Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and histone acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methylation-specific PCR, and the acetylated status of the histones associated with the p21wAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry.We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21 WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73,c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines

BACKGROUND Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers.It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeatcontaining G protein-coupled receptor 5(LGR5)-expressing stem cells,these socalled“tumour-initiating”cells,reminiscent in their properties of the normal intestinal stem cells(ISCs),may explain the apparent heterogeneity of colon cancer cell lines.Also,colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5.Furthermore,in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIM To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODS A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines(Caco-2 and LoVo).These cells were then characterized according to their proliferation capacity,gene expression signatures of ISC markers,and their tumorigenic properties in vivo and in vitro.RESULTS The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2,but not from the LoVo,cell lines,in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog(+4 crypt)stem cells.This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny.Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein(EYFP)from this cell line exhibit tumorigenic-like properties in vivo and in vitro.In contrast,cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties.Thus,cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells,but not LoVo cells.CONCLUSION Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines.Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data.We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.

Growth hormone releasing peptide 2 reverses anorexia associated with chemotherapy with 5-fluoruracil in colon cancer cell-bearing mice

The cancer-associated anorexia-cachexia syndrome is observed in 80% of patients with advanced-stage cancer, and is one of the major obstacles in chemo- therapy. Ghrelin is a orexigenic hormone that has been proposed to prevent anorexia. Aim of the study was to determine whether the addition of the ghrelin agonist growth hormone releasing peptide 2 (GHRP-2) to cytotoxic therapy with 5-fluoruracil (5-FU) prevents the anorexia associated with chemotherapy in cancer cachectic mice. Thirty-three BALB/c female tumourbearing mice were randomized to receive a solution containing: (a) placebo; (b) GHRP-2; (c) 5-FU; or (d) 5-FU + GHRP-2. Ten BALB/c no tumour-bearing mice received placebo solution. Food intake and survival were checked. Six hours after the drug injection the cumulative food intake was signifi cantly increased in mice treated with the combination of 5-FU + GHRP-2 versus the 5-FU alone (P = 0.0096). On day 3, the cumulative food intake of mice treated with GHRP-2,5-FU and 5-FU + GHRP-2 signifi cantly increased com- pared with naive and vehicle groups (P = 0.0007, P = 0.0038 and P = 0.0166, respectively). The median survival time was longer in 5-FU + GHRP-2 treated mice than in those with 5-FU, although it was not signifi cant (18 d versus 15.5 d, P = 0.7). For the fi rst time, we demonstrated that the addition of GHRP-2 to cytotoxic therapy with 5-FU improved appetite in tumour-bearing mice with anorexia/cachexia syndrome in early stage. These data suggest that GHRP-2 may improve the effi cacy of therapy and the quality of life of cancer patients thank to the amelioration of their nutritional state.

Oxidative stress-induced mitochondrial dysfunction in a normal colon epithelial cell line

AIM To determine how a normal human colon cell line reacts to microbial challenge as a way to study oxidative stress-induced responses associated with inflammatory bowel disease.METHODS Normal human colon epithelial cells(ATCC?CRL.1790?)were stimulated with either heat killed E.coli or heat killed murine cecal contents(HKC)and examined for several relevant biomarkers associated with inflammation and oxidative stress including cytokine production,mitochondrial autophagy and oxidant status.TNFα,IL-1βand IL-8 protein concentrations were measured within the supernatants.Fluorescent microscopy was performed to quantify the production of reactive oxygen species(ROS)using an oxidation responsive fluorogenic probe.Mitochondrial morphology and mitochondrial membrane potential was assessed by dual staining using COXIV antibody and a dye concentrating in active mitochondria.Mitochondrial ROS scavenger was used to determine the source of ROS in stimulated cells.Autophagy was detected by staining for the presence of autophagic vesicles.Positive controls for autophagy and ROS/RNS experiments were treated with rapamycin and chloroquine.Mitochondrial morphology,ROS production and autophagy microscopy experiments were analyzed using a custom acquisition and analysis microscopy software(Image J).RESULTS Exposing CRL.1790 cells to microbial challenge stimulated cells to produce several relevant biomarkers associated with inflammation and oxidative stress.Heat killed cecal contents treatment induced a 10-12fold increase in IL-8 production by CRL.1790 cells compared to unstimulated controls at 6 and 12 h(P<0.001).Heat killed E.coli stimulation resulted in a4-5 fold increase in IL-8 compared to the unstimulated control cells at each time point(P<0.001).Both heat killed E.coli and HKC stimulated robust ROS production at 6(P<0.001),and 12 h(P<0.01).Mitochondrial morphologic abnormalities were detected at 6 and12 h based on reduced mitochondrial circularity and decreased mitochondrial membrane potential,P<0.01.Microbial stimulation also induced significant autophagy at 6 and 12 h,P<0.01.Lastly,blocking mitochondrial ROS generation using mitochondrial specific ROS scavenger reversed microbial challenge induced mitochondrial morphologic abnormalities and autophagy.CONCLUSION The findings from this study suggest that CRL.1790cells may be a useful alternative to other colon cancer cell lines in studying the mechanisms of oxidative stress events associated with intestinal inflammatory disorders.

INHIBITORY EFFECT OF DEMO ON THE GROWTH OF TRANSPLANTED HUMAN COLON CANCER IN NUDE MICE

A human colon cancer cell line Hce- 8693 was heterotransplanted in nude mice. Polyamine blosythesis Inhibitor a- dlfiuoromethylomithine (DFMO ) show a marked reproducible inhibition in this model. The size and weight of transplanted tumor In DFMO group were smaller than those of the control group and the average inhibition rate was 72.8% (P < 0.001) . DFMO showed higher tumor inhibitory rate than 5-Fu (35. 4%) (P<0. 001) . Furthermore. DFMO demonstrated less severe bone marrow inhibition in the nude mice than 5-Fu (20. 0% Vs 53. 2%. P<0. 001) .There was no synergistic action in these two drugs at the experimental dose. The concentration of putrescine and spermidine in the plasma and tumor tissue in the DFMO group were 70% lower than those of the control group (P<0. 001) . These results indicate that the anti-tumor effect of DFMO might be explained by the inhibition of polyamine biosynthesis and this study provides an experimental basis for future clinical application of DFMO.

Change in expression of apoptosis genes after hyperthermia,chemotherapy and radiotherapy in human colon cancer transplanted into nude mice

AIM:To investigate the change in expression of p53 ,Bcl-2 ,and Bax genes in human colon cancer cells transplanted into nude mice after hyperthermia,chemotherapy,radiotherapy,thermochemotherapy,thermoradiotherapy and thermochemoradiotherapy. METHODS:Human colon cancer cell line (HT29) was transplanted into the hind limbs of nude mice. Under laboratory simulated conditions of hyperthermia (43℃,60 min),the actual radiation doses and doses of mitomycin C (MMC) were calculated in reference to the clinical radiotherapy for human rectal cancer and chemotherapy prescription for colon cancer. The mice were divided into 6 groups according to the treatment approaches:hyperthermia,chemotherapy,radiotherapy,thermochemotherapy,thermoradiotherapy,and thermochemoradiotherapy. The mice were sacrificed at different time points and the tumor tissue was taken for further procedures. The morphologic changes in membrane,cytoplasm and nuclei of tumor cells of p53,Bcl-2,and Bax after treatment,were observed by immunohistochemistry staining. RESULTS:All of the six treatment modalities down-regulated the expression of p53,Bcl-2 and up-regulated the expression of Bax at different levels. The combined therapy of hyperthermia,with chemotherapy,and/or irradiation showed a greater effect on down-regulating the expression of p53 (0.208 ± 0.009 vs 0.155 ± 0.0115,P < 0.01) and Bcl-2 (0.086 ± 0.010 vs 0.026 ± 0.0170,P < 0.01) and up-regulating Bax expression (0.091 ± 0.0013 vs 0.207 ± 0.027,P < 0.01) compared with any single therapy.CONCLUSION:Hyperthermia enhances the effect of radio-and chemotherapy on tumors by changing the expression of apoptosis genes,such as p53,Bcl-2 and Bax.

Tetrandrine:A Potent Abrogator of G_2 Checkpoint Function in Tumor Cells and Its Mechanism

Objective To assess the ability of tetrandrine （Tet） to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice. Methods MCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry. Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo. Results Clonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio （SER） of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells, whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis （M phase）. Endosomatic experiment showed that tetrandrine caused tumor growth delay in irradiated mice. Conclusion Tetrandrine boosts the cell killing activity of irradiation both in vitro and in vivo. Tetrandrine is a potent abrogator for G2 checkpoint control and can sensitize the cells to radiation.

罗哌卡因对人结肠癌顺铂耐药细胞株顺铂敏感性的增强作用及其机制

目的观察罗哌卡因对人结肠癌顺铂耐药细胞株顺铂敏感性的增强作用,并进一步探讨其作用机制。方法通过浓度递增法将人结肠癌LOVO细胞暴露于顺铂中诱导顺铂耐药株LOVO/DDP形成。将LOVO和LOVO/DDP两种结肠癌细胞株分别分为罗哌卡因联合顺铂组、罗哌卡因组、顺铂组、对照组。对照组加入细胞培养液,顺铂组加入细胞培养液和1.0μmol/L浓度顺铂,罗哌卡因联合顺铂组加入细胞培养液后再同时加入1.0μmol/L浓度顺铂及1 mmol/L浓度罗哌卡因培养。培养14 d,采用平板克隆实验观察细胞增殖能力。培养48 h后,采用Tran⁃swell和流式细胞术观察细胞迁移、凋亡情况;JC-1和ROS实验测定细胞线粒体膜电位和活性氧(ROS);Western blot⁃ting法、免疫荧光法检测细胞株中MMP-9及铁死亡相关蛋白GPX4、Nrf-2、SLC7A11;亚铁离子染色法检测结肠癌细胞株中亚铁离子。结果LOVO细胞中,与对照组比较,顺铂组LOVO细胞株克隆体的形成减少、迁移细胞株数减少、MMP-9蛋白水平降低、凋亡率升高、存活率降低,罗哌卡因组克隆体形成数、迁移细胞株数、MMP-9蛋白水平、细胞凋亡率、细胞存活率变化不明显,顺铂联合罗哌卡因组细胞株克隆体数减少、迁移细胞数减少、MMP-9蛋白水平下降、凋亡率升高、存活率降低(P均<0.05);与顺铂组比较,顺铂联合罗哌卡因组细胞株克隆体数少、迁移细胞数少、MMP-9蛋白水平低、凋亡率高、存活率低(P均<0.05)。在LOVO/DDP细胞株中,与对照组比较,顺铂组、罗哌卡因组细胞株克隆体数、迁移细胞株数、MMP-9蛋白水平变化不明显,顺铂联合罗哌卡因组细胞株克隆体数、迁移细胞株数减少、MMP-9蛋白水平降低、细胞凋亡率降低、细胞存活率升高(P均<0.05)。在LOVO细胞株和LOVO/DDP细胞株中,与对照组比较,其他三组JC-1多聚体红色荧光与单体绿色荧光比值[JC-1(Red/Green)]均降低,ROS水平升高,GPX4蛋白水平下降,GPX4相对荧光强度减弱,Fe2+荧光强度增强,以罗哌卡因联合顺铂组最显著(P均<0.01)。在LOVO/DDP细胞株中,与对照组相比,顺铂组及罗哌卡因组Nrf-2、SLC7A11蛋白表达水平无明显变化(P均>0.05),罗哌卡因联合顺铂组Nrf-2、SLC7A11蛋白水平均下降(P均<0.01)。结论罗哌卡因可增强人结肠癌顺铂耐药细胞株顺铂敏感性,其作用机制可能与由Nrf-2、GPX4、SLC7A11及线粒体膜电位水平降低、ROS水平升高诱导的铁死亡有关。

circ_0000376靶向miR-876-3p调控结肠癌细胞进程

目的 探讨circ_0000376对结肠癌细胞SW480迁移、侵袭和凋亡的影响及其分子机制。方法 选取30例结肠癌组织及其相应的癌旁组织;体外培养SW480细胞,根据实际转染蛋白情况分别设si-NC组、si-circ_0000376组、miR-NC组、miR-876-3p组、si-circ_0000376和anti-miR-876-3p共转染组以及sicirc_0000376和anti-miR-NC共转染组。采取RT-qPCR检测circ_0000376和miR-876-3p的表达水平、Transwell检测细胞迁移和侵袭、流式细胞术检测细胞凋亡、Western blot检测相关蛋白的表达、双荧光素酶报告基因实验检测circ_0000376和miR-876-3p的靶向关系。结果 结肠癌组织中circ_0000376较癌旁组织呈高表达(P<0.05),miR-876-3p较癌旁组织呈低表达(P<0.05);沉默circ_0000376或过表达miR-876-3p可有效降低癌细胞恶性行为活性,MMP-2、MMP-9、Bcl-2蛋白也呈更低表达,细胞凋亡活性Bax蛋白呈明显高表达(P<0.05);circ_0000376靶向调控miR-876-3p表达(P<0.05),并发现使miR-876-3p呈现低表达会造成circ_0000376对SW480细胞迁移、侵袭和凋亡形成逆转作用(P<0.05)。结论 沉默circ_0000376可通过靶向上调miR-876-3p抑制SW480细胞迁移和侵袭,并促进凋亡。

绿茶水提取物诱导体外培养的大肠癌LOVO细胞的凋亡

目的：探讨绿茶水提取物对体外培养的大肠癌ＬｏＶｏ细胞的抑制和诱导细胞发生凋亡的作用。方法：用ＭＴＴ法、琼脂糖凝胶电泳、透射电镜和流式细胞仪的方法，观察绿茶水提取物处理ＬｏＶｏ细胞后其生化和形态学等指标的改变。结果：ＭＴＴ法证实绿茶水提取物对ＬｏＶｏ细胞有抑制作用，抑制率与绿茶水提取物的浓度呈正相关；透射电镜可见ＬｏＶｏ细胞在形态学上出现典型的细胞核固缩、碎裂等凋亡细胞的形态学改变；琼脂糖凝胶电泳呈典型的“梯状（ＤＮＡｌａｄｄｅｒ）”带。流式细胞仪检测结果示：在绿茶水提取物处理ＬｏＶｏ细胞后０～１２ｈＧ１期前无亚二倍体峰出现，而在３６ｈＧ１期前出现一亚二倍体峰，即凋亡峰。结论：绿茶水提取物对大肠癌ＬｏＶｏ细胞有抑制和杀灭作用，而这种作用的机制之一是通过诱导大肠癌细胞发生凋亡，即诱导大肠癌细胞发生凋亡是绿茶杀灭肿瘤细胞的重要机制之一。

螺旋藻藻蓝蛋白对癌激光疗法增敏作用的实验研究

用藻蓝蛋白处理2种癌细胞作体内外激光治癌试验。人大肠癌细胞株HR_(8348)培养后分别用100μg,50μg和25μg的藻蓝蛋白处理,经光波为630nm的铜激光辐照12J/cm^2,用MTT法检测培养癌细胞存活率分别为22.2％,37.6％和89.7％,显示良好的剂量效应。用S_(180)移植瘤小鼠,分别给予藻蓝蛋白注射2mg或口服20mg后,经铜激光辐照瘤体15d后,有效率分别为50％和53％,与对照组比较,具显著差异(P=0.007)。体内外试验证实藻蓝蛋白确具有光敏作用,且其无毒,无副作用,是一种理想的光敏剂。

瘦素对5-氟尿嘧啶损伤结肠癌细胞的影响

目的探讨不同浓度的瘦素对5-氟尿嘧啶(5-FU)损伤结肠癌细胞的影响。方法同时用5-FU及不同浓度的瘦素进行体外干预结肠癌HT-29细胞株。MTT法检测5-FU50%细胞生长抑制率(IC50)的变化。流式细胞仪、原位末端转移酶标记法(TUNEL)进行周期及凋亡分析。以RT-PCR方法检测caspase-9,caspase-3mRNA的表达。结果瘦素抑制5-FU对HT-29的杀伤作用。抑制5-FU诱导的细胞周期阻滞及细胞凋亡。RT-PCR表明加入瘦素后caspase-9,caspase-3mRNA的表达均下降,且呈剂量依赖性。结论瘦素通过下调caspase-9,caspase-3的表达促进结肠癌细胞产生凋亡抵抗,抑制5-FU的损伤作用。

人参皂苷Rg3对人结肠癌细胞株SW480增殖的影响及其作用机制

目的观察人参皂苷Rg3对人结肠癌细胞株SW480增殖的影响,并探讨其可能作用机制。方法取对数生长期SW480细胞,分别加入含160、80、40、0μmol/L人参皂苷Rg3的储备液(分别计为A、B、C、D组)培养24 h,采用MTT法检测人参皂苷Rg3对SW480细胞增殖活性(OD值)的影响,倒置显微镜观察人参皂苷Rg3诱导SW480细胞凋亡形态学改变,流式细胞术检测SW480细胞凋亡率,RT-PCR法检测细胞间黏附分子1(ICAM-1)、闭锁蛋白(Occludin)mRNA,Western blotting法检测ICAM-1、Occludin蛋白。结果与D组比较,A、B、C组细胞OD值下降,凋亡率增加,ICAM-1 mRNA、蛋白表达降低,Occludin mRNA、蛋白表达升高(P均<0.05)。结论人参皂苷Rg3可抑制SW480细胞增殖,促进其凋亡,机制可能与ICAM-1表达下调、Occludin表达上调有关。

刚地弓形虫细胞培养上清对人结肠癌细胞sw480增殖与凋亡的影响

目的观察刚地弓形虫RH株速殖子与人结肠癌sw480细胞系共培养上清对人结肠癌sw480细胞增殖的影响和诱导其凋亡与坏死的情况。方法取对数生长期的人结肠癌sw480细胞(1×106),建立弓形虫RH株速殖子数目分别为2×106、4×106、8×106、16×106的共培养模型,观察弓形虫速殖子在sw480细胞中的寄生,提取共同孵育72h后的培养上清,以新鲜培养基稀释为半量,体外作用人结肠癌sw480细胞12h、24h、48h、72h,CKK-8法检测吸光度(A450值)并计算抑制率;Annexin-v-FITC/PI染色细胞后上流式细胞仪检测细胞凋亡与坏死率;琼脂糖凝胶电泳观察细胞凋亡DNA条带;透射电镜和荧光显微镜观察细胞形态学改变。结果弓形虫RH株速殖子可在人结肠癌sw480细胞内寄生和增殖。CKK-8法检测结果显示上述共培养上清对sw480细胞增殖抑制率随上清作用时间延长均明显增大,48h最大抑制率达44.55%。流式细胞仪检测显示实验组sw480细胞早期凋亡率和晚期凋亡与坏死率随上清作用时间延长均明显增加,48h早期凋亡率达最高值(11.54%),48h以后早期凋亡率下降,晚期凋亡和坏死率显著上升,72h总死亡率可达46.11%,细胞杀伤效果明显;琼脂糖凝胶电泳显示典型的DNA云梯状条带;荧光显微镜和透射电镜观察到细胞凋亡和坏死典型形态。结论弓形虫RH株速殖子与人结肠癌sw480细胞共培养上清对体外培养的sw480细胞增殖有明显的抑制作用,并可诱导sw480细胞凋亡与坏死。

放线菌酮和硫酸锌对儿茶素诱导大肠癌细胞凋亡的拮抗作用

目的：探讨儿茶素诱导体外培养的ＬｏＶｏ大肠癌细胞株凋亡及放线菌酮和硫酸锌对儿茶素诱导细胞凋亡的拮抗作用。方法：用儿茶素处理ＬｏＶｏ细胞，加或不加放线菌酮和硫酸锌，用琼脂糖凝胶电泳、荧光显微镜和流式细胞仪方法，观察ＬｏＶｏ细胞生化和形态学方面的改变。结果：荧光显微镜下可见ＬｏＶｏ细胞在形态学上出现典型的细胞核固缩、碎裂等凋亡细胞的形态学改变；琼脂糖凝胶电泳呈典型的“梯状”（ＤＮＡｌａｄｄｅｒ）带，在同时加于放线菌酮或硫酸锌时，“梯状”带消失；ＰＩ染色的流式细胞仪直方图上出现亚二倍体峰，并具有明显的时间和剂量效应关系。结论：儿茶素能诱导大肠癌ＬｏＶｏ细胞的凋亡，这种凋亡作用能被放线菌酮或硫酸锌所拮抗。

6种鸢尾黄素曼尼希碱衍生物的合成及其抗肿瘤活性研究

目的:对鸢尾黄素进行结构修饰,寻求新的具有抗肿瘤活性的化合物。方法:以鸢尾黄素为先导化合物,分别加入乙醇胺、甲胺、乙胺、二甲胺、二乙胺、正丙胺等胺类试剂和甲醛溶液,经曼尼希(Mannich)反应得到鸢尾黄素曼尼希碱衍生物,根据红外光谱、紫外光谱、质谱、核磁共振等确定其结构。采用溶解度试验法考察鸢尾黄素及其衍生物的水溶性;采用MTT法考察鸢尾黄素及其衍生物对人结肠癌细胞株HCT116、人肺癌细胞株A549、人肝癌细胞株HepG2的增殖抑制作用,并计算半数抑制浓度(IC50);以H22肝癌荷瘤小鼠为模型,考察鸢尾黄素及其衍生物(剂量均为100 mg/kg)的体内抑瘤率。结果:共合成6个鸢尾黄素曼尼希碱衍生物,分别为8-(N-羟乙基)-亚甲基胺基-5,7,4′-三羟基-6-甲氧基异黄酮、8-(N-甲基)-亚甲基胺基-5,7,4′-三羟基-6-甲氧基异黄酮、8-(N,N-二乙基)-亚甲基胺基-5,7,4′-三羟基-6-甲氧基异黄酮、8-(N,N-二甲基)-亚甲基胺基-5,7,4′-三羟基-6-甲氧基异黄酮、8-(N-乙基)-亚甲基胺基-5,7,4′-三羟基-6-甲氧基异黄酮、8-(N-正丙基)-亚甲基胺基-5,7,4′-三羟基-6-甲氧基异黄酮(依次记为化合物1~6)。与鸢尾黄素比较,6个衍生物的水溶性明显提高,溶解度是鸢尾黄素的5~20倍;其中化合物1、3、5对HCT116细胞的IC50分别为(34.82±3.27)、(16.21±4.13)、(33.12±3.25)μmol/L,均强于鸢尾黄素的IC50[(45.23±5.74)μmol/L];对A549细胞的IC50分别为(37.05±5.74)、(26.88±4.52)、(30.13±6.23)μmol/L,均强于鸢尾黄素的IC50[(53.24±6.34)μmol/L];对HepG2细胞的IC50分别为(23.74±1.45)、(18.96±2.34)、(30.95±2.87)μmol/L,均强于鸢尾黄素的IC50[(48.98±2.58)μmol/L];对H22肝癌荷瘤小鼠的抑瘤率分别55.51%、57.20%、49.15%,且均高于鸢尾黄素的抑瘤率(33.05%),其余3个化合物相比鸢尾黄素的抗肿瘤活性无明显优势。结论:在本研究合成的6个鸢尾黄素曼尼希碱衍生物中,化合物1、3、5均具有强于鸢尾黄素的抗肿瘤活性。

癌相关基因在结肠癌中的表达及其甲基化调控

目的 探讨癌相关基因APC、p16 INK4A、p2 1WAF1和c myc等在结肠癌中的表达及其受甲基化的调控。 方法 培养结肠癌细胞SW 1116、Colo 32 0、HT2 9,分别用不同浓度的 5 aza dC干预细胞。提取细胞的RNA ,用RT PCR的方法检测p16 INK4A、p2 1WAF1、APC和c myc等多种基因的表达情况 ;同时以流式细胞仪分析SW1116和Colo 32 0的细胞周期。结果 (1)三种细胞在干预前有较弱的 p16 INK4A和APC的表达 ,而在用 5 aza dC干预后表达增强 ,且在不同的结肠癌细胞株 5 aza dC作用发挥最佳的时间与浓度不同。 (2 ) p2 1WAF1在干预前后均不表达 ,c myc在干预前后表达无明显变化。 结论 三株人结肠癌细胞中 ,5 aza dC均可诱导 p16 INK4A和APC的表达 ,但对 p2 1WAF1和c myc无明显影响。

人参皂苷Rg3对HT-29细胞株MMP-1表达和迁移能力的影响

目的:研究人参皂苷Rg3对结肠癌细胞株HT-29基质金属蛋白酶-1(MMP-1)表达和细胞迁移能力的影响。方法:体外培养HT-29细胞,分为人参皂苷Rg3高剂量组(100μg/mL)、中剂量组(50μg/mL)、低剂量组(25μg/mL)和空白对照组,培养HT-29细胞24、48、72h,RT-PCR法测MMP-1 mRNA表达。利用划痕实验观察Rg3对HT-29细胞迁移能力的影响。结果:24h高剂量组与对照组相比,有显著的统计学差异(P<0.01);48h低、中、高剂量组与对照组相比均有显著的统计学差异(P<0.05、P<0.01、P<0.01);72h低、中、高剂量组与对照组相比均有显著的统计学差异(P<0.01、P<0.01、P<0.01)。划痕实验中空白对照组过河时间为(47.33±2.49)h,低、中、高剂量组过河时间分别为(55.33±2.49)h(P<0.01)、(64.00±1.63)h(P<0.01)和(73.33±1.89)h(P<0.01)。结论:人参皂苷Rg3能抑制HT-29细胞MMP-1的表达和抑制HT-29细胞的迁移能力,并且随着药物浓度的增加及时间的延长,抑制作用增强。

p38MAPK在结肠癌细胞凋亡中的作用及与COX-2的关系

目的探讨结肠癌细胞p38MAPK介导celecoxib(COX-2选择性抑制剂)抗肿瘤的作用及与COX-2的关系。方法用MTT法检测celecoxib对人结肠癌HT-29细胞生长的作用,用Western blot法测定各组细胞COX-2和Phosph-p38MAPK蛋白表达量,采用流式细胞术检测celecoxib和SB203580(p38MAPK特异性抑制剂)作用后HT-29细胞凋亡和细胞周期分布。结果p38MAPK和COX-2蛋白表达量与对照组(0.23±0.12)(0.95±0.14)相比,celecoxib可使p38MAPK蛋白表达水平明显升高(0.62±0.11),而使COX-2蛋白表达水平降低(0.44±0.11);SB203580使p38MAPK(0.12±0.05)及COX-2蛋白(0.23±0.13)表达水平均降低;SB203580和celecoxib共同作用后,p38MAPK表达量介于celecoxib和SB203580作用之间(0.43±0.12),COX-2表达量下降最为显著(0.15±0.10)。celecoxib和celecoxib+SB203580均可显著诱导HT-29细胞凋亡(P<0.01和P<0.05),与对照组(4.31%)相比,其凋亡率分别为40.95%、26.24%。结论在HT-29细胞中,celecoxib可通过活化p38MAPK而诱导结肠癌细胞凋亡,p38MAPK是COX-2的上游激酶,COX-2的表达水平受p38MAPK调控,并且COX-2可能对p38MAPK有负反馈调节作用。celecoxib是通过COX-2及其以外的p38MAPK通路诱导肿瘤细胞凋亡而发挥抗肿瘤作用的。

mTOR信号通路介导黄芩苷抑制人结肠癌细胞的增殖

目的探讨黄芩苷抑制人结肠癌细胞(HCT116)增殖的分子机制。方法采用MTT比色法检测不同浓度的黄芩苷(0.01～100μg/ml)对体外培养的HCT116细胞增殖的抑制作用;采用Western blot分析检测被黄芩苷干预的HCT 116细胞的增殖相关蛋白的表达。结果与对照组比较,浓度为1～100μg/ml的黄芩苷处理组明显抑制HCT 116细胞的增殖(P<0.05)。用100μg/ml黄芩苷处理后,增殖相关的蛋白mTOR、p70S6K、S6、eIF4E表达明显下调(P<0.05),4E-BP1表达明显上调。p-mTOR(Ser2448)、p-S6(Ser235/236)、p-4E-BP1(Thr37/46)的磷酸化水平下降(P<0.05)。结论黄芩苷能通过抑制mTOR信号通路来抑制人结直肠癌HCT116细胞的增殖。