Tumoricidal properties of thymoquinone on human colorectal adenocarcinoma cells via the modulation of autophagy.

Colorectal cancer (CRC) is deadly anaplastic changes in the gastrointestinal tract with high-rate mortality. In recent years, the application of phytocompounds has been extended along with different therapeutic protocols. Here, we monitored the effects of Thymoquinone (TQ) on autophagy via mitochondrial function after modulation of the Wnt/ß-catenin signaling pathway.Human colorectal adenocarcinoma HT-29 cells were treated with TQ (60 µM) and 15 µM Wnt3a inhibitor (LGK974) for 48 h. The survival rate was evaluated using an MTT assay. The expression of Wnt-related factors (c-Myc, and Axin), angiogenesis (VE-Cadherin), and mitophagy-related factors (PINK1, OPTN) was assessed using real-time PCR assay. Protein levels of autophagy factors (Beclin-1, LC3, and P62) were monitored using western blotting. Using flow cytometry analysis, the intracellular accumulation of Rhodamine 123 was evaluated. The migration properties were analyzed using a scratch wound healing assay.Data indicated that TQ can reduce the viability of HT-29 cells compared to the control cells (p < 0.05). The expression of VE-Cadherin was inhibited while the expression of PINK1 was induced in treated cells (p < 0.05). Both LGK974 and TQ-treated cells exhibited activation of autophagy flux (Beclin-1↑, LC3II/I↑, and p62↓) compared to the control group (p < 0.05). TQ can increase intracellular accumulation of Rhodamine 123, indicating the inhibition of efflux mechanisms in cancer cells. Along with these changes, the migration of cells was also reduced (p < 0.05).TQ is a potential phytocompound to alter the dynamic growth of human colorectal HT-29 cells via the modulation of autophagy, and mitophagy-related mechanisms.

Design, synthesis, and bioactivity evaluation of novel indole-selenide derivatives as P-glycoprotein inhibitors against multi-drug resistance in MCF-7/ADR cell.

The inhibition of P-glycoprotein (P-gp) has emerged as an intriguing strategy for circumventing multidrug resistance (MDR) in anticancer chemotherapy. In this study, we have designed and synthesized 30 indole-selenides as a new class of P-gp inhibitors based on the scaffold hopping strategy. Among them, the preferred compound H27 showed slightly stronger reversal activity (reversal fold: 271.7 vs 261.6) but weaker cytotoxicity (inhibition ratio: 33.7% vs 45.1%) than the third-generation P-gp inhibitor tariquidar on the tested MCF-7/ADR cells. Rh123 accumulation experiments and Western blot analysis demonstrated that H27 displayed excellent MDR reversal activity by dose-dependently inhibiting the efflux function of P-gp rather than its expression. Besides, UIC-2 reactivity shift assay revealed that H27 could bind to P-gp directly and induced a conformation change of P-gp. Moreover, docking study revealed that H27 matched well in the active pockets of P-gp by forming some key H-bonding interactions, arene-H interactions and hydrophobic contacts. These results suggested that H27 is worth to be a starting point for the development of novel Se-containing P-gp inhibitors for clinic use.

Nimbin (N1) and analog N3 from the neem seeds suppress the migration of osteosarcoma MG-63 cells and arrest the cells in a quiescent state mediated via activation of the caspase-modulated apoptotic pathway.

BACKGROUND: Natural products are considered effective sources for new therapeutic research and development. The numerous therapeutic properties of natural substances in traditional medicine compel us to investigate the anti-cancer properties of Nimbin (N1) and its semi-natural analog Nimbic acid (N3) from Azadirachta indica against MG-63 Osteosarcoma cells. MATERIALS AND METHODS: The therapeutic efficacy of N1 and N3 were screened for their toxicity and cytotoxic activity using L6 myotubes, zebrafish larvae and MG-63 osteosarcoma cells. The mitochondrial membrane potential was evaluated using the Rhodamine 123 stain. Further, the nuclear and cellular damage was distinguished using Hoechst and Acridine orange/EtBr stain. The mechanism of cell cycle progression, cellular proliferation and caspase cascade activation was screened using scratch assay, flow cytometry, and mRNA expression analysis. RESULTS: The Nimbin and analogue N3 were found to be non-toxic to normal L6 cells (Rat skeletal muscles), exhibited cytotoxicity in MG-63 cells, and were exposed to be an active inhibitor of cell proliferation and migration. Analogs N1 and N3 induced negative mitochondrial membrane potential when stained with Rhodamine 123, leading to nuclear damage and apoptosis stimulation using AO/EtBr and Hoechst. Further, N1 and N3 induced cell cycle arrest in G0/G1 phase in flow cytometry using PI staining and induced apoptosis by activating the caspase cascade and upregulated Caspase 3 and caspase 9. CONCLUSION: The study demonstrated cytotoxic activity against MG-63 osteosarcoma cells while being non-toxic to normal L6 cells. These compounds inhibited cell proliferation and migration, induced mitochondrial dysfunction, nuclear damage, and apoptosis stimulation. Furthermore, N1 and N3 caused cell cycle arrest and activated the caspase cascade, ultimately leading to apoptosis. These findings indicate that N1 and N3 hold promise as potential candidates used alone or combined with existing drugs for further investigation and development as anti-cancer agents.

Carnosine Potentiates Doxorubicin-Induced Cytotoxicity in Resistant NCI/ADR-RES Cells by Inhibiting P-Glycoprotein-In Silico and In Vitro Evidence.

The activity of the P-glycoprotein (P-gp) transporter encoded by the ABCB1 gene confers resistance to anticancer drugs and contributes to cancer-related mortality and morbidity. Recent studies revealed the cytotoxic effects of the endogenous dipeptide carnosine. The current study aimed to investigate the role of carnosine as a potential inhibitor of P-gp activity. We used molecular docking and molecular dynamic simulations to study the possible binding and stability of carnosine-P-gp interactions compared with verapamil. In vitro assays using doxorubicin-resistant NCI/ADR-RES cells were established to test the effects of carnosine (10-300 µM) on P-gp activity by the rhodamine-123 efflux assay and its effect on cell viability and doxorubicin-induced cytotoxicity. Verapamil (10 µM) was used as a positive control. The results showed that carnosine binding depends mainly on hydrogen bonding with GLU875, GLN946, and ALA871, with a higher average Hbond than verapamil. Carnosine showed significant but weaker than verapamil-induced rhodamine-123 accumulation. Carnosine and verapamil similarly inhibited cell viability. However, verapamil showed a more significant potentiating effect on doxorubicin-induced cytotoxicity than a weaker effect of carnosine at 300 µM. These results suggest that carnosine inhibits P-gp activity and potentiates doxorubicin-induced cytotoxicity at higher concentrations. Carnosine might be a helpful lead compound in the fight against multidrug-resistant cancers.

Forsythiaside Protected H9c2 Cardiomyocytes from H2O2-Induced Oxidative Stress and Apoptosis via Activating Nrf2/HO-1 Signaling Pathway.

Forsythiaside, one of the main bioactive components of Chinese medicine Lian Qiao, exerts antioxidant, anti-bacterial, and anti-inflammatory effects. To date, the mechanism of Forsythiaside in cardiomyocyte injury remains unclear. However, the antioxidant effects of Forsythiaside on cardiac cells are currently unknown. This study investigated the effect and mechanism of Forsythiaside on oxidative stress in H9c2 cardiomyocytes. H9c2 cells were treated with H2O2 and Forsythiaside and then transfected with small-interfering RNA against nuclear factor erythroid 2-related factor 2 (siNrf2). Cell viability, apoptosis, accumulation of reactive oxygen species (ROS), and mitochondrial membrane potential were measured using methyl thiazolyl tetrazolium (MTT), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123, respectively. The levels of oxidative stress-related markers were determined using their respective detection kits. Furthermore, the levels of apoptosis- and Nrf2 pathway-related molecules were determined via Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Forsythiaside had no obvious toxicity on H9c2 cells. H2O2 suppressed the viability, and reduced the levels of mitochondrial membrane potential, B-cell lymphoma-2 (Bcl-2), glutathione peroxidase (GSH-Px) and catalase (CAT) and superoxide dismutase (SOD), while promoted apoptosis, ROS accumulation, and elevated the levels of cleaved caspase 3, BCL2-Associated X (Bax) and malondialdehyde (MDA) in H9c2 cells. Contrarily, Forsythiaside reversed the aforementioned effects. H2O2 advanced the levels of cytoplasm Nrf2, heme oxygenase-1 (HO-1), and nucleus Nrf2 in H9c2 cells, whereas Forsythiaside enhanced these effects. SiNrf2 reversed the functions of H2O2 or Forsythiaside in cell viability, MDA, SOD, GSH-Px, CAT, Nrf2, and HO-1 in H9c2 cells, whereas Forsythiaside reversed the aforementioned effects of siNrf2. In sum, Forsythiaside protected H9c2 cells from oxidative stress and apoptosis induced by H2O2 by activating the Nrf2/HO-1 pathway.

Comparison of Chemotherapeutic Activities of Rhodamine-Based GUMBOS and NanoGUMBOS.

Rhodamine derivatives have been widely investigated for their mitochondrial targeting and chemotherapeutic properties that result from their lipophilic cationic structures. In previous research, we have found that conversion of Rhodamine 6G into nanoGUMBOS, i.e., nanomaterials derived from a group of uniform materials based on organic salts (GUMBOS), led to selective chemotherapeutic toxicity for cancer cells over normal cells. Herein, we investigate the chemotherapeutic activity of GUMBOS derived from four different rhodamine derivatives, two bearing an ester group, i.e., Rhodamine 123 (R123) and SNAFR-5, and two bearing a carboxylic acid group, i.e., rhodamine 110 (R110) and rhodamine B (RB). In this study, we evaluate (1) relative hydrophobicity via octanol-water partition coefficients, (2) cytotoxicity, and (3) cellular uptake in order to evaluate possible structure-activity relationships between these different compounds. Intriguingly, we found that while GUMBOS derived from R123 and SNAFR-5 formed nanoGUMBOS in aqueous medium, no distinct nanoparticles are observed for RB and R110 GUMBOS. Further investigation revealed that the relatively high water solubility of R110 and RB GUMBOS hinders nanoparticle formation. Subsequently, while R123 and SNAFR-5 displayed selective chemotherapeutic toxicity similar to that of previously investigated R6G nanoGUMBOS, the R110 and RB GUMBOS were lacking in this property. Additionally, the chemotherapeutic toxicities of R123 and SNAFR-5 nanoGUMBOS were also significantly greater than R110 and RB GUMBOS. Observed results were consistent with decreased cellular uptake of R110 and RB as compared to R123 and SNAFR-5 compounds. Moreover, these results are also consistent with previous observations that suggest that nanoparticle formation is critical to the observed selective chemotherapeutic properties as well as the chemotherapeutic efficacy of rhodamine nanoGUMBOS.

Reversal effect of FW-04-806, a macrolide dilactone compound, on multidrug resistance mediated by ABCB1 and ABCG2 in vitro and in vivo.

BACKGROUND: Overexpression of ATP-binding cassette (ABC) transporters, such as ABCB1 and ABCG2, has been proved to be a major trigger for multidrug resistance (MDR) in certain types of cancer. A promising approach to reverse MDR is the combined use of nontoxic and potent ABC transporters inhibitor with conventional anticancer drugs. We previously reported that FW-04-806 (conglobatin) as a novel Hsp90 inhibitor with low toxicity, capable of attenuating Hsp90/Cdc37 /clients interactions and producing antitumor action in vitro and in vivo. Our early activity screening found that FW-04-806 at non-cytotoxic concentration was able to enhance the cytotoxicity of chemotherapeutic agents on the ABCB1 overexpressing cells. Therefore, we speculated that FW-04-806 might be a promising MDR reversal agent. In the present study we further investigated its reversal effect of MDR induced by ABC transporters in vitro and in vivo. METHODS: MTT assay in vitro and xenograftes in vivo were used to investigate reversal effect of FW-04-806 on MDR in ABCB1 or ABCG2 overexpressing cancer cells. To understand the mechanisms for the MDR reversal, we examined the effects of FW-04-806 on intracellular accumulation of doxorubicin (DOX, adriamycin, adr)/Rhodamine 123 (Rho 123), efflux of doxorubicin, expression levels of gene and protein of ABCB1 or ABCG2 and ATPase activity of ABCB1, and carried out molecular docking between FW-04-806 and human ABCB1. RESULTS: The results indicated that FW-04-806 significantly enhanced the cytotoxicity of substrate chemotherapeutic agents on the ABCB1 or ABCG2 overexpressing cells in vitro and in vivo suggesting its reversal MDR effects. FW-04-806 increased the intracellular accumulation of DOX or Rho123 by inhibiting the efflux function of ABC transporters in MDR cells rather than in their parental sensitive cells. However, unlike other ABC transporter inhibitors, FW-04-806 had no effect on the ATPase activity nor on the expression of ABCB1 or ABCG2 on either mRNA or protein level. Molecular docking suggested that FW-04-806 may have lower affinity to the ATPase site, which was consistent with its no significant effect on the ATPase activity of ABCB1; However FW-04-806 may bind to substrate binding site in TMDs more stably than substrate anticancer drugs therefore obstruct the anticancer drugs pumped out of the cell. CONCLUSIONS: FW-04-806 is a compound that has both anti-tumor and reversal MDR effects, and its antitumor clinical application is worth further study.

Dielectrophoretic trapping of single leukemic cells using the conventional and compact optical measurement systems.

Here, we report a microfluidic same-single-cell analysis to study the inhibition of multidrug resistance due to drug efflux on single leukemic cells. Drug efflux inhibition was investigated in the microfluidic chip using two different fluorescence detection systems, namely, a compact single-cell bioanalyzer and the conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, a compact signal generator was used to conduct dielectrophoretic cell trapping together with the compact SCB. By using the DEP force, a single acute myeloid leukemia cell was trapped in the cell retention structure of the chip. This allowed us to detect dye accumulation in the MDR leukemic cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 were used as the P-glycoprotein inhibitor and fluorescent dye, respectively. The result showed that the Rh123 fluorescence signal in a single-cell increased dramatically over its same-cell control on both fluorescence detection systems due to the inhibition by CsA.

Tenulin and isotenulin inhibit P-glycoprotein function and overcome multidrug resistance in cancer cells.

BACKGROUND: Multidrug resistance (MDR) in cancer is one of the main obstacles in treatment with chemotherapy. Drug efflux through P-glycoprotein is the major mechanism involved in MDR. A potential strategy to provide the best possible clinical outcomes is to develop P-glycoprotein (P-gp) inhibitors from natural products. PURPOSE: The present study investigated the effects of the natural sesquiterpene lactone tenulin and its derivative isotenulin on human P-gp; the mechanisms of kinetic interactions were also explored. METHODS: The human P-gp (ABCB1/Flp-In™-293) stable expression cells were established by using the Flp-In™ system. The effects of tenulin and isotenulin on cell viability were evaluated by SRB assays in established cell lines, sensitive cancer cell line (HeLaS3), and resistant cancer cell line (KB-vin). The transporter inhibition ability was evaluated by calcein-AM uptake assays. The P-gp inhibition kinetics of tenulin and isotenulin were evaluated by rhodamine123 and doxorubicin efflux assays. The ATPase activity was evaluated with the Pgp-Glo™ Assay System. RESULTS: Tenulin and isotenulin significantly inhibited the P-gp efflux function by stimulating P-gp ATPase activity. Tenulin and isotenulin interacted with the effluxes of rhodamine 123 and doxorubicin through a competitive and noncompetitive mechanism, respectively. The combinations of tenulin and isotenulin with chemotherapeutic drugs significantly resensitized MDR cancer cells. CONCLUSION: These results suggested that tenulin and isotenulin are potential candidates to be developed for synergistic treatment of MDR cancers.

Reversal Effect of Oxypeucedanin on P-glycoprotein-mediated Drug Transport.

P-glycoprotein affects the transport of numerous drugs including chemotherapeutic drugs vincristine sulfate (VCR) and docetaxel (DTX), and is one of the main causes for multidrug resistance. Our previous studies have shown that oxypeucedanin (OPD) can enhance the intestinal transit of puerarin and VCR. However, the underlying mechanism is unclear. This study investigated the potential mechanism by which OPD improves P-gp-mediated drug transport. Molecular docking was performed to predict the binding force between OPD and P-gp and the contribution of OPD on P-gp activity. We observed the effect of OPD on the transport of VCR in MDCK-MDR1 cell monolayer and also measured the plasma pharmacokinetic parameters of DTX in the presence and absence of OPD by LC-MS/MS. Moreover, we further investigated the reversal mechanism of OPD on P-gp-mediated drug transport by determining the intracellular accumulation of Rhodamine-123 (Rh123) and P-gp ATPase activity as well as protein expression and mRNA level of P-gp. Our molecular docking results revealed that the binding force between OPD and P-gp was much lower than that between P-gp and verapamil (a P-gp substrate). The transport study in vitro indicated that OPD increased the flux of VCR across MDCK-MDR1 cell monolayer. The in vivo pharmacokinetic parameters data showed OPD increased the absorption of DTX. OPD activated P-gp ATPase activity and enhanced intracellular accumulation of Rh123 in MDCK-MDR1 cells. Western blotting and qRT-PCR outcomes indicated that OPD suppressed P-gp protein expression as well as downregulated P-gp mRNA level. Thus, OPD reverse P-gp-mediated drug transport via inhibition of P-gp activity and P-gp protein expression as well as downregulation of P-gp mRNA level. Our results suggest that OPD could reverse P-gp-mediated drug resistance in tumor cells.

A multi-chamber microfluidic intestinal barrier model using Caco-2 cells for drug transport studies.

This paper presents the design and fabrication of a multi-layer and multi-chamber microchip system using thiol-ene 'click chemistry' aimed for drug transport studies across tissue barrier models. The fabrication process enables rapid prototyping of multi-layer microfluidic chips using different thiol-ene polymer mixtures, where porous Teflon membranes for cell monolayer growth were incorporated by masked sandwiching thiol-ene-based fluid layers. Electrodes for trans-epithelial electrical resistance (TEER) measurements were incorporated using low-melting soldering wires in combination with platinum wires, enabling parallel real-time monitoring of barrier integrity for the eight chambers. Additionally, the translucent porous Teflon membrane enabled optical monitoring of cell monolayers. The device was developed and tested with the Caco-2 intestinal model, and compared to the conventional Transwell system. Cell monolayer differentiation was assessed via in situ immunocytochemistry of tight junction and mucus proteins, P-glycoprotein 1 (P-gp) mediated efflux of Rhodamine 123, and brush border aminopeptidase activity. Monolayer tightness and relevance for drug delivery research was evaluated through permeability studies of mannitol, dextran and insulin, alone or in combination with the absorption enhancer tetradecylmaltoside (TDM). The thiol-ene-based microchip material and electrodes were highly compatible with cell growth. In fact, Caco-2 cells cultured in the device displayed differentiation, mucus production, directional transport and aminopeptidase activity within 9-10 days of cell culture, indicating robust barrier formation at a faster rate than in conventional Transwell models. The cell monolayer displayed high TEER and tightness towards hydrophilic compounds, whereas co-administration of an absorption enhancer elicited TEER-decrease and increased permeability similar to the Transwell cultures. The presented cell barrier microdevice constitutes a relevant tissue barrier model, enabling transport studies of drugs and chemicals under real-time optical and functional monitoring in eight parallel chambers, thereby increasing the throughput compared to previously reported microdevices.

New insights in the in vitro characterisation and molecular modelling of the P-glycoprotein inhibitory promiscuity.

The presence of several binding sites for both substrates and inhibitors is yet a poorly explored thematic concerning the assessment of the drug-drug interactions risk due to interactions of multiple drugs with the human transport protein P-glycoprotein (P-gp or MDR1, gene ABCB1). In this study we measured the inhibitory behaviour of a set of known drugs towards P-gp by using three different probe substrates (digoxin, Hoechst 33,342 and rhodamine 123). A structure-based model was built to unravel the different substrates binding sites and to rationalize the cases where drugs were not inhibiting all the substrates. A separate set of experiments was used to validate the model and confirmed its suitability to either detect the substrate-dependent P-gp inhibition and to anticipate proper substrates for in vitro experiments case by case. The modelling strategy described can be applied for either design safer drugs (P-gp as antitarget) or to target specific sub-site inhibitors towards other drugs (P-gp as target).

Design, synthesis and biological evaluation of novel tetrahydroisoquinoline derivatives as P-glycoprotein-mediated multidrug resistance inhibitors.

Multidrug resistance (MDR) is one of the main obstacles of clinical chemotherapy. A great deal of research shows that the occurrence of drug resistance in various malignant tumors is closely related to the expression of P-glycoprotein (P-gp) on the surface of the cell membrane. In this paper, based on the structure-activity relationship of phenylethyl tetrahydroisoquinoline, we choose tariquidar as the lead compound for the design and synthesis of 17 novel tetrahydroisoquinoline P-gp inhibitors. Additionally, in vitro and in vivo cytotoxicity assays and reversed MDR activity assays were evaluated. Among them, compound 3 had a good reversal of MDR activity and the reversal mechanism study of it was carried out. All of these results demonstrated that compound 3 was considered to be a promising P-gp-mediated MDR reversal candidate.

N-Deoxycholic acid-N,O-hydroxyethyl Chitosan with a Sulfhydryl Modification To Enhance the Oral Absorptive Efficiency of Paclitaxel.

Currently, the most prominent barrier to the success of orally delivered paclitaxel (PTX) is the extremely limited bioavailability of delivered therapeutic. In light of this issue, an amphiphilic sulfhydrylated N-deoxycholic acid-N,O-hydroxyethyl chitosan (TGA-DHC) was synthesized to improve the oral bioavailability of PTX. First, TGA-DHC demonstrated substantial loading of PTX into the inner hydrophobic core. A desirable enhancement in the bioavailability of PTX by TGA-DHC was verified by pharmacokinetic studies on rats against Taxol and non-sulfhydrylated DHC micelles. Moreover, cellular uptake studies revealed significant accumulation of TGA-DHC micelles encapsulating PTX or rhodamine-123 into Caco-2 cells via clathrin/caveolae-mediated endocytosis and inhibition of P-gp efflux of substrates. The results of the Caco-2 transport study further confirmed the mechanistic basis of TGA-DHC efficacy; which was attributed to permeabilized tight junctions, clathrin-mediated transcytosis across the endothelium, and inhibition of P-gp. Finally, in vitro mucoadhesion investigations on freshly excised rat intestine intuitively confirmed increased intestinal retention of drug-loaded TGA-DHC through thiol-mediated mucoadhesion. TGA-DHC has demonstrated the capability to overcome what is perhaps the most prominent barrier to oral PTX efficacy, low bioavailability, and serves as a prominent platform for oral delivery of P-gp substrates.

Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells.

Melatonin has been known as a neuroprotective agent for the central nervous system (CNS) and the blood-brain barrier (BBB), which is the primary structure that comes into contact with several neurotoxins including methamphetamine (METH). Previous studies have reported that the activation of melatonin receptors (MT1/2) by melatonin could protect against METH-induced toxicity in brain endothelial cells via several mechanisms. However, its effects on the P-glycoprotein (P-gp) transporter, the active efflux pump involved in cell homeostasis, are still unclear. Thus, this study investigated the role of melatonin and its receptors on the METH-impaired P-gp transporter in primary rat brain microvascular endothelial cells (BMVECs). The results showed that METH impaired the function of the P-gp transporter, significantly decreasing the efflux of Rho123 and P-gp expression, which caused a significant increase in the intracellular accumulation of Rho123, and these responses were reversed by the interaction of melatonin with its receptors. Blockade of the P-gp transporter by verapamil caused oxidative stress, apoptosis, and cell integrity impairment after METH treatment, and these effects could be reversed by melatonin. Our results, together with previous findings, suggest that the interaction of melatonin with its receptors protects against the effects of the METH-impaired P-gp transporter and that the protective role in METH-induced toxicity was at least partially mediated by the regulation of the P-gp transporter. Thus, melatonin and its receptors (MT1/2) are essential for protecting against BBB impairment caused by METH.

Original Vinca Derivatives: From P-Glycoprotein Substrates to P-Glycoprotein Inhibitors.

The first example of vinca derivatives 16-18 able to modulate P-glycoprotein (Pgp) efflux activity is reported. They were elaborated in two steps from vinorelbine 3 (VLN) by a modification of the velbenamine moiety. These compounds were able to decrease efficiently Pgp mediated influx and efflux of rhodamine-123 (Rho) and to restore the cytotoxicity of vinorelbine 3 (VLN) and doxorubicin (Dox) on K562R (dox-resistant) cell lines.

The Interactions of P-Glycoprotein with Antimalarial Drugs, Including Substrate Affinity, Inhibition and Regulation.

The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10(-6) cm/sec, followed by amodiaquine around 20 x 10(-6) cm/sec; both mefloquine and artesunate were around 10 x 10(-6) cm/sec. Methylene blue was between 2 and 6 x 10(-6) cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine.

Osthole shows the potential to overcome P-glycoprotein­mediated multidrug resistance in human myelogenous leukemia K562/ADM cells by inhibiting the PI3K/Akt signaling pathway.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) has been reported to play a pivotal role in tumor chemotherapy failure. Study after study has illustrated that the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade is involved in the MDR phenotype and is correlated with P-gp expression in many human malignancies. In the present study, osthole, an O-methylated coumarin, exhibited potent reversal capability of MDR in myelogenous leukemia K562/ADM cells. Simultaneously, the uptake and efflux of Rhodamine-123 (Rh-123) and the accumulation of doxorubicin assays combined with flow cytometric analysis suggested that osthole could increase intracellular drug accumulation. Furthermore, osthole decreased the expression of multidrug resistance gene 1 (MDR1) at both the mRNA and protein levels. Further experiments elucidated that osthole could suppress P-gp expression by inhibiting the PI3K/Akt signaling pathway which might be the main mechanism accounting for the reversal potential of osthole in the MDR in K562/ADM cells. In conclusion, osthole combats MDR and could be a promising candidate for the development of novel MDR reversal modulators.

Long-Term Alteration of Reactive Oxygen Species Led to Multidrug Resistance in MCF-7 Cells.

Reactive oxygen species (ROS) play an important role in multidrug resistance (MDR). This study aimed to investigate the effects of long-term ROS alteration on MDR in MCF-7 cells and to explore its underlying mechanism. Our study showed both long-term treatments of H2O2 and glutathione (GSH) led to MDR with suppressed iROS levels in MCF-7 cells. Moreover, the MDR cells induced by 0.1 µM H2O2 treatment for 20 weeks (MCF-7/ROS cells) had a higher viability and proliferative ability than the control MCF-7 cells. MCF-7/ROS cells also showed higher activity or content of intracellular antioxidants like glutathione peroxidase (GPx), GSH, superoxide dismutase (SOD), and catalase (CAT). Importantly, MCF-7/ROS cells were characterized by overexpression of MDR-related protein 1 (MRP1) and P-glycoprotein (P-gp), as well as their regulators NF-E2-related factor 2 (Nrf2), hypoxia-inducible factor 1 (HIF-1α), and the activation of PI3K/Akt pathway in upstream. Moreover, several typical MDR mediators, including glutathione S-transferase-π (GST-π) and c-Myc and Protein Kinase Cα (PKCα), were also found to be upregulated in MCF-7/ROS cells. Collectively, our results suggest that ROS may be critical in the generation of MDR, which may provide new insights into understanding of mechanisms of MDR.

P-glycoprotein inhibitors: synthesis and in vitro evaluation of a preactivated thiomer.

The aim of this study was to synthesize the preactivated thiomer poly(acrylic acid)-cyteine-2-mercaptonicotinic acid (PAA-Cys-2MNA) and to evaluate its P-glycoprotein (P-gp) inhibitory properties. The thiomer (PAA-Cys) was synthesized by covalent immobilization of thiol groups on poly(acrylic acid) (PAA) with a molecular mass of 250 kDa followed by immobilization of 2-mercaptonicotinic acid (2MNA) to thiol groups via disulfide bond formation resulting in PAA-Cys-2MNA. P-gp inhibitory effect of this preactivated thiomer was evaluated on Caco-2 cells. Transports of rhodamine 123 at 37 °C with and without verapamil and at 4 °C were performed to evaluate P-gp function of cells. In total, 1571.81 ± 156.18 µmol thiol groups were immobilized per gram of polymer that were in the next step by 99.88% preactivated. The enhancement ratios of Papp calculated from the ratio between Papp of rhodamine 123 in the presence of P-gp inhibitors and Papp of rhodamine 123 alone were 2.36, 2.09, and 1.84-fold in the presence of PAA-Cys-2MNA, PAA-Cys, and PAA, respectively. Because of its pronounced P-gp inhibitory effect, PAA-Cys-2MNA could be considered as promising macromolecular P-gp inhibitor for various drug delivery systems.