Metabolic deactivation by cytochrome P450 (CYP) is considered a potential mechanism of anticancer drug resistance. However, this hypothesis is predominantly based on indirect pieces of evidence and/or is influenced by interfering factors such as the use of multienzymatic models. Thus, an experimental approach for its verification is needed. In the present work, we employed HepG2 cells transduced with CYP enzymes involved in docetaxel, paclitaxel and vincristine metabolism to provide mechanistic evidence on their possible roles in resistance to these chemotherapeutic agents. Using MTT proliferation tests, we showed that overexpression of CYP3A4 resulted in decreased antiproliferative activity of 1 μM docetaxel (by 11.2, 23.2 and 22.9% at 24, 48 and 72 h intervals, respectively), while the sensitivity of CYP3A4-transduced cells was restored by co-administration of ketoconazole. Paclitaxel exhibited differential efficacy in CYP2C8- and empty vector-transduced cells (significant differences between 10.9 and 24.4% for 0.01, 0.1 and 1 μM concentrations), but neither montelukast nor clotrimazole was capable of affecting this asymmetry. Finally, the pharmacological activity of vincristine was not influenced by CYP3A4 or CYP3A5 overexpression. In the follow-up caspase activation assays, docetaxel was confirmed to be a victim of CYP3A4-mediated resistance, which is, at least partly, brought by impaired activation of caspases 3/7, 8 and 9. In summary, our data demonstrate that CYP3A4-mediated metabolic deactivation of docetaxel might represent a significant mechanism of pharmacokinetic resistance to this drug. In contrast, the possible role of CYPs in resistance to paclitaxel and vincristine has been disconfirmed. Importantly, the expression of CYP3A4 in HepG2_CYP3A4 cells is comparable to that in primary hepatocytes and HepaRG cells, which suggests that our results might be relevant for in vivo conditions, e.g., for hepatocellular carcinoma. Thus, our data may serve as a valuable in vitro background for future in vivo studies exploring the area of intratumoural metabolism-based drug resistance.
- MeSH
- buňky Hep G2 MeSH
- chemorezistence fyziologie MeSH
- cytochrom P-450 CYP3A metabolismus MeSH
- cytochrom P450 CYP2C8 metabolismus MeSH
- cytostatické látky farmakologie MeSH
- hepatocyty účinky léků metabolismus MeSH
- lidé MeSH
- metabolická clearance účinky léků MeSH
- metabolická inaktivace účinky léků MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- protinádorové látky farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Brivanib, a promising tyrosine kinase inhibitor, is currently undergoing advanced stages of clinical evaluation for solid tumor therapy. In this work, we investigated possible interactions of this novel drug candidate with ABC drug efflux transporters and cytochrome P450 (CYP450) drug-metabolizing enzymes that participate in cancer multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). First, in accumulation experiments with various model substrates, we identified brivanib as an inhibitor of the ABCB1, ABCG2, and ABCC1 transporters. However, in subsequent combination studies employing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide proliferation assays in both Madin-Darby canine kidney II (MDCKII) and A431 cellular models, only ABCG2 inhibition was revealed to be able to synergistically potentiate mitoxantrone effects. Advantageous to its possible use as MDR antagonist, brivanib's chemosensitizing properties were not impaired by activity of any of the MDR-associated ABC transporters, as observed in comparative viability assay in the MDCKII cell sublines. In incubation experiments with eight recombinant CYP450s, we found that brivanib potently inhibited CYP2C subfamily members and the CYP2B6 isoform. Finally, in induction studies, we demonstrated that brivanib upregulated ABCB1 and CYP1A2 messenger RNA levels in systemic cell models, although this interaction was not significantly manifested at a functional level. In conclusion, brivanib exhibits potential to cause clinically relevant pharmacokinetic DDIs and act as a modulator of ABCG2-mediated MDR. Our findings might be used as an important background for subsequent in vivo investigations and pave the way for the safe and effective use of brivanib in oncological patients.
- MeSH
- ABC transportér z rodiny G, člen 2 antagonisté a inhibitory MeSH
- alanin analogy a deriváty farmakologie MeSH
- biotransformace účinky léků MeSH
- buněčné linie MeSH
- buňky MDCK MeSH
- chemorezistence účinky léků MeSH
- inhibitory cytochromu P450 farmakologie MeSH
- lékové interakce fyziologie MeSH
- lidé MeSH
- mnohočetná léková rezistence účinky léků MeSH
- nádorové proteiny antagonisté a inhibitory MeSH
- P-glykoproteiny metabolismus MeSH
- psi MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- triaziny farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Alectinib is a tyrosine kinase inhibitor currently used as a first-line treatment of anaplastic lymphoma kinase-positive metastatic nonsmall cell lung cancer (NSCLC). In the present work, we investigated possible interactions of this novel drug with ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 (P450) biotransformation enzymes that play significant roles in the phenomenon of multidrug resistance (MDR) of cancer cells as well as in pharmacokinetic drug-drug interactions. Using accumulation studies in Madin-Darby canine kidney subtype 2 (MDCKII) cells alectinib was identified as an inhibitor of ABCB1 and ABCG2 but not of ABCC1. In subsequent drug combination studies, we demonstrated the ability for alectinib to effectively overcome MDR in ABCB1- and ABCG2-overexpressing MDCKII and A431 cells. To describe the pharmacokinetic interaction profile of alectinib in a complete fashion, its possible inhibitory properties toward clinically relevant P450 enzymes (i.e., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) were evaluated using human P450-expressing insect microsomes, revealing alectinib as a poor interactor. Advantageously for its use in pharmacotherapy, alectinib further exhibited negligible potential to cause any changes in expression of ABCB1, ABCG2, ABCC1, CYP1A2, CYP3A4, and CYP2B6 in intestine, liver, and NSCLC models. Our in vitro observations might serve as a valuable foundation for future in vivo studies that could support the rationale for our conclusions and possibly enable providing more efficient and safer therapy to many oncological patients.
- MeSH
- ABC transportéry účinky léků MeSH
- biotransformace MeSH
- buňky MDCK MeSH
- chemorezistence účinky léků MeSH
- inhibitory proteinkinas farmakokinetika farmakologie MeSH
- karbazoly farmakokinetika farmakologie MeSH
- lidé MeSH
- mnohočetná léková rezistence účinky léků MeSH
- piperidiny farmakokinetika farmakologie MeSH
- psi MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
1. Purine cyclin-dependent kinase inhibitors have recently been recognised as promising candidates for the treatment of various cancers. While pharmacodynamic properties of these compounds are relatively well understood, their pharmacokinetics including possible interactions with placental transport systems have not been characterised to date. 2. In this study, we investigated transplacental passage of olomoucine II and purvalanol A in rat focusing on possible role of p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and/or multidrug resistance-associated proteins (ABCCs). Employing the in situ method of dually perfused rat term placenta, we demonstrate transplacental passage of both olomoucine II and purvalanol A against the concentration gradient in foetus-to-mother direction. Using several ATP-binding cassette (ABC) drug transporter inhibitors, we confirm the participation of ABCB1, ABCG2 and ABCCs transporters in the placental passage of olomoucine II, but not purvalanol A. 3. Transplacental passage of olomoucine II and purvalanol A from mother to foetus is significantly reduced by active transporters, restricting thereby foetal exposure and providing protection against harmful effects of these xenobiotics. Importantly, we demonstrate that in spite of their considerable structural similarity, the two molecules utilise distinct placental transport systems. These facts should be kept in mind when introducing these prospective anticancer candidates and/or their analogues into the clinical area.
- MeSH
- ABC transportéry metabolismus MeSH
- adenosintrifosfát chemie MeSH
- aktivní transport MeSH
- krysa rodu rattus MeSH
- matka - expozice noxám MeSH
- P-glykoproteiny metabolismus MeSH
- placenta účinky léků metabolismus MeSH
- potkani Wistar MeSH
- proteiny spojené s mnohočetnou rezistencí k lékům metabolismus MeSH
- puriny aplikace a dávkování farmakokinetika MeSH
- těhotenství u zvířat MeSH
- těhotenství MeSH
- trofoblasty účinky léků MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- xenobiotika chemie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Paclitaxel (PTX), docetaxel (DTX), 5-fluorouracil (5-FU), cyclophosphamide (CYC) or tamoxifen (TMX) are combined with doxorubicin (DOX) in first-line chemotherapy regimens that are indicated for breast cancer patients. Although the efficacies of these drugs in combination treatments have been demonstrated in clinical practice, their possible interference with DOX metabolism has not been described in detail to date. In the present study, we investigated the possible interactions of human carbonyl reducing enzymes with 5-FU, PTX, DTX, CYC and TMX. First, the reducing activities of carbonyl reducing enzymes toward DOX were tested using incubations with purified recombinant enzymes. In the subsequent studies, we investigated the possible effects of the tested anticancer agents on the DOX-reducing activities of the most potent enzymes (AKR1C3, CBR1 and AKR1A1) and on the DOX metabolism driven by MCF7, HepG2 and human liver cytosols. In both of these assays, we observed that CYC and its active metabolites inhibited DOX metabolism. In the final study, we tracked the changes in AKR1C3, CBR1 and AKR1A1 expression levels following exposure to the tested cytostatics in MCF7 and HepG2 cells. Consequently, no significant changes in the expression levels of tested enzymes were detected in either cell line. Based on these findings, it is feasible to presume that inhibition rather than induction plays a role in the interactions of the tested anticancer agents with DOX-reducing enzymes. In conclusion, our results describe important molecular events that occur during combination breast cancer therapies and might modulate pharmacokinetic DOX resistance and/or behaviour.
- MeSH
- 3-hydroxysteroid dehydrogenasy genetika metabolismus MeSH
- aldehydreduktasa genetika metabolismus MeSH
- alkoholoxidoreduktasy genetika metabolismus MeSH
- biotransformace MeSH
- buňky Hep G2 MeSH
- cyklofosfamid farmakologie MeSH
- doxorubicin metabolismus farmakologie MeSH
- fluoruracil farmakologie MeSH
- hydroxyprostaglandindehydrogenasy genetika metabolismus MeSH
- izoenzymy genetika metabolismus MeSH
- játra účinky léků enzymologie MeSH
- kinetika MeSH
- lékové interakce MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- oxidace-redukce MeSH
- protinádorové látky farmakologie MeSH
- protokoly protinádorové kombinované chemoterapie MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- tamoxifen farmakologie MeSH
- taxoidy farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pharmacokinetic drug resistance is a serious obstacle that emerges during cancer chemotherapy. In this study, we investigated the possible role of aldo-keto reductase 1C3 (AKR1C3) in the resistance of cancer cells to anthracyclines. First, the reducing activity of AKR1C3 toward anthracyclines was tested using incubations with a purified recombinant enzyme. Furthermore, the intracellular reduction of daunorubicin and idarubicin was examined by employing the transfection of A549, HeLa, MCF7 and HCT 116 cancer cells with an AKR1C3 encoding vector. To investigate the participation of AKR1C3 in anthracycline resistance, we conducted MTT cytotoxicity assays with these cells, and observed that AKR1C3 significantly contributes to the resistance of cancer cells to daunorubicin and idarubicin, whereas this resistance was reversible by the simultaneous administration of 2'-hydroxyflavanone, a specific AKR1C3 inhibitor. In the final part of our work, we tracked the changes in AKR1C3 expression after anthracycline exposure. Interestingly, a reciprocal correlation between the extent of induction and endogenous levels of AKR1C3 was recorded in particular cell lines. Therefore, we suggest that the induction of AKR1C3 following exposure to daunorubicin and idarubicin, which seems to be dependent on endogenous AKR1C3 expression, eventually might potentiate an intrinsic resistance given by the normal expression of AKR1C3. In conclusion, our data suggest a substantial impact of AKR1C3 on the metabolism of daunorubicin and idarubicin, which affects their pharmacokinetic and pharmacodynamic behavior. In addition, we demonstrate that the reduction of daunorubicin and idarubicin, which is catalyzed by AKR1C3, contributes to the resistance of cancer cells to anthracycline treatment.
- MeSH
- 3-hydroxysteroid dehydrogenasy antagonisté a inhibitory genetika metabolismus MeSH
- antracykliny agonisté metabolismus farmakologie MeSH
- biotransformace MeSH
- chemorezistence * účinky léků MeSH
- daunomycin agonisté metabolismus farmakologie MeSH
- doxorubicin metabolismus farmakologie MeSH
- enzymová indukce účinky léků MeSH
- flavanony farmakologie MeSH
- hydroxyprostaglandindehydrogenasy antagonisté a inhibitory genetika metabolismus MeSH
- idarubicin agonisté metabolismus farmakologie MeSH
- inhibitory enzymů farmakologie MeSH
- karcinom farmakoterapie MeSH
- kinetika MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny antagonisté a inhibitory genetika metabolismus MeSH
- oxidace-redukce MeSH
- protinádorová antibiotika agonisté metabolismus farmakologie MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- synergismus léků MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Artificial soil is an important standard medium and reference material for soil ecotoxicity bioassays. Recent studies have documented the significant variability of their basic properties among different laboratories. Our study investigated (i) the variability of ten artificial soils from different laboratories by means of the fate, extractability and bioavailability of phenanthrene and lindane, and (ii) the relationships of these results to soil properties and ageing. Soils were spiked with (14)C-phenanthrene and (14)C-lindane, and the total residues, fractions extractable by hydroxypropyl-β-cyclodextrin, and the fractions of phenanthrene mineralizable by bacteria were determined after 1, 14, 28 and 56 days. Significant temporal changes in total residues and extractable and mineralizable fractions were observed for phenanthrene, resulting in large differences between soils after 56 days. Phenanthrene mineralization by indigenous peat microorganisms was suggested as the main driver of that, outweighing the effects of organic matter. Lindane total residues and extractability displayed much smaller changes over time and smaller differences between soils related to organic matter. Roughly estimated, the variability between the artificial soils was comparable to natural soils. The implications of such variability for the results of toxicity tests and risk assessment decisions should be identified. We also suggested that the sterilization of artificial soils might reduce unwanted variability.
Dendritic poly(L-lysines) (DGL) constitute promising nanomaterials applicable as a nonviral gene-delivery vector. In this study, we evaluate the transfection abilities of four DGL generations with special emphasis on the systematic description of the relationship of how generation (i.e., molecule size) affects the transfection efficacy. Using Hep2 cells, we demonstrated that the capability of unmodified DGL to deliver plasmid is of a magnitude lower than that of jetPEI. On the other hand, employing the Hep2 cell line stably transduced with eGFP, we observed that DGL G5 delivers the siRNA oligonucleotide with the same efficiency as Lipofectamine 2000. In further experiments, it was shown that DGL affords excellent ability to bind DNA, protect it against DNase I attack, and internalize it into cells.
- MeSH
- buněčné linie MeSH
- deoxyribonukleasa I metabolismus MeSH
- lidé MeSH
- lipidy MeSH
- malá interferující RNA MeSH
- molekulární sekvence - údaje MeSH
- oligonukleotidy metabolismus farmakokinetika MeSH
- plazmidy farmakokinetika MeSH
- polylysin chemie farmakokinetika MeSH
- sekvence nukleotidů MeSH
- transfekce metody MeSH
- viabilita buněk účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zelené fluorescenční proteiny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.
- MeSH
- ABC transportéry metabolismus MeSH
- biologický transport MeSH
- buněčné linie MeSH
- chemorezistence účinky léků MeSH
- nádorové proteiny metabolismus MeSH
- P-glykoprotein metabolismus MeSH
- psi MeSH
- puriny farmakokinetika MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
