Dill Shows Potential for Herb-Drug Interactions via Up-Regulation of CYP1A2, CYP2C19, SULT1A1, NAT2 and ABCB1 in Caco-2 Cells.

<b>Background and Objective:</b> Dill<i> </i>(<i>Anethum graveolens</i> L.) has the potential to develop as a new alternative medicine due to its pharmacological activities. However, studies into its safety regarding herb-drug interactions have been neglected. This study investigated the risk of dill-induced herb-drug interactions (HDI) by examining its effect on the expression of phase I and II drug-metabolizing enzyme and transporter genes in Caco-2 cells. <b>Materials and Methods:</b> Caco-2 cells (5×10⁵ cells/well) were treated with 10 µM ketoconazole, 20 µM rifampicin or dill extract (60-240 µg mL¹) for 72 hrs. Cell viability was assessed using the resazurin assay and reactive oxygen species (ROS) content was determined with 2 ,7 -dichlorofluorescein diacetate. Aspartate (AST) and alanine aminotransferase (ALT) levels were measured using L-aspartate and L-alanine with α-ketoglutarate as substrate. Expression of phase I (<i>CYP1A2</i>, <i>CYP2C19</i>, <i>CYP2D6</i>, <i>CYP2E1 </i>and <i>CYP3A4</i>) and II (<i>UGT1A6</i>,<i> SULT1A1</i>,<i> NAT1</i>,<i> NAT2 </i>and<i> GSTA1/2</i>) metabolizing genes and transporters (<i>ABCB1</i>,<i> ABCC2</i>,<i> ABCG2 </i>and <i>SLCO1B1</i>) were determined by RT/qPCR. <b>Results:</b> All tested concentrations of dill did not affect cell viability or AST and ALT levels. The highest concentration of dill extract (240 µg mL¹) significantly lowered the ROS level. Expression of <i>CYP1A2</i>, <i>CYP2C19</i>, <i>SULT1A1</i>, <i>NAT2 </i>and <i>ABCB1 </i>mRNA was significantly up-regulated by dill extract. <b>Conclusion:</b> Dill extract did not directly damage Caco-2 cells but prolonged use of dill may increase the risk of HDI via the up-regulation of the drug-metabolizing genes <i>CYP1A2</i>, <i>CYP2C19</i>, <i>SULT1A1</i>, <i>NAT2 </i>and the transporter <i>ABCB1</i>.

Effect of Vitamin K-Mediated PXR Activation on Drug-Metabolizing Gene Expression in Human Intestinal Carcinoma LS180 Cell Line.

The pregnane X receptor (PXR) is the key regulator of our defense mechanism against foreign substances such as drugs, dietary nutrients, or environmental pollutants. Because of increased health consciousness, the use of dietary supplements has gradually increased, and most of them can activate PXR. Therefore, an analysis of the interaction between drugs and nutrients is important because altered levels of drug-metabolizing enzymes or transporters can remarkably affect the efficiency of a co-administered drug. In the present study, we analyzed the effect of vitamin K-mediated PXR activation on drug metabolism-related gene expression in intestine-derived LS180 cells via gene expression studies and western blotting analyses. We demonstrated that menaquinone 4 (MK-4), along with other vitamin Ks, including vitamin K1, has the potential to induce MDR1 and CYP3A4 gene expression. We showed that PXR knockdown reversed MK-4-mediated stimulation of these genes, indicating the involvement of PXR in this effect. In addition, we showed that the expression of MDR1 and CYP3A4 genes increased synergistically after 24 h of rifampicin and MK-4 co-treatment. Our study thus elucidates the importance of drug-nutrient interaction mediated via PXR.

(-)-Epigallocatechin Gallate (EGCG) Enhances the Sensitivity of Colorectal Cancer Cells to 5-FU by Inhibiting GRP78/NF-κB/miR-155-5p/MDR1 Pathway.

Green tea accounts for approximately 20% of the world's total tea yield. (-)-Epigallocatechin gallate (EGCG) is an active catechin in green tea, which suppresses tumor growth and enhances drug sensitivity in various cancers, but the molecular mechanism is still unclear. Chemotherapy drugs, such as 5-fluorouracil (5-FU), are a common strategy for clinical treatment of cancer patients; however, the lower response rate caused by prolonged use becomes the main reason for tumor recurrence. Therefore, discovering a safe and effective chemo-sensitizer is an urgent task required to be solved. Here, we report that EGCG reinforces the sensitivity of colon cancer cells to 5-FU, and the IC50 values of 5-FU is decreased from 40 ± 4.2 µM to 5 ± 0.36 µM in one human colon carcinoma cell line-HCT-116, and from 150 ± 6.4 µM to 11 ± 0.96 µM in the other human colon carcinoma cell line-DLD1 when these cells are cotreated with 50 µM EGCG. Consistently, compared to 5-FU or EGCG treatment alone, the combination of both significantly promotes cancer cell apoptosis and DNA damage. Further mechanism research reveals that treatment of colorectal cancer (CRC) with 50 µM EGCG inhibits GRP78 expression, activates the NF-κB (2.55 ± 0.05-fold for HCT-116 and 2.27 ± 0.08-fold for DLD1) pathway, and enhances miR-155-5p (2.12 ± 0.02-fold for HCT-116 and 2.01 ± 0.01-fold for DLD1) level. The elevated miR-155-5p strongly suppresses target gene MDR1 expression, which blocks the efflux of 5-FU. The accumulation of 5-FU resulted in caspase-3 and PARP activation, Bcl-2 reduction, and Bad increase, which ultimately lead to cancer cell apoptosis. Overall, our data show that EGCG may be act as a novel chemo-sensitizer, and the GRP78/NF-κB/miR-155-5p/MDR1 pathway plays a vital role in EGCG enhancing the sensitivity of colorectal cancer to 5-FU.

Reversal of multidrug resistance by icaritin in doxorubicin-resistant human osteosarcoma cells.

Multidrug resistance (MDR) is one of the major obstacles in cancer chemotherapy. Our previous study has shown that icariin could reverse MDR in MG-63 doxorubicin-resistant (MG-63/DOX) cells. It is reported that icariin is usually metabolized to icariside II and icaritin. Herein, we investigated the effects of icariin, icariside II, and icaritin (ICT) on reversing MDR in MG-63/DOX cells. Among these compounds, ICT exhibited strongest effect and showed no obvious cytotoxicity effect on both MG-63 and MG-63/DOX cells ranging from 1 to 10 µmol·L-1. Furthermore, ICT increased accumulation of rhodamine 123 and 6-carboxyfluorescein diacetate and enhanced DOX-induced apoptosis in MG-63/DOX cells in a dose-dependent manner. Further studies demonstrated that ICT decreased the mRNA and protein levels of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). We also verified that blockade of STAT3 phosphorylation was involved in the reversal effect of multidrug resistance in MG-63/DOX cells. Taken together, these results indicated that ICT may be a potential candidate in chemotherapy for osteosarcoma.

In vitro modulation of the cytochrome P450 and ABCB1/P-glycoprotein activities of the aqueous extract of Allophylus cominia (L) Sw. leaves.

BACKGROUND: The aqueous extract of the Allophylus cominia (L) Sw (Sapindaceae) leaves has shown anti-diabetic, anti-obesity and anti-inflammatory properties. In the Caribbean region, it is typically used for the treatment of type-2 diabetes. METHODS: Considering the herb-drug interaction, the aim of this study was to evaluate the potential effects of the A. cominia extract on the cytochrome P450 (CYP) (rat hepatocyte model) and P-glycoprotein (P-gp) (4T1 cell line) systems. RESULTS: The extract did not decrease the cell viability after being assayed by the MTT test at up to 1500 µg/mL for 72 h. The exposure of the cultured rat hepatocytes to the product (up to 250 µg/mL) for 48 h increased the activities of CYP-1A2, 2C9, and 2E1 by 1.46-, 1.60-, and 1.51-fold, respectively, compared with the controls. The activities of CYP-2B6, 2D6, and 3A4 were not significantly altered, whereas the activity of P-gp decreased by 2- and 4-fold. In addition, the extracts at 100 and 200 µg/mL significantly increased doxorubicin cytotoxicity in these cells 24 h after treatment. CONCLUSIONS: The findings indicate that the A. cominia extract modulates the CYP and P-gp systems increasing sensitivity to doxorubicin. Further studies are necessary to evaluate the potential herb-drug interaction or chemosensitive properties.

Targeting T Cell Bioenergetics by Modulating P-Glycoprotein Selectively Depletes Alloreactive T Cells To Prevent Graft-versus-Host Disease.

T lymphocytes play a central role in many human immunologic disorders, including autoimmune and alloimmune diseases. In hematopoietic stem cell transplantation, acute graft-versus-host-disease (GVHD) is caused by an attack on the recipient's tissues from donor allogeneic T cells. Selectively depleting GVHD-causing cells prior to transplant may prevent GVHD. In this study, we evaluated 24 chalcogenorhodamine photosensitizers for their ability to selectively deplete reactive T lymphocytes and identified the photosensitizer 2-Se-Cl, which accumulates in stimulated T cells in proportion to oxidative phosphorylation. The photosensitizer is also a potent stimulator of P-glycoprotein (P-gp). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria and protects resting lymphocytes that are essential for antipathogen and antitumor responses. To evaluate the selective depletion of alloimmune responses, donor C57BL/6 splenocytes were cocultured for 5 d with irradiated BALB/c splenocytes and then photodepleted (PD). PD-treated splenocytes were infused into lethally irradiated BALB/c (same-party) or C3H/HeJ (third-party) mice. Same-party mice that received PD-treated splenocytes at the time of transplant lived 100 d without evidence of GVHD. In contrast, all mice that received untreated primed splenocytes and third-party mice that received PD-treated splenocytes died of lethal GVHD. To evaluate the preservation of antiviral immune responses, acute lymphocytic choriomeningitis virus infection was used. After photodepletion, expansion of Ag-specific naive CD8(+) T cells and viral clearance remained fully intact. The high selectivity of this novel photosensitizer may have broad applications and provide alternative treatment options for patients with T lymphocyte-mediated diseases.

Jatrophane diterpenes and cancer multidrug resistance - ABCB1 efflux modulation and selective cell death induction.

BACKGROUND: Modulation of P-glycoprotein (ABCB1) and evaluation of the collateral sensitivity effect are among the most promising approaches to overcome multidrug resistance (MDR) in cancer. In a previous study, two rare 12,17-cyclojatrophanes (1-2) and other novel jatrophanes (3-4), isolated from Euphorbia welwitschii, were screened for collateral sensitivity effect. Herein, the isolation of another jatrophane (5) is presented, being the broader goal of this work to investigate the role of euphowelwitschines A (1) and B (2), welwitschene (3), epoxywelwitschene (4) and esulatin M (5) as ABCB1 modulators and/or collateral sensitivity agents. METHODS: Compounds 1-5 were evaluated for ABCB1 modulation ability through combination of transport and chemosensitivity assays, using a mouse T-lymphoma MDR1-transfected cell model. Moreover, the nature of interaction of compound 4 with ABCB1 was studied, using an ATPase assay. The MDR-selective antiproliferative activity of compound 5 was evaluated against gastric (EPG85-257) and pancreatic (EPP85-181) human cancer cells and their drug-selected counterparts (EPG85-257RDB, EPG85-257RNOV, EPP85-181RDB, EPP85-181RNOV). The drug induced cell death was investigated for compounds 4 and 5, using the annexin V/PI staining and the active caspase-3 assay. RESULTS: The jatrophanes 1-5 were able to modulate the efflux activity of ABCB1, and at 2µM, 3-5 maintained the strong modulator profile. Structure activity results indicated that high conformational flexibility of the twelve-membered ring of compounds 3-5 favored ABCB1 modulation, in contrast to the tetracyclic scaffold of compounds 1 and 2. The effects of epoxywelwitschene (4) on the ATPase activity of ABCB1 showed it to interact with the transporter and to be able to reduce the transport of a second subtrate. Drug combination experiments also corroborated the anti-MDR potential of these diterpenes due to their synergistic interaction with doxorubicin (combination index <0.7). Esulatin M (5) showed a strong MDR-selective antiproliferative activity against EPG85-257RDB and EPP85-181RDB cells, with IC50 of 1.8 and 4.8 µM, respectively. Compounds 4 and 5 induced apoptosis via caspase-3 activation. A significant discrimination was observed between the resistant cell lines and parental cells. CONCLUSIONS: This study strengthens the role of jatrophane diterpenes as lead candidates for the development of MDR reversal agents, higlighting the action of compounds 4 and 5.

Permeability of rhynchophylline across human intestinal cell in vitro.

Rhynchophylline (Rhy) is the major component of Uncaria species, which is used in Chinese traditional medicine for the treatment of central nervous system disorders. However, its oral bioavailability has not been known. This study aims to investigate the intestinal permeability and related mechanisms of Rhy using cultured human epithelial Caco-2 cells. The cytotoxicity of Rhy on Caco-2 cells was evaluated with MTT assay. The effect of Rhy on the integrity of Caco-2 cell monolayer was assayed with transepithelial electrical resistance. The permeability of Rhy across cell monolayer was assayed by measuring Rhy quantity in received side with HPLC. The effect of Rhy on the expression of P-glycoprotein and MDR1 was detected with Western blot and flow cytometry, respectively. In the concentration of Rhy, which did not produce toxicity on cell viability and integrity of Caco-2 cell monolayer, Rhy crossed the monolayer with velocity 2.76~5.57×10^-6 cm/sec and 10.68~15.66×10^-6 cm/sec from apical to basolateral side and from basolateral to apical side, respectively. The permeability of Rhy was increased by verapamil, a P-glycoprotein inhibitor, or rhodamine123, a P-glycoprotein substrate. Rhy revealed an induction effect on P-glycoprotein expression in Caco-2 cells. These results demonstrate the low permeability of Rhy in intro, and suggest that P-glycoprotein may underlie the mechanism.

Evaluation of in vitro absorption, distribution, metabolism, and excretion (ADME) properties of mitragynine, 7-hydroxymitragynine, and mitraphylline.

Mitragyna speciosa (kratom) is a popular herb in Southeast Asia, which is traditionally used to treat withdrawal symptoms associated with opiate addiction. Mitragynine, 7-hydroxymitragynine, and mitraphylline are reported to be the central nervous system active alkaloids which bind to the opiate receptors. Mitraphylline is also present in the bark of Uncaria tomentosa (cat's claw). Several therapeutic properties have been reported for these compounds but limited information is available on the absorption and distribution properties. This study focuses on evaluating the absorption, distribution, metabolism, and excretion (ADME) properties of these compounds and their effect on major efflux transporter P-glycoprotein, using in vitro methods. Quantitative analysis was performed by the Q-TOF LC-MS system. Mitragynine was unstable in simulated gastric fluid with 26 % degradation but stable in simulated intestinal fluid. 7-Hydroxymitragynine degraded up to 27 % in simulated gastric fluid, which could account for its conversion to mitragynine (23 %), while only 6 % degradation was seen in simulated intestinal fluid. Mitraphylline was stable in simulated gastric fluid but unstable in simulated intestinal fluid (13.6 % degradation). Mitragynine and 7-hydroxymitragynine showed moderate permeability across Caco-2 and MDR-MDCK monolayers with no significant efflux. However, mitraphylline was subjected to efflux mediated by P-glycoprotein in both Caco-2 and MDR-MDCK monolayers. Mitragynine was found to be metabolically stable in both human liver microsomes and S9 fractions. In contrast, both 7-hydroxymitragynine and mitraphylline were metabolized by human liver microsomes with half-lives of 24 and 50 min, respectively. All three compounds exhibited high plasma protein binding (> 90 %) determined by equilibrium dialysis. Mitragynine and 7-hydroxymitragynine inhibited P-glycoprotein with EC50 values of 18.2 ± 3.6 µM and 32.4 ± 1.9 µM, respectively, determined by the calcein-AM fluorescent assay, while no inhibition was seen with mitraphylline. These data indicate the possibility of a drug interaction if mitragynine and 7-hydroxymitragynine are coadministered with drugs that are P-glycoprotein substrates.

Farnesiferol A from Ferula persica and galbanic acid from Ferula szowitsiana inhibit P-glycoprotein-mediated rhodamine efflux in breast cancer cell lines.

In multidrug resistance (MDR), cancer cells exposed to anticancer agents develop resistance to a wide variety of chemicals and chemotherapeutic agents. Sesquiterpene coumarins are reported to inhibit P-glycoprotein and/or increase cytotoxicity of anticancer drugs in P-gp-overexpressing cell lines. In the current study, we investigated the effects of galbanic acid (from the roots of Ferula szowitsiana) and farnesiferol A (from the roots of Ferula persica) on functionality of the drug transporter P-glycoprotein (P-gp) using a rhodamine 123 efflux assay in a doxorubicin resistant breast cancer cell line (MCF7/Adr). Compared to verapamil, the well-known inhibitor of P-gp, galbanic acid (5, 10, and 25 µg/mL), significantly inhibited the P-gp activity. In inhibition of the P-gp transporter, farnesiferol A (0.5 µg/mL) was more potent than verapamil at 15 min exposure. Our results indicate that the plant derived sesquiterpene coumarins, farnesiferol A and galbanic acid, may be promising candidates to be considered for further studies on the reversal of multidrug resistance phenotype in chemotherapy of cancer patients.

Opioids and efflux transporters. Part 3: P-glycoprotein substrate activity of 3-hydroxyl addition to meperidine analogs.

Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-glycoprotein (P-gp), suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of opioids. Addition of a 3-OH to meperidine and the ketone analog of meperidine yielding bemidone and ketobemidone, respectively, significantly increased P-gp substrate affinity. The results of this study have implications in the development of novel analgesics to be utilized as tools to study the contribution of P-gp on the development of central tolerance to opioids.

Herbal medicine and epilepsy: proconvulsive effects and interactions with antiepileptic drugs.

The use of complementary and alternative medicine is on the rise, including among patients with epilepsy. Herbal medicine, one of the most popular forms of CAM, is considered to be both safe and effective by most consumers. Yet many herbs may increase the risk for seizures, through intrinsic proconvulsant properties or contamination by heavy metals, as well as via effects on the cytochrome P450 enzymes and P-glycoproteins, altering antiepileptic drug (AED) disposition. Herb-drug interactions may be difficult to predict, especially since the quality and quantity of active ingredients are often unknown. Since most patients do not inform their physicians that they are taking herbal medicines, health care professionals must initiate a dialogue in order to prevent complications with the combined regimen. At the same time, further research is required regarding the effect of herbs on seizure activity and interactions with AED treatment.

Induction of hepatic antioxidant enzymes by phenolic acids in rats is accompanied by increased levels of multidrug resistance-associated protein 3 mRNA expression.

Phenolic acids are widespread in plant foods; they contain important biological and pharmacological properties, some of which were shown to be effective in preventing cancer. We investigated the modulatory effects of phenolic acids on an antioxidant system in male Sprague-Dawley rats. Rats were orally administrated gentisic acid (GEA), gallic acid (GA), ferulic acid (FA), and p-coumaric acid (p-CA) at a dosage of 100 mg/kg body weight for 14 consecutive days. At this dose, the activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase were greater after administration of all 4 phenolic acids compared with the control group (P < 0.05). The activities of these enzymes in the small intestine of rats were also significantly greater after GA and p-CA treatment compared with controls. The changes in hepatic CuZnSOD, GPx, and catalase mRNA levels induced by phenolic acids were similar to those noted in the enzyme activities. Oxidized glutathione levels were lower (P < 0.05) in the liver of all phenolic acid-supplemented rats, whereas reduced glutathione was markedly higher than in control rats, especially after administration of GA and p-CA. The liver homogenates obtained from rats that had been administered phenolic acids had higher oxygen radical absorbance capacity than those obtained from control rats. Immunoblot analysis revealed an increased total level of Nrf2, a transcription factor governing the antioxidant response element in phenolic acid-supplemented rats. Phenolic acid-mediated antioxidant enzyme expression was accompanied by upregulation of multidrug resistance-associated protein Mrp3. These experiments show that modulation of phase II antioxidant enzymes and oxidative status in the liver by phenolic acids may play an important role in the protection against adverse effects related to mutagenesis and oxidative damage.

Reversal of P-glycoprotein and multidrug-resistance protein-mediated drug resistance in KB cells by 5-O-benzoylated taxinine K.

A newly synthesized taxoid originally from the Japanese yew Taxus cuspidata, 5-O-benzoylated taxinine K (BTK) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein (MRP)-mediated multidrug resistance. BTK reversed the resistance to paclitaxel, doxorubicin (ADM), and vincristine (VCR) of KB-8-5 and KB-C2 cells that overexpress P-gp by directly interacting with P-gp. BTK also moderately reversed the resistance to ADM of KB/MRP cells that overexpress MRP. However, BTK neither inhibited the transporting activity of MRP nor reduced intracellular glutathione levels in KB/MRP cells. BTK shifted the distribution of ADM in KB/MRP cells from punctate cytoplasmic compartments to the nucleoplasm and cytoplasm by inhibiting acidification of cytoplasmic organelles. These two functions of BTK make it able to reverse both P-gp- and MRP-mediated MDR. BTK in combination with ADM should be useful for treating patients with tumors that overexpress both P-gp and MRP.