Three-dimensional, but not two-dimensional, culture results in tumor growth enhancement after exposure to anticancer drugs.

Previously, we have adapted a recently developed three-dimensional chemosensitivity test, the collagen gel droplet embedded culture drug sensitivity test (CD-DST), for evaluation of chemosensitivity of 12 anticancer drugs against colorectal adenocarcinoma, and surprisingly, it was found that tumor growth enhancement was occasionally observed even after exposure to anticancer drugs. In this study, the CD-DST was applied for human cervical carcinoma cell line HeLa-Ohio (HeLa) cells and its MDR1/P-glycoprotein-overexpressing subline, Hvr100-6 cells, and 12 anticancer drugs were assessed in terms of chemosensitivity and deterioration of tumor, and the results were compared with those by two-dimensional WST-1 assay. Growth enhancement was observed in Hvr100-6 cells, not in HeLa cells, for mitomycin C with the ratio of total volume of colonies in the treated group to that in the untreated group (T/C%) of 135.0%, doxorubicin with T/C% of 162.5% and cyclophosphamide with T/C% of 122.0%, and this was not observed in WST-1 assay. Multidrug resistance was detected both for CD-DST and WST-1 assay. The values of T/C% in CD-DST were comparable with or higher than those of the survival fraction (%) in WST-1 assay, and modification of WST-1 assay procedure gave similar results, suggesting a higher resistance in three-dimensional than in two-dimensional culture. Further investigations should be addressed to the association of MDR1/P-glycoprotein with tumor growth enhancement.

Reversal effects of Ca2+ antagonists on multidrug resistance via down-regulation of MDR1 mRNA.

In previous reports, the effects of 12 Ca2+ antagonists on a multidrug resistant transporter, P-glycoprotein/MDR1, were evaluated in terms of those on MDR1-mediated transport of [3H]digoxin and the sensitivity of vinblastine sulfate or paclitaxel, and they were able to be classified into 4 subgroups based on their actions, as those with transport inhibition and sensitivity recovery, those with or without transport inhibition but marginal sensitivity recovery, and those without both. In this study, our previous findings were confirmed by the resistance against doxorubicin hydrochloride and daunorubicin hydrochloride, and by the recovery of [3H] vinblastine sulfate accumulation. Furthermore, it was found that the effects of 12 Ca2+ antagonists on the sensitivity recovery were also explained by the down-regulation of MDR1 mRNA, suggesting a novel mechanism to reverse the MDR1-mediated multidrug resistance.

Knock-down of sorcin induces up-regulation of MDR1 in HeLa cells.

In our previous study, the MDR1/Pglycoprotein-overexpressing multidrug resistant subline, Hvr100-6, was established from the human cervical carcinoma cell line HeLa-Ohio (HeLa) by stepwise exposure to an anti-microtubule agent, vinblastine sulfate, a typical substrate of MDR1. Their gene and protein expression profiles were analyzed herein, and 148 genes were identified to be differentially expressed by cDNA microarray analysis. The up-regulation of sorcin, a soluble resistance-related calcium-binding protein of 22 kDa, was confirmed in Hvr100-6 cells by the proteome analysis. To clarify the relationship between MDR1 and sorcin, HeLa cells were treated with small interfering RNAs (siRNAs) targeted for theirs mRNAs. The siRNA for MDR1 mRNA resulted in its decrease by 86% and 61% on the days 1 and 2 after the treatment, whereas the expression level of sorcin mRNA was not changed. On the other hand, the siRNA for sorcin mRNA suppressed its expression by 80-90% on days 1-3 after the treatment. Interestingly; suppression of sorcin induced a more than 3-fold increase in the expression level for MDR1 mRNA. An efflux function of MDR1 evaluated with using rhodamine 123 as a probe showed a tendency to be increased in HeLa cells treated with siRNA for sorcin, compared with that in the cells treated with scramble siRNA. The activity and the expression of caspase-3 in the sorcin knock-down HeLa cells were relatively higher than those in the cells treated with scramble siRNA. Thus, we demonstrated that sorcin might be a partial suppressor of MDR1 expression. Furthermore, the present study suggested that sorcin repressed apoptosis via dysfunction of caspase-3.

MDR1 T-129C polymorphism can be predictive of differentiation, and thereby prognosis of colorectal adenocarcinomas in Japanese.

The expression level of MDR1 mRNA was evaluated in colorectal adenocarcinomas and adjacent noncancerous colorectal tissues obtained from 21 Japanese patients. It was lower in the former than in the latter (p=0.012), suggesting its down-regulation as a consequence of malignant transformation of colorectal tissues, possibly with the suppression of differentiation. Relatively lower expression was suggested in moderately-differentiated colorectal adenocarcinomas than well-differentiated ones, but there was no statistical difference (p=0.111). MDR1 mRNA up-regulation was found in a colorectal adenocarcinoma cell line, HCT-15, after treatment with two typical differentiating agents, sodium butyrate and all-trans retinoic acid, suggesting its involvement in the cellular events, resulting in differentiation without malignant transformation. MDR1 T-129C, but not G2677A,T and C3435T, was associated with the lower expression of MDR1 mRNA both in colorectal adenocarcinomas (p=0.040) and adjacent noncancerous colorectal tissues (p=0.023), possibly being an useful invasive marker predicting poorly-differentiated colorectal adenocarcinomas and thereby the poor prognosis of the patients, especially when no extra biopsy samples will be obtained. Further investigations with relatively large number of patients should be undertaken to confirm these preliminary results.

MDR1 haplotype frequencies in Japanese and Caucasian, and in Japanese patients with colorectal cancer and esophageal cancer.

The genotype frequencies of MDR1 T-129C, C1236T, G2677A,T and C3435T SNPs were compared in 154 healthy Japanese and 100 healthy Caucasians to provide basic information on the inter-ethnic differences of pharmacotherapeutic outcome. The variants were found at allelic frequencies of 5.5%, 65.6%, 16.6%, 40.6% and 40.6%, for T-129C, C1236T, G2677A, G2677T and C3435T, respectively, in Japanese, and at 5.1%, 45.9%, 3.6%, 46.4% and 56.6%, respectively, in Caucasians, with a statistically significant difference for C1236T, G2677A,T and C3435T (p<0.001). G2677A was about 5-fold more frequent in Japanese than Caucasians. These genotype frequencies were also investigated in 95 Japanese patients with colorectal cancer (CRC) and esophageal squamous cell carcinoma (ESCC), but no significant difference was detected, when compared with healthy Japanese subjects. The haplotype frequency reached a total of about 85% in Japanese with the following 4 major haplotypes; T(-129)-T1236-T2677-T3435 (36.1%), T(-129)-T1236-G2677-C3435 (22.5%), T(-129)-C1236-G2677-C3435 (14.2%) and T(-129)-C1236-A2677-C3435 (13.3%). The second and fourth haplotypes were hardly inferred in Caucasian, whereas T(-129)-C1236-G2677-T3435 (12.8%) was found to be Caucasian-specific. There was a tendency for higher frequencies of the T(-129)/C-(129)-C1236-A2677-C3435 haplotype in Japanese CRC patients and T(-129)-T1236-T2677-T3435 haplotype in Japanese ESCC patients, compared with that in healthy Japanese subjects.

Effects of acid and lactone forms of eight HMG-CoA reductase inhibitors on CYP-mediated metabolism and MDR1-mediated transport.

PURPOSE: With the growing clinical usage of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), the number of reports concerning serious drug-drug interaction has been increasing. Because recent studies have shown that conversion between acid and lactone forms occurs in the body, drug-drug interaction should be considered on both acid and lactone forms. Thus, we investigated the inhibitory effects of acid and lactone forms of eight statins, including one recently withdrawn, cerivastatin, and two recently developed, pitavastatin and rosuvastatin, on cytochrome P450 (CYP) 2C8, CYP2C9, and CYP3A4/5 metabolic activities and multidrug resistance protein 1 (MDR1) transporting activity. METHODS: The inhibitory effects of statins on CYP metabolic activities and MDR1 transporting activity were investigated using human liver microsomes and MDR1-overexpressing LLC-GA5-COL150 cells, respectively. RESULTS: The acid forms had minimal inhibitory effects on all CYP activities tested, except for fluvastatin on CYP2C9-mediated tolbutamide 4-hydroxylation (IC50 = 1.7 microM) and simvastatin on CYP3A4/5-mediated paclitaxel 3-hydroxylation (12.0 microM). Lactone forms showed no or minimal inhibitory effects on CYP2C8, CYP2C9, and CYP2C19 activities, except for rosuvastatin on the CYP2C9 activity (20.5 microM), whereas they showed stronger inhibitory effects on the CYP3A4/5 activity with the rank order of atorvastatin (5.6 microM), cerivastatin (8.1 microM), fluvastatin (14.9 microM), simvastatin (15.2 microM), rosuvastatin (20.7 microM), and lovastatin (24.1 microM). Pitavastatin and pravastatin had little inhibitory effect, and a similar order was found also for testosterone 6beta-hydroxylation. MDR1-mediated transport of [3H]digoxin was inhibited only by lactone forms, and the rank order correlated with that of inhibitory effects on both CYP3A4/5 activities. Inhibitory effects on MDR1 activity, and on both CYP3A4/5 activities, could be explained by the lipophilicity; however, a significant correlation was found between the lipophilicity and inhibitory effects on CYP2C8-mediated paclitaxel 6alpha-hydroxylation. CONCLUSIONS: We showed the difference between the acid and lactone forms in terms of drug interaction. The lipophilicity could be one of the important factors for inhibitory effects. In the case of statins, it is important to examine the effects of both forms to understand the events found in clinical settings, including the pleiotropic effects.

Haloperidol is an inhibitor but not substrate for MDR1/P-glycoprotein.

The involvement of the multidrug resistant transporter MDR1/P-glycoprotein in the penetration of haloperidol into the brain and absorption in the intestine was investigated to examine its role in inter/intra-individual variability, using the porcine kidney epithelial cell line LLC-PK(1) and its MDR1-overexpressing transfectant, LLC-GA5-COL150. The inhibitory effect of haloperidol on other MDR1 substrates was also investigated in terms of the optimization of haloperidol-based pharmacotherapy. The transepithelial transport of [(3)H]haloperidol did not differ between the two cell lines, and vinblastine, a typical MDR1 substrate, had no effect on the transport, suggesting that haloperidol is not a substrate for MDR1, and it is unlikely that MDR function affects haloperidol absorption and brain distribution, and thereby the response to haloperidol. However, haloperidol was found to have an inhibitory effect on the MDR1-mediated transport of [(3)H]digoxin and [(3)H]vinblastine with an IC50 value of 7.84+/-0.76 and 3.60+/-0.64 microM, respectively, suggesting that the intestinal absorption, not distribution into the brain, of MDR1 substrate drugs could be altered by the co-administration of haloperidol in the clinical setting, although further clinical studies are needed.

Simultaneous determination of single nucleotide polymorphisms of MDR1 genes by electrochemical DNA chip.

The on-chip genotyping system ("the electrochemical DNA chip") has been developed as a more cost-effective genotyping system and was applied to MDR1 genotyping in the present study, which is required for wide use in clinical application and for personalized medication based on genotype. The electrochemical DNA chip was optimized and applied to simultaneous genotyping of four MDR1 polymorphisms (T-129C, C1236T, G2677(A,T) and C3435T) using synthetic model oligonucleotide DNA and human genomic DNA. The electrochemical DNA chip successfully gave the T-129C, C1236T, G2677(A,T) and C3435T genotypes, which were completely consistent with those determined by direct sequencing. In conclusion, the electrochemical DNA chip is useful for simultaneous determination of some genotypes and haplotypes, and efficient genotyping using this system can support future genotype-phenotype studies at a large scale.

Gene expression profiles of ABC transporters and cytochrome P450 3A in Caco-2 and human colorectal cancer cell lines.

PURPOSE: The mRNA levels of MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), cytochrome P450 3A (CYP3A) and villin in human colorectal cell lines (HCT-15, LoVo DLD-1, HCT-116 and SW620) were quantitatively compared with those in Caco-2 cells. METHODS: The mRNA levels were determined by real time quantitative polymerase chain reaction and expressed as the relative concen trations of MDR1 mRNA to glyceraldehyde-3-phosphate dehydro genase (GAPDH) mRNA. RESULTSl MDR1 mRNA was expressed in HCT-15 LoVo and DLD-1 cells at similar or lower level to Caco-2. The expression of MRP mRNA in the cell lines tested was comparable with Caco-2. MRP. mRNA was detected only in HCT-116 and SW620 at significantly lower level than Caco-2. CYP3A mRNA was detected in HCT-15 LoVo, DLD-1 and SW620 at similar level to Caco-2. CONCLUSIONS: HCT-15 LoVo and DLD-1 cells express proteins important for regulating the intestinal absorption of drugs, i.e., MDR1 MRP1 and CYP3A, whereas HCT-116 and SW620 cells were not acceptable for evaluation of absorption properties of drug candidates

Effects of reactive oxygen species on cell proliferation and death in HeLa cells and its MDR1-overexpressing derivative cell line.

In this paper, the effects of H2O2, a typical reactive oxygen species (ROS), on cell proliferation or death were examined using the human cervical carcinoma cell line HeLa and its MDR1-overexpressing subline, Hvr100-6, which was established by stepwise exposure to vinblastine. It was confirmed that the growth of HeLa cells was enhanced by H2O2 at relatively low concentrations in a concentration-dependent manner, and the growth enhancement was suppressed by antioxidants. Doxorubicin and daunorubicin also enhanced the growth of HeLa cells at concentrations lower than IC50 values, and the antioxidants suppressed this effect, being consistent with the fact that both anticancer drugs generate ROS. The growth enhancement by H2O2 or doxorubicin and daunorubicin was not observed in Hvr100-6 cells. In addition, it was suggested that antioxidants had no effect on MDR1 mRNA expression in HeLa and Hvr100-6 cells, and thereby hardly reverse multidrug resistance in tumor cells.