Reduction of cytotoxicity of the alkaloid emetine through P-glycoprotein (MDR1/ABCB1) in human Caco-2 cells and leukemia cell lines

The cytotoxicity of the alkaloid emetine was determined in six human cell lines that differ in the expression of ABC transporters, such as multiple drug resistance protein 1 (MDR1/ABCB1) and multidrug resistance associated protein 1 (MRP1/ABCC1). Emetine reveals a substantial cytotoxicity due to apoptosis that is inversely correlated with the expression of MDR1. Confluent Caco-2 cells with high MDR1 activity and the MDR1 over-expressing leukemia cell line CEM/ADR5000 are more resistant towards emetine (EC (50) 250 microM and 2 microM, respectively) than cells with a low expression of MDR1 (Jurkat cells, CCRF-CEM cells, HL-60 cells) or cells which over-express MRP1 (HL-60/AR) (EC (50) between 0.05 microM for CCRF-CEM and 0.17 microM for Jurkat cells). Apparently emetine is a substrate for MDR1 but not for MRP1. Furthermore, emetine is able to up-regulate the expression of MDR1 as shown IN VITRO by real-time PCR and transport activity studies.     

Caco-2细胞中药物转运蛋白的mRNA表达及活力评价

目的 系统研究Caco-2细胞中各药物转运蛋白的mRNA表达水平及转运活力,对比其与人正常肠道中药物转运蛋白表达的差异。方法 实时荧光定量PCR（qRT-PCR）方法测定Caco-2细胞中人肠道相关转运蛋白MDR1、BCRP、MRP2、OATP1A2、OATP2B1和PEPT1的表达水平;将Caco-2细胞接种于Transwell板内培养21 d并给予不同药物转运蛋白的底物及抑制剂,评价Caco-2细胞中相关转运蛋白的转运活性。结果 qRT-PCR结果表明,药物转运蛋白MDR1、MRP2、BCRP和OATP2B1在Caco-2细胞中均有相对高的表达,表达量的顺序为：MDR1 > MRP2 > OATP2B1 > BCRP,在正常人肠道表达量顺序为BCRP > MDR1 > MRP2 > OATP2B1;转运蛋白活力评价表明,各药物转运蛋白的活力测试结果均为阳性,验证了基因的表达水平结果。结论 Caco-2细胞中表达正常人体肠道表达的部分药物转运蛋白（MDR1、MRP2、BCRP和OATP2B1）,表达水平与正常人体肠道中大致相当,但也存在一定差异。     

Influence of t-butylhydroquinone and beta-naphthoflavone on formation and transport of 4-methylumbelliferone glucuronide in Caco-2/TC-7 cell monolayers

Human Caco-2 cells have been established as a model system for intestinal biotransformation and permeability. When grown on Transwell polycarbonate filters they develop morphologic and biochemical characteristics of enterocytes with well separated apical and basolateral surfaces. In addition, Caco-2/TC-7 cells have proven to be useful to study regulation of human UDP-glucuronosyltransferases (UGTs) by Ah receptor agonists and antioxidant-type inducers such as beta-naphthoflavone (BNF) and t-butylhydroquinone (TBHQ). In the present investigation, formation and transport of 4-methylumbelliferone glucuronide was studied in intact Caco-2 cell monolayers. The following results were obtained: when loaded with 50-200 microM MUF either apically or basolaterally, MUF-GA was the major metabolite which was mostly released (80%) at the basolateral surface, probably via the multidrug resistance protein isoform MRP3; MUF sulfate formation was low (5 +/-2%). Pretreatment of cells with 80 microM TBHQ or 50 microM BNF for 72 hr before addition of 100 microM MUF enhanced basolateral secretion of MUF-GA 1.4- and 1.7-fold, respectively. However, at >200 microM MUF, MUF-GA secretion and induction was smaller, probably due to inhibition of intracellular UGT activity. MRP3 protein was localized to the basolateral surface of Caco-2 cells but was not induced by TBHQ or BNF. The results suggest that MUF-GA is mostly secreted basolaterally in Caco-2 cell monolayers. Treatment with TBHQ or BNF significantly enhanced MUF-GA formation in the intact cell.     

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 concentrations of MDR1 mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Results. MDR1 mRNA was expressed in HCT-15 LoVo and DLD-1 cells at similar or lower level to Caco-2. The expression of MRP1 mRNA in the cell lines tested was comparable with Caco-2. MRP2 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.     

Real-time quantitative polymerase chain reaction for MDR1, MRP1, MRP2, and CYP3A-mRNA levels in Caco-2 cell lines, human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas.

The expression levels of mRNAs for MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), and cytochrome P450 3A (CYP3A) in Caco-2 cells were quantitatively compared with those in human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas. Caco-2 cells (passages 36-88) were kindly supplied by several laboratories in Japan. Human duodenal enterocytes were obtained from five healthy male volunteers. Normal colorectal tissues and colorectal adenocarcinomas were simultaneously obtained from seven patients with primary colorectal adenocarcinoma. MDR1, MRP1, MRP2, and CYP3A mRNA levels were determined by real-time quantitative polymerase chain reactions (PCR). Relative concentrations of mRNAs for target proteins (MDR1, MRP1, MRP2, and CYP3A) and glyceraldehyde-3-phosphate dehydrogenase in Caco-2 cells were 1.00 +/- 0.15, 1.02 +/- 0.06, 0.94 +/- 0.10, and 0.68 +/-0.60, respectively, and those in human enterocytes were about 12-, 3-, 7-, and 8000-fold higher than in the Caco-2 cells, respectively. In contrast, MDR1, MRP1, and CYP3A mRNA levels in Caco-2 cells were comparable to those in normal colorectal tissue and colorectal adenocarcinoma.     

Effect of cell differentiation and passage number on the expression of efflux proteins in wild type and vinblastine-induced Caco-2 cell lines.

The mRNA level expression of MDR1, MRP1-6, BCRP and CYP3A4 was determined by quantitative PCR in wild type (Caco-2WT) and vinblastine-treated (Caco-2VBL) Caco-2 cells at different passage levels (32-53). Differentiation increased the mRNA levels of MDR1, BCRP and all the MRPs except MRP4. Corresponding mRNA levels were observed in Caco-2WT and Caco-2VBL, except that the expression of MRD1 was higher in Caco-2VBL than in Caco-2WT cells. CYP3A4 was barely detected in either cell line. MDR1 functionality was studied using rhodamine123 and verapamil as a substrate-inhibitor pair. Corresponding to the observed differences in mRNA levels, MDR1 activity was higher in the Caco-2VBL cells. In Caco-2WT, MDR1 functionality was elevated at low passage numbers (32-35) compared to higher ones (49-53). Verapamil inhibited MDR1 efflux except at higher passage Caco-2WT cells, where no MDR1 activity could be observed. The results support the use of Caco-2VBL cells in MDR1 screening. The functional expression is higher than in Caco-2WT and remains consistent across the studied passages without major differences in mRNA levels of other efflux proteins. As both the passage number and the level of cell differentiation affect the expression profile of efflux proteins, short-term cell growth protocols should be evaluated accordingly.     

Effects of continuous exposure to digoxin on MDR1 function and expression in Caco-2 cells

The Caco-2 cell line has been used widely for studying intestinal permeability and several transport functions, and express the multidrug resistance transporter MDR1/P-glycoprotein. Previously, the transient exposure to digoxin for 24 h was found to induce MDR1 mRNA in Caco-2 cells. Here, a digoxin-tolerant Caco-2 subline (Caco/DX) was newly established by the continuous exposure of Caco-2 cells to digoxin, and the effects of continuous exposure to digoxin on MDR1 were examined. The 50% growth inhibitory concentration (IC(50)) values for digoxin in Caco-2 and Caco/DX cells were 17.2 and 81.4 nM, respectively. The IC(50) values for paclitaxel, an MDR1 substrate, were 1.0 and 547 nM, respectively, whereas the cytotoxicity of 5-fluorouracil was comparable in both cells. The uptake and efflux of Rhodamine123, an MDR1 substrate, in Caco/DX cells were significantly less and greater, respectively, than those in Caco-2 cells, and these transports were affected by the addition of ciclosporin. The expression of MDR1 mRNA in Caco/DX cells was approximately 2- and 1.7-fold compared with Caco-2 cells and Caco-2 cells treated with 100 nM digoxin for 24 h, respectively. On the other hand, MRP1 mRNA in Caco/DX cells was unchanged. These observations confirmed that the continuous exposure to digoxin, as well as the transient exposure, induced MDR1 in Caco-2 cells.     

Multiple pathways for fluoroquinolone secretion by human intestinal epithelial (Caco-2) cells

1. Human intestinal epithelial Caco-2 cells, T84 cells, and MDCKII cells transfected with human MDR1, were used to investigate the mechanistic basis of transintestinal fluoroquinolone secretion. 2. The fluoroquinolone grepafloxacin was secreted across Caco-2 monolayers by a saturable process (V(max)=16.9 +/- 3.4 nmol.cm(-2).h(-1)). Net secretion was reduced by 2-deoxyglucose/azide treatment to reduce intracellular ATP. 3. Grepafloxacin inhibited [(14)C]-ciprofloxacin (100 microM) secretion across Caco-2 monolayers (K(0.5)=0.8 mM), and concurrently increased the cellular accumulation of ciprofloxacin from the basal medium, indicating inhibition of export across the apical membrane. 4. The unconjugated bile acid, cholic acid, was secreted across Caco-2 monolayers, and this secretion was sensitive to inhibition by the MRP-selective inhibitor MK-571, suggesting MRP2 involvement. Secretion of cholic acid (10 microM) across the apical membrane was also inhibited by grepafloxacin (K(0.5)=0.3 mM), but not by ciprofloxacin. 5. In MDCKII-MDR1 monolayers, net secretion of grepafloxacin was increased by 3.5 fold compared with untransfected controls. Neither ciprofloxacin nor cholic acid showed net secretion in either MDCKII or MDCKII-MDR1 monolayers, showing that in contrast to grepafloxacin, neither are substrates for MDR1. 6. In T84 monolayers, which express MDR1 but not MRP2, neither ciprofloxacin nor cholic acid was secreted, whilst the V(max) for grepafloxacin secretion was lower than in Caco-2 cells, which express both MDR1 and MRP2. 7. In conclusion, the transepithelial secretion of grepafloxacin is mediated by both MRP2 and MDR1, whereas ciprofloxacin is a substrate for neither. Grepafloxacin also competes for the ciprofloxacin-sensitive pathway, which remains to be elucidated.     

Effects of grapefruit juice and orange juice components on P-glycoprotein- and MRP2-mediated drug efflux

We investigated the effects of grapefruit juice (GFJ) and orange juice (OJ) on drug transport by MDR1 P-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2), which are efflux transporters expressed in human small intestine. We examined the transcellular transport and uptake of [(3)H]vinblastine (VBL) and [(14)C]saquinavir in a human colon carcinoma cell line (Caco-2) and in porcine kidney epithelial cell lines transfected with human MDR1 cDNA and human MRP2 cDNA, LLC-GA5-COL150, and LLC-MRP2, respectively. In Caco-2 cells, the basal-to-apical transports of [(3)H]VBL and [(14)C]saquinavir were greater than those in the opposite direction. The ratio of basal-to-apical transport to apical-to-basal transport of [(3)H]VBL and [(14)C]saquinavir by Caco-2 cells was reduced in the presence of MK571 (MRPs inhibitor), verapamil (P-gp inhibitor), cyclosporin A (inhibitor of both), 50% ethyl acetate extracts of GFJ and OJ, or their components (6',7'-dihydroxybergamottin, bergamottin, tangeretin, hepatomethoxyflavone, and nobiletin). Studies of transport and uptake of [(3)H]VBL and [(14)C]saquinavir with MDR1 and MRP2 transfectants showed that VBL and saquinavir are transported by both P-gp and MRP2. GFJ and OJ components inhibited the transport by MRP2 as well as P-gp. However, their inhibitory potencies for P-gp or MRP2 were substrate-dependent. The present study has revealed that GFJ and OJ interact with not only P-gp but also MRP2, both of which are expressed at apical membranes and limit the apical-to-basal transport of VBL and saquinavir in Caco-2 cells.     

Effect of chlorpyrifos on efflux transporter gene expression and function in Caco-2 cells.

The effect of chlorpyrifos (CPF) and its metabolite, chlorpyrifos-oxon (CPO), on multidrug resistance-1 (MDR1) gene expression and efflux transporter function in Caco-2 cells was determined. The effect of CPF and CPO on gene expression in Caco-2 cells was tested as a function of time using RT-PCR and competitive PCR (compPCR) techniques. The RT-PCR results depicted a maximal effect of CPF exposure on MDR1 expression at 8 h, which decreased at 24 h. Studies with CPO displayed an initial increase in expression at 4 h only. The compPCR assays were conducted with the CPF-treated group to quantify the changes in gene expression levels. The compPCR data confirmed and quantitated the results from the time-course study using semiquantitative RT-PCR. In addition to the gene expression studies, changes in efflux transporter function were investigated using Caco-2 cells grown on semipermeable membranes in Transwell plates. The permeability of verapamil was determined in cells treated for 8 h with CPF. Efflux ratios demonstrated that verapamil was effluxed at a higher rate from the CPF-treated cells as compared to the control group, confirming the inductive action of CPF on transporter function. These results suggest that CPF has the potential to modulate the bioavailability of drugs via changes in expression and function of membrane efflux transporters.     

Expression of multidrug resistance proteins and accumulation of cisplatin in human non-small cell lung cancer cells

In order to understand and overcome multidrug resistance (MDR) of human non-small cell lung cancer (NSCLC), mRNA and protein expression levels of P-glycoprotein (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung resistance-related protein (LRP) were investigated and compared with the chemosensitivity and the intracellular/intranuclear cisplatin accumulation of three NSCLC cell lines (Ma-10, Ma-31, and Ma-46). Ma-31 was more resistant than Ma-10 and Ma-46 to cisplatin, carboplatin, etoposide, and paclitaxel. The mRNA level of MDR1 was extremely low, and MDR1 protein was not detected in all cell lines. MRP1 mRNA expression was highest in Ma-31 and lowest in Ma-10, but there was no notable difference between the MRP1 protein expression in three cell lines. LRP mRNA/protein was equally expressed in Ma-10 and Ma-31, but was nominal in Ma-46. The intracellular/intranuclear cisplatin accumulation of the cells was determined to be Ma-31>Ma-46>Ma-10. Thus, MDR1, MRP1, and LRP mRNA and protein expression levels were not correlated with the chemosensitivity or the intracellular/intranuclear cisplatin accumulation of each cell line. The present results indicate that MDR proteins (MDR1, MRP1, and LRP) may not play an important role in the chemoresistance and drug efflux of NSCLC cells.     

Inhibit multidrug resistance and induce apoptosis by using glycocholic acid and epirubicin.

Cancer-cell resistance to chemotherapy limits the efficacy of cancer treatment. The primary mechanisms of multidrug resistance (MDR) are "pump" and "non-pump" resistance. We evaluated the effects and mechanisms of glycocholic acid (GC), a bile acid, on inhibiting pump and non-pump resistance, and increasing the chemosensitivity of epirubicin in human colon adenocarcinoma Caco-2 cells and rat intestine. GC increased the cytotoxicity of epirubicin, significantly increased the intracellular accumulation of epirubicin in Caco-2 cells and the absorption of epirubicin in rat small intestine, and intensified epirubicin-induced apoptosis. GC and epirubicin significantly reduced mRNA expression levels of human intestinal MDR1, MDR-associated protein (MRP)1, and MRP2; downregulated the MDR1 promoter region; suppressed the mRNA expression of Bcl-2; induced the mRNA expression of Bax; and significantly increased the Bax-to-Bcl-2 ratio and the mRNA levels of p53, caspase-9 and -3. This suggests that GC- and epirubicin-induced apoptosis was mediated through the mitochondrial pathway. We conclude that simultaneous suppression of pump and non-pump resistance dramatically increased the chemosensitivity of epirubicin. A combination of anticancer drugs with GC can control MDR via a mechanism that involves modulating P-gp and MRPs as well as regulating apoptosis-related pathways.     

Variability in mRNA expression of ABC- and SLC-transporters in human intestinal cells: comparison between human segments and Caco-2 cells.

The Caco-2 cell monolayer model is widely used as a tool for evaluating human intestinal permeability and interaction with transporters. Therefore, we determined mRNA levels for 15 of the most frequently studied uptake and efflux transporters (MDR1, MRP2-3, BCRP, OCTN2, PepT1, OATP-B, OATP8, OCT1-3, OAT1-3, MCT1) using real-time PCR in Caco-2 cells and in human jejunum and colon. The expression levels in the Caco-2 cells did not significantly vary between different passages (p29-43) and batches for any of the genes measured. However, levels increased with culture time (1-5 weeks) for PepT1, MDR1, MRP2, OATP-B and BCRP. The general rank order of the gene expression levels in Caco-2 cells was established as follows: MRP2>OATP-B>PepT1>MDR1>MCT1 approximately MRP3 approximately BCRP approximately OCTN2>OCT3>OCT1>OAT2. Four genes were absent: OATP8, OCT2, OAT1, and OAT3. Ranking of 11 expressed genes showed a significant correlation between human jejunum and 2-5-week-old Caco-2 cells. The expression profile in colon was, however, very different compared to both Caco-2 cells and jejunum. We conclude that the Caco-2 cells in our hands express similar transporters as the human jejunum, but are different from colon, indicating their usefulness for obtaining small intestinal transport data. In addition, we also suggest that cells with a well-defined range of culture ages should be used to minimize variability in data from experiments and even erroneous conclusions.     

Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is a widely used method in assessment of cytotoxicity and cell viability, and also in anti-cancer drug studies with tumour cells. These cells often express efflux proteins, such as P-glycoprotein (MDR1) or multidrug resistance (MDR) protein 1 (MRP1). MDCKII cells that overexpress these proteins (MDCKII-MDR1 or MDCKII-MRP1) and normal cells (MDCKII-wt) were used to investigate the effects of efflux pump activity on the results of MTT assay. Efflux protein activity was confirmed with calcein-AM efflux assay, and MTT assay was compared to another cytotoxicity test, the LDH release assay. Inhibition of MRP and MDR1 efflux proteins in MDCKII cell lines was associated paradoxically with increased reduction of MTT, implying an apparent increase in cell viability. This effect was seen when MK 571 (MRP1 and MRP2 inhibitor) or verapamil (MRP1 and MDR1 inhibitor) were used to block efflux protein activity. The calcein-AM efflux assay also showed that the MTT reagent inhibits the function of MDR1 in the MDCKII-MDR1 cell line. This study shows that MDR1 and possibly MRP proteins interfere with the MTT assay. Due to wide substrate specificity of efflux proteins and popularity of the MTT assay this interference is not trivial. Presence of any efflux protein substrate may therefore lead to underestimated results in MTT assay, thereby causing potential bias and erroneous conclusions in cytotoxicity studies.     

Evidence for Different ABC-Transporters in Caco-2 Cells Modulating Drug Uptake

Purpose. Secretory systems contribute to drug absorption in the gastrointestinal tract. The purpose of this study was the identification of members of the ATP binding cassette superfamily of secretory transport proteins that may potentially modulate drug absorption in Caco-2 cells, which are an important cellular model predicting enteral absorption of drugs. Methods. Kinetic studies as well as PCR- and Western blot studies with confluent epithelial layers of human Caco-2 cells. Results. The study demonstrates functional expression of multidrug resistance related protein (MRP) and P-glycoprotein (P-gp) in Caco-2 cells: 1) Efflux studies with the MRP specific substrate glutathion-methylfluorescein (GS-MF) showed functional activity of MRP in Caco-2 cells preloaded with the metabolic precursor of GS-MF, chloro-methylfluoresceine-diacetate, CMFDA. Excretion of GS-MF was decreased in presence of the MRP-blocker MK-571.2) Transport experiments with cyclosporin A demonstrated the functional activity of P-gp. Cellular accumulation was increased in presence of the P-gp blocking agent SDZ-PSC 833.3) The expression of the 190 kDa protein MRP and the 170 kDa protein P-gp in Caco-2 cells was shown by Western blot analysis with specific monoclonal antibodies. 4) The expression of MRP-mRNA in Caco-2 cells was detected by RT-PCR and compared with the MRP over-expressing cell line H69AR. MRP primers recognize specifically human MRP1 (GenBank accession number L05628), but not all other published sequences of MRP (MRP2-MRP6). P-gp expression on mRNA-level was also confirmed by RT-PCR. Conclusions. The data demonstrate that besides P-gp, multidrug resistance related protein (MRP) is functionally expressed in Caco-2 cells and contributes to the active excretion of substrates in this cell line.     

48例非小细胞肺癌MDR1及MRP基因表达结果分析

目的：探讨多药耐药（MDR1）基因,多药耐药相关蛋白（MRP）基因在非小细胞肺癌（NSCLC）中的表达情况及其与组织类型的关系。方法：用逆转录多聚酶链反应（RT－PCR）方法检测新鲜肺癌组织标本。结果：48例NSCLC标本MDR1,MRP基因阳性表达率分别为62．5％,66．7％,二种基因共同表达率为43．8％,肺鳞癌MRP基因表达明显高于腺癌（P＜0．01）,而鳞癌与腺癌之间MDR1基因表达无显著性差异（P＞0．05）,结论：MDR1,MRP基因可以共同或分别存在于肺癌组织中,肺鳞癌MRP基因表达率较腺癌高。