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Drug resistance to cisplatin (CDDP) would represent a major obstacle for cancer therapy. The adenosine triphosphate (ATP) binding cassette (ABC) family of transport proteins, such as the 170 kDa P-glycoprotein (multidrug resistance gene-1; MDR-1) and the 190 kDa multidrug resistance-associated proteins (MRPs), are associated with multidrug resistance, including resistance to CDDP. The purpose of the present study was to investigate the relationship between cyclooxygenase-2 (COX-2) expression and the level of chemosensitivity to CDDP. We established the COX-2-overexpressed colon cancer cell line TR-5 from HCT-15 cells. We quantified the expression of m-RNA for MRP-1 and MDR-1 by a real-time PCR method, determining that the values of each gene/standardized GAPDH in HCT-15 and TR-5 were 23+/-0.4 and 6.1+/-0.5 in MRP-1 (p<0.02) and 9.0+/-4.8 and 3.6+/-0.5 in MDR-1, respectively. With respect to chemosensitivity, survival rates for 3 microg/ml and 10 microg/ml of CDDP were 81.5+/-12.2% and 26.1+/-11.7% (IC50=6.5 microg/ml) for HCT-15 and 96.6+/-1.7% and 77.4+/-4.9% (IC50=18.5 microg/ml) for TR-5, respectively, thus TR-5 showed higher resistance to CDDP than HCT-15 did with statistical differences. We also demonstrated a successful re-sensitization to CDDP toxicity in TR-5 by means of the COX-2 selective inhibitor JTE-522, 4-(4-cyclohexyl-2-methyl-1, 3-oxazol-5-yl)-2-fluorobenzene sulfonamide, which markedly decreased the IC50 of CDDP for TR-5 (from 17.3+/-2.6 microg/ml to 8.6+/-2.5 microg/ml). In conclusion, COX-2 overexpression induced increased MRP-1 expression in a colon cancer cell line, TR-5, resulting in chemoresistance to CDDP that was approximately triple the level of chemoresistance observed in the original HCT-15 cells line, as measured by calculation of the IC50. We also confirmed the efficacy of pretreatment of TR-5 cells with the COX-2 selective inhibitor JTE-522 in restoring chemosensitivity of these cells to CDDP, suggesting a strategy for overcoming drug resistance to CDDP.

We investigated the dual modulation by l-leucovorin (LV) and recombinant human interferon-alpha 2a (IFN-alpha 2a) of 5-fluorouracil (5-FU) antitumor activity against human colon carcinoma cells (Co-4) using a nude mouse system. 5-FU was administered intraperitoneally (IP) at 10 or 90 mg/kg. 5-FU (10 mg/kg) was administered daily for 10 days, and 90 mg/kg was administered once. LV was administered IP 1 and 0 h before 5-FU treatment at 200 mg/kg. IFN-alpha 2a was administered subcutaneously (SC) daily for 14 days at 60,000 IU/mouse. When 5-FU was administered at 10 or 90 mg/kg with these two modulators, the antitumor effect was increased significantly, with T/C ratios of 18.1 and 6.1, respectively. These modulatory effects were assessed as synergistic, without associated severe side effects or death during the experimental period. LV augmented the antitumor activity of 5-FU through increment of thymidylate synthetase (TS) inhibition, and IFN-alpha 2a showed a modulatory effect in elevating the intratumoral concentration of fluorouridine without change in TS inhibition. These results suggest that 5-FU antitumor activity against human colon carcinoma could be significantly potentiated without severe side effects by these two modulators, which possess different modes of action.

We have evaluated the antitumor activity of Altretamine (hexamethylmelamine, HMM) on human carcinoma xenografts serially transplanted in nude mice. Five human breast carcinoma xenografts, MX-1, T-61, MCF-7, R-27 and Br-10, were inoculated subcutaneously into female nude mice. Two human stomach carcinoma xenografts, SC-1-NU and St-4, and three human colon carcinoma xenografts, Co-3, Co-4 and Co-6, were inoculated subcutaneously into male nude mice. One pellet of 17 beta-estradiol (0.1 mg/mouse) was inoculated subcutaneously in the mice transplanted with MCF-7 when the tumors were inoculated. HMM was administered per os daily for 4 weeks. MX-1 and T-61 tumors regressed completely after treatment with HMM at a dose of 75 mg/kg (the maximum tolerated dose: MTD) for MX-1 and 25 mg/kg for T-61. Br-10 was sensitive, whereas MCF-7 and R-27 were resistant to HMM at its MTD. HMM exerted the most potent antitumor effect against T-61. Against MX-1, it exerted an antitumor effect equivalent to that of cisplatin or cyclophosphamide. In addition, this agent was effective against all stomach and colon carcinoma xenografts, in particular St-4 (T/C% = 10.7: the mean tumor weight of treated group/the mean tumor weight of control group) and Co-3 (T/C% = 31.5%) which are insensitive to presently available agents. HMM seems worthy of further clinical investigation as a candidate agent to treat breast, stomach, colon and other carcinomas.

We investigated the modulating effect of recombinant human interferon alpha-2a (IFN-alpha) on the antitumor activity of 5-fluorouracil (5-FU) against a human colon carcinoma xenograft (Co-4) in nude mice with reference to changes in the pharmacokinetic pattern of 5-FU. Mice bearing Co-4 received 5-FU ip at a dose of 90 mg/kg once with or without IFN-alpha, which was administered sc at a dose of 60.000 IU/mouse daily for 7 days before 5-FU treatment. When the area under the curve (AUC) and peak plasma concentration (Cmax) of 5-FU with or without IFN-alpha were measured as pharmacokinetic parameters, the pharmacokinetics of 5-FU was not changed by IFN-alpha administration. This result suggests that the modulating effect of IFN-alpha on 5-FU does not involve augmentation of 5-FU pharmacokinetic parameters.

The attachment of 7 human colon cancer lines transplantable into nude mice, and primary tumors and liver metastases from 30 patients with colon cancer to 4 extracellular matrix proteins (EMPs)--Matrigel, laminin, fibronectin, and type IV collagen--was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H (MTT) assay. Cancer cells from the 4 established tumor lines which produced experimental liver metastases in vivo showed significantly greater attachment to each EMP than those from the other 3 tumor lines which did not. Although there were no significant differences between attachment to EMPs of cancer cells from 15 clinical primary tumors with liver metastases and those without, attachment to each EMP of cells derived from liver metastases was significantly greater than that of the cells from the corresponding primary tumors in 8 cases for which liver metastases and primary tumors were examined simultaneously. Attachment to EMPs, which could be determined simply and rapidly using the MTT assay, is thus considered a significant factor in experimental and clinical liver metastases of human colon cancers.

A metastatic model of human colon cancer has been previously established using orthotopic onplantation of histologically intact in tissue nude mice. In this study, effects of immunochemotherapy using OK-432, 5-fluorouracil (5-FU) and mitomycin C (MMC) on Col-2-JCK, a human colon cancer xenograft, were evaluated using this model. When 5-FU and MMC were administered without OK-432, liver metastases were not reduced even at maximum tolerated doses of both drugs, although cecal tumor growth was significantly reduced. On the other hand, when combined with OK-432, both 5-FU and MMC reduced liver metastases with synergistic reduction of cecal tumor growth, demonstrating the potential of combining immunotherapy with chemotherapy against metastases.

We have developed a liver metastatic model of human colon cancer using severe combined immunodeficient (SCID) mice. Liver metastases were observed in all the SCID mice on day 28 after intrasplenic injection with 5 x 10(6) dissociated tumor cells of COL-2-JCK, a human colon cancer strain serially transplanted in nude mice. When this model was applied for chemotherapeutic experiments, 5-fluorouracil (5-FU) demonstrated significant antitumor effects in preventing liver metastases, whereas the efficacy of 5-FU was limited in the currently used sc-ip chemosensitivity assay in nude mice. When the human LDH-5 isozyme was evaluated in the homogenized metastatic liver tissue of SCID mice, a good correlation was obtained between the liver tumor weights and LDH-5 isozyme, suggesting that it could be a promising quantitative indicator for metastases. This model would be useful for further studies on the treatment of liver metastases of colon cancer.

Studies on liver metastasis of human colon cancer are limited because of a lack of suitable animal models. In this study, the usefulness of mice with severe combined immunodeficiency (SCID), which congenitally lack functional T and B lymphocytes, was evaluated in comparison with currently available nude mice. Three human colon cancer xenografts transplantable into nude mice were disaggregated enzymatically to obtain tumor cell suspensions, and implanted intrasplenically into SCID and nude mice. The incidence of splenic tumorigenesis and of liver metastases were significantly greater in SCID mice for all xenografts, in comparison with nude mice. In total, 33 of 36 SCID mice and 17 of 43 nude mice developed liver metastases. On the basis of this result, we conclude that SCID mice would be a more suitable model than nude mice for studying liver metastasis of human colon cancer.

COL-2-JCK, a human colon cancer xenograft line able to be transplanted into nude mice, was implanted in the subserosal layer of the cecum, either as cancer tissue or as a single cell suspension. When cancer tissue was used for the cecal implantation, 100% extensive local tumor growth and a high incidence of metastases to the regional lymph nodes, peritoneum, liver, and lung was observed. In contrast, when the cell suspension of this line was injected into the cecal wall, no metastases were observed, with significantly reduced local tumor growth. The use of cancer tissue maintaining the original cancer tissue structure is therefore considered imperative for allowing full expression of the biological characteristics of cancer cells. This nude mouse model using the cecal implantation of cancer tissue should thus prompt further study on the biology of human colon cancer.

Experimental biochemical modulation of 1-hexylcarbamoyl-5-fluorouracil (HCFU) with l-leucovorin (LV) was carried out using human gastric (H-111) and colon (Co-4) carcinoma xenografts serially transplanted into nude mice. Thirty-five or 70 mg/kg HCFU dissolved in 0.2 ml of 1% hydroxymethyl cellulose was administered po daily for 3 weeks except Sundays, and 50, 100, 200 or 300 mg/kg LV dissolved in 0.2 ml physiological saline was administered po 30 min before administration of HCFU. The biochemically modulated antitumor activity was evaluated in terms of actual tumor weight, the relative mean tumor weight and the degree of inhibition of thymidylate synthetase (TS) in the tumors at the end of the experiments, assayed according to the method of Spears et al. Although 35 mg/kg HCFU was ineffective against gastric carcinoma H-111, combination with 200 or 300 mg/kg LV resulted in a positive antitumor effect of HCFU on this strain without any increase of side effects in terms of body weight loss and mouse mortality. The colon carcinoma strain Co-4 showed marginal sensitivity to HCFU (35 mg/kg) alone, but 50 or 100 mg/kg LV modulated the antitumor activity of HCFU on Co-4 to produce a significant positive effect without any increase in toxicity, and HCFU administered with 100 mg/kg LV was more effective than the maximum tolerated dose of HCFU (70 mg/kg) alone. The TS inhibition rate was closely related to the biochemical modulation of HCFU antitumor activity by LV, suggesting that the modulation involves an increase of the ternary complex of TS, 5,10-methylene tetrahydrofolate from LV and 5-fluorodeoxyuridine 5'-monophosphate (FdUMP). Combination of HCFU and LV is therefore thought to be useful in increasing the antitumor activity of HCFU on gastrointestinal carcinomas without enhancing its toxicity.

We have conducted a three-dimensional cellular growth assay using collagen gel matrix with an endpoint of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay, on four human gastric and colonic cancer cell lines. Three-dimensionally growing cells in collagen gel matrix were 2- to 180-fold more resistant to mitomycin C, doxorubicin, 5-fluorouracil, and cisplatin than those in monolayer culture, and these resistances were increased further when the cells increased their three-dimensionality. Furthermore, the influence of fibroblasts in the collagen gel matrix on the chemosensitivity of cancer cells was less than that in monolayer culture. Since this new assay using collagen gel matrix with an endpoint of the MTT assay was able to detect the increase of drug resistance of human cancer cell lines by three-dimensional cellular growth using a simple and convenient procedure, it was considered to be more useful than conventional monolayer cultures for evaluating the chemosensitivity of cancer cells.