短链脂肪酸对结肠肿瘤细胞增殖分化的影响

短链脂肪酸(SCFA)由抗性淀粉、纤维多糖等在结肠腔内经厌氧菌酵解生成,主要包括乙酸、丙酸、丁酸等,是结肠腔内重要的有机酸阴离子。作为结肠黏膜首选能源底物,SCFA有增进钠吸收,促进结肠上皮细胞增殖与黏膜生长,提供代谢能量,调节肠免疫功能,对肠黏膜有营养和保护作用。丁酸是主要的抑制结肠肿瘤细胞增殖、诱导肿瘤细胞分化和凋亡、影响原癌基因表达的物质。

结肠肿瘤前细胞系的建立及应用

结肠肿瘤前细胞即永生化结肠上皮细胞系(IMCE),来源于Immorto小鼠和腺瘤性结肠息肉病基因(Apc)min/+小鼠杂交后子代小鼠的结肠上皮细胞,其表型正常,可发生恶性转化,已成为肠道肿瘤发生发展相关研究较为理想的细胞模型。此文主要就该细胞系的建立及其在肠道肿瘤发生机制和筛选防治药物等领域中的应用进展作一综述。

半枝莲通过Hedgehog非经典信号通路抑制结肠肿瘤干细胞自我更新和分化

目的:探究半枝莲醇提物对结直肠干细胞自我更新和分化的影响并阐明其分子作用机制。方法:慢病毒转染结肠肿瘤HT-29细胞,磁珠抗体标记法分选结肠干细胞;用CCK-8法检测半枝莲对肿瘤干细胞的活性;3D培养法、干细胞成球实验、琼脂糖集落实验探究半枝莲对HT-29Smo-CSC的生长更新及其转化的影响。实时荧光定量PCR和Western blot技术检测半枝莲对非经典Hedgehog信号通路基因和蛋白Shh、Ptch1、Gli1、Sufu及其标记基因CD133、靶基因c-Myc、分化基因CK20、增殖基因Ki67、更新基因Nanog的影响。结果:半枝莲能抑制HT-29Smo-CSC活性、生长转化及成球能力,显著下调Gli1、c-Myc、Shh mRNA及蛋白的表达,上调Sufu,下调Ptch1、CD133、CK20、Ki67、Nanog mRNA表达。结论:半枝莲醇提物能抑制HT-29Smo-CSC的增殖和分化,对非经典Hedgehog信号通路关键靶基因和蛋白有显著抑制作用,提示半枝莲能通过非经典Hedgehog信号通路抑制结直肠肿瘤干细胞自我更新和分化。

蛋白酶体抑制剂MG-132逆转人结肠癌细胞获得性TRAIL耐药

目的:探讨蛋白酶体抑制剂MG-132逆转人结肠癌细胞获得性TRAIL耐药的作用及其可能的机制。方法:在MG-132和TRAIL蛋白联合处理获得性TRAIL耐药的人结肠癌细胞DLD1-TRAIL/R后,MTT法检测细胞的存活率,流式细胞术检测细胞凋亡率,Westernblotting检测细胞中各种凋亡相关蛋白的表达和JNK激酶的磷酸化水平。结果:MG-132联合TRAIL蛋白处理DLD1-TRAIL/R细胞后,其细胞存活率明显下降(P<0.01),而细胞凋亡率则明显增加(P<0.01)。Westernblotting检测显示,联合处理后DLD1-TRAIL/R细胞中各种凋亡信号分子包括caspase-8、caspase-9、caspase-3、Bid和PARP蛋白均明显活化,线粒体中细胞色素C和Smac蛋白大量释放;进一步的Westernblotting检测显示,死亡受体DR5和凋亡诱导蛋白Bik的表达水平明显增高,而其他凋亡信号分子包括DR4、Bax、Bak、Bcl-XL、XIAP和Survivin等则无明显改变;检测结果还显示,MG-132能诱导JNK激酶发生磷酸化,使用JNK激酶抑制剂SP600125能够阻断MG-132诱导的DR5表达,但不影响Bik的表达,并且不能减弱MG-132和TRAIL蛋白联合处理对DLD1-TRAIL/R细胞的致凋亡效应(P<0.05)。结论:蛋白酶体抑制剂MG-132能逆转人结肠癌细胞DLD1-TRAIL/R的获得性TRAIL耐药,其机制可能与Bik蛋白上调后启动线粒体凋亡途径有关,与JNK通路激活无关。

5氟尿嘧啶上调干细胞标记物CD133在结肠癌细胞中的表达

目的:研究通过5氟尿嘧啶(5-FU)对结肠癌干细胞标记物CD133表达的影响,探讨5-FU对结肠癌干细胞的影响。方法:用流式细胞仪检测CD133在结肠癌细胞株表面的表达,磁珠细胞分离的方法分离结肠癌细胞株DLD1中CD133阳性和阴性的细胞群,细胞克隆形成实验检测2群细胞的自我更新能力,新型四唑氮盐方法(MTS)检测2群细胞对5-FU敏感性的差异,qPCR方法检测5-FU处理结肠癌细胞后CD133 mRNA水平的变化。结果:结肠癌细胞株DLD1、HT29、SW480、HCT116、Lovo、RKO细胞表面CD133的表达率分别为30.20%、82.00%、0.34%、91.80%、85.30%、0.28%。DLD1细胞中以CD133为标记有2群明显的细胞,MACS方法分离后阳性细胞群中CD133为87.21%±5.33%,而阴性细胞群中阴性细胞的比例为84.30%±4.65%;CD133阳性的细胞与未分离及CD133阴性细胞相比,克隆形成能力强(46.33%±4.44%vs31.00%±2.00%,P<0.05),对5-FU的敏感性下降20%,P<0.01。在DLD1和HT29细胞中,5-FU1mg/L上调CD133 mRNA水平的表达,从1升为1.684±0.012(P<0.01)、HT29细胞从30.702±0.284升为49.379±0.460(P<0.01)。结论:与CD133阴性细胞相比CD133阳性细胞克隆形成能力强,对5-FU的敏感性下降;5-FU上调干细胞标记物CD133 mRNA水平的表达,CD133阳性的结肠癌干细胞在5-FU的治疗过程中被富集。

Id2在SGC-7901和Colo-205肿瘤干细胞相关亚群中表达的研究

目的:探讨Id2(inhibitor of DNAbinding or inhibitor of differentiation)在SGC-7901和Colo-205细胞株中小亚群(sidepopulation,SP)细胞中的表达,并鉴定其是否为胃肠道癌干样细胞的特异性标志。方法:用Hoechest33342染色检测SGC-7901和Colo-205细胞株中SP细胞的比例;通过荧光免疫细胞化学染色比较Id2在SP细胞与非SP细胞中表达的差异。结果:胃癌SGC-7901、结肠癌Colo-205细胞中SP细胞的比例分别为3.8%和4.3%;Id2在SGC-7901细胞SP中的阳性表达率为34.2%,显著低于在非SP中的阳性表达率97.7%,P<0.001,而Id2在Colo-205细胞SP中的阳性表达率达77.4%,显著高于在非SP中的阳性表达率4.8%,P<0.001。结论:Id2可能是胃癌SGC-7901细胞中SP细胞的阴性分子标志,同时可能是结肠癌Colo-205细胞中SP细胞的阳性分子标志。

外源性Muc1、Survivin和Nanog基因启动子在结肠癌干细胞中的表达活性

目的探讨结肠癌干细胞中不同基因启动子的活性,寻找可能用于结肠癌干细胞靶向治疗的启动子。方法应用流式细胞仪分选人结肠癌HCT116细胞系中CD133+CD44+标记的肿瘤干细胞。采用PCR技术获取Nanog、Muc1和Survivin基因启动子并分别克隆入pGL3-basic质粒。将含有启动子的pGL3-basic质粒和pGL3-control质粒分别与pRL-sv40共转染结肠癌细胞(HCT116、SW620、HT29)、结肠癌干细胞(CD133+CD44+HCT116)及人正常肝细胞(QSG7701),通过检测双荧光素酶活性以测定启动子活性。结果pGL3-basic-Nanog、pGL3-basic-Muc1、pGL3-basic-Survivin经测序鉴定正确。HCT116细胞系中CD133+CD44+细胞比率约占42.2%。双荧光素酶活性检测显示:在HCT116、SW620、HT29和CD133+CD44+HCT116细胞中,Muc1与Survivin均表现出了较强的活性,而在正常细胞QSG7701中活性较低。结论 Muc1、Survivin启动子可能是用于靶向结肠癌干细胞治疗的有效候选启动子。

结肠癌基因

结肠癌基因１９９３年仅５月一个月就发现了亨廷顿氏疾病和卢·格里克氏疾病的基因。上周，一个有１４位研究人员的小组宣布发现了结肠癌基因的证据，在美国每年有５７０００人死于这种病，这种病是癌症死亡的第二大原因。这些遗传学家估计，在美国每２００人中就有１人携...

Oncoprotein expression and inhibition of apoptosis during colorectal tumorigenesis

AIMS To study bcl-2 and P53 protein expression and inhibition of apoptosis during colorectal tumorigenesis. METHODS Expression of bcl -2 and p53 in 45 colorectal ade- nomas and 61 colorectal carcinomas was detected by immunohis- tochemical staining. RESULTS The bcl-2 and P53 protein expression was uniformly negative in normal mucosa,whereas bcl-2 and p53 positive rates were significantly higher in adenoma and carcinoma than in nor- reals(P<0.01 ).The area with strong bcl-2 expression was of- ten the area with severely dysplasia.In colorectal adenoma,ex- pression of p53 increased with the increasing size and dysplasia, in adenomas≥20 mm being higher than adenomas<10 mm(77, 8% vs 35.0%,P<0.05).p53 was relevant to differentiation and Duke's staging.A significant inverse correlation was found between bcl-2 and p53 in immunostaining in the adenomas,but not in the carcinomas.Furthermore,carcinomas with a high per- centage of bcl-2 positive cells were significantly more likely to have low rates of apoptosis. CONCLUSIONS These results suggest that bcl-2 gene appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis,p53 expression plays an important role in the develop- ment and malignant change of colorectal adenoma,bcl-2 and p53 may be used as a good marker relating to cell apoptosis.

一次精神的朝圣——访金华艾克医院院长孙尚见

肿瘤能治吗?带了这样一个题目,本刊特派记者专程赴浙江金华走访了中华中医药学会肿瘤委员会副主任委员、浙江金华艾克医院院长孙尚见。从孙尚见的行医经历看,似乎对这个题目有了一个答案。金华之行,专访艾克医院孙尚见院长,那才是一次精神的朝圣。这个时代具有一种精神,是难能可贵的。

Changing patterns of colorectal cancer in China over a period of 20 years

AIM： To determine whether any changes have occurred on the patterns of colorectal cancer in China. METHODS： Data from 21 Chinese articles published from 1980 to 1999, were used to analyze the time trend of colorectal cancer according to the patients＇age at diagnosis, sex, the site of the tumor, stage, and the pathology. RESULTS： From 1980s to 1990s, the mean age of the colorectal cancer patients has increased. The percentage of the female patients rose. The distribution of colorectal carcinoma shows a predominance of rectal cancer. However, the proportion of proximal colon cancer （induding transverse and ascending colon） increased significantly accompanied by a decline in the percentage of rectal cancer. Similarity in the percentage of distal colon cancer between two decades was revealed. In the 1990s, statistically more Stage B patients were found than those in 1980s. In addition, databases show a significant decrease in the Stage D cases. The proportion of adenocarcinoma increased, but the mucinous adenocarcinoma decreased during two decades. CONCLUSION： These findings indicate that the pattern of colorectal cancer in China has been changing. Especially, a proximal shift due to the increasing proportion of ascending and transverse colon cancer has occurred in China.

Amygdalin inhibits genes related to cell cycle in SNU-C4 human colon cancer cells

AIM： The genes were divided into seven categories according to biological function; apoptosis-related, immune response-related, signal transduction-related, cell cyclerelated, cell growth-related, stress response-related and transcription-related genes. METHODS： We compared the gene expression profiles of SNU-C4 cells between amygdalin-treated （5 mg/mL, 24 h） and non-treated groups using cDNA microarray analysis. We selected genes downregulated in cDNA microarray and investigated mRNA levels of the genes by RT- PC R. RESULTS： Microarray showed that amygdalin downregulated especially genes belonging to cell cycle category： exonuclease 1 （EXO1）, ATP-binding cassette, sub-family F, member 2 （ABCF2）, MRE11 meiotic recombination 11 homolog A （MRE11A）, topoisomerase （DNA） Ⅰ （TOP1）, and FK506 binding protein 12-rapamycin-associated protein 1 （FRAP1）. RT-PCR analysis revealed that mRNA levels of these genes were also decreased by amygdalin treatment in SNU-C4 human colon cancer cells. CONCLUSION： These results suggest that amygdalin have an anticancer effect via downregulation of cell cycle-related genes in SNU-C4 human colon cancer cells, and might be used for therapeutic anticancer drug.

Promoter hypomethylation and reactivation of MAGE-A1 and MAGE-A3 genes in colorectal cancer cell lines and cancer tissues

AIM： To verify the expression and methylation status of the MAGE-A1 and MAGE-A3 genes in colorectal cancer tissues and cancer cell lines. METHODS： We evaluated promoter demethylation status of the MAGE-A1 and MAGE-A3 genes by RT-PCR analysis and methylation-specific PCR （MS-PCR）, as well as sequencing analysis, after sodium bisulfite modification in 32 colorectal cancer cell lines and 87 cancer tissues. RESULTS： Of the 32 cell lines, MAGE-A1 and MAGE-A3 expressions were observed in 59% and 66%, respectively. Subsequent to sodium bisulfite modification and MSPCR analysis, the promoter hypomethylation of MAGE-A1 and MAGE-A3 was confirmed in both at 81% each. Promoter hypomethylation of MAGE-A1 and MAGE-A3 in colorectal cancer tissues was observed in 43% and 77%, respectively. Hypomethylation of MAGE-A1 and MAGE-A3 genes in corresponding normal tissues were observed in 2% and 6%, respectively. CONCLUSION： The promoter hypomethylation of MAGE genes up-regulates its expression in colorectal carcinomas as well as in gastric cancers and might play a significant role in the development and progression of human colorectal carcinomas.

Hydrogen sutfide protects colon cancer cells from chemopreventative agent β-phenylethyl isothiocvanate induced apoptosis

AIM:Hydrogen sulfide (H2S) is a prominent gaseous constituent of the gastrointestinal (GI) tract with known cytotoxic properties. Endogenous concentrations of H2S are reported to range between 0.2-3.4 mmol/L in the GI tract of mice and humans. Considering such high levels we speculate that, at non-toxic concentrations, H2S may interact with chemical agents and alter the response of colonic epithelium cells to such compounds. The GI tract is a major site for the absorption of phytochemical constituents such as isothiocyanates, flavonoids, and carotenoids, with each group having a role in the prevention of human diseases such as colon cancer. The chemopreventative properties of the phytochemical agent p-phenyethyl isothiocyanate (PEITC) are well recognized. However, little is currently known about the physiological or biochemical factors present in the GI tract that may influence the biological properties of ITCs. The current study was undertaken to determine the effects of H2S on PEITC mediated apoptosis in colon cancer cells. METHODS: Induction of apoptosis by PEITC in human colon cancer HCT116 cells was assessed using classic apoptotic markers namely SubGl population analysis, caspase-3 like activity and nuclear fragmentation and condensation coupled with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) viability assay and LDH leakage. RESULTS: PEITC significantly induced apoptosis in HCT116 cells as assessed by SubGl population formation, nuclear condensation, LDH leakage and caspase-3 activity after 24 h, these data being significant from control groups (P<0.01). In contrast, co-treatment of cells with physiological concentrations of H2S (0.1-1 mmol/L) prevented PEITC mediated apoptosis as assessed using the parameters described. CONCLUSION: PEITC effectively induced cell death in the human adenocarcinoma cell line HCT116 in vitro through classic apoptotic mechanisms. However, in the presence of H2S, apoptosis was abolished. These data suggest that H2S may play a significant role in the response of colonic epithelial cells to beneficial as well as toxic agents present within the GI tract.

Antithrombin reduces reperfusion-induced hepatic metastasis of colon cancer cells

AIM： To examine whether antithrombin （AT） could prevent hepatic ischemia/reperfusion （I/R）-induced hepatic metastasis by inhibiting tumor necrosis factor （TNF）-α-induced expression of E-selectin in rats. METHODS： Hepatic I/R was induced in rats and mice by clamping the left branches of the portal vein and the hepatic artery. Cancer cells were injected intrasplenically. The number of metastatic nodules was counted on day 7 after I/R. TNF-α and E-selectin mRNA in hepatic tissue, serum fibrinogen degradation products and hepatic tissue levels of 6-keto-PGF1α, a stable metabolite of PGI2, were measured. RESULTS： AT inhibited increases in hepatic metastasis of tumor cells and hepatic tissue mRNA levels of TNF-α and E-selectin in animals subjected to hepatic I/R. Argatroban, a thrombin inhibitor, did not suppress any of these changes. Both AT and argatroban inhibited I/R-induced coagulation abnormalities. I/R-induced increases of hepatic tissue levels of 6-keto-PGF1α were significantly enhanced by AT. Pretreatment with indomethacin completely reversed the effects of AT. Administration of OP-2507, a stable PGI2 analog, showed effects similar to those of AT in this model. Hepatic metastasis in congenital AT-deficient mice subjected to hepatic I/R was significantly increased compared to that observed in wild-type mice. Administration of AT significantly reduced the number of hepatic metastases in congenital AT-deficient mice. CONCLUSION： AT might reduce I/R-induced hepatic metastasis of colon cancer cells by inhibiting TNF-α-induced expression of E-selectin through an increase in the endothelial production of PGI2. These findings also raise the possibility that AT might prevent hepatic metastasis of tumor cells if administered during the resection of liver tumors.

Radiosensitivity of human colon cancer cell enhanced by immunoliposomal docetaxel

AIM:To enhance the radiosensitivity of human colon cancer cells by docetaxel. METHODS: Immunoliposomal docetaxel was prepared by coupling monoclonal antibody against carcinoembryonic antigen to cyanuric chloride at the PEG terminus of liposome. LoVo adenocarcinoma cell line was treated with immunoliposomal docetaxel or/and irradiation. MTT colorimetric assay was used to estimate cytotoxicity of immunoliposomal docetaxel and radiotoxicity. Cell cycle redistribution and apoptosis were determined with flow cytometry. Survivin expression in LoVo cells was verified by immunohistochemistry. D801 morphologic analysis system was used to semi-quantify immunohistochemical staining of survivin. RESULTS: Cytotoxicity was induced by immunoliposomal docetaxel alone in a dose-dependent manner. Immunoliposomal docetaxel yielded a cytotoxicity effect at a low dose of 2 nmol/L. With a single dose irradiation, the relative surviving fraction of LoVo cells showed a dose-dependent response, but there were no significant changes as radiation delivered from 4 to 8 Gy. Compared with liposomal docetaxel or single dose irradiation, strongly radiopotentiating effects of immunoliposomal docetaxel on LoVo cells were observed. A low dose of immunoliposomal docetaxel could yield sufficient radiosensitivity. Immunoliposomal docetaxel were achieved both specificity of the conjugated antibody and drug radiosensitization. Combined with radiation, immunoliposomal docetaxel significantly increased the percentage of G2/M cells and induced apoptosis, but significantly decreased the percentage of cells in G2/G1 and S phase by comparison with liposomal docetaxel. Immunohistochemical analysis showed that the brown stained survivin was mainly in cytoplasm of LoVo cells. Semi-quantitative analysis of the survivin immunostaining showed that the expression of survivin in LoVo cells under irradiation with immunoliposomal docetaxel was significantly decreased. CONCLUSION: Immunoliposomal docetaxel is strongly effective for target radiosensitation in LoVo colon carcinoma cells, and may offer the potential to improve local radiotherapy.

Expression of a novel apoptosis inhibitor-survivin in colorectal carcinoma

AIM： To investigate the role of survivin expression in the pathogenesis of colorectal carcinoma. METHODS： Immunohistochemistry S-P method and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNE） were used to detect the expression of survivin and apoptotic cell in situ in colorectal cancerous tissues, para-cancerous tissues and normal tissues of 48 cases of colorectal carcinoma. RESULTS： The survivin positive unit （PU） was higher in cancerous tissues （38.76±5.14） than in para-cancerous （25.17±7.26） or normal tissues （0.57±0.03） （P〈0.05）. The apoptosis index （AI） of para-cancerous tissues was （7.51±2.63%） higher than cancerous tissues （4.65±1.76%）. The expression of survivin was associated with pathological grade, lymph node metastasis and Dukes stage of colorectal carcinoma. CONCLUSION： Survivin expression may play an important role in carcinogenesis of colorectal carcinoma and may be associated with malignant biological behaviors of colorectal carcinoma.

Reduced expression ofβ-catenin inhibitor Chibby in colon carcinoma cell lines

AIM： To analyse the Chibby expression and its function in colon carcinoma cell lines and colorectal carcinoma （CRC）. METHODS： Chibby expression levels were investigated by quantitative RT-PCR in a panel of seven different colon carcinoma cell lines. By sequencing, we analysed mutational status of Chibby. To test whether Chibby exhibited effects on β-catenin signalling in colon carcinoma cells, we transfected SW480 cells with Chibby expression plasmid and, subsequently, analysed activity of β-catenin and tested for alterations in cellular phenotype. In addition, we examined Chibby mRNA levels in samples of colorectal carcinomas and adjacent normal tissues by using quantitative RT-PCR and hybridised gene chips with samples from CRC and normal tissues. RESULTS： Chibby mRNA expression was strongly downregulated in colon carcinoma cell lines in comparison to normal colon epithelial cells and no mutation in any of the examined colon carcinoma cell lines was found. Further, we could show that Chibby inhibited β-catenin activity in TOPflash assays when over-expressed in SW480 cells. Proliferation and invasion assays with Chibby transfected SW480 cells did not reveal profound differences compared to control cells. In contrast to these in vitro data, quantitative RT-PCR analyses of Chibby mRNA levels in CRC tumor samples did not show significant differences to specimens in adjacent non-cancerous tissue. Consistent with these findings, gene chips analysing tissue samples of tumors and corresponding normal tissue did not show altered Chibby expressionCONCLUSION： Altered Chibby expression might be observed in vitro in different colon carcinoma cell lines. However, this finding could not be confirmed in vitro in CRC tumors, indicating that Chibby is not likely to promote CRC tumor development or progression. As Chibby is an important inhibitor of β-catenin signalling, our data implicate that the usability of colon carcinoma cell lines for in vitro studies analysing the Wnt/β-catenin pathway in colorectal carcinoma needs extensive verification.

The expression of glucose regulated protein-94 in colorectal carcinoma cells treated by sodium butyrate

The expression of glucose regulated protein 94 (GRP94)during the treatment of human colorectal carcinoma cell lineClone A cells with sodium butyrate was studied. Sodium butyrate (SB) can cause functional and morphological effects on Clone A cells including growth arrest at Go/G1 stage and cell differentiation as observed by morphological changes, MTT and flow cytometry assays, as well as reduced Grp94 gene expression as shown by Northern blot and Western blot assays. The possible mechanism of the correlation between Grp94 gene expression and tumor growth inhibition and cell differentiation is briefly discussed.

Effects of inositol hexaphosphate on proliferation of HT-29 human colon carcinoma cell line

AIM： To investigate the effects of inositol hexaphosphate （IP6） on proliferation of HT-29 human colon carcinoma cell line. METHODS： Cells were exposed to various concentrations （0, 1.8, 3.3, 5.0, 8.0, 13.0 mmol/L） of IP6 for a certain period of time. Its effect on growth of HT-29 cells was measured by MTT assay. The expressions of cell cycle regulators treated with IP6 for 2 d were detected by imrnunocytochemistry. RESULTS： IP6 inhibited the HT-29 cell growth in a dose- and time-dependent manner. Analysis of cell cycle regulator expression revealed that IP6 reduced the abnormal expression of P53 and PCNA and induced the expression of P21. CONCLUSION： IP6 has potent inhibitory effect on proliferation of HT-29 cells by modulating the expression of special cell cycle regulators.