Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh-123). The sorted cells were then assayed for their in vivo spleen colony-forming ability (day -8, -12, and -16 CFU-S) and their ability to repopulate the bone marrow or spleen over a 13-day period with CFU-S-12, CFU-GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU-S-12 or CFU-GM, had low affinity for Rh-123. These cells showed minimal spleen colony-forming ability, and the ratio of MRA to CFU-S-12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU-S-16, CFU-S-12, and CFU-S-8 retained increasing amounts of Rh-123, respectively, and CFU-S-8 were almost exclusively found among cells with high Rh-123 affinity. These cells also included about half of all day-12 CFU-S, and the ratio of MRA to day-12 CFU-S was 0. The results show that MRA cells, SRA cells, CFU-S-16, CFU-S-12, and CFU-S-8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre-CFU-S.     

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.     

Histone deacetylase inhibitor apicidin-mediated drug resistance: involvement of P-glycoprotein

Multidrug resistance (MDR), which is a significant impediment to the success of cancer chemotherapy, is attributable to the overexpression of membrane transport proteins, such as P-glycoprotein (P-gp), resulting in an increased drug efflux. In this study, we show that the histone deacetylase (HDAC) inhibitor apicidin leads to resistance of HeLa cells to paclitaxel through the induction of P-gp expression. Furthermore, apicidin dramatically increases the release of a fluorescent P-gp substrate, rhodamine 123, from cells. In parallel, apicidin resistance to the apoptotic potential of paclitaxel is associated with induction of P-gp expression in HeLa cells, as evidenced by specific inhibition of P-gp function using either the pharmacological inhibitor verapamil or RNA silencing. We also demonstrate the contribution of apicidin-induced functional P-gp expression to drug resistance using KB cells. Failure of P-gp induction by apicidin does not reverse paclitaxel-induced cytotoxicity in the cells. Although HDAC inhibitors are widely appreciated as a new class of anti-tumor agent, our findings clearly demonstrate that apicidin treatment may lead to P-gp-mediated resistance to other anti-tumor agents, suggesting a need for careful design of clinical applications using HDAC inhibitors.     

Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues.

BACKGROUND: Chloromethyl-X-rosamine (CMXRos) and MitoTracker Green (MTG) have proved to be useful dyes with which to measure mitochondrial function. CMXRos is a lipophilic cationic fluorescent dye that is concentrated inside mitochondria by their negative mitochondrial membrane potential (MMP). MTG fluorescence has been used as a measure of mitochondrial mass independent of MMP. The fluorescence ratio of the two dyes is a relative measure of the MMP independent of mitochondrial mass. Because MTG was recently reported to be sensitive to MMP, we have reevaluated the effects of loss of MMP on MTG and CMXRos fluorescence, using both flow cytometry and laser scanning confocal microscopy (LSCM). METHODS: Using flow cytometry, the relative fluorescence of CMXRos, R123, and MTG was determined in human lymphoblastoid cell lines (LCLs) with or without carbonyl cyanide p-trifluoromethoxylphenyl-hydrazone (FCCP), used to collapse the MMP. LSCM analysis was also used to evaluate the effect of FCCP on MTG and CMXRos fluorescence of mouse cells and viable lenses in culture. The cytotoxicity of the dyes was determined using flow analysis of endogenous NADH fluorescence. The sensitivity of MTG fluorescence to H(2)O(2) was also evaluated using flow cytometry. RESULTS: CMXRos fluorescence was dependent on MMP, whereas MTG fluorescence was not affected by MMP, using either flow or LSCM. Specific staining of mitochondria was seen with both dyes in all cell types tested, without evidence of cytotoxicity, as determined by NADH levels. H(2)O(2) damage slightly increased MTG staining of cells. CONCLUSIONS: Our results indicate that CMXRos is a nontoxic sensitive indicator of relative changes in MMP, whereas MTG is relatively insensitive to MMP and oxidative stress, using both flow and LSCM analyses, provided optimal staining conditions are used. In addition, these dyes can be useful for the study of mitochondrial morphology and function in whole tissues, using LSCM.     

Isolation and enrichment of murine spermatogonial stem cells using rhodamine 123 mitochondrial dye

Stem cells possess enormous therapeutic potential in tissue replacement. To study stem cells further, they must be isolated. Techniques are available for enrichment and study of hematopoietic stems cells, but thus far, techniques for purification of spermatogonial stem cells have not been described. Enrichment techniques for hematopoietic stem cells include the use of fluorescence-activated cell sorter analysis with Hoechst 33342 and rhodamine 123 (Rho) dyes. Use of Hoechst dye to isolate spermatogonial stem cells has been unsuccessful in our laboratory, and our results have conflicted with those from other laboratories. Taking advantage of the differential staining of the Rho dye, we report a novel method to enrich murine spermatogonial stem cells. Testicular cells are harvested from cryptorchid ROSA26 male mice. Populations of these cells are then stained with the Hoechst and Rho dyes, allowing them to be sorted by flow cytometry into a side population (SP) of Hoechst low-intensity cells and populations of low (Rho(low)) or high (Rho(hi)) fluorescent intensity. Sterile recipients, W/W(v) mice, with an intrinsic germ cell deficiency were transplanted with the Hoechst SP cells, Rho(low), Rho(hi), and nonsorted donor cells. No spermatogonial stem cell colonies were derived from the Hoechst SP cells. The number of spermatogonial stem cell colonies from transplanted Rho(low) cells showed a 17- and 20-fold enrichment over those of Rho(hi) and nonsorted cells, respectively.     

Tetrandrine and fangchinoline,bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.     

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.     

Effects of plant sterols on human multidrug transporters ABCB1 and ABCC1

The effects of dietary plant sterols on human drug efflux transporters P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-gp-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytosterols found in foods, herbs, and dietary supplements such as β-sitosterol, campesterol, stigmasterol, fucosterol, and z-guggulsterone were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-gp, increased in the presence of guggulsterone in KB-C2 cells. The efflux of rhodamine 123 from KB-C2 cells was inhibited by guggulsterone. Guggulsterone also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-gp and MRP1 were stimulated by guggulsterone. These results suggest that guggulsterone, a natural dietary hypolipidemic agent have dual inhibitory effects on P-gp and MRP1 and the potencies to cause food-drug interactions.     

Direct flow cytometric quantification of alkaline phosphatase activity in rat bone marrow stromal cells.

Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.     

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.     

Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters

An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC(2)(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.     

Use of fluorescent dyes as molecular probes for the study of multidrug resistance

Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine. Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl. The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells. All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells. The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium. Sensitive cells retained the dyes for several hours. The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium. The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells. These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.     

MDR1 gene expression enhances long-term engraftibility of cultured bone marrow cells

Primitive hematopoietic stem cells are responsible for long-term engraftment in irradiated host. Here, we report that multi-drug resistance 1 (mdr1) gene expressing primitive hematopoietic cells were multiplied in ex vivo culture, with the support of extracellular matrix components and cytokines. About 20-fold expansion of total nucleated cells was achieved in a 10-day culture. Lin(-)Sca-1(+) and long-term culture-initiating cells were increased by 54- and 26-fold, respectively. Expanded cells were long-term multi-lineage engraftible in sub-lethally irradiated mice. Donor-derived peripheral blood chimerism was significantly higher (73.2+/-9.1%, p<0.01) in expanded cells than in normal and 5-flurouracil-treated bone marrow cells. Most interestingly, the expression of mdr1 gene was significantly enhanced in cultured cells than in other two sources of donor cells. The mdr1 gene was functional since expanded cells effluxed Hoechst 33342 and Rh123 dyes. These results suggest that primitive engraftible stem cells can be expanded in the presence of suitable microenvironments.     

Participation of bone marrow derived cells in cutaneous wound healing

Bone marrow has long been known to be a source of stem cells capable of regeneration of the hematopoeitic system. Recent reports, however, have indicated that bone marrow might also contain early stem cells that can differentiate into other organ tissues such as skin. While these studies have illustrated that bone marrow stem cells could find their way to the skin, they have not addressed the dynamics of how bone marrow stem cells might participate in the homeostatis and regeneration of skin. In this report we followed green fluorescent protein (GFP) labeled bone marrow transplanted into non-GFP mice in order to determine the participation of bone marrow stem cells in cutaneous wounds. Our results indicate that there are a significant number of bone marrow cells that traffic through both wounded and non-wounded skin. Wounding stimulated the engraftment of bone marrow cells to the skin and induced bone marrow derived cells to incorporate into and differentiate into non-hematopoietic skin structures. This report thus illustrates that bone marrow might be a valuable source of stem cells for the skin and possibly other organs. Wounding could be a stimulus for bone marrow derived stem cells to travel to organs and aid in the regeneration of damaged tissue.     

Role of cytokines in experimentally induced lung cancer and chemoprevention by COX-2 selective inhibitor, etoricoxib

This study explored the role of pro- and anti-inflammatory cytokines in dimethyl benz(a)anthracene (DMBA)-induced lung cancer and its subsequent correction with a COX-2 inhibitory NSAID, etoricoxib. A single dose of DMBA (20?mg/kg body weight) in 0.9?% NaCl administered intratracheally was used to induce tumors in the rat lungs in 20?weeks. The study of pro-inflammatory cytokines like IL-1??, TNF-??, and IFN-?? revealed their upregulation by DMBA administration and restoration of their levels toward normal by the treatment with etoricoxib, while the anti-inflammatory cytokine IL-2 was found to be down-regulated with carcinogen administration and corrected with etoricoxib treatment. Apoptosis was studied by mitochondrial Bcl-2/Bax ratio and staining with fluorescent dyes acridine orange/ethidium bromide. The results showed a decreased apoptotic level with DMBA which was corrected with etoricoxib. Also, mitochondrial membrane potential was studied using JC-1 and rhodamine-123, which are membrane permeant fluorescent dyes, and generate information about cells at lower and higher mitochondrial membrane potential (???_M). The results showed the presence of maximum number of cells with higher ???_M in the DMBA group and their number was considerably lowered in the other three groups.     

Kinetic analyses as a critical parameter in defining the side population (SP) phenotype

The side population (SP) phenotype has been reported as a method to identify hematopoietic stem cells in the bone marrow based upon differential staining with the fluorescent dye, Hoechst 33342. This technique has drawn great interest in the stem cell community, as it may provide a simple approach to the enrichment of progenitor cells from a variety of normal and malignant tissues. The frequency of these cells and their performance in functional assays has varied considerably within the literature. To investigate mechanisms that may contribute to the SP phenotype, we measured the fluorescence emission of Hoechst-stained bone marrow cells as a function of both time and dye concentration using a custom flow cytometer and data acquisition software. These measurements demonstrate that all nucleated cells within the bone marrow undergo an identical staining pattern at varying rates, even under conditions previously reported to abrogate the SP. Therefore, the SP phenotype is not unique to stem cells, but rather represents a transient feature of marrow cells exposed to Hoechst 33342 for varying amounts of time. We propose that heterogeneity of SP-defined populations may be a consequence of the rate at which differing cell populations accumulate Hoechst 33342. Further, we suggest that dye uptake kinetics will likely be an important factor for optimal use of Hoechst 33342 in isolating stem cells.     

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.     

Accumulation of daunomycin and fluorescent dyes by drug-transporting Malpighian tubule cells of the tobacco hornworn, Manduca sexta

Insect Malpighian tubules actively transport a variety of xenobiotics, and it has been proposed that P-glycoprotein (P-gp), or the multidrug transporter, is involved. To test this idea, we observed the interaction of known P-gp substrates with isolated, living Malpighian tubules from tobacco hornworm (Manduca sexta) larvae. Specifically, the fluorescent drugs daunomycin, rhodamine 123, acridine orange and Hoechst 33342 were applied to the basal side of tubules (proximal portion) in a well of fluid on a coverslip; the subsequent distribution of the drugs was monitored by laser scanning confocal microscopy. Contrary to expectation, none of the drugs appeared in the lumen even after 1-2 h of incubation, although the cells of the tubule were intensely stained within 1 min. For daunomycin, neither verapamil, a P-gp inhibitor, nor nicotine, an alkaloid which appears to be transported by a P-gp-like mechanism in this species, had any effect on the pattern of staining. In sharp contrast to the fast and intense staining of Malpighian tubules, portions of muscle, nerve cord and body fat showed only light staining with daunomycin, and only after prolonged periods. The results suggest that for some drugs, Malpighian tubules act as xenobiotic scavengers, and that this property is unrelated to P-gp-mediated transport.     

Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols

Many beneficial proprieties have been associated with polyphenols from green tea, such as chemopreventive, anticarcinogenic, antiatherogenic and antioxidant actions. In this study, we investigated the effects of green tea polyphenols (GTPs) and their principal catechins on the function of P-glycoprotein (P-gp), which is involved in the multidrug resistance phenotype of cancer cells. GTPs (30 microg/ml) inhibit the photolabeling of P-gp by 75% and increase the accumulation of rhodamine-123 (R-123) 3-fold in the multidrug-resistant cell line CH(R)C5, indicating that GTPs interact with P-gp and inhibit its transport activity. Moreover, the modulation of P-gp transport by GTPs was a reversible process. Among the catechins present in GTPs, EGCG, ECG and CG are responsible for inhibiting P-gp. In addition, EGCG potentiates the cytotoxicity of vinblastine (VBL) in CH(R)C5 cells. The inhibitory effect of EGCG on P-gp was also observed in human Caco-2 cells, which form an intestinal epithelial-like monolayer. Our results indicate that, in addition to their anti-cancer properties, GTPs and more particularly EGCG inhibit the binding and efflux of drugs by P-gp. Thus, GTPs or EGCG might be potential agents for modulating the bioavailability of P-gp substrates at the intestine and the multidrug resistance phenotype associated with expression of this transporter in cancer cells.