Effects of alpha-difluoromethylornithine and 5-fluorouracil on the proliferation of a human colon adenocarcinoma cell line

Because alpha-difluoromethylornithine (DFMO) reduces the incidence of experimental colon cancers, inhibits the growth of human lung cancer cells and human leukemia cells in culture, and in combination with methylglyoxal (bis)guanylhydrazone induces remission in children with leukemia, its effectiveness against a human colon adenocarcinoma cell line (Colo 205) was tested alone and in combination with 5-fluorouracil (5-FU). Both DFMO (2 X 10(-4) M) and 5-FU (10(-6) M) inhibited Colo 205 cell proliferation. Above 5 X 10(-4) M DFMO (p less than 0.001) and at 10(-4) M 5-FU (p less than 0.001), Colo 205 growth was completely inhibited. Although DFMO did not sensitize Colo 205 cells to a noninhibitory concentration of 5-FU, the effectiveness of inhibitory concentrations of 5-FU and DFMO in reducing Colo 205 cell growth was additive. DFMO (2 X 10(-4) M) caused 89 to 93% inhibition of ornithine decarboxylase activity (p less than 0.001) and reduced levels of putrescine (93%; p less than 0.01) and spermidine (57%; p less than 0.02). Growth rate and the intracellular putrescine and spermidine contents were restored by 10(-6) M putrescine. DFMO could be an effective chemotherapeutic agent against human colonic cancer because of its effects at such unusually low concentrations in vitro.     

Supraadditive effect of 2′,2′-difluorodeoxycytidine (gemcitabine) in combination with oxaliplatin in human cancer cell lines

PURPOSE: This study assessed the cytotoxic effects of the nucleoside analog gemcitabine in combination with the diaminocyclohexane platinum compound oxaliplatin. METHODS: Growth inhibition studies were performed using the human CEM leukemia cell line and the colon-cancer cell lines HCT 116 and Colo 320 DM. Gemcitabine-oxaliplatin combinations were compared with gemcitabine-cisplatin combinations in the same cell lines using similar experimental settings. Cells were exposed for 2 h to gemcitabine and then for 24 h to oxaliplatin or cisplatin, and vice versa. RESULTS: The 50% inhibitory concentrations (IC50 values) in single-drug experiments using 2 h of exposure to gemcitabine and 24 h of exposure to oxaliplatin or cisplatin were, respectively, 89 pM, 11.1 microM, and 10.3 microM for CEM cells; 46 pM, 10.2 microM, and 2.7 microM for HCT 116 cells; and 102 pM, 4.6 microM, and 8.6 microM for Colo 320 DM cells. Gemcitabine-oxaliplatin combinations displayed supraadditive effects in human leukemia and colon-cancer cell lines. The sequence of gemcitabine followed by oxaliplatin was more effective than the opposite sequence in HCT 116 and Colo 320 DM colon-cancer cell lines, whereas the sequence of oxaliplatin followed by gemcitabine yielded to synergistic effects in CEM cells. The cytotoxic effects of gemcitabine-oxaliplatin combinations were better than (HCT 116 cells) or equal to (CEM and Colo 320 DM cells) those of gemcitabine-cisplatin combinations. CONCLUSION: Our data show that the combination of gemcitabine with oxaliplatin exerts potent antiproliferative effects in human leukemia and colon cancer cells, warranting further investigations in the framework of phase I-II trials as an alternative for the treatment of solid malignancies.     

Macrophages from cancer patients: analysis of TRAIL,TRAIL receptors,and colon tumor cell apoptosis

BACKGROUND: Tumor-infiltrating macrophages secrete cytokines, including Fas ligand, tumor necrosis factor-alpha (TNF-alpha), and TNF-related apoptosis-inducing ligand (TRAIL). TRAIL induces apoptosis in tumor cells but not in normal cells; however, regulation of TRAIL and its receptors in cancer patients is relatively uncharacterized. We investigated whether macrophages from cancer patients produce TRAIL and whether apoptosis in cultured colon adenocarcinoma cells involves TRAIL and its receptors. METHODS: Macrophages isolated from pleural effusions of nine cancer patients and five control patients with congestive heart failure (whose effusions contained no tumor cells) were cultured. Levels of TRAIL, TNF-alpha, interferon alpha, and Fas ligand in conditioned medium were measured by enzyme-linked immunosorbent assays. Apoptosis of human colon adenocarcinoma cell lines, including Colo 205, was determined by the Annexin V method and terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick-end labeling (TUNEL). Cell-surface TRAIL receptors were measured by flow cytometry. RESULTS: Conditioned culture medium from macrophages isolated from pleural effusions containing 1%-5% tumor cells (CM-A) contained TRAIL at 980-1300 pg/mL, whereas that from macrophages from pleural effusions containing more than 50% tumor cells or containing no tumor cells (CM-B) contained TRAIL at 0-50 pg/mL. When cultured with medium containing 50% CM-A, 40% (95% confidence interval [CI] = 30% to 50%) of Colo 205 cells underwent apoptosis; when cultured with 50% CM-B, 8% (95% CI = 3% to 13%) underwent apoptosis. When Colo 205 cells were cultured with 50% CM-A, cell-surface expression of TRAIL death receptors DR5 and DR4 increased 13-fold and sixfold, respectively, compared with that of untreated Colo 205 cells. Recombinant TRAIL induced 90% (95% CI = 85% to 95%) of Colo 205 cells to undergo apoptosis and acted synergistically with TNF-alpha to induce apoptosis. CONCLUSION: Macrophages from cancer patients appear to be activated by tumor cells to produce TRAIL and to increase the expression of TRAIL death receptors DR4 and DR5 on tumor cells.     

促胃液素对结肠癌细胞侵袭力的影响

目的研究促胃液素对人结肠癌细胞侵袭力的影响,以及促胃液素-促胃液素受体-黏着斑激酶(FAK)信号通路在此过程中的作用。方法使用促胃液素受体的真核表达载体pCR3．1／GR转染人结肠癌细胞株Colo320(Colo320／GR),使其高表达促胃液素受体,达到上调信号通路的作用;使用反义寡核苷酸阻断FAK的表达,达到下调信号通路的作用。使用递增剂量的促胃液素(0,0．1,1,10,100nmol／L)刺激Colo320和Colo320／GR细胞,用Boyden小室检测各组细胞侵袭力变化;使用免疫沉淀和蛋白质印迹法,柃测细胞FAK第397位酪氨酸(tyr-397)磷酸化的状况。使用免疫沉淀、蛋白质印迹法和Boyden小室法检测正常细胞、Colo320／GR、转染反义寡核苷酸组细胞和对照组细胞在接受促胃液素刺激前后,细胞侵袭力和FAK tyr-397磷酸化的变化。结果促胃液素能够增强Colo320和Colo320／GR细胞侵袭力和FAK tyr-397磷酸化,具有剂量依赖性。促胃液素受体表达增加可以增强细胞的侵袭力和FAK tyr-397磷酸化。使用FAK反义寡核苷酸对FAK的表达进行阻断后,促胃液素的作用明显下降。结论促胃液素可有效地增强结肠癌细胞的侵袭力,其作用是通过促胃液素-促胃液素受体-FAK通路实现的。     

PHA-767491对结直肠癌Colo320细胞增殖和凋亡的影响

目的:探讨PHA-767491对结直肠癌细胞系Colo320细胞增殖和凋亡的影响。方法:配置不同浓度的PHA-767491作用于Colo320细胞，利用CCK-8法检测细胞的增殖能力；流式细胞术Annexin V/PI双染色法检测细胞的凋亡情况。结果:CCK-8结果显示PHA-767491能显著抑制Colo320细胞的增殖能力。流式细胞术检测结果显示PHA-767491能诱导Colo320细胞的凋亡。结论:PHA-767491能够抑制Colo320细胞的增殖并且诱导凋亡。     

Inhibition of mitogen stimulated growth of human colon cancer cells by interferon

Recombinant human interferon alpha inhibits growth of a human colon cancer cell line, Colo 205. To explore the mechanisms of IFN induced growth inhibition, quiescent Colo 205 cells were stimulated to proliferate in serum-free media by defined growth factors. Addition of insulin, transferrin and selenium (ITS) stimulated DNA synthesis, as measured by 3H-thymidine incorporation, in a dose-dependent manner. IFN-alpha (at concentrations greater than 100 U ml-1) inhibited ITS stimulated DNA synthesis by 63%. Inhibition of cell cycle traverse was confirmed by flow cytometric analysis. Although IFN inhibited growth of ITS-treated cells, steady state levels of c-myc mRNA remained above levels observed in unstimulated cells. IFN inhibited DNA synthesis only when added prior to mitogen stimulation. IFN, added 6 h after exposure of quiescent cells to ITS, failed to inhibit cell growth. Addition of increasing concentrations of ITS failed to overcome the IFN-induced growth inhibition. These results suggest IFN may inhibit cell growth in part by antagonizing the action of growth factors.     

Hedgehog signaling pathway is inactive in colorectal cancer cell lines

The Hedgehog (Hh) signaling pathway plays an important role in human development. Abnormal activation of this pathway has been observed in several types of human cancers, such as the upper gastro-intestinal tract cancers. However, activation of the Hh pathway in colorectal cancers is controversial. We analyzed the expression of the main key members of the Hh pathway in 7 colon cancer cell lines in order to discover whether the pathway is constitutively active in these cells. We estimated the expression of SHH, IHH, PTCH, SMO, GLI1, GLI2, GLI3, SUFU and HHIP genes by RT-PCR. Moreover, Hh ligand, Gli3 and Sufu protein levels were quantified by western blotting. None of the cell lines expressed the complete set of Hh pathway members. The ligands were absent from Colo320 and HCT116 cells, Smo from Colo205, HT29 and WiDr. GLI1 gene was not expressed in SW480 cells nor were GLI2/GLI3 in Colo205 or Caco-2 cells. Furthermore the repressive form of Gli3, characteristic of an inactive pathway, was detected in SW480 and Colo320 cells. Finally treatment of colon cancer cells with cyclopamine, a specific inhibitor of the Hh pathway, did not downregulate PTCH and GLI1 genes expression in the colorectal cells, whereas it did so in PANC1 control cells. Taken together, these results indicate that the aberrant activation of the Hh signaling pathway is not common in colorectal cancer cell lines.     

Insulin receptor regulation in cultured human tumor cells

Insulin binding to monolayer cell cultures of human fibroblasts, human colon carcinoma (HCT-8, HT-29), human breast carcinoma (MCF-7, T-47D), and melanoma (MM-96) was measured using 125I-insulin. Binding was time and temperature dependent in all cell lines, and only one cell line (MM-96) degraded 125I-insulin. High-affinity insulin-binding sites (Kd = 1.4 X 10(-9) M to 0.4 X 10(-10) M) were detected in all cell lines, and insulin-binding capacity ranged from 0.6 to 14 fmol/10(6) cells. Receptor down-regulation was studied by exposing cells to increasing concentrations of unlabeled insulin, dissociating surface-bound insulin and measuring residual receptors by 125I-insulin uptake. Exposure of tumor cells to greater than 10(-6) M insulin for 2 hr at 37 degrees led to a decrease in the number of insulin binding sites in MM-96 and colon cell lines only, with maximum down-regulation ranging from 58% (MM-96) to 88% (HCT-8) receptor loss. The decrease in insulin binding was due to a decreased number of receptors per cell with no change in affinity. Monolayers exposed to 1.7 X 10(-5) M unlabeled insulin for 7 hr at 37 degrees invariably showed greater than 50% receptor loss. However, monolayers exposed to 1.7 X 10(-8) M unlabeled insulin for 7 hr at 37 degrees showed less marked (0 to 39%) down-regulation. In comparison, human fibroblasts showed 57% receptor loss after exposure to 3.5 X 10(-9) M unlabeled insulin for 7 hr. Thus, markedly supraphysiological concentrations of insulin are required to down-regulate insulin receptors in tumor cell lines compared with normal cells. This suggests a tumor-associated resistance to receptor down-regulation.     

靶向siRNA对人直肠癌Colo320细胞的抑制作用

目的:利用小分子干扰RNA(siRNA)技术抑制人直肠癌Colo320细胞c-my基因的表达,探讨c-myc基因在人直肠癌Colo320细胞中的作用.方法 :设计人直肠癌Colo320细胞基因特异性小分子干扰RNA,用体外转录方法合成人直肠癌Colo320细胞的小分子干扰RNA并转染该细胞,培养48～96 h后,收集细胞RNA,应用实时荧光定量PCR方法检测转染细胞中c-myc基因mRNA水平变化,Western blotting检测c-myc表达的蛋白,四甲基偶氮唑蓝(MTT)法和集落形成试验检测细胞增殖活性.结果:转染siRNA后,与对照组相比,实验组pGensil-c-myc-1、2、3、4的c-myc基因mRNA水平明显降低,c-myc的蛋白表达也明显降低.MTT法和集落形成试验检测表明,实验组的细胞增殖速率明显低于对照组.结论:在人直肠癌Colo320 细胞中存在RNA干扰的机制,特异性siRNA能够有效地抑制c-myc基因的表达,从而抑制Colo320 细胞的增殖.     

hFRNK基因转移对结肠癌细胞中E-钙黏素/β-连环素复合物的影响

目的研究hFRNK基因对结肠癌细胞Colo320WT中E-钙黏素（E-cadherin）／β-连环素（β-catenin）复合物的影响。方法利用AdEasy^TM系统在大肠肝菌内同源重组构建表达人FRNK基因的腺病毒载体pAdhFRNK。脂质体转染pCR3．1／GR质粒于结肠癌细胞Colo320中,G418筛选出稳定表达CCK-2R的阳性克隆,RT-PCR鉴定。用10^-8 mol／L胃泌素干预Colo320WT细胞12 h和pAdhFRNK体外感染Colo320WT细胞2 d后,再用10^-8 mol／L的胃泌素干预细胞12 h,然后用免疫共沉淀观察TX-100可溶性部分和不溶性部分的E-cadherin和β-catenin的表达。利用细胞化学方法观察E-cadherin和β-catenin在Colo320WT细胞中的分布情况。结果在胃泌素干预12 h后的TX-100可溶性部分中,E-cadherin和β-catenin的表达量明显降低,而TX-100不溶解部分表达增加。pAdhFRNK感染胃泌素干预后的细胞,在TX-100可溶性部分中,E-cadherin和β-catenin的表达量增加,而TX-100不溶性部分中表达降低。细胞化学方法显示,E-cadherin和β-catenin在胃泌素干预12 h后,由胞膜向胞内和胞核转移,而胃泌素干预pAdhFRNK感染后的细胞中,两者分布又逆转。结论hFRNK基因可明显对抗外源性胃泌素引起Colo320WT细胞中E-cadherin和β-catenin的分布异常,其机制可能是通过阻断FAK磷酸化及其通路而实现。     

转移相关基因CD44V6在不同恶性肿瘤细胞系中的表达及意义

目的 寻找具有高转移潜能的肿瘤细胞系。方法 采用免疫组织化学染色ＳＰ法,检测ＣＤ４４Ｖ６在不同恶性肿瘤细胞系中表达。结果 仅有Ｃｏｌｏ－２０５细胞表达ＣＤ４４Ｖ６,其表现为细胞膜表面有呈连续线状分布的棕色颗粒。结论 大肠癌Ｃｏｌｏ－２０５细胞可能具有较高的转移能力。     

Apoptosis Induced by NS-398, a Selective Cyclooxygenase-2 Inhibitor, in Human Colorectal Cancer Cell Lines

Recent studies have suggested that apoptosis is a key phenomenon in the chemopreventive action of nonsteroidal antiinflammatory drugs (NSAIDs), which exhibit cancer-preventive and tumor-regressive effects in the human colon. The effect of NS-398, N -(2-cyclohexyloxy-4-nitrophenyl)methanesul-fonamide, which is a selective inhibitor of cyclooxygenase-2 (COX-2), on the induction of apoptosis in two human colorectal cancer cell lines (Colo320 and THRC) was determined. The apoptotic ratios (-fold vs. control value) of Colo320 in the presence of 100 μM indomethacin and NS-398 were 3.3 ± 1.5 and 9.0±0.94, and those of THRC were 2.3±0.46 and 7.4±0.87, respectively. The ability of NS-398 to induce apoptosis is greater than that of indomethacin. Both indomethacin and NS-398 reduced the cell proliferation in a concentration-dependent manner. The IC₅₀ values of NS-398 (54.8+3.6 and 77.2±4. 9μM ) were significantly lower than those of indomethacin (206.3±43.0 and 180.3±22.6/ μM ) at P<0.01 in Colo320 and THRC cell lines, respectively. These findings suggest that NS-398, a selective inhibitor of COX-2, is a possible candidate for a chemopreventive agent with a potent apoptosis-inducing effect and low nlcerogenic activity.     

The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-kappa B inhibition

Addressing the puzzling role of amidated gastrin(17) (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis, we analysed potential candidate genes involved in G17-dependent NF-kappaB inhibition and apoptosis. The colorectal carcinoma cell line Colo320 overexpressing the wild-type CCK2 receptor (Colo320wt) underwent G17-induced apoptosis along with suppressed NF-kappaB activation and decreased expression of the antiapoptotic NF-kappaB target genes cIAP1 and cIAP2, whereas G17 was without effect on Colo320 cells expressing a CCK2 receptor bearing a loss of function mutation (Colo320mut). Gene microarray analysis revealed an elevated expression of the stress response gene IEX-1 in G17-treated Colo320wt but not Colo320mut cells. Quantitative real-time PCR and conventional RT-PCR confirmed this G17-dependent increase of IEX-1 expression in Colo320wt cells. If these cells were subjected to IEX-1 knockdown by small interfering RNA transfection, the apoptosis-inducing effect of G17 was abolished. Moreover, tumor necrosis factor alpha (TNFalpha)- or 5-FU-induced apoptosis that is greatly enhanced by G17 treatment in Colo320wt cells was prevented if IEX-1 expression was repressed. Under these conditions of blocked IEX-1 expression, the NF-kappaB activity remained unaffected by G17, in particular in Colo320wt cells co-treated with TNFalpha and also the suppressive effect of G17 on cIAP1 and cIAP2 expression was not observed anymore if IEX-1 expression was blocked. Conversely, IEX-1 overexpression in Colo320mut cells caused an increase of basal and TNFalpha- or 5-FU-induced apoptosis, an effect not further triggered by G17 treatment. Using a xenograft tumor model in severe combined immune deficiency mice, we could show that experimental systemic hypergastrinemia induced by the administration of omeprazole led to enhanced apoptosis as well as to a marked increase of IEX-1 expression in Colo320wt tumors, but not in Colo320mut tumors. These observations indicate that the proapoptotic effect of G17 on human colon cancer cells expressing the wild-type CCK2 receptor is mediated by IEX-1, which modulates NF-kappaB-dependent antiapoptotic protection and thereby exerts tumor-suppressive potential.     

Antitumor effect of 2'-deoxy-5-fluorouridine conjugates against a murine thymoma and colon carcinoma xenografts.

The conjugation of antineoplastic drugs to monoclonal antibodies reactive with tumor associated antigens conveys selective cytotoxicity, overcoming the systemic toxicities caused by drugs during standard chemotherapy. 2'-Deoxy-5-fluorouridine, a more potent derivative of 5-fluorouracil, is an antimetabolite which exerts its cytotoxic action by inhibiting the enzyme thymidylate synthetase and as a result inhibits DNA synthesis. 2'-Deoxy-5-fluorouridine was successfully conjugated to anti-Ly-2.1 reactive with the murine thymoma ITT(1)75NS E3, I-1, and 250-30.6 reactive with human colon cancer cells using the active ester of 2'-deoxy-5-fluoro-3'-O-succinoyluridine (5FdUrdsucc). In vitro, 5Fd-Urdsucc-anti-Ly-2.1 was selectively cytotoxic against ITT(1)75NS E3 murine thymoma cells at nanomolar concentrations. The human colon carcinoma cell LIM1899 was inhibited by 5FdUrdsucc-I-1 conjugates in the range of 10(-7)-10(-8) M, as were Colo 205 cells by 5FdUrdsucc-250-30.6 conjugates. In vivo, 5FdUrdsucc conjugates were more effective than equivalent amounts of free 5FdUrdsucc. Against the ITT(1)75NS E3 murine thymoma, a single dose of 100 micrograms (5FdUrdsucc equivalents) 5FdUrdsucc-anti-Ly-2.1 resulted in 85% tumor inhibition compared to mean tumor size of control mice. Irrelevant 5FdUrdsucc conjugates failed to inhibit tumor growth. Multiple doses of 5FdUrdsucc-I-1 conjugate produced 50% growth inhibition of the moderately differentiated tumor LIM1899. In contrast, the human colon carcinoma Colo 205 was relatively resistant to free 5FdUrdsucc and 5FdUrdsucc-250-30.6 conjugates.     

Increased EGF receptors on human squamous carcinoma cell lines

Characterisation and quantitation of epidermal growth factor receptors (EGFR) have been carried out on eight human squamous carcinoma cell lines and the results compared with those from simian virus transformed keratinocytes and normal keratinocytes grown under similar conditions. All cells tested possess both high and low affinity receptors with dissociation constants ranging from 2.4 X 10(-10) M to 5.4 X 10(-9) M. When epidermal growth factor (EGF) binds to its receptor it is internalised and degraded and the receptor is down regulated. Malignant cells and virally transformed cells possess 5-50 times more EGF receptors than normal keratinocytes and one cell line LICR-LON-HN-5 possesses up to 1.4 X 10(7) receptors per cell, which is the highest number yet reported for a cell line. These results are discussed in the context of recent data that suggest that the increased expression of EGF receptors in epidermoid malignancies may be an important component of the malignant phenotype in these tumours.     

Conjugated linoleic acid inhibits peritoneal metastasis in human gastrointestinal cancer cells

The effect of conjugated linoleic acid (CLA) on peritoneal metastasis was examined by in vitro treatment of cancer cells and mouse peritoneal metastasis models. First, cell growth of MKN28 human gastric cancer cells and Colo320 human colon cancer cells was suppressed by CLA in a dose-dependent manner with an increment in apoptosis. CLA significantly inhibited invasion into type IV collagen-coated membrane of MKN28 and Colo320 cells (p < 0.05). CLA-induced growth inhibition was recovered by the exposure to antisense S-oligodeoxynucleotide for peroxisome proliferator-activated receptor (PPAR)-gamma in both cell lines. BALB/c nu-nu mice were inoculated with MKN28 and Colo320 cells into their peritoneal cavity, and administrated with CLA intraperitoneally (weekly, 4 times). CLA treatment did not affect food intake or weight gain of mice. CLA treatment significantly decreased metastatic foci of both cells in the peritoneal cavity (p < 0.005). Survival rate in mice inoculated with MKN28 or Colo320 cells was significantly recovered by CLA treatment (p = 0.0025 and 0.0052, respectively). Protein production in MKN28 and Colo320 cells treated with CLA showed a decrease in epidermal growth factor receptor and transforming growth factor-alpha and an increase in Bax. These findings suggest that CLA inhibits metastasis of human gastric and colon cancer cells.     

5-aminosalicylic acid enhances anchorage-independent colorectal cancer cell death

Resistance to anoikis, the cell death triggered by the loss of anchorage to the substratum, is an essential prerequisite in the proliferation and diffusion of colorectal cancer (CRC) cells. We examined whether 5-aminosalicylic acid (5-ASA), a drug that seems to reduce the risk of colitis-associated CRC, enhances CRC cell anoikis. To this end, Colo205 cells were treated with 5-ASA in the presence or absence of inhibitors of caspases (zVAD-fmk) and reactive oxygen species (ROS). We demonstrate that 5-ASA enhances Colo205 cell death. Although 5-ASA induces dissipation of mitochondrial transmembrane potential and caspase-3 activation, zVAD-fmk does not completely prevent the 5-ASA-induced cell death. 5-ASA also enhances the synthesis of ROS. However, inhibitors of ROS reduce the fraction of 5-ASA-induced Colo205 cell death but do not confer protection. In contrast, the 5-ASA-mediated Colo205 cell death is preventable by Bcl-2 over-expression. These data suggest a mechanism by which 5-ASA interferes with colon carcinogenesis.     

The Hollow Fibre Assay as a model for in vivo pharmacodynamics of fluoropyrimidines in colon cancer cells

The Hollow Fibre Assay (HFA) is usually applied as an early in vivo model for anti-cancer drug screening, but is potentially an excellent model for short-term in vivo pharmacodynamic studies. We used the model to study the in vivo role of thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) in the cytotoxicity and pharmacodynamics of TAS-102 in colon cancer cells. TAS-102 is a new oral drug formulation, which is composed of trifluorothymidine (TFT) and thymidine phosphorylase inhibitor (TPI), which prevents TFT degradation. We compared the activity with Xeloda (capecitabine), which is activated by TP into 5FU. Hollow fibres filled with human Colo320 or Colo320TP1 colorectal cancer cells with deficient or high TP expression, respectively, were implanted subcutaneously (s.c.) at both flanks of BALB/c mice. The mice were treated orally over 5 days with TAS-102, TFT alone, 5'DFUR+/-TPI or capecitabine at their maximum tolerated dose (MTD). The cells were retrieved from the fibres and assayed for growth (MTT assay), cell cycle distribution (flow cytometry) and apoptosis induction (FragEL method). TAS-102 induced considerable growth inhibition (50%, P<0.01) to both cell lines, which was completely abolished in the absence of TPI. Capecitabine and its metabolite 5'DFUR reduced proliferation of Colo320TP1 cells in the fibres significantly (down to 25-40%), but much less in Colo320 cells, whereas addition of TPI reduced the effect of 5'DFUR, although not completely. These differences in cytotoxic effects were reflected in the pharmacodynamic evaluation. TAS-102 induced a G2M-phase arrest (from 25 to 40%) and apoptosis (>8-fold), which was more pronounced in Colo320 than in Colo320TP1. Again, omission of TPI neutralised the effect of TAS-102. Similarly, 5'DFUR and capecitabine induced a significant G2M-phase arrest (up to 45%) in the Colo320TP1 cell line, but less pronounced in the parental Colo320. Addition of TPI to 5'DFUR reduced this effect to control levels. Also induction of apoptosis was reduced in the presence of TPI. The data demonstrated that the HFA is excellently suited for studying short-term pharmacodynamic effects of fluoropyrimidines in vivo. TAS-102 is only effective in inducing cytotoxicity when systemic TPI is present, but acts against both low and high TP expressing colon cancer cells, while 5'DFUR needs cellular TP to exert significant activity.     

Epidermal growth factor prolongs survival time of tumor-bearing mice

We observed that human epidermal growth factor (hEGF) alone prolonged the survival time of mice bearing various murine syngeneic tumors as well as athymic nude mice bearing human xenografts. No changes in the subcutaneous solid tumor mass volume were observed. Prolongation of survival time by hEGF was observed in mice bearing murine epidermoid carcinoma (BSC) and human gastric carcinoma (KATO III), but not in murine epidermoid carcinoma (KLN205) or human epidermoid carcinoma (A431). Human tumor cells such as A431, KATO III, and murine tumor cells, KLN205, BSC had roughly 2 X 10(6), 3 X 10(4), 1.3 X 10(3) and 1 X 10(3) EGF receptors/cell, respectively. Although KLN205 and BSC tumor cells maintained nearly the same number of EGF receptors, the effects of hEGF were very different. Although A431 tumor cells had nearly 100 times more receptors than KATO III cells, the prolongation of survival time of mice bearing A431 by hEGF was no better than that of mice bearing KATO III. Accordingly, it appears that this prolongation of survival time by hEGF is independent of the number of EGF receptors on tumor cells. In addition, hEGF was shown to inhibit experimental pulmonary metastasis of murine BSC tumor, but was ineffective with murine KLN205 tumor. These results suggest that prolongation of survival time by hEGF may result from the inhibition of tumor cell metastasis and EGF may play a role in preventing the metastasis of certain malignant neoplasms unrelated to its effects through the EGF receptor on tumor cells.     

PHA-767491对结直肠癌Colo320细胞迁移和侵袭的影响

目的:探讨PHA-767491对结直肠癌细胞系Colo320侵袭和转移的作用.方法:配置不同浓度的PHA-767491作用于Colo320细胞.利用CCK-8法检测细胞的增殖能力确定IC50.Westem blot法检测PHA-767491对Colo320细胞Cdc-7表达的影响.利用划痕实验检测细胞的迁移能力.Transwell小室法检测细胞的侵袭能力.结果:CCK-8法结果显示PHA-767491能显著抑制Colo320细胞的增殖能力,24小时IC50为4.51 μmol./L.Western blot结果显示PHA-767491能显著抑制Colo320细胞Cdc-7的表达.划痕实验结果显示PHA-767491能显著抑制Colo320细胞的迁移能力.Transwell结果显示PHA-767491能抑制Colo320细胞的侵袭能力.结论:PHA-767491能够抑制Colo320细胞的迁移和侵袭.