THE LACTATE RECEPTOR (HCAR1/GPR81) CONTRIBUTES TO DOXORUBICIN CHEMORESISTANCE VIA ABCB1 TRANSPORTER UP-REGULATION IN HUMAN CERVICAL CANCER HeLa CELLS

L-lactate functions as a hormone via the hydroxycarboxylic acid receptor 1 (HCAR1, also referred to as HCA1 or GPR81) in adipose tissue (1) or brain cells (2). Recently, an important role of HCAR1 and its natural agonist, lactate, in cancer biology was also demonstrated. Previously, our group showed the essential role of HCAR1 and monocarboxylate transporters (MCT4) in the lactate-mediated enhancement of cellular DNA repair capacity in cervical cancer cell lines (3). Similar observations have also been reported for pancreatic and breast cancer cells (4, 5).

HCAR1, is a member of the G-protein-coupled receptor (GPCR) family involved in tumor progression, invasion and metastasis (6, 7). Li and coworkers demonstrated that, upon agonist binding, HCAR1 receptors activate Gi protein that subsequently induces Erk1/2 activation, primarily through a PKC-dependent pathway (8). Interestingly, PKC signaling pathway has been reported to modulate drug resistance by up-regulation of ABC transporters in cancers (9-14).

The ATP-binding cassette (ABC) protein family in normal physiological conditions is responsible for preventing over accumulation of toxins within the cell by efflux of many xenobiotics, including anthracyclines, taxanes, vinca alkaloids, etc. While the broad substrate specificity is a desirable feature in normal cells, the efflux mechanisms may protect cancer cells from first line cytotoxic drugs and be responsible for ineffectiveness of chemotherapy. Thus, their presence in many tumors often resulted in unsuccessful cancer therapy outcomes (15). The most extensively characterised transporter within ABC protein family is ATP-binding cassette sub-family B member 1 (ABCB1), associated with resistance to doxorubicin, paclitaxel and vincristine (15). Another well known proteins mediated multidrug-resistance phenotype are ABCG2-implicated in resistance to camptothecin analogues and mitoxantrone (15, 16), and ABCC1, that confers resistance to folate-based antimetabolites, anthracyclines, vinca-alkaloids, and anti-androgens (15, 17).

Herein, we showed another mechanism of HCAR1-mediated resistance to doxorubicin in cancer cells. We demonstrated for the first time that cancer cells exposed to HCAR1 agonists (L-lactate, D-lactate or 3,5-dihydroxybenzoic acid (DHBA) showed increased, while HCAR1-silenced cells decreased, both expression and activity of ABCB1. The observed up-regulation of ABCB1 was accompanied by decrease in doxorubicin accumulation in HeLa cells and a subsequent apoptosis decrease. In addition, we could observe reduced survival of HCAR1-deprived cells after treatment with doxorubicin. Finally, we showed that PKC pathway is involved in regulation of ABCB1 activity in HeLa cells.

MATERIALS AND METHODS

Chemicals

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Go6983 was dissolved in DMSO and added to cells at a final DMSO concentration of 0.1% (v/v). Control cells were incubated in 0.1% DMSO alone.

Cell culture

The human cancer cell line HeLa was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and was authenticated by short tandem repeat profiling (LGC Standards, UK) before the study began. The HCAR1 shRNA-expressing HeLa cells were obtained as described previously (3). Cells were cultured in DMEM (Life Technologies) supplemented with 10% foetal bovine serum (PAA Laboratories GmbH, Pasching, Austria) and antibiotics (Life Technologies) at 37°C in a humidified atmosphere containing 5% CO2. HCAR1-silenced cells were additionally supplemented with 7.5 µg/ml of puromycin. The cells were routinely tested for mycoplasma contamination and were passaged every three or four days using TrypLE Express (Life Technologies).

Real time-PCR

A detailed experimental procedure for real-time PCR and a list of DNA repair gene primers (including housekeeping genes) were described by Wagner et al. (3). The following primers were used for the human ABC transporter genes:

ABCB1 F:5’-GCTCCTGACTATGCCAAAGC-3’,

R: 5’-TCTTCACCTCCAGGCTCAGT-3’;

ABCC1 F:5’-AGTGGAACCCCTCTCTGTTTAAG-3’,

R: 5’-CCTGATACGTCTTGGTCTTCATC-3’;

ABCG2 F:5’-CACCTTATTGGCCTCAGGAA-3’,

R:5’-CCTGCTTGGAAGGCTCTATG-3’.

All gene expression values were normalised to the housekeeping genes HMBS and HPRT before calculating the ratios.

Western blot analysis

The experimental design for evaluating HCAR1, ABCB1, ABCC1 and ABCG2 protein levels was described previously (3). The following primary antibodies were used: anti-GPR81/FKSG80 (SAB2501267, Sigma-Aldrich), anti-MDR1(ABCB1), anti-MRP1(ABCC1), and anti-β-actin conjugated with HRP (sc-55510, sc-365635, sc-642, and sc-1616, respectively, Santa Cruz Biotechnology, Inc.), and anti-BCRP/ABCG2 (ab3380, Abcam, Cambridge, UK).

ABCB1 immunocytochemistry

Cells grown on Lab-Tek chamber slides (Nunc, Thermo Fisher Scientific, Inc.) were fixed with 4% formaldehyde and blocked with 3% BSA in PBST for 1 hour. Then, cells were stained with an anti-MDR1 (ABCB1) antibody overnight at 4°C. Primary antibody binding was visualised using an Alexa Fluor 546-conjugated goat anti-mouse antibody (Life Technologies) followed by nuclear staining with 1 µg/ml Hoechst 33342 for 20 min. Images were acquired using a confocal microscope (Nikon D-Eclipse C-1 Plus) equipped with a 63× objective.

Functional analysis of ABCB1 transporter

Accumulation assays engaging the following cell-permeant, fluorescent ABCB1 substrates: calcein-AM, doxorubicin (DOX), X-rhod-1 (Life Technologies) and rhodamine 123 were used to evaluate ABCB1 transport function using flow and image cytometry. For flow cytometry analysis, cells were trypsinized and suspended at 106 cells/ml density. Two fluorescent substrate-based transport assays were performed using calcein-AM and DOX. For each transport experiment with either calcein-AM or DOX the cell samples were split into two separate fractions - one with fluorescence dye and fairly specific inhibitor of ABCB1 (verapamil) and second with fluorescent dye only. Calcein-AM and verapamil were added at final concentration 100 nM and 10 µM, respectively. Time-course experiments were conducted for 20 minutes and fluorescence signal was measured every 4 minutes using BD LSRII flow cytometer (excitation 488 nm, detection in FITC channel). Similar conditions were used for doxorubicin accumulation assay (DOX concentration was 1 µM, and fluorescent signal was detected in PerCP channel). The resulting fluorescence values (arbitrary units, au) of each sample were plotted against time (min) and the curves obtained were used to calculate area under the curve (auc (au/min)) parameter describing substrate transport (GraphPad Prism software). Specific activity of ABCB1 transporter (Δauc) was calculated as a difference between auc of sample with fluorochrome and inhibitor, and auc of sample with fluorochrome only. Experiments were performed three times.

For image cytometry analysis, cells grown on a 96-well plate were incubated in the presence of ABCB1 substrates: 0.35 µM X-rhod-1, 0.5 µM rhodamine123 or 10 µM DOX alone or with verapamil (20 µM; followed by 1 hour incubation in verapamil containing medium) for 45 min (X-rhod-1, rhodamine123) or 90 min (DOX). After washing with PBS and nuclei staining with Hoechst 33342, cellular fluorescence resulting from X-rhod-1, rhodamine123 or DOX intracellular accumulation, was immediately measured using an ArrayScan VTI HCS Reader equipped with a 10 × objective. Images of five fields per well were acquired, and 150 cells/well were analysed using Molecular Translocation Bioapplication V3 software. The results were shown as a mean cellular fluorescence. Experiments were performed three times, each in six replicates.

DNA cell content analysis using image cytometry

Control and HCAR1-silenced cells grown on a 96-well plate were treated with DOX (100, 250, 500, or 1000 nM) for 30 min, washed and further incubated for 24 hours. After washing with ice-cold PBS, cells were fixed with 4% formaldehyde for 20 min and stained with 200 ng/ml Hoechst 33342 for 45 min at RT. Images of 16 fields per well were acquired using an ArrayScan VTI HCS Reader (Thermo Fisher Scientific, Inc.) equipped with a 10× objective. The cells number and fluorescence intensity was analysed using Cell Cycle Bioapplication V3 software. Experiments were performed three times, each in six replicates.

Growth Inhibition Assay (GIA) and detection of cell apoptosis

For GIA, control and HCAR1-silenced cells were seeded in 96-well plates at 1000 cells/well in 100 µl of medium. Twenty-four hours after seeding, cells were exposed for 30 min to various concentrations of doxorubicin and washed and cultured in drug-free medium for 3 days. Cell growth inhibition was assessed by neutral red uptake assay.

Early apoptosis in doxorubicin treated cells was measured using Annexin V binding assay. Cells grown on a 96-well plate were treated with 1 µM DOX alone or with DHBA (500 µM; followed by 24 h incubation in DHBA containing medium) for 30 min. After 72 hours, cells were stained with 2 µg/ml Hoechst 33342 for 45 min, washed with staining buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4), and incubated with Annexin V conjugated with Alexa Fluor 488 (Life Technologies) for 15 min at RT. The plate was analysed using an ArrayScan VTI HCS Reader equipped with a 10× objective. Images of 16 fields per well were acquired, and cellular fluorescence intensity was analysed using Cell Health Profiling Bioapplication V3 software. Experiments were performed three times, each in six replicates. The data are presented as the mean ± S.E.M. of the percentage of cells exhibiting greater cellular fluorescence than the untreated population.

Statistical analysis

The experiments were conducted in three or six replicates and performed in at least three independent experiments. The data are presented as the mean ± S.E.M. GraphPad Prism software was used to analyse and plot data. Statistical significance was evaluated using a Student’s t-test or one-way ANOVA followed by Tukey’s test.

RESULTS

HCAR1 agonists induce ABCB1 expression

Recently, we have shown that lactate through histone deacetylase inhibition and HCAR1 activation participate in the resistance of cervical carcinoma cells to anticancer drugs (3). To further explore the potential mechanism of HCAR1-mediated resistance to DOX, we determined whether the effects of HCAR1 agonists was related to induction of the multidrug resistance (MDR) phenomenon. One of the key protein conferring resistance to clinically used anticancer drugs is ABCB1. The ABCB1 expression was measured after 24 h incubation of HeLa cells with L-lactate, D-lactate, or DHBA. We observed that DHBA up-regulated ABCB1 both, at the mRNA and protein level (2.9-fold and 1.3-fold, respectively) (Fig. 1A, 1B). L- and D-lactate exposure resulted in less pronounced changes as they increased ABCB1 mRNA level by 1.6- and 2-fold. However, the lactate effect on ABCB1 protein level was only slightly visible (Fig. 1B).

Fig. 1. Effects of HCAR1 stimulation on ABCB1 expression in HeLa cells. Relative levels of ABCB1 mRNA (A) and ABCB1 protein (B) in HeLa cells incubated in the absence or presence of 20 mM L-lactate, 20 mM D-lactate, or 500 µM DHBA for 24 hours. The real-time PCR results are presented as fold change in mean gene expression ± S.E.M. relative to untreated cells from at least three independent experiments. Gene expression values were normalised to the housekeeping genes HMBS and HPRT before calculating the ratios. The representative Western blot of three independent experiments is shown. The ABCB1 protein level was quantified using densitometry, normalised to β-actin and presented as mean fold change ± S.E.M. relative to untreated cells from at least three independent experiments. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s test. *P < 0.05 and **P < 0.01 indicate significant differences compared to the untreated cells.

HCAR1 is required for ABCB1 expression

To assess the role of HCAR1 in regulation of ABCB1 expression, we analysed ABCB1 expression in established HCAR1 shRNA-expressing cells, that revealed down-regulation of mRNA and protein level of HCAR1 by 80 and 40%, respectively (Fig. 2A, 2B). Interestingly, these results indicated a correlation between ABCB1 and HCAR1 expression as the mRNA and protein levels for ABCB1 were affected similarly to HCAR1. Additionally, the obtained results were confirmed by a microscopic study of HCAR1-silenced cells stained with an anti-ABCB1 antibody (Fig. 2C).

Fig. 2. Silencing of HCAR1 diminishes ABCB1 expression in HeLa cells. (A) The relative mRNA level of HCAR1 and ABCB1 in control and HCAR1 shRNA-expressing HeLa cells. The real-time PCR results are presented as fold change in mean gene expression ± S.E.M. relative to control shRNA cells from at least three independent experiments. All gene expression values were normalised to the housekeeping genes HMBS and HPRT before calculating the ratios. (B) The relative protein level of HCAR1 and ABCB1 in control and HCAR1 shRNA-expressing HeLa cells. The representative Western blot of at least three independent experiments is shown. HCAR1 protein was visible on the blot as two bands at 40-55 kDa, corresponding to unglycosylated and glycosylated variants of the receptor as described by Lauritzen and others (30). The ABCB1 and HCAR1 protein levels were quantified using densitometry, normalised to β-actin and presented as mean fold change ± S.E.M. relative to control shRNA cells from at least three independent experiments. (C) Immunocytochemical staining of ABCB1 in control and HCAR1 shRNA-expressing HeLa cells. Statistical significance was evaluated using Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.01 indicate significant differences compared to the corresponding counterparts.

HCAR1 stimulation and HCAR1-silencing affect transport of ABCB1 substrates

Next, we investigated the transport activity of ABCB1 transporter by assessing cellular accumulation of fluorescent ABCB1 substrates using two complimentary techniques: flow and image cytometry. Doxorubicin (DOX) is an ABCB1 substrate and frequently used in the cancer treatment (18). Using flow cytometry technique and utilising calcein and DOX as a ABCB1 substrates we showed that incubation of cells with DHBA resulted in 2- and 1.45-fold increase in ABCB1 transport activity, respectively (Fig. 3A). In the next step, we evaluated the effects of HCAR1-silencing on ABCB1 substrates accumulation in cells using another known specific fluorescent substrate for the ABCB1 protein - X-rhod-1 (19). As expected, HCAR1-silenced HeLa cells characterised by compromised ABCB1 expression accumulated 85% more X-rhod-1 dye in comparison to control shRNA cells (Fig. 3B). Finally, we confirmed that HCAR1-silenced cells accumulate significantly more DOX and do not response to DHBA stimulation as compared to control shRNA cells (Fig. 3C). The specificity of this processes was confirmed by parallel incubations with verapamil (Ver), a potent ABCB1 inhibitor (18).

In order to demonstrate that dyes transport is mainly related to ABCB1 status, we assessed expression levels of other transporters that may be involved in the studied processes (20, 21). We evaluated the expression of ABCC1 and ABCG2 in control shRNA and HCAR1-silenced cells and did not find any differences in the proteins level (Fig. 4A-4D). The observed increase of ABCG2 mRNA level in HCAR1-silenced did not translate into higher protein level (Fig. 4A, 4B).

Fig. 3. Effects of HCAR1 stimulation with DHBA and compromised HCAR1 expression on ABCB1 activity and intracellular accumulation of fluorescent ABCB1 substrates in HeLa cells. (A) Cells untreated or incubated with DHBA were loaded with calcein or DOX in the presence or absence of verapamil, mixed and proceeded for time-course measurement of cellular fluorescence changes. The resulting fluorescence values (arbitrary units, au) of each sample were plotted against time (min) and the curves obtained were used to calculate area under the curve [auc (au/min)] parameter describing substrate transport. Specific activity of ABCB1 protein (Δauc) was calculated as a difference between auc of sample with fluorochrome and inhibitor, and auc of sample with fluorochrome only. (B, C) Effects of HCAR1 silencing on ABCB1 substrates accumulation. (B) Control and HCAR1 shRNA-expressing HeLa cells were incubated in the absence or presence of 20 µM verapamil (Ver) for 1 hour followed by 1 hour incubation in the presence of 350 nM X-rhod-1. After washing with PBS and nuclei staining with Hoechst 33342, cellular fluorescence as a result of X-rhod-1 intracellular accumulation was measured and analysed using an ArrayScan VTI HCS Reader. (C) Cells untreated or pretreated with 20 µM verapamil were loaded with 10 µM DOX for 90 min, washed and followed by nuclei staining with Hoechst 33342. Doxorubicin accumulation was showed as nuclear fluorescence measured and analysed at 90 min after DOX loading using an ArrayScan VTI HCS Reader. The results are presented as mean fluorescence fold change ± S.E.M. relative to control shRNA cells from at least three independent experiments. Statistical significance was evaluated using Student’s t-test or one-way ANOVA followed by Tukey’s test. *P < 0.05, **P < 0.05, and ***P < 0.01 indicate significant differences compared to the corresponding counterparts.

Fig. 4. Effects of HCAR1 silencing on ABCG2 and ABCC1 expression in HeLa cells. (A, C) mRNA level for ABCG2 and ABCC1 in control and HCAR1 shRNA-expressing HeLa cells. Real-time PCR results are presented as the mean fold change in gene expression ± S.E.M. relative to control shRNA cells from at least three independent experiments. All gene expression values were normalised to the housekeeping genes HMBS and HPRT before calculating the ratios. (B, D) ABCG2 and ABCC1 protein levels in control and HCAR1 shRNA-expressing HeLa cells. The representative Western blot of at least three independent experiments is shown. The ABCG2 and ABCC1 levels were quantified using densitometry, normalised to β-actin and presented as the mean fold change ± S.E.M. relative to control shRNA cells. Statistical significance was evaluated using Student’s t-test.

HCAR1-silencing sensitises cells to doxorubicin treatment

DNA cell content analysis and growth inhibition assay (GIA) were performed to investigate the effect of HCAR1 on doxorubicin-mediated cytotoxity. For DNA cell content analysis control shRNA and HCAR1-silenced HeLa cells were incubated in the presence of DOX (100 – 1000 nM) for 30 min, followed by incubation in a DOX-free medium for 24 hours. Doxorubicin is believed to act primarily through immobilizing topoisomerase IIa (Topo-IIα)-DNA complexes, thereby inducing lethal cellular damage by inhibition of re-ligation. Since Topo-IIα is expressed primarily in the S and G2 phases, cell cycle arrest after DOX treatment is observed predominantly in G2/M (4N DNA content) (22). We used the DNA content 2N/4N ratio to measure a cell cycle distribution. As is shown in Fig. 5A, treatment with DOX primarily affected HCAR1 shRNA-expressing cells while the 2N/4N index of control shRNA cells only slightly decreased within the 100 – 500 nM DOX concentration range. The most protective effect of HCAR1 was observed at 500 nM DOX (84% of control versus 54% of receptor silenced cells).

The protective effects of HCAR1 on cell cycle arrest after DOX treatment translated into higher HeLa proliferation rate as shown by GIA results (Fig. 5B). Decreased receptor expression in HCAR1 shRNA-expressing cells resulted in a 5-fold increase in sensitivity to cell growth inhibition by DOX treatment (IC50 = 0.10 µM; 95% CI 0.078 – 0.13) in comparison to control shRNA cells (IC50 = 0.53 µM; 95% CI 0.36 – 0.77).

We also examined the effects of HCAR1 stimulation with DHBA on doxorubicin-induced death in HeLa cells. DHBA alone had no significant effect on cell apoptosis (16.8 ± 1.6% compared to untreated cells 16.2 ± 1.5%). The 72 hours after DOX treatment, positive staining with annexin V exhibited 27% of DHBA stimulated cells, while control cells 39% (Fig. 5C).

Fig. 5. Presence of HCAR1 enhances the resistance of HeLa cells to doxorubicin. 2N/4N DNA index (A) and cell viability (B) of control shRNA (white columns or white circles) and HCAR1 shRNA-expressing HeLa cells (black columns or black circles) incubated with DOX. (C) HeLa cells were either untreated or pretreated with 500 µM DHBA for 24 hours and then incubated with 1 µM DOX for 30 min. After 72 hours, cells were stained with Annexin V - Alexa Fluor 488 conjugate. The results are presented as mean ± S.E.M. from at least three independent experiments. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s test or Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences compared to the corresponding counterparts.

Effects of PKC inhibitor Go6983 on DHBA-mediated resistance to doxorubicin

PKC signalling is known to regulate diverse cellular responses, including apoptosis and survival through extracellular signal integration (23). Because PKC plays a crucial role in HCAR1-mediated signal transduction, we examined whether a PKC inhibitor reverses the modulatory effects of DHBA. HeLa cells were preincubated with the PKC specific inhibitor Go6983, followed by stimulation with DHBA. We observed, that Go6983 completely abolished the observed DHBA-mediated up-regulation of ABCB1 protein level (Fig. 6A). DHBA-dependent decrease in accumulation of ABCB1 substrate: rhodamine123 was also eliminated by preincubation with Go6983 (Fig. 6B).

Fig. 6. DHBA stimulated up-regulation of ABCB1 is compromised by PKC inhibitor Go6983. HeLa cells were preincubated in the absence or presence of 10 µM Go6983 for 1 hour and either stimulated with 500 µM DHBA for 24 hours or left untreated. (A) The representative Western blot of at least three independent experiments is shown. The ABCB1 protein level was quantified using densitometry, normalised to β-actin and presented as the mean fold change ± S.E.M. relative to untreated cells from at least three independent experiments. Statistical significance was evaluated using Student’s t-test. *P < 0.05 and **P < 0.01 indicate significant differences compared to the untreated cells. (B) Cells untreated or pretreated with 20 µM verapamil were loaded with 0.5 µM rhodamine123 for 45 min. Cellular fluorescence of rhodamine123 was measured and analysed using an ArrayScan VTI HCS Reader. The results are presented as mean fluorescence fold change ± S.E.M. relative to untreated cells from at least three independent experiments. Statistical significance was evaluated using Student’s t-test or one-way ANOVA followed by Tukey’s test. *P < 0.05 and **P < 0.05 indicate significant differences compared to the corresponding counterparts.

DISCUSSION

Recent evidence has identified lactate as an active metabolite that acts as an autocrine and paracrine pseudo-hormone via the hydroxycarboxylic acid receptor 1. There is growing evidence indicating interplay of HCAR1 and its agonist, L-lactate, in driving cancer cell growth, metastasis, and resistance to anticancer therapy (3-5). Previously, we demonstrated that both L- and D-lactate, acting as HDAC inhibitors, promote epigenetic chromatin rearrangement, resulting in enhanced DNA repair and cell survival (3). Interestingly, we observed the most pronounced enhancement of DNA repair by lactate after treatment with doxorubicin, as compared to two other drugs, cisplatin and neocarzinostatin. Doxorubicin is a valuable clinical chemotherapeutic agent demonstrating a broad spectrum of antineoplastic activity against cancers. The main mechanism of doxorubicin action is attributed to intercalation into DNA and disruption of topoisomerase-II-mediated DNA repair and generation of free radicals and their damage to cellular membranes, DNA and proteins (22). The latter feature of doxorubicin play an indisputable role in the development of clinical side-effects such as cardiotoxicity, neurotoxicity and blood cells toxicity. In order to overcome these shortcomings and increase doxorubicin therapeutic index, supplementation with various antioxidants e.g. pyrroline nitroxide or resveratrol along with doxorubicin therapy are being extensively studied (24, 25).

Herein, we demonstrate the currently unknown mechanism of HCAR1 mediated resistance of cervical cancer HeLa cells to the anticancer drug doxorubicin. Studied HeLa cell line represents cervix-originated cells which are constantly exposed to lactate receptor stimulation with L- and D-lactate produced by symbiotic lactic acid bacteria in lower female genital tract, under physiological conditions. One of the key proteins responsible for multidrug resistance (MDR) to clinically used anticancer drugs is the ABCB1 transporter. Incubating HeLa cells with DHBA or lactate revealed up-regulation of ABCB1, with the most potent effects observed following DHBA exposure (Fig. 1). Thus, earlier observations of lactate-driven accelerated processing of DNA damage could be, in part, related to enhanced expression of ABCB1 via HCAR1 stimulation. Indeed, HCAR1 silencing resulted in down-regulation of ABCB1, suggesting the presence of direct relationship between both transmembrane proteins (Fig. 2). Furthermore, diminished expression of ABCB1 in HCAR1-compromised HeLa cells translated into higher accumulation of doxorubicin in cells, while DHBA-treated cells exhibited lower accumulation of ABCB1 substrates (Figs. 3 and 6). Moreover, our study confirmed positive regulation of ABCB1 only, rather than other ABC transporters (ABCC1 and ABCG2) capable of doxorubicin efflux (Fig. 4). Although we found ABCG2 mRNA in HCAR1-deprived cells was two-fold higher than in control cells (Fig. 4A), this phenomenon was not confirmed at the protein level (Fig. 4B). Interestingly, such expression linkage between ABCB1 and ABCG2 was previously described as an inverse relationship observed in vitro (26) and in vivo (27, 28). We think that this observation might be explained by activation of compensatory mechanisms among these transporters that overlap their substrate specificity in order to counteract drug toxicity.

ABCB1 is one the most studied drug transporters, and its enhanced expression can lead to therapeutic failure as a result of insufficient intracellular drug concentration (15). Our study showed that cells acquiring higher ABCB1 expression upon DHBA treatment were less prone to apoptosis after doxorubicin treatment in comparison to untreated counterparts (Fig. 5C). Furthermore, cells exhibiting reduced expression of ABCB1 as a consequence of HCAR1-deprivation showed pronounced sensitivity to growth inhibition (5-fold) after exposition to doxorubicin in comparison to control cells (Fig. 5B). Thus, our results are in line with previous reports published by Roland et al. and Staubert et al. (4, 5). These authors reported that cells with silenced HCAR1 showed a dramatic decrease in growth and metastasis (pancreatic cancer cells) or induced considerable cell death (breast cancer).

There is emerging data indicating involvement of PKC signalling pathway in hyperactivation of ABCB1 in cancer cells and enhanced resistance to anticancer therapy. Recently, endothelin receptor A (ETAR) has been reported to activate ABCB1 expression in small cell lung cancer (29) and normal brain capillary endothelial cells via activation of PKC. According to Kim et al. (13), ABCB1-mediated drug efflux in human MDR breast cancer cells might be also regulated through transporter phosphorylation by activated PKCα. Finally, widespread intrinsic feature of elevated PKC level in cancer cells often underlies unresponsiveness to anticancer therapy due to ABCB1 expression stimulation (9). In our study, we also examined whether a PKC inhibitor could reverse the stimulatory effects of DHBA and HCAR1-driven resistance to doxorubicin. Pretreatment of HeLa cells with the PKC specific inhibitor Go6983 abolished most of the effects of receptor stimulation with DHBA (Fig. 6). As a consequence, up-regulation of ABCB1 protein level and the functional improvement of transporter substrate efflux were ceased (Fig. 6B). Thus, the demonstrated results suggest that HCAR1-enhanced resistance to anticancer drugs also utilises PKC-dependent signalling pathway in HeLa cells.

To sum up, our findings support a critical role for HCAR1 in cancer cell biology. Moreover, the present study provides new insight into the mechanism underlying the regulation of cellular resistance to genotoxins/chemotherapeutics through HCAR1-ABCB1 axis. Most importantly, we demonstrated that HCAR1 may account for development of MDR phenomenon and increased resistance of tumours to clinically used drugs.

Acknowledgements: We thank Katarzyna Sobierajska for her help in revising the manuscript and thoughtful comments.

This project was supported by The National Science Centre in Poland under grant number UMO-2011/03/B/NZ4/00046.

Conflict of interests: None declared.

REFERENCES

1. Liu C, Wu J, Zhu J, et al. Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81. J Biol Chem 2009; 284: 2811-2822.
2. Morland C, Lauritzen KH, Puchades M, et al. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1: Expression and action in brain. J Neurosci Res 2015; 93: 1045-1055.
3. Wagner W, Ciszewski WM, Kania KD. L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation. Cell Commun Signal 2015; 13: 36. doi: 10.1186/s12964-015-0114-x
4. Roland CL, Arumugam T, Deng D, et al. Cell surface lactate receptor GPR81 is crucial for cancer cell survival. Cancer Res 2014; 74: 5301-5310.
5. Staubert C, Broom O, Nordstrom A. Hydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolism. Oncotarget 2015; 14: 19706-19720.
6. Liu Y, An S, Ward R, et al. G protein-coupled receptors as promising cancer targets. Cancer Lett 2016; 376: 226-239.
7. O’Hayre M, Degese MS, Gutkind JS. Novel insights into G protein and G protein-coupled receptor signaling in cancer. Curr Opin Cell Biol 2014; 27: 126-135.
8. Li G, Wang HQ, Wang LH, Chen RP, Liu JP. Distinct pathways of ERK1/2 activation by hydroxy-carboxylic acid receptor-1. PLoS One 2014; 9: e93041. doi: 10.1371/journal.pone.0093041
9. Miklos W, Pelivan K, Kowol CR, et al. Triapine-mediated ABCB1 induction via PKC induces widespread therapy unresponsiveness but is not underlying acquired triapine resistance. Cancer Lett 2015; 361: 112-120.
10. Mayati A, Le Vee M, Moreau A, et al. Protein kinase C-dependent regulation of human hepatic drug transporter expression. Biochem Pharmacol 2015; 98: 703-717.
11. Zhao LJ, Xu H, Qu JW, Zhao WZ, Zhao YB, Wang JH. Modulation of drug resistance in ovarian cancer cells by inhibition of protein kinase C-alpha (PKC-α) with small interference RNA (siRNA) agents. Asian Pac J Cancer Prev 2012; 13: 3631-3636.
12. Sun Q, Li Y. The inhibitory effect of pseudolaric acid B on gastric cancer and multidrug resistance via Cox-2/PKC-α/P-gp pathway. PLoS One 2014; 9: e107830. doi: 10.1371/journal.pone.0107830
13. Kim CW, Asai D, Kang JH, Kishimura A, Mori T, Katayama Y. Reversal of efflux of an anticancer drug in human drug-resistant breast cancer cells by inhibition of protein kinase Ca (PKCa) activity. Tumour Biol 2016; 37: 1901-1908.
14. Lili X, Xiaoyu T. Expression of PKCa, PKCe, and P-gp in epithelial ovarian carcinoma and the clinical significance. Eur J Gynaecol Oncol 2015; 36: 181-185.
15. Shukla S, Ohnuma S, Ambudkar SV. Improving cancer chemotherapy with modulators of ABC drug transporters. Curr Drug Targets 2011; 12: 621-630.
16. Noguchi K, Katayama K, Sugimoto Y. Human ABC transporter ABCG2/BCRP expression in chemoresistance: basic and clinical perspectives for molecular cancer therapeutics. Pharmgenomics Pers Med 2014; 7: 53-64.
17. Debska S, Owecka A, Czernek U, Szydlowska-Pazera K, Habib M, Potemski P. Transmembrane transporters ABCC - structure, function and role in multidrug resistance of cancer cells. Postepy Hig Med Dosw 2011; 65: 552-561.
18. Shen F, Chu S, Bence AK, et al. Quantitation of doxorubicin uptake, efflux, and modulation of multidrug resistance (MDR) in MDR human cancer cells. J Pharmacol Exp Ther 2008; 324: 95-102.
19. Bartosz G, Blauz A, Rychlik B, Wieczorek K. A method of determining the level of transport activity of the ABCB1 protein in a tested mammalian cell. Patent pending No. 404000.
20. Sukowati CH, Rosso N, Pascut D, et al. Gene and functional up-regulation of the BCRP/ABCG2 transporter in hepatocellular carcinoma. BMC Gastroenterol 2012; 12: 160. doi:10.1186/1471-230X-12-160
21. Zhang YK, Wan YJ, Gupta P, Chen ZS. Multidrug resistance proteins (MRPs) and cancer therapy. AAPS J 2015; 17: 802-812.
22. Potter AJ, Gollahon KA, Palanca BJ, et al. Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin. Carcinogenesis 2002; 23: 389-401.
23. Bluwstein A, Kumar N, Leger K, et al. PKC signaling prevents irradiation-induced apoptosis of primary human fibroblasts. Cell Death Dis 2013; 4: e498. doi: 10.1038/ cddis.2013.15
24. Hamlaoui S, Mokni M, Limam N, et al. Resveratrol protects against acute chemotherapy toxicity induced by doxorubucin in rat erythrocyte and plasma. J Physiol Pharmacol 2012; 63: 293-301.
25. Tabaczar S, Czepas J, Koceva-Chyla A, Kilanczyk E, Piasecka-Zelga J, Gwozdzinski K. The effect of the nitroxide pirolin on oxidative stress induced by doxorubicin and taxanes in the rat brain. J Physiol Pharmacol 2017; 68: 295-308.
26. Bark H, Xu HD, Kim SH, Yun J, Choi CH. P-glycoprotein down-regulates expression of breast cancer resistance protein in a drug-free state. FEBS Lett 2008; 582: 2595-600.
27. Cisternino S, Mercier C, Bourasset F, Roux F, Scherrmann JM. Expression, up-regulation, and transport activity of the multidrug-resistance protein Abcg2 at the mouse blood-brain barrier. Cancer Res 2004; 64: 3296-3301.
28. Agarwal S, Uchida Y, Mittapalli RK, Sane R, Terasaki T, Elmquist WF. Quantitative proteomics of transporter expression in brain capillary endothelial cells isolated from P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and P-gp/Bcrp knockout mice. Drug Metab Dispos 2012; 40: 1164-1169.
29. Englinger B, Lotsch D, Pirker C, et al. Acquired nintedanib resistance in FGFR1-driven small cell lung cancer: role of endothelin-A receptor-activated ABCB1 expression. Oncotarget 2016; 7: 50161-50179.
30. Lauritzen KH, Morland C, Puchades M, et al. Lactate receptor sites link neurotransmission, neurovascular coupling, and brain energy metabolism. Cereb Cortex 2014; 24: 2784-2795.