AHR/CYP1A1 interplay triggers lymphatic barrier breaching in breast cancer spheroids by inducing 12(S)-HETE synthesis.

A causal link between overexpression of aryl hydrocarbon receptor (AHR) and its target cytochrome P450 1A1 (CYP1A1) and metastatic outgrowth of various cancer entities has been established. Nevertheless, the mechanism how AHR/CYP1A1 support metastasis formation is still little understood. In vitro we discovered a potential mechanism facilitating tumour dissemination based on the production of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Utilising a three-dimensional lymph endothelial cell (LEC) monolayer & MDA-MB231 breast cancer cell spheroid co-culture model in combination with knock-down approach allowed elucidation of the molecular/biochemical basis of AHR/CYP1A1-induced tumour breaching through the LEC barrier. Enzyme immunoassay evidenced the potential of recombinant CYP1A1 to synthesise 12(S)-HETE in vitro and qPCR and Western blotting measured gene and protein expression in specific experimental settings. In detail, AHR induced CYP1A1 expression and 12(S)-HETE secretion in tumour spheroids, which caused LEC junction retraction thereby forming large discontinuities allowing transmigration of the tumour. This was enforced by the activating AHR ligand 6-formylindolo (3,3-b)carbazole (FICZ), or inhibited by the AHR antagonist 3,3'-diindolylmethane (DIM) as well as by siRNA against AHR and CYP1A1. AHR and NF-κB were negatively cross talking and therefore, the inhibition of AHR (but not CYP1A1) induced RELA, RELB, NFKB1, NFKB2 and the NF-κB target MMP1, which itself promotes tumour intravasation by a mechanism that is different from 12(S)-HETE. Conversely, the inhibition of NFKB2 induced AHR, CYP1A1 and 12(S)-HETE synthesis. The approved clinical drugs guanfacine and vinpocetine, which inhibit CYP1A1 and NF-κB, respectively, significantly inhibited LEC barrier breaching in vitro indicating an option to reduce metastatic dissemination.

Interplay of drug metabolizing enzymes with cellular transporters.

Many endogenous and xenobiotic substances and their metabolites are substrates for drug metabolizing enzymes and cellular transporters. These proteins may not only contribute to bioavailability of molecules but also to uptake into organs and, consequently, to overall elimination. The coordinated action of uptake transporters, metabolizing enzymes, and efflux pumps, therefore, is a precondition for detoxification and elimination of drugs. As the understanding of the underlying mechanisms is important to predict alterations in drug disposal, adverse drug reactions and, finally, drug-drug interactions, this review illustrates the interplay between selected uptake/efflux transporters and phase I/II metabolizing enzymes.

Organic anion­transporting polypeptides contribute to the uptake of curcumin and its main metabolites by human breast cancer cells: Impact on antitumor activity.

Curcumin is a natural polyphenolic compound with pronounced anticancer properties, despite its low bioavailability caused by extensive glucuronidation and sulfation. Information on the cellular uptake mechanisms and their contribution to the anticancer effects of curcumin and its biotransformation products is limited. The present study, therefore, investigated the role of organic anion­transporting polypeptides (OATPs) in the cellular uptake of curcumin and its major metabolites in OATP­expressing Chinese hamster ovary (CHO) and human ZR­75­1 breast cancer cells. The uptake rates for curcumin in OATP1B1­, OATP1B3­ and OATP2B1­transfected CHO cells were 2­ to 3­fold higher than wild­type cells. Curcumin sulfate was transported by all three OATPs, although to a much lesser extent, while uptake of tetrahydrocurcumin was the highest but only via OATP1B1 and OATP1B3. Notably, curcumin glucuronide did not exhibit any affinity for these OATPs. The increased mRNA levels of OATP1B1 in wild­type human breast cancer ZR­75­1 cells compared with OATP1B1 knockdown cells was associated with a higher initial uptake of curcumin and tetrahydrocurcumin leading to decreased IC50 values. In conclusion, our data revealed that OATPs act as cellular uptake transporters for curcumin and its major metabolites, and this may also be applicable to patients undergoing cancer therapy.

Involvement of UDP-Glucuronosyltransferases and Sulfotransferases in the Excretion and Tissue Distribution of Resveratrol in Mice.

Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. It is unknown whether the expression of metabolizing enzymes correlates with resveratrol levels in organs and tissues. Therefore, we investigated the metabolism and tissue distribution of resveratrol in mice and assessed its association with the expression of UDP-glucuronosyltransferase (Ugt) and sulfotransferase (Sult) genes. Plasma, urine, feces, and various organs were analyzed using high-performance liquid chromatography at up to 8 h after intragastric resveratrol administration. The metabolism of resveratrol was pronounced, leading to the formation of resveratrol glucuronides and sulfates. Concentrations of resveratrol and its metabolites were high in the gastrointestinal organs, urine, and feces, but low in the liver and kidneys. In lung, heart, thymus, and brain tissues, parent resveratrol levels exceeded the sulfate and glucuronide concentrations. The formation of resveratrol conjugates correlated with the expression of certain Ugt and Sult genes. Reverse transcription quantitative PCR (RT-qPCR) analysis revealed high mRNA expression of Ugt1a1 and Ugt1a6a in the liver, duodenum, jejunum, ileum, and colon, leading to high concentrations of resveratrol-3-O-glucuronide in these organs. Strong correlations of resveratrol-3-O-sulfate and resveratrol-3-O-4'-O-disulfate formation with Sult1a1 mRNA expression were also observed, particularly in the liver and colon. In summary, our data revealed organ-specific expression of Sults and Ugts in mice that strongly affects resveratrol concentrations; this may also be predictive in humans following oral uptake of dietary resveratrol.

12(S)-HETE increases intracellular Ca(2+) in lymph-endothelial cells disrupting their barrier function in vitro; stabilization by clinical drugs impairing calcium supply.

Secretion of 12(S)-HETE by breast cancer emboli provokes "circular chemorepellent induced defects" (CCIDs) in the adjacent lymphatic vasculature facilitating their intravasation and lymph node metastasis which determines prognosis. Therefore, elucidating the mechanism of lymph endothelial cell (LEC) wall disintegration may provide cues for anti-metastatic intervention. The role of intracellular free Ca(2+) for CCID formation was investigated in LECs using MCF-7 or MDA-MB231 breast cancer cell spheroids in a three-dimensional cell co-culture model. 12(S)-HETE elevated the Ca(2+) level in LEC by activating PLC/IP3. Downstream, the Ca(2+)-calmodulin kinase MYLK contributed to the phosphorylation of Ser19-MLC2, LEC contraction and CCID formation. Approved clinical drugs, lidoflazine, ketotifen, epiandrosterone and cyclosporine, which reportedly disturb cellular calcium supply, inhibited 12(S)-HETE-induced Ca(2+) increase, Ser19-MLC2 phosphorylation and CCID formation. This treatment strategy may reduce spreading of breast cancer through lymphatics.

The effect of organic anion-transporting polypeptides 1B1, 1B3 and 2B1 on the antitumor activity of flavopiridol in breast cancer cells.

The contribution of organic anion transporting polypeptides (OATPs) to the cellular uptake of flavopiridol was investigated in OATP1B1-, OATP1B3- and OATP2B1-expressing Chinese hamster ovary (CHO) cells. Uptake of flavopiridol into these cells showed typical Michaelis-Menten kinetics with much higher transport capacity for OATP1B3 compared to OATP1B1 and OATP2B1 (Vmax/Km, 33.9 vs. 8.84 and 2.41 µl/mg/min, respectively). The predominant role of OATPs was further supported by a dramatic inhibition of flavopiridol uptake in the presence of the OATP substrate rifampicin. Uptake of flavopiridol by OATPs also seems to be an important determinant in breast cancer cells. The much higher mRNA level for OATP1B1 found in wild-type compared to ZR-75-1 OATP1B1 knockdown cells correlated with higher flavopiridol initial uptake leading to 4.6-fold decreased IC50 values in the cytotoxicity assay (IC50, 1.45 vs. 6.64 µM). Cell cycle profile also showed a clear incidence for a stronger cell cycle arrest in the G2/M phase for ZR-75-1 wild-type cells compared to OATP1B1 knockdown cells, further indicating an active uptake via OATP1B1. In conclusion, our results revealed OATP1B1, OATP1B3 and OATP2B1 as uptake transporters for flavopiridol in cancer cells, which may also apply in patients during cancer therapy.

Effects of anthocyans on the expression of organic anion transporting polypeptides (SLCOs/OATPs) in primary human hepatocytes.

Anthocyans (anthocyanins and their aglycones anthocyanidins) are colorful pigments, naturally occurring in fruits. They exhibit many biological effects and have potent health benefits. Anthocyans are widely used as dietary supplements and the safety of products containing them is of great importance. To investigate whether anthocyans influence the expression of hepatic uptake transporters from the organic anion transporting polypeptide (SLCO gene/OATP protein) family, we carried out studies on primary cultures of human hepatocytes. The hepato-cellular accumulation of widely used drugs such as statins and some anticancer drugs is mediated by the liver-specific OATP1B1 and OATP1B3, thus any interference with expression of these particular transporters might influence therapeutic outcomes. We evaluated the effects of 21 anthocyanins and their corresponding 6 anthocyanidins on the expression levels of SLCO1B1/SLCO1B3 by RT-qPCR. Changes in OATP protein levels were confirmed by western blotting. Our data show that OATP1B1 responds differently to anthocyans compared with OATP1B3. We observed the induction of SLCO1B1 gene and OATP1B1 protein in four hepatocyte samples by the anthocyanins malvin chloride, malvidin-3-O-galactoside chloride and cyanidin-3-O-sophoroside chloride. For SLCO1B3, a reduction in the expression levels was seen with delphin chloride and the anthocyanidin pelargonidin. Although the values varied considerably between primary hepatocyte isolates from different individuals, a mean induction of SLCO1B1 (up to 60%) and reduction of SLCO1B3 (by less than 25%) were detected. We propose that the effects of anthocyans derived from high dose dietary supplements may have to be taken into account in patients undergoing a therapy with drugs transported by OATP1B1 and OATP1B3.

Specific expression of OATPs in primary small cell lung cancer (SCLC) cells as novel biomarkers for diagnosis and therapy.

The expression of organic anion transporting polypeptides (OATPs) was elucidated in cell lines from small cell lung cancer (SCLC) and lung carcinoids and in paraffin-embedded samples from primary and metastatic SCLCs. We found a strong relationship between OATP expression and the origin of the cells, as cells from primary or metastatic SCLC and carcinoid tumors differ with respect to OATP levels. OATP4A1 is most prominent in non-malignant lung tissue and in all SCLC and carcinoid cell lines and tissues, OATP5A1 is most prominent in metastatic cells, and OATP6A1 is most prominent in SCLC cell lines and tumors. Treatment with topotecan, etoposide and cisplatin caused significant changes in the expression patterns of OATP4A1, OATP5A1, OATP6A1, chromogranin and synaptophysin. This effect was also evident in GLC-14 cells from an untreated SCLC patient before chemotherapy compared to GLC-16/-19 chemoresistant tumor cells from this patient after therapy. mRNA expression of OATP4A1, 5A1 and 6A1 correlates with protein expression as confirmed by quantitative microscopic image analysis and Western blots. OATPs might be novel biomarkers for tumor progression and the development of metastasis in SCLC patients.

Resveratrol and its major sulfated conjugates are substrates of organic anion transporting polypeptides (OATPs): impact on growth of ZR-75-1 breast cancer cells.

SCOPE: Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. These effects are observed despite its low bioavailability, which is particularly caused by extensive phase II metabolism. It is unknown whether resveratrol and its metabolites can accumulate to bioactive levels in organs and tissues through protein-mediated transport mechanisms. Because organic anion transporting polypeptides (OATPs) mediate the uptake of many clinically important drugs, we investigated their role in the cellular transport of resveratrol and its major glucuronides and sulfates. METHODS AND RESULTS: Uptake experiments were performed with resveratrol and its glucuronides and sulfates in OATP-expressing Chinese hamster ovary (CHO) and breast cancer (ZR-75-1) cells. The uptake rates for resveratrol in OATP1B1-, OATP1B3-, and OATP2B1-transfected Chinese hamster ovary cells were four- to sixfold higher compared to wild-type cells. Resveratrol-3-O-4'-O-disulfate was transported by OATP1B1 and OATP1B3, while resveratrol-3-O-sulfate was exclusively transported by OATP1B3. However, resveratrol-4'-O-sulfate, resveratrol-3-O-glucuronide, and resveratrol-4'-O-glucuronide did not show any affinity for these OATPs. OATP-dependent uptake of resveratrol was also confirmed in ZR-75-1 cells. CONCLUSION: Our data revealed that OATPs act as cellular uptake transporters for resveratrol and its major sulfates, which must be considered in humans following oral uptake of dietary resveratrol.

Interplay between metabolism and transport of resveratrol.

Resveratrol exhibits a variety of biological and pharmacological activities despite its extensive metabolism to sulfates and glucuronides in the intestine and liver. The metabolism of resveratrol is cell specific and strongly correlates with enzyme expression levels. However, a high rate of biotransformation, in concert with the action of the efflux transporters MRP2, MRP3, and ABCG2, reduces intracellular resveratrol concentrations, and may thereby decrease its pharmacological activity. Interestingly, biotransformation is also dependent on disease status. For example, significantly greater sulfation of resveratrol occurs in human breast tumor tissue than in adjacent nonmalignant tissue. The observed differences, however, do not correlate with the expression of sulfotransferases responsible for catalyzing resveratrol sulfation, but rather with significantly higher steroid sulfatase mRNA levels. The in vitro activity of resveratrol sulfates may not necessarily reflect their in vivo function, given the fact that ubiquitously existing human sulfatases can convert the metabolites back to active resveratrol in humans.

Organic anion transporting polypeptides (OATPs): regulation of expression and function.

Eleven members of the human organic anion transporter (OATP) family (grouped into six families) facilitate the Na(+)- independent transmembrane transport of various endo- and xenobiotics (bile acids, bilirubin, steroid hormone conjugates, thyroid hormones, prostaglandins, clinically used drugs, and toxins). OATPs are 12-transmembrane glycoproteins (643-722 amino acids) and contain many conserved structural features, for example, eleven cysteines in the large extracellular loop 5. They are important for proper transport, for which translocation of substrates through a central, positively-charged pore in a rocker-switch-type mechanism has been proposed. Although OATPs are expressed in various cells and tissues, some members show a more restricted pattern (well-studied OATP1B1/OATP1B3 in liver, OATP4C1 in kidney, and OATP6A1 in testis). In cancer, the distribution pattern is no longer maintained, and OATPs, like OATP1B3, become upregulated in malignant tissues (colon, breast, prostate). Studies in cell lines and animal models further revealed that the expression of OATPs is regulated in a cell- and tissue-specific way by cytokines and activation of nuclear receptors (LXR, FXR, PXR, CAR, HNF4). Also epigenetic mechanisms and postranslational modifications influence their expression and function. Therefore, changes in the expression of OATPs under pathological conditions will influence transport processes causing an altered accumulation of OATP substrates in cells of excretory organs (intestine, liver, kidney) and on various blood/organ barriers (such as brain, testis, placenta). For drugs, this may result in increased toxicity and adverse drug reactions. Therefore, it is important to improve the knowledge on the regulation and function of individual OATPs, and to apply it for therapeutic considerations.

The analysis of organic anion transporting polypeptide (OATP) mRNA and protein patterns in primary and metastatic liver cancer.

Organic anion transporting polypeptides (OATP, SLCO genes) mediate the uptake of endobiotics and drugs. Thus, their expression levels and pattern could be of relevance for cancer therapy. This prompted us to investigate the expression of poorly characterized OATPs, namely OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in hepatic cancer of different origin. First, mRNA levels of all eleven OATPs were determined in paired (cancerous and adjacent non-cancerous) specimens from 43 patients with primary liver cancer (hepatocellular carcinoma, HCC; cholangiocellular carcinoma, CCC) and liver metastases from colon tumors (MLT). Real-time RT-PCR analysis revealed that all OATPs, except OATP1C1 and OATP6A1, are extensively expressed in nearly all samples. In contrast to downregulated OATP1B1, OATP1B3, OATP1A2 and OATP2B1 in cancerous vs. non-cancerous samples, an increase in OATP2A1, OATP3A1, OATP4A1 and OATP5A1 mRNA levels was seen in tumors (up to 40-fold for OATP5A1 in the MLT group). Therefore, OATP2A1, OATP3A1, OATP4A1 and OATP5A1 were further investigated by immunofluorescence microscopy on paraffin-embedded cancerous and non-cancerous sections (seven per group). OATP-derived immunoreactivity was observed in plasma membranes and cytosol of hepatic tumor cells, and additionally, in various cytokeratin 19 positive bile ducts. An increased percentage of immunoreactive cells and a higher staining intensity in cancerous vs. non-cancerous paraffin sections paralleled higher mRNA levels of OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in cancerous tissues of HCC, CCC and MLT patients. The extensive expression of OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in hepatic tumors of different origin suggests that these transporters might be further exploited for the discovery of novel anticancer agents.