Inhibition of glycosphingolipid biosynthesis reverts multidrug resistance by differential modulation of ABC transporters on chronic myeloid leukemias

Multidrug resistance (MDR) in cancer manifests due to cross-resistance to chemotherapeutic drugs with neither structural nor functional relationship, markedly by increased expression and activity of ABC superfamily transporters. Evidences indicate sphingolipids as substrates to ABC proteins in processes such as cell signaling, membrane biosynthesis and inflammation, and products of its biosynthetic route were shown to favor cancer progression. Glucosylceramide (GlcCer) is a ubiquitous glycosphingolipid (GSL) generated by glucosylceramide synthase, a key cell regulator enzyme encoded by the UDP-glucose ceramide glucosyltransferase (UGCG) gene. Under stress, cells increase de novo biosynthesis of ceramides, which return to sub-toxic levels after assimilation into GlcCer by UGCG. Given that cancer cells seem to mobilize UGCG and increase GSL contents for the clearance of ceramides ultimately contributing to treatment failure, we studied how inhibiting GSL biosynthesis would affect the MDR phenotype of chronic myeloid leukemias. Results indicate that MDR associates to higher expression of UGCG and to a complex GSL profile. Inhibition of this glucosyltransferase greatly reduced GM1 expression, and cotreatment with standard chemotherapeutics sensitized cells leading to mitochondrial membrane potential loss and apoptosis. Despite reducing ABCB1 expression, only the ABCC-mediated efflux activity was affected. Consistently, efflux of C6-ceramide, one byproduct of UGCG downregulation, was reduced after inhibition of ABCC-mediated transport. Overall, UGCG inhibition impaired the malignant glycophenotype of MDR leukemias, overcoming drug resistance through distinct mechanisms. This work brings more comprehension about the involvement of GSL for chemotherapy failure, and modulation of its contents emerges as an intervention targeted to MDR leukemias.


ABSTRACT
Multidrug resistance (MDR) in cancer manifests due to cross-resistance to chemotherapeutic drugs with neither structural nor functional relationship, markedly by increased expression and activity of ABC superfamily transporters. Evidences indicate sphingolipids as substrates to ABC proteins in processes such as cell signaling, membrane biosynthesis and inflammation, and products of its biosynthetic route were shown to favor cancer progression. Glucosylceramide (GlcCer) is a ubiquitous glycosphingolipid (GSL) generated by glucosylceramide synthase, a key cell regulator enzyme encoded by the UDP-glucose ceramide glucosyltransferase (UGCG) gene. Under stress, cells increase de novo biosynthesis of ceramides, which return to sub-toxic levels after assimilation into GlcCer by UGCG. Given that cancer cells seem to mobilize UGCG and increase GSL contents for the clearance of ceramides ultimately contributing to treatment failure, we studied how inhibiting GSL biosynthesis would affect the MDR phenotype of chronic myeloid leukemias. Results indicate that MDR associates to higher expression of UGCG and to a complex GSL profile. Inhibition of this glucosyltransferase greatly reduced GM1 expression, and cotreatment with standard chemotherapeutics sensitized cells leading to mitochondrial membrane potential loss and apoptosis. Despite reducing ABCB1 expression, only the ABCC-mediated efflux activity was affected. Consistently, efflux of C6-ceramide, one byproduct of UGCG downregulation, was reduced after inhibition of ABCC-mediated transport. Overall, UGCG inhibition impaired the malignant glycophenotype of MDR leukemias, overcoming drug resistance through distinct mechanisms. This work brings more comprehension about the involvement of GSL for chemotherapy failure, and modulation of its contents emerges as an intervention targeted to MDR leukemias.
As a multifactorial sum of diseases, cancer presents great challenges to the development of safe, successful therapies. Distinct mechanisms employed by transformed cells to avoid toxicity generated by chemotherapy often crosstalk, leading to an adapted phenotype comprising both intrinsic and acquired drug resistance. Multidrug resistance (MDR) is the main hurdle to chemotherapy success, as stress-adapted molecular mechanisms including reduced influx, increased efflux and accelerated metabolism of xenobiotics work in tandem reducing the effective concentration at the molecular target (1). Efflux transporters such as ATP-binding cassette (ABC) proteins actively detoxify cells and tissues from both xenobiotics and toxic metabolites, playing major roles in MDR. Regardless of the diversity of ABC subfamilies and isoforms two proteins are mostly associated to MDR, ABCB1 (P-glycoprotein, Pgp) and ABCC1 (Multidrug Resistance Protein 1, MRP1) (1,2). ABCB1 and ABCC1 share similarities and differences when cellular localization and substrate specificity are considered. The latter is mostly located on plasma membrane, the first is present on any cell membrane, and both were shown to associate to microdomains such as lipid rafts depending on cell subtype (3,4). Both actively extrude a variety of non-related chemotherapeutic drugs, but ABCC1 is able to transport substrates in conjugation with glutathione (GSH) or in cotransport as well (5), playing complementary roles for the MDR phenotype. ABCB1 interacts with liposoluble or amphipathic molecules that are prone to accumulate in the intramembrane space (6), and ABCC1 exhibit higher affinity for negatively-charged glucoronates, sulfates, GSHconjugated compounds and products from lipid metabolism (7).
Sphingolipids such as ceramides and its phosphorylated or glycosylated forms are directly involved in cell fate, assuming active parts on either cell proliferation or death (8). Ceramides, whether originated from sphingomyelin remodeling or synthesized de novo on the endoplasmic reticulum (ER) are transferred to cis-Golgi, where they are employed as substrates to UDP-glucose ceramide glucosyltransferase (UGCG) to form glucosylceramide (GlcCer), the precursor to all glycosphingolipids (GSL). Endogenous ceramides have been directly linked to cancer treatment, given that chemotherapeutic agents with unrelated mechanisms for example paclitaxel, daunorubicin, etoposide (9)(10)(11) and the tyrosine kinase inhibitors sorafenib and imatinib (12) rely on ceramides to drive the intrinsic pathway of apoptosis through caspase activation or caspase-and p53-independent mitotic catastrophe (11,13). Second to their structural role on the organization of lipid rafts (14), GSL relates to development of drug resistance considering that cancer cells often present increased UGCG expression, being able to incorporate ceramides on GSL (15). Concerning MDR, a close crosstalk of ABCB1 and GSL has been observed;

ABCB1
and UGCG were coincidently overexpressed in drug-resistant breast, ovary, cervical, colon cancer and on chronic myeloid leukemias (16,17); GlcCer regulates ABCB1 expression through Wnt/-catenin and cSrc signaling (18); and this transporter is able to act as a flippase on the transfer of GlcCer from the cis-Golgi to trans-Golgi during GSL biosynthesis (18). Despite its capacity of translocating sphingolipids such as sphingosine-1-phosphate (19) and GlcCer on polarized cells (20) and its coexpression with UGCG on colon cancer (21), similar relationship involving ABCC1 activity and GSL is not clear.
Considering the diversity of mechanisms MDR cancer cells resort to avoid and adapt to chemotherapeutic stress and the prime involvement of UGCG on the generation of GSL (22), the fate of endogenous ceramides is critical to successful cancer chemotherapy on a molecular level. Several studies evaluated the expression of ABCB1 and reversal of drug sensitivity on solid tumors and its association with GSL; nevertheless, our work focused on leukemic cells that express both ABCB1 and ABCC1, extending to the functional evaluation of those proteins after UGCG inhibition, which finds little coverage from the literature. In this context, we report the distinct ways ABCB1 and ABCC1 expression and activity were modulated after impairment of GSL biosynthesis on clinically relevant models of drug-resistant chronic myeloid leukemias.

MDR chronic myeloid leukemias overexpress UGCG along with a complex GSL profile, which is reverted after treatment with a ceramide analogue
De novo ceramide synthesis on Golgi increases during stress, and cancer cells are able to upregulate ceramide glycosylation ultimately changing GSL contents on cell membranes. To determine if selection with standard chemotherapeutics would alter these processes on human leukemias, the expression of UGCG, and profiles of GSL and GM1 were evaluated on K562 (drug-sensitive), and on MDR derivatives Lucena-1 (K562/VCR) and FEPS (K562/DNR) cells. Results on Fig. 1A and 1B indicate that the glucosyltransferase that catalyzes the addition of UDP-glucose to ceramide, UGCG, is upregulated on the MDR leukemia models, notably on the FEPS line. Next, the three cell lines were treated with a specific UGCG inhibitor, the ceramide analogue EtDO-P4, and their effects on viability and GSL contents are depicted on Fig. 1C, 1D and 1E. Based on these results, a sub-toxic concentration of this inhibitor was employed for further assays. MDR cells showed increased contents and more complex profiles of GSL after thin-layer chromatography when compared to the parental, and treatment with 1 M EtDO-P4 for 24 h significantly reduced GSL expression on all three leukemias. Since total extraction does not distinguish the GSL on plasma or intracellular membranes, log-phase growing, viable cells were stained with cholera toxin (CHT-FITC). CHT specifically binds GM1 on the extracellular leaflet, and an increase in its fluorescence could be observed on the MDR cells Lucena-1 and FEPS after flow cytometry. In accordance, UGCG inhibition reduced GM1 contents in 55% and 75% on these cells, contrasting to a reduction of 35% on drug-sensitive K562 ( Fig.  1F and 1G).

Pharmacological UGCG inhibition induces cytotoxicity and decreases drug resistance
Disruption of the GSL biosynthesis machinery could lead to accumulation of sphingolipid mediators, altering cell signaling and leading to either survival or death depending on the intrinsic properties of each cell subtype. Thus, the effects of UGCG inhibition on cellular viability are depicted on Table 1. Results indicate that the MDR phenotype and the increased UGCG expression translated into a modest resistance to EtDO-P4, since only FEPS presented higher viability and IC50 after a 72 h treatment ( Fig. 1C and Table 1).
Given that UGCG is the first enzyme on the GSL biosynthesis pathway, inhibition of its activity would impair cell responses to stress. In this context, results on Table 1 suggest that GSL depletion synergizes with the cytotoxicity caused by chemotherapeutic drugs with diverse mechanisms of action, as sub-lethal cotreatment with EtDO-P4 reduced the IC50 for vincristine (VCR) and daunorubicin (DNR) on Lucena-1 and the IC50 for DNR and cisplatin (CDDP) on FEPS cells. This reduction did not manifest on K562, which in line with earlier results, suggests that GSL depletion showed minimal effects on sensitive cells. Noteworthy, treatment with one subproduct from UGCG inhibition, N-hexanoyl-D-erythro-sphingosine (C6-ceramide, C6-cer), induced opposite effect to EtDO-P4 on these cells. The lower IC50 values for C6-cer on Lucena-1 and FEPS than on K562 suggest that this sphingolipid induces collateral sensitivity, a hypersensitivity towards secondary agents that arises from the development of resistance towards an unrelated primary drug.

UGCG
inhibition reduces mitochondrial membrane potential (m) and induces apoptotic cell death Among sphingolipids, ceramides are linked to apoptotic cell death through the intrinsic pathway due to direct or indirect interaction with the mitochondrial permeability transition pore. To investigate if the mitochondria would be involved on the cell death observed after GSL depletion, the MDR leukemias Lucena-1 and FEPS were treated with EtDO-P4, DNR or C6-cer for 24 h and incubated with rhodamine 123 (Rho123). This fluorescent dye accumulates within energized mitochondria, and this retention is progressively lost as m is reduced. Fig. 2A and 2B indicate that only 2 M EtDO-P4 significantly reduced mitochondrial Rho123 fluorescence, a similar result obtained after C6-cer treatment. As expected, DNR was not able to change mitochondria polarization; however, when combined with 1 M EtDO-P4, m was reduced to similar levels to after 2 M EtDO-P4 or 20 M C6-cer treatment. On par with previous results, the stress after UGCG inhibition led MDR cells to apoptosis in a dosedependent fashion, given that 57.09% and 34.06% of Lucena-1 and FEPS, respectively, underwent early or late apoptosis (upper right + lower right quadrants) after 72 h when treated with 2 M EtDO-P4, and over 90% after 4 M ( Fig. 2C and  2D).

Differential contribution of ABC transporters to the reversal of the MDR phenotype after UGCG inhibition
Results so far showed that depletion of GSL led to increased cell death, and this synergizes with chemotherapeutic drugs likely due to accumulation of ceramides on MDR leukemias. ABCB1 and ABCC1 transporters are key drivers to MDR phenotype, actively extruding xenobiotics thus reducing cell death. In this context, the expression of these proteins was evaluated in conditions matching previous assays. Treatment with EtDO-P4 produced, as demonstrated on Fig. 3, distinct effects on expression of ABCB1 and ABCC1. Sub-toxic UGCG inhibition altered the expression of neither ABC protein, whereas treatment with a 2 M concentration of EtDO-P4 for 24 h reduced only ABCB1 expression on both cell lines. It should be pointed that despite 2 M EtDO-P4 induced cell stress on diverse assays after 72 h, cells were still viable after 24 h (Fig. S1).
Regardless of not altering ABCB1 or ABCC1 expression on mild conditions, the ABCmediated transport after pre-treatment with EtDO-P4 was investigated as well. For this, cells were then incubated with specific fluorescent substrates for ABCB1 and ABCC transporters, and dye retention after free or inhibited efflux were analyzed by the median fluorescence intensity (MFI). Again, results in Fig. 4 indicate that GSL depletion did not impair ABCB1-mediated transport, since profiles of Rho123 MFI were similar irrespective of EtDO-P4 treatment. ABCC activity, on the other hand, was significantly modulated after treatment, since carboxyfluorescein (CF) MFI was higher after both free and inhibited efflux for Lucena-1 ( Fig. 5A and 5C) and for FEPS ( Fig. 5B and 5D). It is important to note that CF may be transported by other ABCC subfamily members than ABCC1, but not by ABCB1, given that ABCB1 inhibition with VP did not affect CF MFI, despite the MDR leukemias expressing both ABCB1 and ABCC1 (Fig. S2).

ABCC but not ABCB1 transports one byproduct of a compromised GSL biosynthesis pathway
The roles of GSL and ABC transporters for the adaptation to cellular stresses are well discussed, and pharmacological impairment of UGCG was demonstrated to efficiently modulate the main features of the MDR phenotype. However, when analyzed as whole, results point to MDR leukemias being able to actively reduce ceramide levels by mechanisms apart from glycosylation. To examine this possibility, the efflux assays of ABCB1 and ABCC substrates were performed on untreated cells in the presence of C6-cer as competitive inhibitor, and the resultant MFI and percentages of cells loaded with either Rho123 or CF after the efflux phases were evaluated. In agreement with previous experiments, one more time the ABCB1 efflux of Rho123 was not altered in the presence of increasing C6-cer concentrations ( Fig. 6A-D and Fig. S3A). An opposite outcome was observed when C6-cer was co-incubated during the CF efflux assay, as results indicate CF retention in both Lucena-1 and FEPS. MFI was dose-dependently increased on both MDR cells, notably when 40 M C6-cer was employed as a competitor to ABCC-mediated efflux ( Fig. 7A-D). This profile could be better appreciated when observing the percentages of CF+ cells, which reached 50% in the presence of 40 M C6-cer (Fig.  S3B). These results suggest that C6-cer could be transported by ABCC proteins.
To further delineate if ceramides would be directly transported out by ABC proteins, the activity assay was once more performed on untreated cells, this time with a fluorescent, nitrobenzodiaxole-labeled ceramide derivative (C6-NBD-cer) as substrate. Results on Fig. 8 corroborate that ABCC transporters mediate the efflux of C6-NBD-cer, owing to fact that the MFI and percentages of C6-NBD-cer+ cells significantly increased in the presence of MK-571 but not VP when compared to controls (Fig. 8A-D and Fig. S3C).

Discussion
A number of studies successfully demonstrated associations linking glycosphingolipids and multidrug resistance in several solid tumor types through overexpression of UGCG (23), though few addressed how would its inhibition affect both the expression and efflux activity of ABCB1 and ABCC1 on non-polarized cells. There is evidence that the transport of membrane lipid analogs varies in cellular localization; although ABCB1 translocates C6-NBD-sphingolipids across the apical plasma membrane, ABCC1 seems to transport those analogs to the basolateral plasma membrane on the polarized kidney cell LLC-PK1 (20). Observations that are more recent indicate that ABCB1 and ABCC1 differ in cellular localization, and ABCB1 was shown to transport ceramides along Golgi membranes as well (18). Albeit these observations suggest a similar aptitude to transport sphingolipids, the cellular and organ expressions of ABCB1 and ABCC1 likely take parts on how diverse cells manage sphingolipid levels during proliferation, differentiation, apoptosis and response to cellular stresses. Cells from epithelial origin may express ABC proteins in opposite sides of the cell membrane in a way that, for both bloodbrain barrier cells and in the placenta, ABCB1 and ABCC1 are considered, respectively, apical and basolateral transporters (24,25). Our study proposes, given that peripheral leukemic cells from hematopoietic origins do not present this organization, that ABCB1 and ABCC1 might play complementary yet diverse roles in dealing with xenobiotics and/or sphingolipid mediators.
In a normal bone marrow, it has been demonstrated that GSL levels vary among the distinct stages of erythrocyte differentiation, in a way that GM3 is increased on more differentiated cells such as megakaryocytes (26). Likewise, the efflux activity mediated by ABC transporters is important for the development of hematopoietic progenitors, since ABCB1 and ABCC1 are present on cells with undifferentiated phenotypes on bone marrow or peripheral blood from human (27,28) and murine (29,30) origins. Chronic myeloid leukemias result from a reciprocal translocation of BCR and ABL genes among chromosomes 9 and 22 during the erythroblast stage, forming the Philadelphia chromosome. This abnormality contains the chimeric BCR-ABL oncogene, which encodes a protein with constitutive tyrosine kinase activity (31) that sustains proliferative cell signaling and evasion of apoptosis through increased membrane GM1 among other phenotypes (32). Therapy is performed with tyrosine kinase inhibitors such as imatinib mesylate in combination with standard chemotherapeutic drugs for remission (33). Among 30% of patients display some degree of resistance to these drugs, which is closely associated to the expression and activity of ABCB1 (34).
Despite originating from the highly undifferentiated erythroblastic K562 cell line, Lucena-1 and FEPS MDR cells used in this work present higher number of megakaryocytes as well as more differentiated profiles (35). Conversely, it was reported that K562 cells selected for resistance with minimal concentrations of vinblastine and epirubicin, drugs that share similarities in structure and in modes of action with VCR and DNR, showed increased ABCB1 expression with no correlation to specific markers of erythroid origin (36). Considering this, we used different assays to assess GSL and UGCG expression. Our results indicated that selection with the chemotherapeutic drugs VCR or DNR led to a complex and diversified GSL profile, despite UGCG was only significantly increased on FEPS. In this context, treatment with the UGCG inhibitor EtDO-P4 dosedependently reduced viability on all three cells, nonetheless only Lucena-1 and FEPS showed significant reductions in GM1 levels. A closer observation of the histograms of treated and control cells suggests that MDR cells are more homogenous in GM1 contents than the parental K562. In parallel, both MDR leukemias were sensitized to DNR, Lucena-1 to VCR and FEPS to CDDP as well. This may relate to the specific mechanism of action exerted by each chemotherapeutic during selection, since small differences in expression and in the ceramide portions of sialylated glycolipids could be observed between Lucena-1 and FEPS. Doxorubicin, a DNR analog, was demonstrated to increase ceramide production and UGCG expression on an assortment of tumor cells with variable degrees of drug resistance (37), thus strengthening the relationship between sphingolipids and drug resistance.
Pharmacological UGCG inhibition could be increasing ceramide levels, which along with the stress caused by chemotherapeutics, would explain the higher sensitivity of MDR cells. K562 would possibly shift ceramide glycosylation to galactosylceramide (GalCer) rather than GlcCer, a fact that was described to happen on U-937 and HL-60 human leukemic cells, when the EtDO-P4 analogs PPMP and PDMP actively protected those cells to DNR toxicity (38). In accordance with our results, MDR cells showed lower mitochondrial membrane potentials (m) when GSL depletion was concomitant with DNR treatment, with toxic EtDO-P4 concentrations or when C6-cer was added to the cultures. In those conditions, ceramides could interact with energized mitochondria through direct physical contact or by inducing conformational changes on Bax pro-apoptotic protein leading to intrinsic pathway of apoptosis or caspaseindependent cell death (39,40). Though our results would not discern those mechanisms the participation of mitochondria in the apoptosis induced after EtDO-P4 is clear, considering that the m loss observed after 24 h would translate into reduced viability, phosphatidylserine exposure and DNA fragmentation after 72 h.
The equilibrium of ceramides and GlcCer, GalCer or GSL may influence cell fate and in extension, the success of therapeutic interventions, and a variety of drug-resistant cell lines present increased cholesterol, sphingomyelin and GSL in comparison to their respective sensitive counterparts (41). The MDR phenotype of cells used in this work is well described, being representative of freshly obtained cells from patients with chemotherapy refractory chronic myeloid leukemia (42,43). In spite of data indicating that GlcCer modulates ABCB1 expression (17), and considering the promiscuity and overlap in recognition of substrates (44), the relevance of GSL to ABC activity is controversial at best (45). As such, we observed that sub-toxic GSL depletion was not able to affect the expression of ABCB1 and ABCC1 on neither MDR cell; a 24 h incubation with 2 M EtDO-P4, however, showed reductions in the expression of ABCB1 exclusively. Conversely, the effect of GSL depletion on the efflux performed by ABCB1 or ABCC subfamily members was the opposite; the fluorescence for CF retained inside Lucena-1 and FEPS pre-treated with EtDO-P4 was consistently higher after the efflux assays, indicating that ABCC transport was partially hindered but not the one mediated by ABCB1.
Lucena-1 and FEPS are cross-resistant to a diversity of compounds with natural and synthetic origins owing to, but not limited to, its high efflux activity mediated by the ABC transporters ABCB1 and ABCC1, and to imatinib mesylate as well, which has been demonstrated to increase ceramide and reduce sphingosine-1-phosphate levels on chronic myeloid leukemias (46). Resistance to that inhibitor associates to expression of ABCB1, however with no clear correlation to its efflux activity (47), which suggests that these cells manage their ceramides in alternative forms, involving or not ABCC1 in specific. In this context, ABCB1-mediated efflux activity was demonstrated to be disconnected from the translocation of ceramides on Golgi (48), and the ABCC1 inhibitor MK-571 was shown to affect retrograde membrane transport and to reduce GlcCer formation (49), evidences that point to at least partial involvement of ABCC in regulating sphingolipid levels. Albeit our results concerning ABCC inhibition could not be directly associated to ABCC1 since CFDA, PRB and MK-571 act as substrates and inhibitors to other members from ABCC subfamily (50), to the best of our knowledge, neither Lucena-1 nor FEPS express ABCC2 or express in significantly lower levels than ABCC1 (51). The participation of other ABCC subfamily members such as ABCC3 and ABCC4, however, could not be discarded and should be taken into account in future studies.
It is somewhat difficult to find suitable combinations of substrates and inhibitors to probe the efflux mediated by each subfamily member and, as such, we opted for the most studied and widely employed ones. CFDA, as stressed in our work, may be transported by other ABCC subfamily members than ABCC1, but not by ABCB1. In the same context, a study performed by Dogan et al. in 2014, observed the same outcome on 11 leukemia cell lines with diverse degrees of drug resistance (50). That study considered CFDA or calcein the best substrates to prime ABCC1 activity, and given that calcein is a well-known ABCB1 susbtrate (52), our choice of substrates is in accordance with the literature. The same logic applies to the inhibitors. VP, shown to present ABCC1 modulation properties (53), was selective to ABCB1 when combined with Rho123 as substrate and did not affect CFDA efflux. MK-571 on the other hand is a potent, specific inhibitor of ABCC subfamily proteins that has no effect on ABCB1 (50). It is important to notice that we employed PRB as opposed to the more specific MK-571 ABCC inhibitor during the efflux assays on EtDO-P4 pretreated cells since it could interfere with ceramide levels (49). As such, when combined to the aforementioned substrates, VP (for ABCB1) and PRB or MK-571 (for ABCC) minimize the possibility of cross-interference.
When C6-cer, one possible subproduct from UGCG inhibition and a known mediator of cell stress, was evaluated as a competitor to either ABCB1 or ABCC transport results were similar to the one after EtDO-P4 treatment, provided that this sphingolipid impaired the ABCC-mediated CF efflux from Lucena-1 and FEPS and increased percentages of CF+ cells after the challenge. One more time, significant changes in neither ABCB1mediated Rho123 efflux nor Rho123+ cells were observed. Finally, when a fluorescent derivative of C6-cer was employed as sole substrate, once more the MFI and the percentages of C6-NBD-cer+ were significantly higher when ABCC activity was inhibited, with a marginal increase in presence of a ABCB1 inhibitor. It is important to disclose that the efflux assays with C6-cer were performed on shorter (1 h) times than viability assays (72 h), and with a higher cell count as well (2×10 5 cells on the first; 2×10 4 cells.mL -1 on the latter).
Noteworthy, the effects of C6-cer and EtDO-P4 on MDR cells seemed inversely correlated to K562. Lucena-1 and FEPS showed greater decreases in GM1 contents, moderate resistance to EtDO-P4 cytotoxicity and sensitivity to C6-cer-induced viability loss. Although sharing a common precursor, the chemotherapy employed during selection resulted in distinctive phenotypes, in a way that a microarray analysis showed 130 genes with altered expression comparing K562 and Lucena-1, 932 between K562 and FEPS, and 1211 between the two MDR lines (51), ABCB1 being the most overexpressed gene in these cells. Considering this, it was beyond our focus to thoroughly investigate the specific way each cell line would respond to UGCG inhibition; nonetheless, our results point to the possibility of exogenous C6-cer as well as the combination of EtDO-P4 and standard drugs inducing collateral sensitivity, a hypersensitivity towards secondary agents that arises from the development of resistance towards an unrelated primary drug. Those agents passively enter the cell and, after being extruded by ABCB1 or ABCC1, repeat this futile cycle, increasing ATP consumption. Replenishment of ATP increases oxidative stress and the demand for glutathione, a prime ABCC1 substrate (44) and the main peptide involved on defense to oxidative stress. This ultimately leads cells to a form of synthetic lethality due to depletion of these molecules (54,55). Downregulation of ABCB1 after UGCG impairment would increase intracellular levels of chemotherapeutics and, as consequence, de novo production of ceramides in the ER. In parallel, ABCC1 would mediate the efflux of those sphingolipids and drugs as well, further depleting cells of glutathione and ATP, resulting in m loss and apoptosis through collateral sensitivity. This possibility is feasible once we consider that treatment with C2-ceramide reduced levels of this peptide on a model of epidermal tumor (56) and induced m loss and apoptosis on models of prostate and colon cancer (40).
In a broader scope, high levels of plasma circulating ceramides can result from a diversity of inflammatory-associated conditions such as diabetes, obesity and cardiovascular diseases, possibly due to changes in remodeling of membrane sphingolipids (57). Concerning cancer, the first study to demonstrate clinical relevance of ceramides to breast cancer, reported higher ceramide levels in the neoplastic tissue than in peritumor region or in the plasma of primary breast cancer patients, associated with better prognostic (58). Strikingly, a few studies related increased long-chain ceramide levels on the plasma from patients with advanced ovarian (59) and pancreatic cancer (60), related to higher malignancy. Therefore, participation of ABC transporters on ceramide homeostasis and in an efflux mechanism to extracellular plasma acceptors such as apolipoproteins, apart from ceramide glycosylation or conversion to sphingomyelin had already been proposed but not demonstrated (61). Given the ubiquitous expression of ABCC1, its active efflux of inflammatory lipid mediators such as leukotriene C4 and sphingosine-1-phospate and its association to poor prognostic in cancer (19,44), ABCC1 emerges as candidate for this role. All things considered, our data indicate that ABCB1 and ABCC subfamily transporters are differentially downregulated following UGCG inhibition.
In this work, our results extend the relationship between sphingolipid glycosylation and acquired resistance mechanisms on models of human MDR neoplastic cells. Further studies addressing the modulation of sphingolipid levels and their cellular fates could translate in the development of strategies directed to MDR hematologic neoplasias, or to the proposal of an adjuvant, off-label use of drugs currently employed against Gaucher's disease, which accumulates GlcCer in multiple organs as in the bone marrow. Comprehension of the distinct roles of ABC proteins on the clearance of ceramides could ultimately return therapeutic possibilities to patients with chemotherapy refractory leukemias.

Experimental procedures Cell lines
The chronic myeloid leukemia cell lines K562, Lucena-1 (K562/VCR) and FEPS (K562/DNR) were cultured in RPMI-1640 medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 25 mM HEPES and 2 g.L -1 sodium bicarbonate adjusted to pH 7.4, 100 U penicillin and 100 g.mL -1 streptomycin (all obtained from Sigma-Aldrich) and with 10% fetal bovine serum (FBS) (Thermo Fischer Scientific, Waltham, MA, USA) inactivated at 56 °C for 1 h prior to use. Dr. Vivian M. Rumjanek kindly donated the MDR cells Lucena-1 and FEPS. Briefly, K562 cells were exposed to increasing concentrations of the chemotherapeutic drugs vincristine sulfate (VCR) and daunorubicin hydrochloride (DNR) (both from Sigma-Aldrich), as described before (62,63). For subcultures, 2×10 4 cells.mL -1 were harvested every 3 days, complete RPMI was added and then maintained at 37 ºC in 5% CO2. Lucena-1 and FEPS were cultured, respectively, in the presence of 60 nM VCR and 500 nM DNR in order to maintain the MDR phenotypes. Prior to all experiments, the MDR Lucena-1 and FEPS were cultured free of drugs to avoid additive effects.

Western blot analysis
K562 and their MDR counterparts Lucena-1 and FEPS were cultured as described before for 48 h, then lysed in lysis buffer (1% Triton X-100; 150 mM NaCl; 25 mM Tris, pH 7.4; 5 mM EDTA; 0.5% sodium deoxycholate; 0.1% SDS; 5 mM tetrasodium pyrophosphate; 50 mM sodium fluoride; 1 mM sodium orthovanadate) containing 1:200 protease inhibitor cocktail (Sigma-Aldrich), and centrifuged at 21000×g for 10 min at 4 ºC. 60 g protein was subjected to SDS-PAGE, followed by transfer to PVDF membranes (Millipore, Burlington, MA, USA). After blocking with 5% skim milk for 30 min, membranes were rinsed with T-TBS and stained with anti-UGCG (clone 1E5; Santa Cruz Biotech, Dallas, TX, USA) and anti-βactin (clone AC-74, Sigma-Aldrich) antibodies overnight at 4 °C. Membranes were rinsed, incubated with anti-mouse IgG-HRP (Cell Signaling Technology, Danvers, MA, USA) for 2 h at room temperature. After rinsing with T-TBS, membranes were developed by use of Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer, Waltham, MA, USA). Densitometry analyses were performed using ImageJ 1.52d software (U.S. National Institutes of Health, Bethesda, MA, USA) (67). The relative expression of UGCG was calculated as the density of the UGCG band divided by the density of the -actin band for each cell line.

Assessment of GM1 expression on plasma membranes
The glycosphingolipid monosialotetrahexosylganglioside (GM1) was assessed on cell surfaces by flow cytometry. K562, Lucena-1 and FEPS cells were cultured for 24 h as described before. Cell density was adjusted to 2×10 5 per well and then incubated with 5 μg.mL -1 fluorescein isothiocyanate-conjugated cholera toxin (CHT-FITC, Sigma-Aldrich) for 30 min at 4 ºC in a light-protected environment. Cell suspensions were centrifuged, resuspended in cold PBS and then analyzed by flow cytometry. Five identical polypeptide B subunits from CHT specifically bind to the GM1 on cell surfaces, and considering that the GSL biosynthesis occurs in a stepwise fashion, the GM1 MFI can relate to the global cell GSL composition (66).

Apoptosis assay
The Annexin V/propidium iodide (PI) assay was performed for apoptosis detection. Lucena-1 and FEPS cells were incubated in 24-well plates in the conditions described above and treated with EtDO-P4. After 72 h, cell density was adjusted to 5×10 5 cells per sample, washed with phosphatebuffered saline (PBS) supplemented with 5% FBS and resuspended in a solution of Annexin V-FITC and propidium iodide (PI) (BD Biosciences, San Diego, CA, USA) in accordance with the manufacturer's protocol. Cells were incubated at room temperature for 15 min and analyzed by flow cytometry. Dot-plots were divided into four quadrants as follows: upper left (PI+/Annexin-V-), necrotic cells; upper right (PI+/Annexin-V+), late apoptotic cells; lower left (PI-/Annexin-V-), viable cells; lower right (PI-/Annexin-V+), early apoptotic cells.

Assessment of mitochondrial membrane potential
Changes in the mitochondrial membrane potential (m) were probed using the rhodamine 123 dye (Rho123; Sigma-Aldrich), which specifically stains energized mitochondria in cultured cells (68). Lucena-1 and FEPS cells were cultured for 24 h with DNR, EtDO-P4 or C6-cer as described before. Following, cell density was adjusted to 2×10 5 per sample and then incubated with 2.5 M Rho123 for 30 min at 37 ºC in 5% CO2 in a light-protected environment. Cell suspensions were centrifuged, resuspended in cold PBS and then analyzed by flow cytometry.

Immunophenotypes of MDR cells
The expression of the ABC transporters ABCB1 and ABCC1 was evaluated by flow cytometry. Lucena-1 and FEPS were treated with EtDO-P4 for 24 h as described before, cell density was adjusted to 2×10 5 per sample at room temperature, then cells were permeabilized and fixed with BD FACS Lysing solution (BD Biosciences) for 10 min and non-specific binding was blocked with PBS 10% FBS for 20 min. Following incubation with anti-ABCB1 (clone D-11) or anti-ABCC1 (clone QCRL-1) human primary antibodies (both from Santa Cruz Biotech, Dallas, Texas, USA) for 30 min at 4 ºC, cells were washed with cold PBS and stained with Alexa488conjugated mouse anti-human IgG secondary antibody (Thermo Fischer Scientific) for 30 min at 4 ºC in a light-protected environment. Cell suspensions were centrifuged, resuspended in cold PBS and then analyzed on a flow cytometer.

ABC-mediated efflux assays
The ABCB1 and ABCC transport assays were performed, respectively, with the use of the Rho123 and 5(6)-carboxyfluorescein diacetate (CFDA) dyes (Sigma-Aldrich). Rho123 and CFDA are able to passively distribute into the cell, and while the first is fluorescent and is actively extruded by ABCB1, the latter undergoes hydrolysis by nonspecific esterases in the cytosol, originates the fluorescent substrate carboxyfluorescein (CF) that only then is transported out by ABCC subfamily members, notably ABCC1 (69). Briefly, assays were performed in two 30-minute steps, sufficient for the accumulation and efflux of dyes, carried on at 37 ºC in 5% CO2 in a light-protected environment. 2×10 4 cells.mL -1 were treated for 24 h with 1 M EtDO-P4 as described prior to the assays. Then, 2×10 5 Lucena-1 or FEPS cells were incubated in 96-well plates with 250 nM Rho 123 or 500 nM CFDA diluted in RPMI medium to allow accumulation of the dyes within cells. Following, cells were centrifuged at 200×g for 7 min and resuspended in fresh RPMI medium to allow efflux of the dyes (free efflux). In parallel, cells were incubated with the ABCB1 inhibitor verapamil (VP) (70) or the ABCC inhibitors probenecid (PRB) or MK-571 (50) (inhibited efflux), respectively at the concentrations of 10 M, 1.25 mM and 25 M. As negative control, cells were exposed to medium only. Next, cells were again centrifuged, resuspended in cold PBS and maintained on ice until acquisition by flow cytometry. Alternatively, C6-cer was employed as a competitive inhibitor during both steps of the assay. Fluorescence histograms were divided into two distinct areas: Rho123 or CF-negative on the left, accounting for 95% of control cells with low MFI for Rho123 or CF, and Rho123 (Rho123+) or CF-positive (CF+) on the right, corresponding to cells still loaded with dyes after efflux phase. A vertical line on each graph indicated those regions.

C6-ceramide efflux assays
Assays were performed in two 30-minute steps, in similar conditions to the ABC-mediated efflux assays. 2×10 5 Lucena-1 or FEPS cells were incubated in 96-well plates with 1 M nitrobenzoxadiazole-labeled C6-ceramide (C6-NBD-cer) (Avanti Polar Lipids) diluted in 0.1% absolute ethanol to allow accumulation of the fluorescent sphingolipid within cells. In parallel, cells were incubated with the ABCB1 inhibitor verapamil (VP) or the ABCC inhibitor MK-571 (inhibited efflux). As negative control, cells were exposed to medium only, and treated as described before for acquisition by flow cytometry. Fluorescence histograms were divided into two distinct areas: C6-NBD-cer-negative on the left, accounting for 95% of control cells with low MFI, and C6-NBD-cer-positive (C6-NBD-cer+) on the right, corresponding to cells still loaded with the fluorescent sphingolipid after efflux phase. A vertical line on each graph indicated those regions.

Flow cytometry
The median fluorescence intensities (MFI) from 15,000 viable cells, gated in accordance with forward (FSC) and side scatter (SSC) parameters representative of cell size and granularity, were acquired using the FL1-H filter on a BD FACSCalibur flow cytometer (BD Biosciences). All post-analyses were performed on Summit version 4.3 software (Dako Colorado, Inc., Fort Collins, CO, USA).

Statistical analysis
Statistical analyses were performed using GraphPad Prism version 7.0 software. For two paired comparisons, statistical significance was calculated by parametric Student's t-tests for normally distributed data, according to the D'Agostino-Pearson test. Otherwise, the nonparametric Wilcoxon test was employed. For more than two comparisons, unpaired one-way ANOVA or Kruskal-Wallis tests were used, respectively, for parametric and nonparametric data. Otherwise, paired one-way ANOVA and the Friedman test were employed for parametric and nonparametric data respectively. Bonferroni's post-test was used for parametric data while the Dunn's post-test was employed for nonparametric data. Null hypotheses were rejected when p-values were lower than 0.05, and significances were represented by (*) for p <0.05, (**) for p <0.01 and (***) for p <0.001.