for24h or not. Flow cytometric analysis revealed that after the treatment, the expression of CD133was reduced (21.3%vs15.1%;5-FU alone vs MET+5-FU; P<0.05).
5. Western Blot Analysis for expression of β-catenin
Treatment of SW620cells with5-FU for48h alone resulted in decreased expression of β-catenin. Compared with control group, this decrease was not significant (5-FU alone vs control:P>0.05). Pre-treatment of cells with MET potentiated the down-regulation of β-catenin expression induced by5-FU (5-FU alone vs MET+5-FU; P<0.05).
[Conclusions]
1.5-FU inhibited the growth of SW620cells, and the inhibition was potentiated by MET pre-treatment, via the reduction of the proportion of cells in S phase and apoptosis induction.
2. The potentiation of the inhibitory effect of5-FU by MET was dependent on the increase of the sensitivity of CD133+cells to5-FU and the reduction of β-catenin protein levels in SW620cells, which is related with the attenuating of Wnt/β-catenin signaling pathway activity.
3. We conclude that the combination therapy with MET might be a novel therapeutic modality to improve efficacy of5-FU based chemotherapy.
引文
[1]Wild S, Roglic G, Green A, et al. Global prevalence of diabetes:estimates for the year 2000 and projections for 2030[J]. Diabetes Care,2004,27:1047-1053.
[2]Yang WY, Lu JM, Weng JP, et al. Prevalence of diabetes among men and women in China[J]. N Engl J Med,2010,362(25):2425-2426.
[3]Inoue M, Iwasaki M, Otani T, et al. Diabetes mellitus and the risk of cancer: results from a large-scale population-based cohort study in Japan[J]. Arch Intern Med,2006,166(17):1871-1877.
[4]Larsson SC, Mantzoros CS, Wolk A. Diabetes mellitus and risk of breast cancer: a meta-analysis[J]. Int J Cancer,2007,121:856-862.
[5]Larsson SC, Orsini N, Wolk A (2005) Diabetes mellitus and risk of colorectal cancer:a meta-analysis. J Natl Cancer Inst 97:1679-1687
[6]Tsugane S, Inoue M. Insulin resistance and cancer:epidemiological evidence[J]. Cancer Sci,2010,101:1073-1079.
[7]Nicolucci A. Epidemiological aspects of neoplasms in diabetes[J]. Acta Diabetol. 2010,47:87-95.
[8]Vgneri P, Frasca F, Sciacca L, et al. Diabetes and cancer[J]. Endocr Relat Cancer, 2009,16:1103-1123.
[9]Park SK, Park MK, Suk JH,et al.Cause-of-death trends for diabetes mellitus over 10 years[J]. Korean Diabetes J,2009,33:65-72.
[10]ALeksandrovski YA. Molecular mechanism of the cross-impact of pathological processes in combined diabetes and cancer. Research and clinical aspects [J]. Biochemistry(Mosc),2002,67(2):1329-1346.
[11]Heuson JC, Legros N. Influence of insulin deprivation on growth of the 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats subjected to alloxan diabetes and food restriction[J]. Cancer Res,1972,32:226-232.
[12]Heuson JC, Legros N, Heimann R. Influence of insulin administration on growth of the 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in intact ophorectomized, and hypophysectomized rats[J]. Cancer Res 1972;32:233-238.
[13]Calle EE, Kaaks R. Overweight, obesity and cancer:epidemiologicalevidence and proposed mechanisms[J]. Nat Rev Cancer,2004,4:579-591.
[14]Papa V, Pezzino V, Costantino A, et al. Elevated insulin receptor content in human breast cancer[J]. J Clin Invest,1990,86:1503-1510.
[15]Frasca F, Pandini G, Scalia P,et al. Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells[J]. Mol Cell Biol,1999,19:3278-3288.
[16]Yang YY, Hemessy S, Lewis JD. Type 2 diabetes mellitus and the risk of colorectal cancer [j]. Clin Gastroenterol Hepalol,2005,3(6):587-594.
[17]Larsson SC, Orsini N, Wolk A. Diabetes mellitus and risk of colorectal cancer:a meta-analysis[J]. J Natl Cancer,2005,97(22):1679-1687.
[18]胡水清,汤哲,张玫.糖尿病与结直肠癌患病危险关系的调查分析[J].世界华人消化杂志2007；15(1)：88-91.
[19]Bowker SL, Majumdar SR, Veugelers P, et al. Increased cancer-related mortality for patients with type 2 diabetes who use sulfonylureas or insulin[J]. Diabetes Care.2006,29:254-258.
[20]Eurich DT, McAlister FA, Blackburn DF et al. Benefits and harms of antidiabetic agents in patients with diabetes and heartfailure:systematic review[J]. BMJ, 2007,335:497-452.
[21]Margolis DJ, Hoffstad O, Strom BL (2008) Association between serious ischemic cardiac outcomes and medications used to treat diabetes. Pharmacoepidemiol Drug Saf 17:753-759
[22]Currie CK, Poole CD, Gale EAM. The influence of glucose-lowering therapies on cancer risk in type2 diabetes[J]. Diabetologia,2009,52:1766-1777.
[23]Evans JM, Donnelly LA, Emslie-Smith AM, et al. Metformin and reduced risk of cancer in diabetic patients[J]. BMJ,2005,330:1304-1305.
[24]Rayter Z, leicester RJ. Adjuvant chemotherapy for colorectal canner[J]. Ann R Coll Surg,1995,77 (4):81-84.
[25]Yang YX, Hennessy S, Lewis JD. Insulin therapy and colorectal cancer risk among type 2 diabetes mellitus patients[J]. Gastroenterology,2004,127: 1044-1050.
[26]Zhang ZL, Zheng ZL, Kan H, et al. Reduced risk of colorectal cancer with metformin therapy in patient with type2 diabetes:a meta-analysis[J]. Diabetes Care,2011,34(10):2323-2328.
[27]Lee JH, Kim TI, Jeon SM, et al. The effects of metformin on the survival of colorectal cancer patients with diabetes mellitus[J]. Int J Cancer,2012,131(3): 752-759.
[28]Lee MS, Hsu CC, Wahlqvist ML, et al. Type 2 diabetes increases and metformin reduces total, colorectal, liver and pancreatic cancer incidences in Taiwanese:a representative population prospective cohort study of 800,000 individuals[J]. BMC Cancer,2011,11:20-29.
[29]李明,顾晋.中国结直肠癌20年来发展模式的变化趋势[J].中华胃肠外科杂志,2004,7(3)：214.
[30]Guastamacchia E, Resta F, Triggiani V, et al. Evidence for a putative relationship between type 2 diabetes and neoplasia with particular reference to breast cancer:role of hormones,growth factors and specific receptors[J]. Immune Endocr Metabol Disord,2004,4:59-66.
[31]Hosono K, Endo H, Takahashi H,et al. Metformin suppresses coloreetal aberrant crypt foci in a short-term clinical trial[J]. Cancer Prev Res,2010,3(9): 1077-1083.
[32]Hosono K, Endo H, Takahashi H, et al. Metformin suppresses azoxymethane indused colorectal aberrant crypt foci by activating AMP-activated protein kinase[J]. Mol Carcinog,2010,49(7):662-671.
[33]Algire C, Amrein L, Aakikhani M, et al. Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase[J]. Endocr Relat Cancer,2010,17(2): 351-360.
[34]Zakikhani M, Aowling RJ, Sonenberg N, et al. The effects of adiponectin and metformin on prostate and colon neoplasia involve activation of AMPK-activated protein kinase[J]. Cancer Prev Res,2008,1(5):369-375.
[35]Tomimoto A, Endo H, Sugiyama M, et al. Metformin suppresses intestinal polyp growth in ApcMin/+mice[J]. Cancer Sci,2008,99(11):2136-2141.
[36]Stumvoll M, Nurjhan N, Perriello G, et al. Metabolic effects of metformin in non-insulin-dependent diabetes mellitus[J]. N Engl J Med,1995,333:550-554.
[37]Hundal HS, Ramlal T, Reyes R, et al. Cellular mechanism of metformin action involves glucose transporter translocation from an intracellular pool to the plasma membrane in L6 muscle cells [J]. Endocrinology,1992,131:1165-1173.
[38]Hadad SM, Fleming S, Thompson AM. Targeting AMPK:A new therapeutic opportunity in breast cancer[J]. Critical Reviews in Oncology Hematology,2008, 67:1-7.
[39]Capano M, Crompton M. Bax translocates to mitochondria of heart cells during simulated ischaemia:involvement of AMP-activated and p38 mitogen-activated protein kinases[J]. Biochem J,2006,395:57-64.
[40]Jones RG, Plas DR, Kubek S, et al. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint[J]. Mol Cell,2005,18:283-293.
[41]Campas C, Lopez JM, Santidrian AF, et al. Acadesine activates AMPK and induces apoptosis in B-cell chronic lymphocytic leukemia cells but not in T lymphocytes[J]. Blood,2003,101:3674-3680.
[42]Kefas BA, Heimberg H, Vaulont S, et al. AICA-riboside induces apoptosis of pancreatic beta cells through stimulation of AMP-activated protein kinase[J]. Diabetologia,2003,46:250-254.
[43]Donnenberg VS, Donnenberg AD. Multiple drug resistanceincancer revisited:the cancer stem cell hypothesis[J]. J Clin Pharmacol,2005,45(8):872-877
[44]Vazquez MA, Oliveras FC, Cufi S, et al. Metformin regulates breast cancer stem cell ontogeny by transcriptional regulation of the epithelial-mesenchymal transition (EMT) status[J]. Cell Cycle,2010,9(18):3807-3814.
[45]Soritau O, Tomuleasa C, Aldea M, et al. Metformin plus temozolomide-based chemotherapy as adjuvant treatment for WHO grade Ⅲ and Ⅳ malignant gliomas[J]. J BUON,2011,16(2):282-289.
[46]Lliopoulos D, Hirsch HA, Struhl K. Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types[J]. Cancer Res,2011,71(9):3196-3201.
[47]Oliveras FC, Cufi S, Vazquez MA, et al. Micro(mi)RNA expression profile of breast cancer epithelial cells treated with the anti-diabetic drug metformin: induction of the tumor suppressor miRNA let-7aand suppression of the TGFβ-induced oncomiR miRNA-181α[J]. Cell Cycle,2011,10(7):1144-1151.
[48]Vazquez MA, Oliveras FC, Cufi S, et al. The anti-diabetic drug metformin suppresses the metastasis-associated protein CD24 in DNA-MB-468 triple-negative breast cancer cells[J]. Oncol Rep,2011,25(1):135-140.
[49]Rattan R, Graham RP, Maguire JL, et al. Metformin suppresses ovarian cancer growth and metastasis with enhancement of cisplatin cytotoxicity in vivo[J]. Neoplasis,2011,13(5):483-491.
[50]Wu N, Du C, Gu H, et al. Metformin induces apoptosis of lung cancer through activating JNK/p38 MAPK pathway and GADD153[J]. Neoplasma,2011,58(6): 482-490.
[51]Hadad S, Iwamoto T, Jordan L, et al. Evidence for biological effects of metformin in operable breast cancer:a preoperative, window-of-opportunity, randomized trial[J]. Breast Cancer Res Treat,2011,128(3):783-94.
[52]Mader RM. Resistance to 5-fluorouracil[J].Gen Pharmacil,1998,31(5):661-665.
[53]Choi S, Ku JL. Resistance of colorectal cancer cells to radiation and 5-FU is associated with MELK expression[J]. Biochem Biophys Res Commun,2011, 412(2):207-213.
[54]Fan X, Ouyang N, Teng H, et al. Isolation and characterization of spheroid cells from the HT29 colon cancer cell line[J]. Int J Colorectal Dis,2011,26(10): 1279-1285.
[55]Bijnsdorp IV, Peters GL, Temmink OH, et al. Differential activation of cell death and autophagy results in an increased cytotoxic potential for trifluorothymidine compared to 5-fluorouracil in colon cancer cells[J]. Int J Cancer,2010,126(10): 2457-2468.
[56]Dallas NA, Xia L, Fan F, et al. Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-1 receptor inhibition[J]. Cancer Res,2009,69(5):1951-1957.
[57]O'Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodifient mice[J]. Nature,2007,445(7123): 106-110.
[58]Tadoro M, Francipane MG, Medema JP, et al. Colon cancer stem cells:promise of targeted therapy [J]. Gastroenterology,2010,138(6):2151-2162.
[59]Coco C, Zannoni GF, Caredda E, et al. Increased expression of CD 133 and reduced dystroglycan expression are strong predictors of poor outcome in colon cancer patients[J]. J Exp Clin Cancer Res,2012,31(1):71-81.
[60]李宝秀 张晓实 刘国龙.CD133在局部晚期结肠癌肿瘤细胞中的异常表达及意义[J].广东医学,2010,31(13)：1678-1681.
[61]Xu W, Lin H, Zhang Y, et al. Compound Kushen injection suppresses human breast cancer stem-like cells by down-regulating the canonical Wnt/beta-catenin pathway[J]. J Exp Clin Cancer Res,2011,30(1):103-108.
[62]Bitaret N, Bandres E, Boni V, et al. MicroRNA-451 is involved in the self-renewal, tumorigenicity, and chemoresistance of colorectal cancer stem cell[J]. Stem Cells,2011,29(11):1661-1671.
[63]Peng C, Zhang X, Yu H, et al. Wnt5a as a predictor in poor clinical outcome of patients and a mediator in chemoresistance of ovarian cancer [J]. Int J Gynecol Cancer,2011,21(2):280-288.
[64]Maugeri SM, Vigneri P, De MR. Cancer stem cells and chemosensitivity[J]. Clin Cancer Res,2011,17(15):4942-4947.
[65]Noda T, Nagano H, Takemasa L, et al. Activation of Wnt/beta-catenin signaling pathway induces chemoresistance to interferon-alpha/5-fluorouracil combination therapy for hepatocellular carcinoma[J]. Br J Cancer,2009,100(10):1647-1658.
[66]Horst D, Kriegl L, Engel J, et al. CD 133 and nuclear beta-catenin:the marker combination to detect high risk cases of low stage colorectal cancer[J]. Eur J Cancer,2009,45(11):2034-2040.
[67]Takatani T, Minagawa M, Takatani R, et al. AMP-activated protein kinase attenuates Wnt/β-catenin signaling in human osteoblastic Saos-2 cells[J]. Mol Cell Endocrinol,2011,339(1-2):114-119.
[68]Kawamoto H, Yuasa T, Kubota Y, et al. Characteristics of CD133(+) human colon cancer SW620 cells[J]. Cell Transplant,2010,19(6):857-864.
[69]Liao Y, Hu X, Huang X, et al. Quantitative analyses of CD 133 expression facilitate researches on tumor stem cells[J]. Biol Pharm Bull,2010,33(5): 738-742.
[70]Chai H, Liu M, Tian R, et al. miR-20a targets BNIP2 and contributes chemotherapeutic resistance in colorectal adenocarcinoma SW480 and SW620 cell lines[J]. Acta Biochim Biophys Sin,2011,43(3):217-225.
[71]钱荣立.关于糖尿病的新诊标准及分型[J].中国糖尿病杂志,2000,8(1)：5-6.
[72]李明,顾晋.中国结直肠癌20年来发展模式的变化趋势[J].中华胃肠外科杂志,2004,7(3)：214.
[73]梁文霞,郎云星.2型糖尿病合并恶性肿瘤22例分析[J].实用糖尿病杂志,2008,4(5)：23-24.
[74]Atlanta, GA. Global Cancer Facts & Figures[M]. American Cancer Society,2007: 12.
[75]Chen S, Song X, Chen Z,et al. CD133 Expression and the Prognosis of Colorectal Cancer:A Systematic Review and Meta-Analysis[J]. PLoS One.2013; 8(2):1371-1381.
[76]Dowling RJ, Zakikhani M, Fantus IG, et al. Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells [J]. Cancer Res,2007,67(22):10804-10812.
[77]Zakikhani M, Dowling R, Fantus IG,et al.Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells [J]. Cancer Res,2006; 66(21):10269-10273.
[78]Algire C, Zakikhani M, Blouin MJ,et al. Metformin attenuates the stimulatory effect of a high-energy diet on in vivo H59 carcinoma growth[J]. Endocr Relat Cancer.2008,15(3):833-839.
[79]Ben Sahra I, Laurent K, Loubat A, et al. The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level [J]. Oncogene,2008,27(25):3576-3586.
[80]Gotlieb WH, Saumet J, Beauchamp MC et al. In vitro metformin anti-neoplastic activity in epithelial ovarian cancer[J]. Gynecol Oncol,2008,110(2):246-250.
[81]Buzzai M, Jones RG, Amaravadi RK, et al. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth[J]. Cancer Res,2007,67(14):6745-6752.
[82]周小智,薛耀明,朱波等.二甲双胍对人结肠癌细胞SW480增殖的影响[J].南方医科大学学报,2010,30(8)：1935-1942.
[83]Deng YH, Pu XX, Huang MJ, et al.5-Fluorouracil upregulates the activity of Wnt signaling pathway in CD133-positive colon cancer stem-like cells[J]. Clin J Cancer,2010,29(9):810-815.
[1]Wild S, Roglic G, Green A, et al. Global prevalence of diabetes:estimates for the year 2000 and projections for 2030[J]. Diabetes Care,2004,27:1047-1053.
[2]胡水清,汤哲,张玫.糖尿病与结直肠癌患病危险关系的调查分析[J].世界华人消化杂志,2007,15(1)：88-91·
[3]Seow A, Yuan JM, KohWP, et al. Diabetesmellitus and risk ofcolorectal cancer in the Singapore Chinese Health Study[J]. J NatlCancer Inst,2006,98(2):135-138.
[4]Coughlin SS, Calle EE, TerasLR, et al. Diabetesmellitus as a predictor of cancermortality in a large cohort of US adults[J]. AM J Epidemio,2004,159(12): 1160-1167.
[5]Larsson SC, Giovannucci E, Wolk A. Diabetes and colorectal cancer incidence in the cohort of Swedish men [J]. Diabetes Care,2005,28(7):1805-1807.
[6]Meyerhardt JA, Catalano PJ, Haller DG, et al. Impact of diabetesmellitus on outcomes in patients with colon cancer[J]. J Clin Onco,2003,21 (3):433-440.
[7]Nilsen TI, Vatten LJ. Prospective study of colorectal cancer risk and physica activity, diabetes, blood glucoseand BMI:exploring the hyperinsulinaemia hypothesis[J]. Br J Cancer,2001,84(3):417-422.
[8]Giovannucci E. Insulin, insulin-like growth factors and colon cancer:a review of the evidence[J]. J Nutr,2001,131(11):3109-3120.
[9]Larsson SC, Orsini N, WolkA. Diabetesmellitus and risk of colorectal cancer:a meta-analysis[J]. J Natl Cancer Inst,2005,97(22):1679-1687.
[10]Vigneri P, Frasca F, Sciacca L, et al. Diabetes and cancer[J]. Endocr Relat Cancer, 2009,16(4):1103-11231.
[11]Davila JA, Morgan RO, Shaib Y, et al. Diabetes increases the risk ofhepatocellular carcinoma in theUnited States:a population based case control study[J]. Gut,2005,54(4):533-539.
[12]EI-Serag HB, Tran T, EverhartJE. Diabetes increases the risk of chronic liver disease and hepatocellular carcinoma[J]. Gastroenterology,2004,126(2): 460-468.
[13]Inoue M, Iwasaki M,Otani T, et al. Diabetesmellitus and the risk of cancer: results from a large scale population based cohort study in Japan[J]. Arch Intern Med,2006,166(17):1871-1877.
[14]Hassan MM,Hwang LY,Hatten CJ,et al. Risk factors for hepatocellular carcinoma: synergism of alcohol with viral hepatitis and diabetes mellitus[J]. Hepatology, 2002,36:1206-1213.
[15]Fujino Y, Mizoue T, Tokui N, et al. A prospective study of blood transfusion history and liver cancer in a high-endemic area of Japan[J]. Diabetes Metab Res Rev,2001,17:374-379.
[16]Fisher WE. Diabetes:risk factor for the development of pancreatic cancer or manifestation of the disease [J]? World J Surg,2001,25:503-508.
[17]Silverman DT, SchiffmanM, Everhart J, et al. Diabetesmellitus, othermedical conditions and familialhistory of cancer as risk factors for pancreatic cancer [J]. Br J Cancer,1999,80(11):1830-1837.
[18]Kuriki K, Hirose K, Tajima K. Diabetes and cancer risk for all and specific sites among Japanese men and women[J]. Eur J CancerPrev,2007,16(1):8389.
[19]Wang F, Herrington M, Larsson J, et al. The relationship between diabetes and pancreatic cancer[J]. MolCancer,2003,2:4.
[20]Michels KB, Solomon CG, Hu FB, et al. Type 2 diabetes and subsequent incidence ofbreast cancer in the nurses'health study[J]. DiabetesCare,2003, 26(6):1752-1758.
[21]Lipscombe LL, Goodwin PJ, Zinman B, et al. The impact of diabetes on surcival following breast cancer[J]. Breast Cancer Res Treat,2008,109(2):389-395.
[22]Stattin P, Bjr O, Ferrari P, et al. Prospective study of hyperglycemia and cancer risk[J]. Diabetes Care,2007,30 (3):561-567.
[23]Jee SH, OhrrH, Sull JW, et al. Fasting serum glucose level and cancer risk in Korean men and women[J]. JAMA,2005,293(2):194-202.
[24]王静.糖尿病与口腔肿瘤的相关性分析[J].中国综合临床,2007,23(6)：486.
[25]Zendehdel K, Nyren O, Ostenson CG, et al. Cancer incidence in patient swith type 1 diabetesmellitus:a population-based cohort study in sweden[J]. J Natl Cancer Inst,2003,95(23):1797-1800.
[26]Shu X, Ji J, Li X, et al. Cancer risk among patients hospitalized for Type 1 Diabetes mellitus:a population-based cohort study in Sweden[J]. Diabet Med, 2010,27(7):791-797.
[27]Kolho L, Jusufovic J, Miettinen, et al. Parietal cell antibodies and helicobacter pylori in children[J]. J Pediatr Gastroenterol Nutr,2000,30(3):265-268.
[28]Oldenburg B, Diepersloot RJ, Hoekstra JB. High prevalence of Helicobacter pylori in diabetes mellitus patients[J]. Dig Dis Sc,1996,41(3):458-461.
[29]Swerdlow AJ, Laing SP, Qiao Z, et al. Cancer incidence and mortality in patients with insulin-treated diabetes:a UK cohort study[J]. Br J Cancer,2005,92(11): 2070-2075.
[30]季尚玮,郭宏华.糖尿病与消化系统肿瘤关系的研究进展[J].国际消化病杂志,2006,26(1)：61-63.
[31]杨前勇,邹大进.糖尿病中的氧化损伤与抗氧化研究进展[J].国际内分泌代谢杂志,2006,26(增刊)：8-10.
[32]Brownlee M. The pathobiology of diabetic complication:a unifying mechanism. Diabetes,2005,3(54):1615-625.
[33]Heuson JC, Legros N. Influence of insulin deprivation on growth of the 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats subjected to alloxan diabetes and food restriction[J]. Cancer Res,1972,32:226-232.
[34]Heuson JC, Legros N, Heimann R. Influence of insulin administration on growth of the 7;12-dimethylbenz(a)anthracene-induced mammary carcinoma in intact oophorectomized, and hypophysectomized rats[J]. Cancer Res,1972;32:233-238.
[35]Calle EE, Kaaks R. Overweight, obesity and cancer:epidemiologicalevidence and proposed mechanisms[J]. Nat Rev Cancer,2004,4:579-591.
[36]Papa V, Pezzino V, Costantino A, et al. Elevated insulin receptor content in human breast cancer[J]. J Clin Invest,1990,86:1503-1510.
[37]Frasca F, Pandini G, Scalia P, et al. Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells[J]. Mol Cell Biol,1999,19:3278-3288.
[38]Song E, Dong Y,Han LN, et al. Diabetic retinopathy:VEGF,bFGF and retinal vascular pathology[J]. Song Chin Med J (Engl),2004,117(2):247-251.
[39]Witmler AN, Vrensen GF, Van Noorden CJ. Vascular endothelial growth factors and angiogenesis in eye disease[J]. Prog Retin Eye Res,2003,22(1):1-29.
[40]Lee MJ, Suh CH, Li ZH. Clinicopathological significance of maspin expression in breastcancer[J]. Korean Med Sc,2006,21(2):309-314.
[41]Ma J, Zhang L, Ru GQ, et al. Upregulation ofhypoxiawith elevated vascular endothelial growth factor expression and excessive an giogenesis and predicts a poorprognosis in gastric carcinoma[J]. World J Gastroentero,2007,13(11): 1680-1686.
[42]Zhang H, Yu DM, Chen Y, et al. Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes[J]. Lab Immu Clin Med,2002,9: 90-94.
[2]Yang WY, Lu JM, Weng JP, et al. Prevalence of diabetes among men and women in China[J]. N Engl J Med,2010,362(25):2425-2426.
[3]Inoue M, Iwasaki M, Otani T, et al. Diabetes mellitus and the risk of cancer: results from a large-scale population-based cohort study in Japan[J]. Arch Intern Med,2006,166(17):1871-1877.
[4]Larsson SC, Mantzoros CS, Wolk A. Diabetes mellitus and risk of breast cancer: a meta-analysis[J]. Int J Cancer,2007,121:856-862.
[5]Larsson SC, Orsini N, Wolk A (2005) Diabetes mellitus and risk of colorectal cancer:a meta-analysis. J Natl Cancer Inst 97:1679-1687
[6]Tsugane S, Inoue M. Insulin resistance and cancer:epidemiological evidence[J]. Cancer Sci,2010,101:1073-1079.
[7]Nicolucci A. Epidemiological aspects of neoplasms in diabetes[J]. Acta Diabetol. 2010,47:87-95.
[8]Vgneri P, Frasca F, Sciacca L, et al. Diabetes and cancer[J]. Endocr Relat Cancer, 2009,16:1103-1123.
[9]Park SK, Park MK, Suk JH,et al.Cause-of-death trends for diabetes mellitus over 10 years[J]. Korean Diabetes J,2009,33:65-72.
[10]ALeksandrovski YA. Molecular mechanism of the cross-impact of pathological processes in combined diabetes and cancer. Research and clinical aspects [J]. Biochemistry(Mosc),2002,67(2):1329-1346.
[11]Heuson JC, Legros N. Influence of insulin deprivation on growth of the 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats subjected to alloxan diabetes and food restriction[J]. Cancer Res,1972,32:226-232.
[12]Heuson JC, Legros N, Heimann R. Influence of insulin administration on growth of the 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in intact ophorectomized, and hypophysectomized rats[J]. Cancer Res 1972;32:233-238.
[13]Calle EE, Kaaks R. Overweight, obesity and cancer:epidemiologicalevidence and proposed mechanisms[J]. Nat Rev Cancer,2004,4:579-591.
[14]Papa V, Pezzino V, Costantino A, et al. Elevated insulin receptor content in human breast cancer[J]. J Clin Invest,1990,86:1503-1510.
[15]Frasca F, Pandini G, Scalia P,et al. Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells[J]. Mol Cell Biol,1999,19:3278-3288.
[16]Yang YY, Hemessy S, Lewis JD. Type 2 diabetes mellitus and the risk of colorectal cancer [j]. Clin Gastroenterol Hepalol,2005,3(6):587-594.
[17]Larsson SC, Orsini N, Wolk A. Diabetes mellitus and risk of colorectal cancer:a meta-analysis[J]. J Natl Cancer,2005,97(22):1679-1687.
[18]胡水清,汤哲,张玫.糖尿病与结直肠癌患病危险关系的调查分析[J].世界华人消化杂志2007；15(1)：88-91.
[19]Bowker SL, Majumdar SR, Veugelers P, et al. Increased cancer-related mortality for patients with type 2 diabetes who use sulfonylureas or insulin[J]. Diabetes Care.2006,29:254-258.
[20]Eurich DT, McAlister FA, Blackburn DF et al. Benefits and harms of antidiabetic agents in patients with diabetes and heartfailure:systematic review[J]. BMJ, 2007,335:497-452.
[21]Margolis DJ, Hoffstad O, Strom BL (2008) Association between serious ischemic cardiac outcomes and medications used to treat diabetes. Pharmacoepidemiol Drug Saf 17:753-759
[22]Currie CK, Poole CD, Gale EAM. The influence of glucose-lowering therapies on cancer risk in type2 diabetes[J]. Diabetologia,2009,52:1766-1777.
[23]Evans JM, Donnelly LA, Emslie-Smith AM, et al. Metformin and reduced risk of cancer in diabetic patients[J]. BMJ,2005,330:1304-1305.
[24]Rayter Z, leicester RJ. Adjuvant chemotherapy for colorectal canner[J]. Ann R Coll Surg,1995,77 (4):81-84.
[25]Yang YX, Hennessy S, Lewis JD. Insulin therapy and colorectal cancer risk among type 2 diabetes mellitus patients[J]. Gastroenterology,2004,127: 1044-1050.
[26]Zhang ZL, Zheng ZL, Kan H, et al. Reduced risk of colorectal cancer with metformin therapy in patient with type2 diabetes:a meta-analysis[J]. Diabetes Care,2011,34(10):2323-2328.
[27]Lee JH, Kim TI, Jeon SM, et al. The effects of metformin on the survival of colorectal cancer patients with diabetes mellitus[J]. Int J Cancer,2012,131(3): 752-759.
[28]Lee MS, Hsu CC, Wahlqvist ML, et al. Type 2 diabetes increases and metformin reduces total, colorectal, liver and pancreatic cancer incidences in Taiwanese:a representative population prospective cohort study of 800,000 individuals[J]. BMC Cancer,2011,11:20-29.
[29]李明,顾晋.中国结直肠癌20年来发展模式的变化趋势[J].中华胃肠外科杂志,2004,7(3)：214.
[30]Guastamacchia E, Resta F, Triggiani V, et al. Evidence for a putative relationship between type 2 diabetes and neoplasia with particular reference to breast cancer:role of hormones,growth factors and specific receptors[J]. Immune Endocr Metabol Disord,2004,4:59-66.
[31]Hosono K, Endo H, Takahashi H,et al. Metformin suppresses coloreetal aberrant crypt foci in a short-term clinical trial[J]. Cancer Prev Res,2010,3(9): 1077-1083.
[32]Hosono K, Endo H, Takahashi H, et al. Metformin suppresses azoxymethane indused colorectal aberrant crypt foci by activating AMP-activated protein kinase[J]. Mol Carcinog,2010,49(7):662-671.
[33]Algire C, Amrein L, Aakikhani M, et al. Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase[J]. Endocr Relat Cancer,2010,17(2): 351-360.
[34]Zakikhani M, Aowling RJ, Sonenberg N, et al. The effects of adiponectin and metformin on prostate and colon neoplasia involve activation of AMPK-activated protein kinase[J]. Cancer Prev Res,2008,1(5):369-375.
[35]Tomimoto A, Endo H, Sugiyama M, et al. Metformin suppresses intestinal polyp growth in ApcMin/+mice[J]. Cancer Sci,2008,99(11):2136-2141.
[36]Stumvoll M, Nurjhan N, Perriello G, et al. Metabolic effects of metformin in non-insulin-dependent diabetes mellitus[J]. N Engl J Med,1995,333:550-554.
[37]Hundal HS, Ramlal T, Reyes R, et al. Cellular mechanism of metformin action involves glucose transporter translocation from an intracellular pool to the plasma membrane in L6 muscle cells [J]. Endocrinology,1992,131:1165-1173.
[38]Hadad SM, Fleming S, Thompson AM. Targeting AMPK:A new therapeutic opportunity in breast cancer[J]. Critical Reviews in Oncology Hematology,2008, 67:1-7.
[39]Capano M, Crompton M. Bax translocates to mitochondria of heart cells during simulated ischaemia:involvement of AMP-activated and p38 mitogen-activated protein kinases[J]. Biochem J,2006,395:57-64.
[40]Jones RG, Plas DR, Kubek S, et al. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint[J]. Mol Cell,2005,18:283-293.
[41]Campas C, Lopez JM, Santidrian AF, et al. Acadesine activates AMPK and induces apoptosis in B-cell chronic lymphocytic leukemia cells but not in T lymphocytes[J]. Blood,2003,101:3674-3680.
[42]Kefas BA, Heimberg H, Vaulont S, et al. AICA-riboside induces apoptosis of pancreatic beta cells through stimulation of AMP-activated protein kinase[J]. Diabetologia,2003,46:250-254.
[43]Donnenberg VS, Donnenberg AD. Multiple drug resistanceincancer revisited:the cancer stem cell hypothesis[J]. J Clin Pharmacol,2005,45(8):872-877
[44]Vazquez MA, Oliveras FC, Cufi S, et al. Metformin regulates breast cancer stem cell ontogeny by transcriptional regulation of the epithelial-mesenchymal transition (EMT) status[J]. Cell Cycle,2010,9(18):3807-3814.
[45]Soritau O, Tomuleasa C, Aldea M, et al. Metformin plus temozolomide-based chemotherapy as adjuvant treatment for WHO grade Ⅲ and Ⅳ malignant gliomas[J]. J BUON,2011,16(2):282-289.
[46]Lliopoulos D, Hirsch HA, Struhl K. Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types[J]. Cancer Res,2011,71(9):3196-3201.
[47]Oliveras FC, Cufi S, Vazquez MA, et al. Micro(mi)RNA expression profile of breast cancer epithelial cells treated with the anti-diabetic drug metformin: induction of the tumor suppressor miRNA let-7aand suppression of the TGFβ-induced oncomiR miRNA-181α[J]. Cell Cycle,2011,10(7):1144-1151.
[48]Vazquez MA, Oliveras FC, Cufi S, et al. The anti-diabetic drug metformin suppresses the metastasis-associated protein CD24 in DNA-MB-468 triple-negative breast cancer cells[J]. Oncol Rep,2011,25(1):135-140.
[49]Rattan R, Graham RP, Maguire JL, et al. Metformin suppresses ovarian cancer growth and metastasis with enhancement of cisplatin cytotoxicity in vivo[J]. Neoplasis,2011,13(5):483-491.
[50]Wu N, Du C, Gu H, et al. Metformin induces apoptosis of lung cancer through activating JNK/p38 MAPK pathway and GADD153[J]. Neoplasma,2011,58(6): 482-490.
[51]Hadad S, Iwamoto T, Jordan L, et al. Evidence for biological effects of metformin in operable breast cancer:a preoperative, window-of-opportunity, randomized trial[J]. Breast Cancer Res Treat,2011,128(3):783-94.
[52]Mader RM. Resistance to 5-fluorouracil[J].Gen Pharmacil,1998,31(5):661-665.
[53]Choi S, Ku JL. Resistance of colorectal cancer cells to radiation and 5-FU is associated with MELK expression[J]. Biochem Biophys Res Commun,2011, 412(2):207-213.
[54]Fan X, Ouyang N, Teng H, et al. Isolation and characterization of spheroid cells from the HT29 colon cancer cell line[J]. Int J Colorectal Dis,2011,26(10): 1279-1285.
[55]Bijnsdorp IV, Peters GL, Temmink OH, et al. Differential activation of cell death and autophagy results in an increased cytotoxic potential for trifluorothymidine compared to 5-fluorouracil in colon cancer cells[J]. Int J Cancer,2010,126(10): 2457-2468.
[56]Dallas NA, Xia L, Fan F, et al. Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-1 receptor inhibition[J]. Cancer Res,2009,69(5):1951-1957.
[57]O'Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodifient mice[J]. Nature,2007,445(7123): 106-110.
[58]Tadoro M, Francipane MG, Medema JP, et al. Colon cancer stem cells:promise of targeted therapy [J]. Gastroenterology,2010,138(6):2151-2162.
[59]Coco C, Zannoni GF, Caredda E, et al. Increased expression of CD 133 and reduced dystroglycan expression are strong predictors of poor outcome in colon cancer patients[J]. J Exp Clin Cancer Res,2012,31(1):71-81.
[60]李宝秀 张晓实 刘国龙.CD133在局部晚期结肠癌肿瘤细胞中的异常表达及意义[J].广东医学,2010,31(13)：1678-1681.
[61]Xu W, Lin H, Zhang Y, et al. Compound Kushen injection suppresses human breast cancer stem-like cells by down-regulating the canonical Wnt/beta-catenin pathway[J]. J Exp Clin Cancer Res,2011,30(1):103-108.
[62]Bitaret N, Bandres E, Boni V, et al. MicroRNA-451 is involved in the self-renewal, tumorigenicity, and chemoresistance of colorectal cancer stem cell[J]. Stem Cells,2011,29(11):1661-1671.
[63]Peng C, Zhang X, Yu H, et al. Wnt5a as a predictor in poor clinical outcome of patients and a mediator in chemoresistance of ovarian cancer [J]. Int J Gynecol Cancer,2011,21(2):280-288.
[64]Maugeri SM, Vigneri P, De MR. Cancer stem cells and chemosensitivity[J]. Clin Cancer Res,2011,17(15):4942-4947.
[65]Noda T, Nagano H, Takemasa L, et al. Activation of Wnt/beta-catenin signaling pathway induces chemoresistance to interferon-alpha/5-fluorouracil combination therapy for hepatocellular carcinoma[J]. Br J Cancer,2009,100(10):1647-1658.
[66]Horst D, Kriegl L, Engel J, et al. CD 133 and nuclear beta-catenin:the marker combination to detect high risk cases of low stage colorectal cancer[J]. Eur J Cancer,2009,45(11):2034-2040.
[67]Takatani T, Minagawa M, Takatani R, et al. AMP-activated protein kinase attenuates Wnt/β-catenin signaling in human osteoblastic Saos-2 cells[J]. Mol Cell Endocrinol,2011,339(1-2):114-119.
[68]Kawamoto H, Yuasa T, Kubota Y, et al. Characteristics of CD133(+) human colon cancer SW620 cells[J]. Cell Transplant,2010,19(6):857-864.
[69]Liao Y, Hu X, Huang X, et al. Quantitative analyses of CD 133 expression facilitate researches on tumor stem cells[J]. Biol Pharm Bull,2010,33(5): 738-742.
[70]Chai H, Liu M, Tian R, et al. miR-20a targets BNIP2 and contributes chemotherapeutic resistance in colorectal adenocarcinoma SW480 and SW620 cell lines[J]. Acta Biochim Biophys Sin,2011,43(3):217-225.
[71]钱荣立.关于糖尿病的新诊标准及分型[J].中国糖尿病杂志,2000,8(1)：5-6.
[72]李明,顾晋.中国结直肠癌20年来发展模式的变化趋势[J].中华胃肠外科杂志,2004,7(3)：214.
[73]梁文霞,郎云星.2型糖尿病合并恶性肿瘤22例分析[J].实用糖尿病杂志,2008,4(5)：23-24.
[74]Atlanta, GA. Global Cancer Facts & Figures[M]. American Cancer Society,2007: 12.
[75]Chen S, Song X, Chen Z,et al. CD133 Expression and the Prognosis of Colorectal Cancer:A Systematic Review and Meta-Analysis[J]. PLoS One.2013; 8(2):1371-1381.
[76]Dowling RJ, Zakikhani M, Fantus IG, et al. Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells [J]. Cancer Res,2007,67(22):10804-10812.
[77]Zakikhani M, Dowling R, Fantus IG,et al.Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells [J]. Cancer Res,2006; 66(21):10269-10273.
[78]Algire C, Zakikhani M, Blouin MJ,et al. Metformin attenuates the stimulatory effect of a high-energy diet on in vivo H59 carcinoma growth[J]. Endocr Relat Cancer.2008,15(3):833-839.
[79]Ben Sahra I, Laurent K, Loubat A, et al. The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level [J]. Oncogene,2008,27(25):3576-3586.
[80]Gotlieb WH, Saumet J, Beauchamp MC et al. In vitro metformin anti-neoplastic activity in epithelial ovarian cancer[J]. Gynecol Oncol,2008,110(2):246-250.
[81]Buzzai M, Jones RG, Amaravadi RK, et al. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth[J]. Cancer Res,2007,67(14):6745-6752.
[82]周小智,薛耀明,朱波等.二甲双胍对人结肠癌细胞SW480增殖的影响[J].南方医科大学学报,2010,30(8)：1935-1942.
[83]Deng YH, Pu XX, Huang MJ, et al.5-Fluorouracil upregulates the activity of Wnt signaling pathway in CD133-positive colon cancer stem-like cells[J]. Clin J Cancer,2010,29(9):810-815.
[1]Wild S, Roglic G, Green A, et al. Global prevalence of diabetes:estimates for the year 2000 and projections for 2030[J]. Diabetes Care,2004,27:1047-1053.
[2]胡水清,汤哲,张玫.糖尿病与结直肠癌患病危险关系的调查分析[J].世界华人消化杂志,2007,15(1)：88-91·
[3]Seow A, Yuan JM, KohWP, et al. Diabetesmellitus and risk ofcolorectal cancer in the Singapore Chinese Health Study[J]. J NatlCancer Inst,2006,98(2):135-138.
[4]Coughlin SS, Calle EE, TerasLR, et al. Diabetesmellitus as a predictor of cancermortality in a large cohort of US adults[J]. AM J Epidemio,2004,159(12): 1160-1167.
[5]Larsson SC, Giovannucci E, Wolk A. Diabetes and colorectal cancer incidence in the cohort of Swedish men [J]. Diabetes Care,2005,28(7):1805-1807.
[6]Meyerhardt JA, Catalano PJ, Haller DG, et al. Impact of diabetesmellitus on outcomes in patients with colon cancer[J]. J Clin Onco,2003,21 (3):433-440.
[7]Nilsen TI, Vatten LJ. Prospective study of colorectal cancer risk and physica activity, diabetes, blood glucoseand BMI:exploring the hyperinsulinaemia hypothesis[J]. Br J Cancer,2001,84(3):417-422.
[8]Giovannucci E. Insulin, insulin-like growth factors and colon cancer:a review of the evidence[J]. J Nutr,2001,131(11):3109-3120.
[9]Larsson SC, Orsini N, WolkA. Diabetesmellitus and risk of colorectal cancer:a meta-analysis[J]. J Natl Cancer Inst,2005,97(22):1679-1687.
[10]Vigneri P, Frasca F, Sciacca L, et al. Diabetes and cancer[J]. Endocr Relat Cancer, 2009,16(4):1103-11231.
[11]Davila JA, Morgan RO, Shaib Y, et al. Diabetes increases the risk ofhepatocellular carcinoma in theUnited States:a population based case control study[J]. Gut,2005,54(4):533-539.
[12]EI-Serag HB, Tran T, EverhartJE. Diabetes increases the risk of chronic liver disease and hepatocellular carcinoma[J]. Gastroenterology,2004,126(2): 460-468.
[13]Inoue M, Iwasaki M,Otani T, et al. Diabetesmellitus and the risk of cancer: results from a large scale population based cohort study in Japan[J]. Arch Intern Med,2006,166(17):1871-1877.
[14]Hassan MM,Hwang LY,Hatten CJ,et al. Risk factors for hepatocellular carcinoma: synergism of alcohol with viral hepatitis and diabetes mellitus[J]. Hepatology, 2002,36:1206-1213.
[15]Fujino Y, Mizoue T, Tokui N, et al. A prospective study of blood transfusion history and liver cancer in a high-endemic area of Japan[J]. Diabetes Metab Res Rev,2001,17:374-379.
[16]Fisher WE. Diabetes:risk factor for the development of pancreatic cancer or manifestation of the disease [J]? World J Surg,2001,25:503-508.
[17]Silverman DT, SchiffmanM, Everhart J, et al. Diabetesmellitus, othermedical conditions and familialhistory of cancer as risk factors for pancreatic cancer [J]. Br J Cancer,1999,80(11):1830-1837.
[18]Kuriki K, Hirose K, Tajima K. Diabetes and cancer risk for all and specific sites among Japanese men and women[J]. Eur J CancerPrev,2007,16(1):8389.
[19]Wang F, Herrington M, Larsson J, et al. The relationship between diabetes and pancreatic cancer[J]. MolCancer,2003,2:4.
[20]Michels KB, Solomon CG, Hu FB, et al. Type 2 diabetes and subsequent incidence ofbreast cancer in the nurses'health study[J]. DiabetesCare,2003, 26(6):1752-1758.
[21]Lipscombe LL, Goodwin PJ, Zinman B, et al. The impact of diabetes on surcival following breast cancer[J]. Breast Cancer Res Treat,2008,109(2):389-395.
[22]Stattin P, Bjr O, Ferrari P, et al. Prospective study of hyperglycemia and cancer risk[J]. Diabetes Care,2007,30 (3):561-567.
[23]Jee SH, OhrrH, Sull JW, et al. Fasting serum glucose level and cancer risk in Korean men and women[J]. JAMA,2005,293(2):194-202.
[24]王静.糖尿病与口腔肿瘤的相关性分析[J].中国综合临床,2007,23(6)：486.
[25]Zendehdel K, Nyren O, Ostenson CG, et al. Cancer incidence in patient swith type 1 diabetesmellitus:a population-based cohort study in sweden[J]. J Natl Cancer Inst,2003,95(23):1797-1800.
[26]Shu X, Ji J, Li X, et al. Cancer risk among patients hospitalized for Type 1 Diabetes mellitus:a population-based cohort study in Sweden[J]. Diabet Med, 2010,27(7):791-797.
[27]Kolho L, Jusufovic J, Miettinen, et al. Parietal cell antibodies and helicobacter pylori in children[J]. J Pediatr Gastroenterol Nutr,2000,30(3):265-268.
[28]Oldenburg B, Diepersloot RJ, Hoekstra JB. High prevalence of Helicobacter pylori in diabetes mellitus patients[J]. Dig Dis Sc,1996,41(3):458-461.
[29]Swerdlow AJ, Laing SP, Qiao Z, et al. Cancer incidence and mortality in patients with insulin-treated diabetes:a UK cohort study[J]. Br J Cancer,2005,92(11): 2070-2075.
[30]季尚玮,郭宏华.糖尿病与消化系统肿瘤关系的研究进展[J].国际消化病杂志,2006,26(1)：61-63.
[31]杨前勇,邹大进.糖尿病中的氧化损伤与抗氧化研究进展[J].国际内分泌代谢杂志,2006,26(增刊)：8-10.
[32]Brownlee M. The pathobiology of diabetic complication:a unifying mechanism. Diabetes,2005,3(54):1615-625.
[33]Heuson JC, Legros N. Influence of insulin deprivation on growth of the 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats subjected to alloxan diabetes and food restriction[J]. Cancer Res,1972,32:226-232.
[34]Heuson JC, Legros N, Heimann R. Influence of insulin administration on growth of the 7;12-dimethylbenz(a)anthracene-induced mammary carcinoma in intact oophorectomized, and hypophysectomized rats[J]. Cancer Res,1972;32:233-238.
[35]Calle EE, Kaaks R. Overweight, obesity and cancer:epidemiologicalevidence and proposed mechanisms[J]. Nat Rev Cancer,2004,4:579-591.
[36]Papa V, Pezzino V, Costantino A, et al. Elevated insulin receptor content in human breast cancer[J]. J Clin Invest,1990,86:1503-1510.
[37]Frasca F, Pandini G, Scalia P, et al. Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells[J]. Mol Cell Biol,1999,19:3278-3288.
[38]Song E, Dong Y,Han LN, et al. Diabetic retinopathy:VEGF,bFGF and retinal vascular pathology[J]. Song Chin Med J (Engl),2004,117(2):247-251.
[39]Witmler AN, Vrensen GF, Van Noorden CJ. Vascular endothelial growth factors and angiogenesis in eye disease[J]. Prog Retin Eye Res,2003,22(1):1-29.
[40]Lee MJ, Suh CH, Li ZH. Clinicopathological significance of maspin expression in breastcancer[J]. Korean Med Sc,2006,21(2):309-314.
[41]Ma J, Zhang L, Ru GQ, et al. Upregulation ofhypoxiawith elevated vascular endothelial growth factor expression and excessive an giogenesis and predicts a poorprognosis in gastric carcinoma[J]. World J Gastroentero,2007,13(11): 1680-1686.
[42]Zhang H, Yu DM, Chen Y, et al. Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes[J]. Lab Immu Clin Med,2002,9: 90-94.