Peptide Transporter Substrate Identification during Permeability Screening in Drug Discovery: Comparison of Transfected MDCK-hPepT1 Cells to Caco-2 Cells

Chong, Sae-Ho,Patel, Karishma,Quan, Yong,Timoszyk, Julita,Han, Yong-Hae,Wang, Bonnie,Vig, Balvinder,Faria, Teresa N.,Balimane, Praveen. V. 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.4

The purpose of this study was to investigate the utility of stably transfected MDCK-hPepT1 cells for identifying peptide transporter substrates in early drug discovery and compare the characteristics of this cell line with Caco-2 cells. MDCK-hPepT1 , MDCK-mock, and Caco-2 cells grown to confluence on 24-well Transwell were used for this study. Expression levels of different transporter proteins (PepT1 , PepT2, P-gp) in these cell lines were assessed by qRT-PCR. Permeability studies were conducted in parallel in all the cells with a diverse set of pep-tide substrates using the optimized experimental condition: 100 ${\mu}$M, apical pH 6.0, basolateral pH 7.4,2 hr incubation at 37${\circ}$C. Permeability studies were also conducted with classical P-gp substrates (tested in hi-directional mode) and paracellularly absorbed probes to investigate the differences between the cell lines. As expected, MDCK-hPepT1 cells express signifcantly higher level of PepT1 mRNA compared to both Caco-2 and MDCK-mock cells. Efflux transporter, P-gp, was expressed adequately in all the cell lines. Permeability studies demonstrated that classical peptide substrates had significantly higher permeability in stably transfected MDCK-hPepT1 cells compared to MDCK-mock and Caco-2 cells. The transfected MDCK-hPepT1 cells were qualitatively similar to Caco-2 cells with respect to functional P-gp efflux activity and paracellular pore activity. Stably transfected MDCK-hPepT1 cells have been domonstrated as a viable alternative to Caco-2 cells for estimating the human absorption potential of peptide transporter substrates. These cells behave similar to Caco-2 cells with regards to P-gp efflux and paracellular pore activity but demonstrate greater predictability of absorption values for classical peptide substrates (for which Caco-2 cells under-estimate oral absorption).

Peptide Transporter Substrate Identification during Permeability Screening in Drug Discovery: Comparison of Transfected MDCK-hPepT1 Cells to Caco-2 Cells

Praveen. V. Balimane,Saeho Chong,Karishma Patel,Yong Quan,Julita Timoszyk,한용해,Bonnie Wang,Balvinder Vig,Teresa N. Faria 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.4

The purpose of this study was to investigate the utility of stably transfected MDCK-hPepT1 cells for identifying peptide transporter substrates in early drug discovery and compare the characteristics of this cell line with Caco-2 cells. MDCK-hPepT1, MDCK-mock, and Caco-2 cells grown to confluence on 24-well Transwell‚ were used for this study. Expression levels of different transporter proteins (PepT1, PepT2, P-gp) in these cell lines were assessed by qRTPCR. Permeability studies were conducted in parallel in all the cells with a diverse set of peptide substrates using the optimized experimental condition: 100 µM, apical pH 6.0, basolateral pH 7.4, 2 hr incubation at 37°C. Permeability studies were also conducted with classical P-gp substrates (tested in bi-directional mode) and paracellularly absorbed probes to investigate the differences between the cell lines. As expected, MDCK-hPepT1 cells express significantly higher level of PepT1 mRNA compared to both Caco-2 and MDCK-mock cells. Efflux transporter, P-gp, was expressed adequately in all the cell lines. Permeability studies demonstrated that classical peptide substrates had significantly higher permeability in stably transfected MDCK-hPepT1 cells compared to MDCK-mock and Caco-2 cells. The transfected MDCKhPepT1 cells were qualitatively similar to Caco-2 cells with respect to functional P-gp efflux activity and paracellular pore activity. Stably transfected MDCK-hPepT1 cells have been demonstrated as a viable alternative to Caco-2 cells for estimating the human absorption potential of peptide transporter substrates. These cells behave similar to Caco-2 cells with regards to Pgp efflux and paracellular pore activity but demonstrate greater predictability of absorption values for classical peptide substrates (for which Caco-2 cells under-estimate oral absorption).

Chemopreventive effect of α-viniferin in azoxymethane-induced mouse colorectal tumor and Caco-2 cells

Dong Hoon Kwak,Sang-Kyung Shin,So-Young Youm,Tae-Wang Kim,Youngsoo Kim,Byeongwoo Ahn 충북대학교 동물의학연구소 2015 Journal of Biomedical and Translational Research Vol.16 No.2

α-Viniferin (AVF), a trimer of resveratrol, is known to have an anti-inflammatory effect via inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). It has been reported that up-regulated COX-2 and iNOS are expressed in colon cancer tissues of humans and rodents as well as pre-neoplastic aberrant crypt foci (ACF) of rodents. In this study, chemopreventive effects of AVF were assessed in Caco-2 cells as well as azoxymethane (AOM)-induced colorectal tumorigenesis in mice. Anti-tumor effect of AVF with regards to apoptotic induction was assessed by TUNEL and caspase-3 expression in human colon cancer Caco-2 cells. For development of ACF, AOM was administered with to mice intraperitoneally at a dose of 10 mg/kg once a week for 3 weeks. To induce colitis-related colon cancer, mice were administered a single dose of AOM (10 mg/kg) and 2% dextran sodium sulfate in drinking water. Mice treated with 0.05 and/or 0.1 mg of AVF by gavage showed significantly reduced development of ACF and colorectal tumors. Immunofluorescence detection in Caco-2 cells showed reduced COX-2 and iNOS expression, whereas cleavage of caspase-3 and apoptotic cell numbers increased upon AVF treatment. Immunostaining showed reduced expression levels of COX-2 and iNOS expression along with increased cleaved caspase-3 expression increased upon AVF treatment. These results suggest that AVF has chemopreventive effects on colorectal cancer via anti-inflammatory potential and pro-apoptotic activity.

Extensive Hepatic Uptake of Pz-peptide, a Hydrophilic Proline-Containing Pentapeptide, into Isolated Hepatocytes Compared with Colonocytes and Caco-2 Cells

Shin, Tae-Ha,Lee, Pung-Sok,Kwon, Oh-Seung,Chung, Youn-Bok The Pharmaceutical Society of Korea 2003 Archives of Pharmacal Research Vol.26 No.1

The objective of the present study was to investigate the uptake process of 4-Phenylazobenzoxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide), a hydrophilic and collagenase-labile pentapeptide, by isolated hepatocytes. For comparison, the uptake of Pz-peptide by Caco-2 cells and colonic cells, two known paracellular routes of Pz-peptide, was also evaluated. A simple and sensitive reversed-phase HPLC assay method using UV detection has been developed. The coefficient of variation for all the criteria of validation were less than 15%. The method was, therefore, considered to be sutable for measuring the concentration of Pz-peptide in the biological cells. Pz-peptide was extensively uptaked into hepatocytes. The initial velocity of Pz-peptide uptake assessed from the initial slope of the curve was plotted as Eadie-Hofstee plots. The maximum velocity ($V_{max}$) and the Michaelis constant ($K_m$) were 0.190$\pm$0.020 $nmol/min/10^6$ cells and 12.1$\pm$3.23 $\mu$M, respectively. The permeability-surface area product ($PS{influx}$) was calculated to be 0.0157 ml/min/10^6$ cells. $V_{max}$ and $K_m$ values for Caco-2 cells were calculated to be 6.22$\pm$0.930 pmol/min/10^6$ cells and 82.8$\pm$8.37 $\mu$M, respectively, being comparable with those of colonocytes (6.04$\pm$1.03 pmol/min/10^6$ cells and 87.8$\pm$13.2 $\mu$M, respectively). $PS_{influx}$ values for Caco-2 cells and colonocytes were calculated to be 0.0751 $\mu$l/min/10^6$ cells and 0.0688 $\mu$l/min/10^6$ cells, respectively. The more pronounced uptake of Pz-peptide by hepatocytes, when compared with Caco-2 cells and colonocytes, is probably due to its specific transporter. In conclusion, Pz-peptide, a paracellularly transported pentapeptide in the intestine and ocular epithelia, was uptaked into hepatocytes extensively. Although Pz-peptide is able to be uptaked into the Caco-2 cells and colonocytes, it is less pronounced when compared with hepatocytes. $PS_{influx}$ values of Caco-2 cells and colonocytes for unbound Pz-peptide under linear conditions were less than 0.4% when compared with that of hepatocytes.

Exposure to Probiotic Lactobacillus acidophilus L-92 Modulates Gene Expression Profiles of Epithelial Caco-2 Cells

Sae Yanagihara,Shinji Fukuda,Hiroshi Ohno,Naoyuki Yamamoto 한국식품영양과학회 2012 Journal of medicinal food Vol.15 No.6

To understand host gastrointestinal response after exposure to probiotic Lactobacillus acidophilus L-92,microarray analysis of cultured epithelial Caco-2 cells was performed. Of the 187 genes down-regulated after 4 h treatment with L-92, 25 were involved in RNA splicing; 12, in cell cycle; 8 were transcriptional regulators; 2 were involved in ubiquitin proteolysis; 2, in adhesion; 2, in meiosis; 2, in splicing; and 2 encoding cytokines. In the RNA splicing group, genes encoding small nuclear RNAs, nuclear pore complex interacting proteins, RNA binding motif proteins, and SMG1 homologs (phosphatidylinositol 3-kinase-related kinase) were identified. Among the only 13 genes up-regulated by the treatment, 5 were involved in histone structure, and 2 were involved in metabolism. Genes belonging to cell adhesion, transmembrane proteins,mitogen-activated protein kinase, immune response, DNA binding, inflammation, and protein synthesis groups were mainly up-regulated after 20 h of treatment, whereas no significantly down-regulated genes were observed. In the present transcriptome analysis, during the early stage of treatment (four hours of treatment) with L-92, genes involved in cell growth and cell meiosis were mainly repressed. During the late phase of treatment (20 h of treatment), the expression of the genes linked to cell adhesion activity and metabolism for cell growth was enhanced. From the present transcriptome analysis, we suggest that Caco-2 cells slow down cell death and turnover of RNA synthesis as an early response to L-92 treatment; at the late stage of treatment, the genes involved in cell proliferation, transcriptional activity, and apoptosis are activated.

Modulation of Tight Junctions does Not Predict Oral Absorption of Hydrophilic Compounds: Use of Caco-2 and Calu-3 Cells

Kamath, Amrita V.,Morrison, Richard A.,Mathias, Neil R.,Dando, Sandra A.,Marino, Anthony M.,Chong, Sae-Ho 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.8

Permeability estimates using Caco-2 cells do not accurately predict the absorption of hydrophilic drugs that are primarily absorbed via the paracellular pathway. The objective of this study was to investigate whether modulation of tight junctions would help differentiation of paracellularly absorbed compounds. Tight junctions in Caco-2 cell monolayers were manipulated using calcium depletion approaches to decrease the transepithelial electrical resistance (TEER) of the monolayers, and permeability of hydrophilic compounds were measured under these conditions. Permeability of these compounds were also measured in Calu-3 cells, which have tighter junctions than Caco-2 cells. Calcium depletion loosened the tight junctions of Caco-2 cells to varying levels as measured by the decrease in TEER values of the monolayers. While the absolute permeability of all the model compounds increased as the tight junctions were loosened, the ratios of their permeability relative to mannitol permeability were similar. The permeability of these compounds in the tighter Calu-3 cells were also found to be similar to each other. Altering the tight junctions of Caco-2 cells to obtain leakier cell monolayers, or using a cell line with tighter junctions like Calu-3 cells, did not improve differentiation between well absorbed and poorly absorbed hydrophilic drugs. Mere manipulation of the tight junctions to increase or decrease transepithelial electrical resistance does not appear to be a viable approach to predict human absorption for hydrophilic compounds that are primarily absorbed via the paracellular pathway.

Modulation of Tight Junctions does Not Predict Oral Absorption of Hydrophilic Compounds: Use of Caco-2 and Calu-3 Cells

Amrita V. Kamath,Richard A. Morrison,Neil R. Mathias,Sandra A. Dando,Anthony M. Marino,Saeho Chong 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.8

Permeability estimates using Caco-2 cells do not accurately predict the absorption of hydrophilic drugs that are primarily absorbed via the paracellular pathway. The objective of this study was to investigate whether modulation of tight junctions would help differentiation of paracellularly absorbed compounds. Tight junctions in Caco-2 cell monolayers were manipulated using calcium depletion approaches to decrease the transepithelial electrical resistance (TEER) of the monolayers, and permeability of hydrophilic compounds were measured under these conditions. Permeability of these compounds were also measured in Calu-3 cells, which have tighter junctions than Caco-2 cells. Calcium depletion loosened the tight junctions of Caco-2 cells to varying levels as measured by the decrease in TEER values of the monolayers. While the absolute permeability of all the model compounds increased as the tight junctions were loosened, the ratios of their permeability relative to mannitol permeability were similar. The permeability of these compounds in the tighter Calu-3 cells were also found to be similar to each other. Altering the tight junctions of Caco-2 cells to obtain leakier cell monolayers, or using a cell line with tighter junctions like Calu-3 cells, did not improve differentiation between well absorbed and poorly absorbed hydrophilic drugs. Mere manipulation of the tight junctions to increase or decrease transepithelial electrical resistance does not appear to be a viable approach to predict human absorption for hydrophilic compounds that are primarily absorbed via the paracellular pathway.

TNF-alpha Downregulates E-cadherin and Sensitizes Response to γ-irradiation in Caco-2 Cells

이재연,정유진,최선심,정은경 대한암학회 2009 Cancer Research and Treatment Vol.41 No.3

Purpose : The purpose of the present study was to assess the biological effects of TNF-alpha in Caco-2 well-differentiated colon adenocarcinoma cells and to determine radiation sensitivity in order to develop TNF-alpha into a cancer therapeutic agent. Materials and Methods : A cell viability test was conducted via a colorimetric and colony forming assay after 1 day and 3 days of incubation with TNF-alpha. Western blotting analysis and immunofluorescence staining were conducted to explore TNF-alpha-induced morphological and molecular changes in the adhesion molecules, E-cadherin and claudin-4. The effects of γ-irradiation at a dose of 2 Gy on cell survival were evaluated by a clonogenic assay. The molecular changes in apoptosis-regulatory proteins were assessed by Western blotting. Results : Caco-2 cells were highly resistant to TNF alpha-induced cell death and 2 Gy of γ-irradiation. However, we observed the downregulation of the adherens junctional protein, E-cadherin and translocation of tight junctional protein, claudin-4 from the membrane to the cytosol induced by TNF-alpha treatment which would indicate cell-cell junction disruptions. These alterations of junctional proteins influenced the regulation of cell death in response to 2 Gy of γ-irradiation. The combined treatment of TNF-alpha with 2 Gy of γ-irradiation reduced the survival of Caco-2 cells by down-regulating bcl-xl and activating JNK pathways. Conclusion : These results suggest that TNF-alpha might be potentially applied as a therapeutic agent in order to enhance sensitivity to 2 Gy of γ-irradiation administered in radiotherapy for the treatment of human colon cancer.

Effect of Particle Size of Zinc Oxides on Cytotoxicity and Cell Permeability in Caco-2 Cells

Chang, Hyun-Joo,Choi, Sung-Wook,Ko, Sang-Hoon,Chun, Hyang-Sook The Korean Society of Food Science and Nutrition 2011 Preventive Nutrition and Food Science Vol.16 No.2

The cell permeability and cytotoxic effects of different-sized zinc oxide (ZnO) particles were investigated using a human colorectal adenocarcinoma cell line called Caco-2. Morphological observation by scanning electron microscopy revealed that three zinc oxides with different mean particle sizes (ZnO-1, 20 nm; ZnO-2, 90~200 nm; ZnO-3, $1\sim5\;{\mu}m$) tended to aggregate, particularly in the case of ZnO-1. When cytotoxicities of all three sizes of zinc oxide particles were measured at concentration ranges of $1\sim1000\;{\mu}g$/mL, significant decreases in cell viability were observed at concentrations of $50\;{\mu}g$/mL and higher. Among the three zinc oxides, ZnO-1 showed the lowest viability at $50\;{\mu}g$/mL in Caco-2 cells, followed by ZnO-2 and ZnO-3. The permeate concentration of ZnO-1 from the apical to the basolateral side in the Caco-2 model system after four hours was about three-fold higher than that of either ZnO-2 or ZnO-3. These results demonstrated that ZnO-1, with a 20 nm mean particle size, had poorer viability and better permeability in Caco-2 cells than ZnO-2 and ZnO-3.

Effect of Particle Size of Zinc Oxides on Cytotoxicity and Cell Permeability in Caco-2 Cells

Hyun-Joo Chang,Sung-Wook Choi,Sanghoon Ko,Hyang-Sook Chun 한국식품영양과학회 2011 Preventive Nutrition and Food Science Vol.16 No.2

The cell permeability and cytotoxic effects of different-sized zinc oxide (ZnO) particles were investigated using a human colorectal adenocarcinoma cell line called Caco-2. Morphological observation by scanning electron microscopy revealed that three zinc oxides with different mean particle sizes (ZnO-1, 20 ㎚; ZnO-2, 90~200 ㎚; ZnO-3, 1~5 ㎛) tended to aggregate, particularly in the case of ZnO-1. When cytotoxicities of all three sizes of zinc oxide particles were measured at concentration ranges of 1~1000 ㎍/㎖, significant decreases in cell viability were observed at concentrations of 50 ㎍/㎖ and higher. Among the three zinc oxides, ZnO-1 showed the lowest viability at 50 ㎍/㎖ in Caco-2 cells, followed by ZnO-2 and ZnO-3. The permeate concentration of ZnO-1 from the apical to the basolateral side in the Caco-2 model system after four hours was about three-fold higher than that of either ZnO-2 or ZnO-3. These results demonstrated that ZnO-1, with a 20 ㎚ mean particle size, had poorer viability and better permeability in Caco-2 cells than ZnO-2 and ZnO-3.