Comparative study on ABCB1-dependent efflux of anthracyclines and their metabolites: consequences for cancer resistance

Abstract 1. ABCB1 (P-glycoprotein, MDR1) is one of the most important transporter involved in cancer multi-drug resistance. It also plays a significant role in cancer resistance against anthracyclines, an anticancer group of drugs, including doxorubicin and daunorubicin. Several intracellular enzymes metabolise anthracyclines to carbonyl-reduced, hydroxy metabolites, which have impaired cytotoxic properties. However, metabolite efflux by ABCB1 transporter is not well characterised, while it may be the mechanism responsible for the metabolites’ lack of activity. 2. In this study recombinant ABCB1 ATPase transporter assay; anthracyclines accumulation assay in resistant cells overexpressing ABCB1; and molecular modelling were used to investigate anthracyclines: doxorubicin and daunorubicin and their carbonyl-reduced metabolites (doxorubicinol, daunorubicinol) susceptibility for ABCB1-dependent efflux. 3. Based on the kinetics parameters of ATPase activity of ABCB1, it was found that daunorubicinol exerted an exceptionally high potential for being effluxed by the ABCB1 transporter. ABCB1 significantly affected the accumulation pattern of studied chemicals in resistant cancer cells. Doxorubicin and daunorubicinol accumulation were influenced by the activity of ABCB1 modulator – valspodar. 4. Results indicate that ABCB1 activity affects not only anthracyclines but also their metabolites. Therefore crosstalk between the process of anthracyclines metabolism and metabolite efflux may be the mechanism of impairing anticancer properties of anthracyclines metabolites.


Introduction
ABC transporters are transmembrane proteins involved in the ATP-dependent translocation of the organic compound across cellular membranes.Among them, ABCB1 protein (Pglycoprotein) is one of the most significant.ABCB1 is a large (170 kDa) single polypeptide composed of 1280 amino acid residues.It contains two transmembrane domains (TMDs) and two cytosolic nucleotide-binding domains (NBDs).Each TMD consists of six highly hydrophobic a-helices.The TMDs form the pathway by which drug molecules cross the membrane, switching between inward-and outward-facing conformations, while NBDs are responsible for binding ATP and utilising its energy.ABCB1 is consistently expressed in most tissues, especially on the cell membrane of epithelial cells, and is responsible for the efflux of lipophilic organic cations, mainly xenobiotics; hence it is a significant participant of the body detoxication system (Grigoreva et al. 2022;Mora Lagares and Novi� c 2022).
On the other hand ABCB1 overexpression, likewise, some other members of the ABC family are involved in cancer resistance.Cancer resistance is a complex biochemical phenomenon, which relies on the development of decreased sensitivity of neoplastic cells to previously effective cytotoxic drugs.Increased expression of ABC transporters intensifies the efflux of anticancer drugs out of cells and lowers their intracellular concentration, decreasing their interaction with molecular targets.Cancer resistance reduces the outcome of therapy and is a cause of treatment failure (Pilotto Heming et al. 2022).ABCB1 transporter is one of the most important proteins involved in transporter-dependent resistance and plays a significant role in cancer resistance against taxanes, Vinca alkaloids, and anthracyclines (Krause 2019;Chen et al. 2021).Modulation of ABCB1 activity is considered a potential therapeutic approach to sensitise cells against cytotoxic agents; however, all P-glycoprotein inhibitors, despite their activity in preclinical models, failed during clinical investigations.Search for novel ABCB1 modulators is still a significant field in the cancer-chemosensitizing concept.This idea is sometimes questionable, because of the probable low druggability of ABCB1 modulators.The tissue selectivity of inhibitors was proposed as a solution.Recently ABCB1 is also considered a marker of chemoresistant and poor prognosis cancer (Robey et al. 2018;Chen et al. 2021).
Anthracyclines, including doxorubicin (DOX) and daunorubicin (DAUN), a group of widely used oncologic drugs -topoisomerase II inhibitors, are classic substrates of ABCB1 and activity of this transporter is believed to be a major contributor of cancer resistance against their cytotoxicity (Capelôa et al. 2020).On the other hand, their biotransformation -two electron side-chain carbonyl reduction, is known to be a mechanism of cancer cell resistance.Carbonyl reduction produces secondary alcohol metabolites, namely doxorubicinol (DOXol) and daunorubicinol (DAUNol), which were found to be less active than parental structures.They have lower DNA binding ability and decreased penetration to cells' nuclei (Piska et al. 2017).There is also some information in the literature, which may suggest the involvement of ABCB1 in metabolite transport.In a clinical trial, DOX and DOXol concentration profiles were significantly altered by the co-administration of a zosuquidar -ABCB1 modulator (Callies et al. 2003).Similarly, cyclosporine as an MDR modulator increased the AUC of DOX and DOXol by 55% and 350%, respectively (Bartlett et al. 1994).Nilotinib, also an ABCB1 modulator, increased DOX and its metabolite accumulation in tissues (Zhou et al. 2016).However, no research would directly and unambiguously define metabolites, as substrates of ABCB1.
In this study, DOX, DAUN, and their respective carbonylreduced metabolites DOXol and DAUNol were characterised as substrates of ABCB1.The interaction was studied using recombinant ABCB1 protein and cancer DOX-resistance cell line co-treated with a specific ABCB1 inhibitor.Moreover, molecular modelling was used to investigate the binding of studied anthracyclines with the transporter.

Resistant cell line establishment
A549 cells were cultured in a culture medium, additionally supplemented with DOX.The starting concentration of DOX was 0.05 mM.Once cells reached decreased sensitivity to DOX, observed by proper proliferation potential in comparison to control, decreased alterations in cell morphology, and cell death, its concentration was increased by the next 0,05 mM.Cells were used in further experiments after 3 months of continuous culturing with DOX, in which the final concentration achieved 0.2 mM.Resistant cell lines were marked as A549/DOX.
The cells on the slides were cultured for 24 h.The cells were then rinsed with PBS with Ca 2þ and Mg 2þ and fixed with 3.7% formaldehyde (15 min at 37 � C).In the next step, the cells were permeabilized with 0.1% Triton X-100 solution (5 min, at 37 � C) and blocked with 3% BSA in PBS (1 h at 37 � C).The cells were then incubated in the presence of a primary mouse antibody to P-gp (C219 clone, Invitrogen, #MA1-26528) at a dilution of 1:500 at 3% BSA and incubated for 90 min.After this time, the antibody was removed, the cells were washed three times with distilled water, and incubated in the presence of a secondary antibody (1:1000) Alexa 488 rabbits against mouse IgG for another 45 min (Invitrogen, #A-11059).Staining was carried out in two independent repetitions.

Cytotoxicity assay
A549 and A549/DOX cells were seeded in 96-multiwell plates at a density of 5 � 10 3 /well.After 24 h cells were incubated with ABCB1 modulator -Valspodar (VAL; 2.5 mM) or vehicle control and DOX or DAUN in a broad concentration range.After 48 h MTT solution was added to cells and once the black crystal of formazan appeared on the bottom of the well (3 h), the medium was aspirated and formazan was dissolved in DMSO.The absorbance of solutions was read on a multiwell plates reader at k ¼ 570 nm.Cell viability was calculated by dividing the absorbance of the experimental by the control (�100%).IC 50 was calculated using GraphPad Prism software 9.5.0 (San Diego, California USA).

Anthracylines accumulation in cells
A549 and A549/DOX cells were seeded in black 24-multiwell plates with coverglass bottom (Eppendorf Cells Imaging Plates) at a density of 2.5 � 10 4 /well.After 48 h cells were incubated with DOX, DAUN, DOXol or, DAUNol (10 lM) and in some cases with VAL (2.5 lM).After 2 h medium was aspirated, cells were washed twice, and FluoroBrite DMEM medium was added to wells.Fluorescence was observed under a microscope Leica DMI6000B.Pictures were taken using LAS-X Software.Fluorescence intensity was determined in Image J 1.53t.

ABCB1-ATPase assay
P-gp Glo (Promega) ATPase kit was used to investigate the interaction of anthracyclines and their metabolites with the transporter.Membranes containing recombinant ABCB1 transporter were incubated in a concentration range of DOX, DAUN, DOXol and DAUNol in supplier buffer in white 96-well plates.MgATP was added to initiate the activity of the transporter.After 40 min detection solution was added and after 20 min luminescence was read on a multiwell plates reader.Verapamil was used as a positive control.ATP consumption was represented as DRLU, which is calculated as a change of luminescence induced by a studied agent against the basal activity of the transporter.Michaelis-Menten kinetics model was used to compare ATPase activation profiles for studied molecules.Michaelis-Menten curves were determined in GraphPad Prism 9.5.0 (San Diego, California USA).

Molecular modelling
Human ABCB1 crystal structure 6C0V (rcsb.org/structure/6C0V)was selected for molecular docking of DOX, DAUN, DOXol, and DAUNol.The structure presents outward-facing conformation with no co-crystalized ligands in binding sites described previously as H-, M-, and R-sites (Kim and Chen 2018).Among all available up-to-date human and chimeric ABCB1 crystals, 6C0V possesses the highest resolution (3.40 Å).Molecular modelling studies were performed using Small-Molecule Drug Discovery Suite (Schr€ odinger, Inc).The structures of analysed anthracyclines and their C13-reduced metabolites were optimised using LigPrep tool, generating the most possible protonation states in the pH range 7 ± 1. ABCB1 crystal structure 6C0V was refined using Protein Preparation Wizard: hydrogen atoms were added and the energy of the whole system was minimised using OPLS3e force field (Sastry et al. 2013).SiteMap was used to identify top-ranked potential binding sites within the optimised protein (Halgren 2009).The indicated sites were then compared with the one described by Ferreira et al. as R-site, in which several compounds, including anthracyclines, bound as ABCB1 ligands (Ferreira et al. 2013).After confirming that the protein area mapped by Ferreira et al. as R-site is also one of the top-ranked sites indicated by SiteMap for 6C0V, Induced-Fit docking (IFD) was applied to dock DOX, DAUN, DOXol and DAUNol and evaluate their binding mode (Sherman et al. 2006).The box centroid was set automatically based on the position of 46 amino acids pointed as the ones forming the Rsite (Ferreira et al. 2013).No constraints were applied during IFD.As an output, 10 best-ranked protein-ligand complexes (ranked by IFDScore) for each compound were obtained and analysed, based on found molecular interactions.Root-meansquare deviation of atomic positions (RMSD) was calculated separately for the ligand of each IFD complex, taking the ligand position from its top-ranked complex as the reference structure.Based on the obtained values, mean RMSD, standard deviation, and median were calculated for the analysed anthracyclines and their derivatives.

Results and discussion
ABC transporters utilise the energy of ATP to translocate organic molecules through a biological membrane.Therefore measurement of ATP consumption is one of the methods to study ABC transporters' activity.In the present study, ATPase activity of ABCB1 transporter was evaluated against DOX, DAUN, and their carbonyl-reduced metabolites: DOXol and DAUNol.ATP utilisation was determined with the use of luciferase and luminescence measurement, resulting in DRLU (DRelative Light Unit) value.Therefore DRLU is related to the drug-induced ATPase activity of ABCB1.ATPase activity of ABCB1 in the presence of DOX and DOXol or DAUN and DAUNol are presented in Figure 1.
To compare anthracyclines-induced activation of ABCB1, the Michaelis-Menten equation was utilised.Parameters generated by nonlinear regression are presented in Table 1.Results show that DOX and DOXol stimulated ATPase activity of ABCB1 with a similar potency.DAUN also showed a comparable manner of activation.Much higher activity was observed for DAUNol, in which Vmax/Km value was ca. 3 three times higher than other anthracyclines.Relative low Km was the main cause of high Vmax/Km.Hence, among anthracyclines, DAUNol was determined to be the most potent compound in stimulating ABCB1.
Next, anthracyclines and their metabolites were studied in a biological assay, including an anthracyclines-resistant cancer cell line.A549 cell line resistant to DOX was acquired by continuous culturing cells with low, increasing in time, concentration of anthracycline.The resistant phenotype was confirmed by a viability assay.The increased IC 50 value for both DOX and DAUN, shows that cells are less sensitive to agents, and submicromolar concentrations of anthracyclines are no longer effective (Table 2).Because both anthracyclines were found to be less active, the same mechanism of resistance was supposed.To confirm the involvement of ABCB1 in the mechanism of resistance, valspodar, as a selective ABCB1 modulator was used as a chemosensitizing agent.Its effective, significant resistance-reversing activity was a reason to recognise ABCB1, as a drug transporter responsible for drug resistance.Overexpression of ABCB1 leading to the increased presence of a transporter on the cell membrane was further  confirmed by immunochemical staining.In A549-resistant cell lines, a much higher level of a protein was detected on cell membranes, than in a native cell line (Figure 2).Resistant cells were then used in an accumulation assay (Figure 3).Anthracylines and their metabolites were incubated with resistant cells with or without the modulator of ABCB1 -valspodar.Because anthracycline derivatives exert fluorescence properties, the accumulation of agents in cells was investigated under a fluorescence microscope.
In resistant A549/DOX cells, DOX and DOXol were presented at a low level.Treatment of cells with VAL, resulted in a significant increase in DOX content, while the influence on DOXol was slight.DAUN was found to be highly accumulated in cells, while the fluorescence signal coming from DAUNol was very low, showing its low intracellular concentrations.VAL did not affect the high accumulation of DAUN in cells, however, showed a significant influence on a DAUNol level.Average cell fluorescence is presented in Supplementary Table S4.Previously, Ross et al. found altered anthracyclines (DAUN, idarubicin) and metabolite accumulation patterns in multidrug resistance cells.However, the study was performed with a non-selective transporter inhibitor (verapamil) (Ross et al. 1995).A similar study was performed by Kuffel and Ames with DOX and idarubicin (Kuffel and Ames 1995).While overexpression of ABCB1 was confirmed in A549/DOX cell lines, other transporters may also be upregulated in studied cells.However, valspodar, as a selective transporter modulator, confirms the contribution of ABCB1 in the observed effect (May et al. 2008).
ABCB1 crystal structure 6C0V was selected for molecular studies as the one with no co-crystalized ligands that could subsequently affect the quality of docking simulations.The only co-crystallized elements were two ATP molecules and two magnesium atoms, placed within the cytoplasmic nucleotide-binding domain (NBD) (Kim and Chen 2018).That region is located relatively far from the putative anthracycline binding site (R-site).Although Ferreira et al. pointed out the amino acid residues that form three main ABCB1 binding sites, the prediction was based on the inward-facing conformation of the protein (Aller et al. 2009;Ferreira et al. 2013).6C0V presents outward-facing conformation and thus first we used SiteMap to check whether those changes of conformation hinder the ligand accessibility to the area described previously as R-site.The first site that was indicated by SiteMap as ligand-accessible corresponded with R-site (Figure 4(a-c)).Hence, that area was used to dock anthracyclines and their reduced metabolites.As a result of IFD, we obtained 40 protein-ligand complexes (10 complexes for each ligand), with a repetitive mode of interaction between anthracycline and selected amino acid residues.An exemplary pose of DOXol, showing the general binding scheme of all anthracyclines docked to ABCB1, is presented in Figure 5(a-b).Rings C and D were oriented extracellularly.In this part of the ligands, two H-bond interactions were observed in some poses: between carbonyl groups and Ser831 or Arg832.Rings A and B, the daunosamine sugar ring, and the fragment with metabolically sensitive carbonyl  group were located towards the inside of the cell.C6 phenolic group formed H-bond with Glu353 in most of the obtained complexes.The most repetitive pattern of interaction was observed in daunosamine (sugar) ring of anthracyclines.Charged Glu782 was the key amino acid forming interactions with both hydroxy (H-bond) and protonated amine (salt bridge) groups of daunosamine.In some complexes, besides the two mentioned interactions, an additional H-bond between the protonated amine group and the main chain of Gly778 was observed (Figure 6).We neither observed any protein-ligand interaction between C13 carbonyl group of DOX, nor DAUN.In terms of the side chain (C14) hydroxy group, the presence of which differentiates DOX and DOXol from DAUN and DAUNol, its main point of interaction was a side chain of Glu184, acting as H-bond acceptor.It played the same role in H-bond interaction with C13-reduced groups of DOXol and DAUNol.In some DOXol complexes, we observed double H-bond interaction with both C13 and C14 hydroxy groups.In our simulations, Glu184 residue seemed to play a crucial role in differentiating the binding mode of the analysed ligands: for DAUN there was no chance for such interaction with Glu184, for DOX and DAUNol we observed one type of interaction with Glu184 (DOX -C14 hydroxy group; DAUNol -C13 hydroxy group), for DOXol we observed one or two types of interactions that were mentioned earlier (Figure 7).However, considering the total amount and type of the observed protein-ligand interactions, especially in the sugar part of the ligands, it does not seem that additional interactions with Glu184 could be a key factor determining different affinities of individual anthracyclines to ABCB1.It is important to note that Glu184 is a part of H-site, therefore anthracyclines also slightly bind with this binding pocket.Apart from the described hydrogen bonds and ionic interactions, we did not notice any p-interactions.The described mode of interaction was observed in a vast majority of the obtained complexes.However, there were some complexes in which the ligand was flipped.In that case, the sugar ring was placed in the vicinity of Glu184 and C13 carbonyl group next to Glu782.This mode was observed among all ligands: DOX (2 complexes), DAUN (1), DOXol (3), and DAUNol (2).However, these were individual cases and therefore we did not consider this binding mode to be correct.Besides the evaluation of the binding mode of selected anthracyclines and their metabolites, we compared ligands' RMSD, taking as reference the position of the ligand in the top-ranked IFD complex.Calculations were performed separately for each ligand.The results are presented in Table 3.Additional analyses of standard deviation and median are also included.Based on the obtained values we assume that a single change in ANT structure, such as an additional C14 hydroxy group or replacement of the carbonyl group with a hydroxy group in C13 does not affect critically ligand's affinity to ABCB1.These structural changes do not affect the number and type of protein-ligand interaction and hence apparent changes in the position of the entire ligand inside the protein.Otherwise, we would expect significant differences in mean RMSD between DOX and DOXol, DAUN and DAUNol, as well as between DOX and DAUN.While it is well known that ABCB1 typically binds with organic compounds in an inward-facing conformation, our study utilised the outward-facing conformation.This choice was motivated by the high-resolution data available from the 6C0V crystal structure and the limited existing literature on the binding of   anthracyclines to the outward-facing conformation of ABCB1.The docking simulations revealed a distinct set of interactions in comparison to those reported by Ferreira et al. for the inward-facing conformation (Ferreira et al. 2013).Nevertheless, it is noteworthy that in the outward-facing conformation, certain amino acids within the R-site still constitute a favoured binding pocket for anthracyclines.Consequently, the translocation of molecules is presumed to be governed by conformational changes in the R-site, resulting in a reduction in binding affinity with these amino acids, thereby altering the number and types of interactions between the molecule and the protein The performed analyses indicate differences between anthracyclines and their metabolites' susceptibility to ABCB1dependent efflux.The most important difference is between DAUN and its 13-hydroxy metabolite DAUNol.In both, ABCB1 ATPase assay and accumulation/efflux assay in ABCB1-dependent resistant cancer cells, DAUNol seems to be a molecule the most dependent on the activity of ABCB1 transporter.This phenomenon may have great biological consequences.DAUNol formation may cause increased efflux of the anthracyclines to pool from intracellular compartments to extracellular space or sequestration in intracellular vesicles.To this time DAUNol formation was described to decrease DAUN activity by decreased cytotoxic properties of a metabolite.In this study novel perspective explaining the decreased activity of 13-hydroxy metabolites of anthracyclines is given.
Additionally, results may have great significance in the search for a novel ABCB1 inhibitor.Since the metabolite is a better substrate than the parent molecule to a transporter, it is reasonable to include DAUNol in the process of searching for DAUN chemosensitizing drugs.Gonzalez et al. found that 94% of DAUN was converted to DAUNol within 2 h in CBR1overexpressing leukaemia cells (Gonzalez et al. 1995), which indicates the potential significance of a metabolite formation for an overall biological effect.Also, further studies are recommended to characterise the phenomenon among other anthracyclines, especially when this group of drugs is currently very commonly applied in clinical oncology and many new anthracyclines are under development.On the other hand, the involvement of other ABC transporters involved in cancer resistance like ABCC2, should be characterised.
With the use of molecular docking, we determined the potential main ABCB1 amino acid residues interacting with DOX, DAUN, DOXol, and DAUNol in the outward-facing conformation of the transporter, however, the use of that in silico technique did not let us find a reason why DAUNol is the best substrate of ABCB1 transporter.The main cause may be the complexity of the protein and multiple conformational states of ABCB1 during the process of anthracyclines translocation through the transporter.An option for clarification of this problem might be the use of more sophisticated simulations, including molecular dynamics (Mora Lagares et al. 2021).Also, a study performed on the inward-faced model may result in finding different binding modes for studied molecules.The possible mechanism of ABCB1 modulation may be similar to one proposed by Nguyen et al. (2020), who found that different rates of DOX and DAUN efflux are caused by inducing changes in the conformational distribution of mouse ABCB1.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Activation of ABCB1 by anthracyclines and their metabolites in ATPase assay.

Figure 2 .
Figure 2. Native (A,B) and resistant (C,D) cells in visible-light microscopy (left) and fluorescence (right) microscopy, ABCB1 labelled with primary mouse antibody to P-glycoprotein and visualised by immunofluorescence method.

Figure 3 .
Figure 3. Anthracyclines (A -DOX, B-DOXol, C -DAUN, D -DAUNol) accumulation in A549/DOX cells in the absence (left column) or the presence (right column) of VAL.Fluorescence intensity may be found in Supplemental Materials.

Figure 4 .
Figure 4. (A) ABCB1 crystal structure 6C0V displayed as grey thin tubes.The grey surface represents the area pointed by SiteMap tool as the one with the highest accessibility for ligands; (B, C) Comparison of the ligand-accessible area indicated by SiteMap (grey surface) with R-site described by Ferreira et al. as the region in which several compounds, including anthracyclines, bind as ABCB1 ligands (amino acids forming R-site marked red; Transmembrane helices 1 and 7 (TM1 and TM7) have been pointed to increase the special orientation of the structures.

Figure 5 .
Figure 5. (A) The general position of anthracyclines (on an example of DOXol displayed as green sticks) docked to ABCB1 crystal structure 6C0V (grey cartoon); (B)The most prevalent predicted binding mode of anthracyclines and their reduced metabolites (on an example of DOXol) within R-site of ABCB1.Amino acid residues engaged in ligand binding throughout H-bonds (dotted yellow lines) and salt bridges (dotted purple lines) are displayed as sticks.

Figure 6 .
Figure 6.Molecular interactions formed between ABCB1 amino acid residues Gly778 and Glu782, and daunosamine ring occurring in all anthracyclines and their reduced metabolites are in the scope of this article.H-bonds are displayed as dotted yellow lines; the salt bridge is displayed as dotted purple lines.

Figure 7 .
Figure 7. Representative poses of DOX, DAUN and their reduced metabolites docked to ABCB1 crystal structure 6C0V.The main attention was put on the ligand aliphatic fragment and differences in the mode of interaction with Glu184.A variety of binding modes were observed for all analysed compounds: DOX (A), DOXol (B), DAUN (C), DAUNol (D).

Table 2 .
Cytotoxic properties of DOX and DAUN in cells and chemosensitive activity of VAL.

Table 3 .
Comparison of RMSD values calculated based on results of Induced-Fit docking (IFD) of anthracyclines and their C13 reduced metabolites.