Adjuvant Effect of NSAIDs on the Cytotoxicity of Colon Cancer Cells to 5-FU

PURPOSE: Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the conversion of arachidonic acid to prostaglandins, is believed to be an important enzyme related to colorectal cancer. A large number of studies have supported the concept that non-steroidal anti-inflammatory drugs (NSAIDs) targeting COX alter the biologic processes of colon carcinogenesis. Although COX-2 inhibitors generally reduce the growth rate of established tumors, tumor regression is rarely observed. Hence, it is reasonable that COX-2 inhibitors be given in conjunction with standard anti-cancer therapy in treating cancer. We investigated whether aspirin and meloxicam not only are cytotoxic but also potentiate the antitumor effect of 5-Fluorouracil (5-FU) against colon cancer cells. METHODS: Expressions of COX-1 and COX-2 were determined by using the reverse transcriptase-polymerase chain reaction (RT-PCR) & Western blotting assay in 9 colon cancer cell lines. The cytotoxicities of NSAIDs and/or 5-FU were determined by using a microculture tetrazolium dye (MTT) assay. RESULTS: COX-1 mRNA and protein, as well as COX-2 mRNA, were variably expressed in all the cell lines tested whereas COX-2 protein was expressed in HT-29 and to a lesser extent in HCT-8, but not in the other cell lines. We selected two representative cell lines, HT-29 expressing COX-2 protein and SNU-C1 not expressing it. The dose-dependent cytotoxicity was observed in both cell lines treated with aspirin and with meloxicam. A combination treatment of aspirin or meloxicam with 5-FU revealed some additive effect, rather than a synergistic effect, for both cells lines. This additive effect was remarkable even for low concentrations of the drugs. Furthermore, the additive effect was highest when the combination was adminstered sequentially, 5-FU followed by aspirin or meloxicam, in both cell lines. CONCLUSIONS: These results suggest that a combination therapy using NSAIDs and 5-FU might be useful in the treatment of colon cancer cells not expressing COX-2, as well as in colon cancer cells expressing COX-2.

Expression and Function of ABC Transporters as Multidrug Resistance Mechansims in Colon Cancer Cells

PURPOSE: Multidrug resistance (MDR) is a phenomenon whereby tumor cells acquire resistance to a broad range of structurally and functionally diverse chemotherapeutic drugs. The most widely implicated mechanism of MDR is that concerned with altered membrane transporters in tumor cells. P-glycoprotein (Pgp), multidrug resistance protein (MRP), and breast-cancer-resistance protein (BCRP) are well-known membrane transporters that pump out antitumor agents by using an ATP-dependent process, the so-called ATP-binding cassette (ABC) superfamily or transporter. This study was undertaken to test the prevalence of each ABC transporter and to determine which transporter has functional acitivity in various colon cancer cells. METHODS: Expressions of Pgp, MRP, and BCRP mRNA were determined in 9 colon-cancer cell lines by using an RT-PCR assay. The sensitivity to anticancer agents substrate for each ABC transporter in the colon cancer cells determined using an MTT assay. The accumulation of fluorescent compounds for functional detection of each ABC transporter was determined by using flow cytometry. RESULTS: Pgp mRNA was variably expressed in 6 of 9 colon cancer cells lines. MRP and BCRP mRNA were expressed in all the 9 cell lines. A smaller cytotoxic effect to paclitaxel and a smaller amount of rhodamine123 accumulation were observed in Colo 320HSR expressing the highest levels of Pgp than in SNU-C5 not expressing Pgp. These effects in Colo320HSR were reversed with the addition of various Pgp inhibitors, but such a reversal did not occur in SNU-C5. The cytotoxic effect to VP-16 was not related to the expression levels of MRP in Colo320HSR and SNU-C, but the amount of calcein-AM accumulation was reversed with addition of probenecid, MRP inhibitor. The cytotoxic effect and the drug accumulation of mitoxantrone were not related to the expression levels of BCRP. CONCLUSIONS: This study suggests that of the ABC transporters, primarily Pgp and MRP have functional activity in colon cancer cell lines.

Expression and Function of ABC Transporters as Multidrug Resistance Mechansims in Gastric Cancer Cells

PURPOSE: Multidrug resistance (MDR) is a phenomenon whereby tumor cell acquire resistance to a broad range of structurally and functionally diverse chemotherapeutic drugs. The most widely implicated mechanism of MDR is that of altered membrane transporter in tumor cells. P-glycoprotein (Pgp), multidrug resistance protein (MRP), and breast cancer-resistance protein (BCRP) are well known membrane transporters, which pump out antitumor agents via an ATP-dependent process, the so called ATP-binding cassette (ABC) superfamily or transporter. This study was undertaken to test the prevalence of each ABC transporter, and which of then exhibit functional activity in various gastric cancer cells. METHODS: The expressions of Pgp, MRP, and BCRP mRNA were determined by RT-PCR assay on 10 gastric cancer cells. The sensitivity to anticancer agents, substrates for each ABC transporter in the gastric cancer cells was determined using the MTT assay. The intracellular accumulation of fluorescent compounds for the functional detection of each ABC transporter was determined using flow cytometry. RESULTS: The Pgp mRNA was expressed at various levels in 9 out of the 10 gastric cancer cells tested, but significantly low. MRP mRNA was constitutively expressed in all the cells. BCRP mRNA was differentially expressed in 5 of the gastric cancer cells. There was no relation between the expressions of Pgp and MRP and the cytotoxicity to each substrate. It was observed that the accumulations of paclitaxel and VP-16 were significantly increased on the additions of PSC833 and probenecid, respectively, in all tested cells. The reversal effect of drug accumulation by each inhibitor was much higher in the MRP than Pgp. With BCRP, the observed cytotoxic effect and amount of mitoxanthrone accumulation were less than in the cells expressing the highest levels of BCRP compared to those that did not. However the mitoxanthrone accumulation was not increased on the addition of FTC in the either cell type. CONCLUSION: This study suggests that of the ABC transporters, MRP has primarily functional activity, whereas that of Pgp is only slight, in the gastric cancer cells. Other possible MDR mechanisms involved will have to be explored in further studies.