鳖甲浸出液对人肠癌细胞（HR－8348）的毒性作用研究

报道鳖甲浸出液对人肠癌细胞系（ＨＲ－８３４８）的体外实验，分别从细胞抑制率、细胞集落形成率、细胞上清液中ＬＤＨ活性的测定，光、电镜下的细胞形态观察，探讨了鳖甲对人肠癌的作用机理。初步结果表明：单纯鳖甲２ｇ／Ｌ浓度以上对肠癌细胞有抑制生长作用，鳖甲加氟脲嘧啶联合用药对肠癌细胞的抑制生长作用较单纯氟脲嘧啶更显著。这一实验结果将为临床应用鳖甲抗肠癌治疗提供了实验依据，并提示：鳖甲加氟脲嘧啶联合用药对肠癌的治疗效果更佳，充分显示了协同抗癌作用。

乌骨藤提取物体外对人肠癌细胞增殖实验研究

目的:研究乌骨藤提取物对体外培养的人肠癌细胞(lovo)增殖的影响。方法:用两种方法(A、B)制备乌骨藤提取物,采用不同浓度的乌骨藤提取物分别作用于培养的lovo细胞,通过细胞形态学和MTT法检测乌骨藤提取物对lovo细胞增殖的影响。结果:乌骨藤提取物A对细胞的最大无毒浓度大约为0.648mg/ml;药物浓度在0.389mg/ml、0.648mg/ml、1.08mg/ml、1.8mg/ml、3mg/ml时的抑制率分别为6.58%、8.24%、13.02%、59.69%、66.23%;用回归法求出乌骨藤提取物A对lovo细胞增殖的半数抑制浓度为1.625mg/ml。乌骨藤提取物A对细胞的最大无毒浓度大约为0.648mg/ml;药物浓度在0.389mg/ml、0.648mg/ml、1.08mg/ml、1.8mg/ml、3mg/ml时的抑制率分别为14.49%、15.48%、29.69%、63.26%、64.05%;用回归法求出乌骨藤提取物B对lovo细胞增殖的半数抑制浓度为1.84mg/ml。结论:乌骨藤提取物对体外培养的人肠癌细胞(lovo)的增殖有明显抑制作用。

中药长必安对人大肠癌细胞CD_(44)表达的意义

目的探讨中药长必安对人大肠癌细胞表面CD44 表达的影响。方法按长必安含药小鼠血清作用浓度 ,将两株大肠癌细胞各自分别分成低、中、高三组 ,同时设阴性对照组、阳性对照组和长必安与5 -Fu联合用药组。将FITC标记鼠抗人CD44 抗体与上述各组细胞共同孵育后 ,用流式细胞仪检测各组细胞表面CD44 阳性百分率。结果中药长必安作用的各组细胞其CD44 的阳性率较阴性对照组和阳性对照组都有显著下降。结论长必安不仅能抑制大肠癌细胞的体外生长 ,还能有效地降低大肠癌细胞表面的CD44 表达 ,提示在临床应用中 ,长必安在直接抑制肿瘤的同时 ,还能调节机体的免疫应答和免疫状态。

黄连小檗碱对人肠癌Caco-2细胞凋亡和细胞周期的影响

目的探讨黄连小檗碱对人肠癌细胞株Caco-2细胞周期和细胞凋亡的影响及其作用机制。方法采用MTT法,检测不同时间点不同浓度黄连小檗碱对Caco-2细胞的增殖抑制作用;应用流式细胞术(Annexin V-FITC/PI双染法),检测不同浓度黄连小檗碱对Caco-2细胞周期和凋亡的影响。结果不同浓度的黄连小檗碱干预肠癌细胞Caco-2不同时间后,均具有一定的增殖抑制作用,在一定浓度范围内,可以剂量和时间依赖性的抑制Caco-2细胞增殖。黄连小檗碱干预细胞24 h后,可使Caco-2细胞停滞在G2/M期,诱导细胞凋亡(P<0.05)。结论黄连小檗碱可以显著抑制Caco-2细胞增殖,其作用机制可能与阻滞细胞周期从而诱导细胞凋亡有关。

T0901317通过法尼醇X受体调节人肠癌细胞载脂蛋白M表达

载脂蛋白M（ApoM）是一种脂质运载蛋白家族的血浆蛋白质，主要存在于HDL中。本研究旨在观察T0901317通过法尼醇x受体调节人肠癌细胞载脂蛋白M表达。

小干扰RNA下调长链非编码RNATUG1表达对肠癌细胞LoVo增殖、迁移能力的影响

我们探讨TUG1在多株人肠癌细胞中表达情况及其与肠癌细胞生物学功能的关系。一、材料与方法1．材料：肠癌细胞株HCT116、HCT8、HT29、LS174T、LoVo和SW620购自美国典型菌种保藏中心（ATCC）。Lipofectamine 2000购自Invitrogene公司；针对TUGl的小干扰RNA（siRNA）及无关序列由深圳吉玛公司合成；反转录-聚合酶链反应（RT-PCR）仪购自美国ABI公司；细胞计数试剂盒（CCK-8）购自上海碧云天生物技术公司；Transwell试剂盒购自B＆D公司。

青龙衣化学成分体内抗肿瘤作用及其急性毒性试验研究

目的:研究青龙衣中6个化学成分对人胃癌BGC-823和人肠癌HCT-15细胞的抑制作用及其急性毒性情况。方法:采用MTT法对从青龙衣中分离获得的6个化合物胡桃醌、3-乙氧基胡桃醌、12β,20(R),24(R)-三羟基达玛烷-25-烯-3-酮、20(S)-原人参二醇、2α,3β,23-三羟基-12-烯-28-熊果酸、Juglanin A进行BGC-823、HCT-15细胞毒活性筛选,并采用寇氏法对上述化合物进行急性毒性试验研究。结果:6种化合物对BGC-823和HCT-15细胞均有抑制作用,其中以胡桃醌的细胞毒作用最强,IC_(50)值分别为(9.6±2.35)μmo1/L和(27.8±2.66)μmo1/L;同时急性毒性试验表明上述化合物均有一定毒性作用,其中胡桃醌毒性较大,LD_(50)值为33.57 mg/kg,毒性的反应时间较快,对小鼠机体的损伤最大。结论:该实验结果为进一步研究青龙衣的体内抗肿瘤作用及其作用机制提供了可行性实验依据和物质基础。

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 historic 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 methyla-tion-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 p21WAF1 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.

Docosahexaenoic acid suppresses arachidonic acid-induced proliferation of LS-174T human colon carcinoma cells

AIM： To investigate the impact of arachidonic acid （AA） and docosahexaenoic acid （DHA） and their combination on colon cancer cell growth. METHODS： The LS-174T colon cancer cell line was used to study the role of the prostaglandin precursor AA and the omega-3 polyunsaturated fatty acid DHA on cell growth. Cell viability was assessed in XTT assays. For analysis of cell cycle and cell death, flow cytometry and DAPI staining were applied. Expression of cyclooxygenase-2 （COX-2）, p21 and bcl-2 in ceils incubated with AA or DHA was examined by real-time RT-PCR. Prostaglandin E2 （PGE2） generation in the presence of AA and DHA was measured using a PGE2- ELISA. RESULTS： AA increased cell growth, whereas DHA reduced viability of LS 174T cells in a time- and dosedependent manner. Furthermore, DHA down- regulated mRNA of bcl-2 and up-regulated p21. Interestingly, DHA was able to suppress AA-induced cell proliferation and significantly lowered AA-derived PGE2 formation. DHA also down-regulated COX-2 expression. In addition to the effect on PGE2 formation, DHA directly reduced PGE2-induced cell proliferation in a dosedependent manner. CONCLUSION： These results suggest that DHA can inhibit the pro-proliferative effect of abundant AA or PGE2.

Inhibition of invasiveness and expression of epidermal growth factor receptor in human colorectal carcinoma cells induced by retinoic acid

Human amniotic basement membrane (HABM) model and agarose drop explant method were used to in-vestigate the effects of retinoic acid(RA) on the invasive-ness alld adhesiveness to the basement membrane, and the migration of a highly invassive human colorectal cancer cell line CCL229. Results showed that 5 ×106 MRA markedly reduced the in vitro invasiveness and adhesiveness to the HABM, and the migration of the CCL229 cells. In addi-tion, to elucidate the relation between expression of epider-mal growth factor receptor (EGFR) and the invasiveness of the colorectal carcinoma cells, two well-differentiated, but with different invasiveness colorectal cancer cell lines were compared at mRNA level for expressioll of EGFR by using EGFR cDNA probe labeled with digoxigenin (DIG). Expression of EGFR was showll to be markedly higher in the highly invassive CCL229 cells than that in the low in- vasive CX-1 cells. Furthermore, expression of EGFR in RA treated CCL229 cells gradually decreased with time,the level being the lowest on day 6 of the RA treatment.

TTYH2,a human homologue of the Drosophila melanogaster gene tweety,is up-regulated in colon carcinoma and involved in cell proliferation and cell aggregation

AIM: To investigate the expression patterns of TTYH2 in the human colon cancer and colon cancer cell lines and to evaluate the inhibitory effect of small interfering RNA (siRNA) on the expression of TTYH2 in colon cancer cell lines.METHODS: We investigated the expression patterns of TTYH2 in colon cancer, adjacent non-tumorous colon mucosa, and cancer cell lines (DLD-1, caco-2, and Lovo) by RT-PCR. Furthermore, a siRNA plasmid expression vector against TTYH2 was constructed and transfected into DLD-1 and Caco-2 with LipofectamineTM 2000. The down regulation of TTYH2 expression was detected by RT-PCR and the role of siRNA in inducing cell proliferation and cell aggregation was evaluated by MTT and aggregation assay.RESULTS: TTYH2 gene expression in colon cancer tissue was significantly up-regulated compared with normal colonic mucosa (1.23 ± 0.404 vs 0.655 ± 0.373, P = 0.0103). Colon cancer derived cell lines including DLD-1, Caco-2, and Lovo also expressed high levels of TTYH2. In contrast, transfection with siRNA-TTYH2 signifi cantly inhibited both proliferation and scattering of these cancer cell lines.CONCLUSION: The present work demonstrates, for the fi rst time, that the TTYH2 gene expression is signifi cantly up-regulated in colon cancer. The TTYH2 gene may play an important role in regulating both proliferating andmetastatic potentials of colorectal cancer.

Ginsenoside Rg3 inhibits colon cancer cell migration by suppressing nuclear factor kappa B activity

OBJECTIVE: To study the mechanism of the inhibitory effect of ginsenoside Rg3 on colon cancer cell migration.METHODS: Transwell migration assays were performed to investigate the inhibitory effect of ginsenoside Rg3 on SW480 cell migration. Electrophoretic mobility shift assays(EMSAs) and dual luciferase reporter assays were used to study the suppression capability of Rg3 on nuclear factor kappa B(NF-κB) activity. Western blotting was adopted to determine protein levels.RESULTS: Two-hundred micromolar ginsenoside Rg3 significantly inhibited SW480 cell migration(P < 0.05). EMSA showed that Rg3 suppressed the DNA binding ability of NF-κB. Dual luciferase reporter assay showed that Rg3 decreased NF-κB-regulated gene transcription(P < 0.01). Western blots indicated that Rg3 down-regulated expression of the NF-κB-regulated matrix metalloproteinase 9,cyclooxygenase-2 and C-Myc. An NF-κB inhibitor,pyrrolidine dithiocarbamate,enhanced the inhibitory effect of Rg3 on SW480 cell migration.CONCLUSION: Ginsenoside Rg3 has a strong antitumor migration capability by suppressing NF-κB activity and expression of NF-κB-regulated gene products. It could be a good adjuvant for colon cancer patients during the course of chemotherapy.