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[beta]-carotene reverses multidrug resistant cancer cells by selectively modulating human P-glycoprotein function.

ABSTRACT

Background: The issue of multidrug resistance (MDR) cancer is one of the major barriers to successful chemotherapy treatment. The ATP-binding cassette (ABC) efflux transporters play an important role in the chemotherapeutic failure. Several generations of ABC efflux transporter inhibitors have been developed, however, none of them could provide better clinical outcome due to systemic toxicides and significant drug-drug interactions. Therefore, the present study focused on identifying the effect of the natural carotenoid on ABC transporters and may provide a safer choice to defeat MDR cancer.

Purpose: The aim of the present study was to evaluate the inhibitory potency of [beta]-carotene on the ABC efflux transporters, as well as the reversal effect of [beta]-carotene toward MDR cancers. The underlying molecular mechanisms and inhibitory kinetics of [beta]-carotene on the major ABC efflux transporter, P-glycoprotein, were further investigated.

Methods: The human P-gp (ABCB1/FIp-In[TM]-293), MRP1 (ABCC1/FIp-In[TM]-293) and BCRP (ABCC2/FIp-In[TM]-293) stable expression cells were established by using the FIp-In[TM] system. The cytotoxicity of [beta]-carotene was evaluated by MTT assay in the established cell lines, sensitive cancer cell lines (HeLaS3 and NCIH460) and resistant cancer cell lines (KB-vin and NCI-H460/MX20). Surface protein detection assay and eFluxx-ID Green Dye assay were applied for confirmation of surface expression and function of the transporters. The transporter inhibition potency of [beta]-carotene was evaluated by calcein-AM uptake assay and mitoxantrone accumulation assay. Further interaction kinetics between [beta]-carotene and P-gp were analyzed by rhodaminel23 and doxorubicin efflux assay. The influence of [beta]-carotene on ATPase activity was evaluated by Pgp-Glo[TM] Assay System.

Results: Among the tested ABC efflux transporters, [beta]-carotene significantly inhibited human P-gp efflux function without altering ABCB1 mRNA expression. Furthermore, [beta]-carotene stimulated both P-gp basal ATPase activity and the verapamil-stimulated P-gp ATPase activity. In addition, [beta]-carotene exerted partially inhibitory effect on BCRP efflux function. The combination of [beta]-carotene and chemotherapeutic agents significantly potentiated their cytotoxicity in both cell stably expressed human P-gp (ABCB1/FIpln[TM]-293) and MDR cancer cells (KB-vin and NCI-H460/MX20).

Conclusion: The present study indicated that [beta]-carotene may be considered as a chemo-sensitizer and regarded as an adjuvant therapy in MDR cancer treatment.

Keywords:

Multidrug resistant cancer

[beta]carotene

P-gP

Reversal effects

Introduction

Despite the advances in cancer treatments, development of multidrug resistance (MDR) remains one of the major clinical obstacles. There are several cellular mechanisms have been proposed to be related to MDR, and the one that is most commonly encountered may be the over-expression of ATP-binding cassette (ABC) efflux transporters (Szakacs et al. 2014).

Among the human ABC transporter superfamily, ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP), are widely recognized to be associated with cancer MDR (Szakacs et al. 2006). P-gp is a transmembrane efflux pump responsible for the elimination of xenobiotics. The substrates of P-gp are mostly hydrophobic compounds and many chemotherapeutic agents, such as paditaxel, vincristine, doxorubicin, docetaxel and kinase inhibitors are efflux by P-gp (Szakacs et al. 2006). Overexpression of P-gp has been well characterized in many poor clinical response solid tumors, such as breast cancer, colon cancer, and sarcoma, as well as refractory hematological malignancies (Szakacs et al. 2014). In terms of the MRP1, its expression has been linked to disease progression in lung, renal, colorectal, ovarian, breast and prostate cancers (Szakacs et al. 2014). MRP1 demonstrated a substrate preference for negatively charged substrates, such as glutathione, glucuronate, and sulfate conjugates, suggesting its role in maintaining cell redox state. The commonly used anticancer agents, vincristine and vinblastine, are also substrates of MRP1 (Szakacs et al. 2006). As for the BCRP transporter, it is a half-transporter and dimerization is essential to be functional. BCRP is highly expressed in cancer stem cells and transports many chemotherapeutic agents (Stacy et al. 2013; Zhou et al. 2001). Several studies indicated that the expression of BCRP correlated to the prognostic outcomes of adult and pediatric acute myeloid leukemia (Benderra et al. 2005; van den Heuvel-Eibrink et al. 2002). These findings led to the investigation of the inhibitors for these transporters and the combination therapies of chemotherapeutic agents with the inhibitors which represented a promising approach to circumvent MDR cancers.

Several generations of P-gp inhibitors have been developed; however, limited success was achieved (Szakacs et al. 2006). There are several potential causes for the lack of clinical benefit of the candidate P-gp inhibitors, including the non-targeted inhibition, pharmacokinetic interaction between chemotherapeutic agents and the candidate P-gp inhibitors, and the patient population was not screened for the P-gp expression level and genotypes (Szakacs et al. 2006). In order to overcome these drawbacks, several alternative approaches have been considered. Some research tried to target ABC transporters by developing monoclonal antibody (Szaloki et al. 2014), whiles others attempted to utilize the liposomes or nanocarriers for selectively targeting MDR cancer cells (Dong and Mumper 2010). Besides these strategies, natural phytochemicals purified from herbs, foods and dietary supplements have drawn much attention. Several natural compounds have been identified as potential P-gp inhibitors, including quercetin, curcuminoids, capsaicin, and carotenoids (Wink et al. 2012). Recent studies revealed that carotenoids exhibited anticancer and cancer preventive activities by induction of apoptosis and inhibition of the expression of oncogenes (Kim et al. 2014; Lee et al. 2013). Some of the carotenoids were shown to be substrates of P-gp and worked synergistically with chemotherapeutic agents in MDR cancer cells (Eid et al. 2012). There were also clinical observational studies which supported the role of [beta]-carotene in cancer prevention (Eliassen et al. 2015; Leoncini et al. 2015). Although the biological activities of carotenoids and in particular their anti-MDR activity in several resistant cancer cells has been reported (Molnar et al. 2004), the influence of [beta]-carotene on cancer MDR reversal was not consistent (Eid et al. 2012). Furthermore, the effect of [beta]-carotene on MRP1 and BCRP efflux function was not considered previously. In addition, the underlying mechanism of [beta]-carotene interfered with these ABC transporters were not investigated in previous studies either.

The present study aimed to evaluate the individual effect of ficarotene on human P-gp, MRP1 and BCRP expression and function. Further in depth biochemical and kinetic investigation was performed to elucidate the underlying mechanisms of the [beta]-carotene mediated transporter inhibition. The effect of [beta]-carotene on the cytotoxicity of chemotherapeutic agents toward MDR cancer cells was also carried out to evaluate the potential of [beta]-carotene to overcome ABC efflux transporter mediated MDR in cancer chemotherapy.

Materials and methods

Chemicals

The following chemicals were purchased from Sigma Chemical Co (St. Louis, MO, USA): [beta]-carotene, calcein-AM, doxorubicin, DMSO, etoposide, hoechst33342, mitoxantrone, MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), paditaxel, rhodaminel23, R-(+)-verapamil, sulforhodamine B, trichloroacetic acid, Tris Base, vincristine, and 5-FU. The FIp-In[TM] system and all cell culture medium were obtained from Invitrogen (Carlsbad, CA, USA). The full-length human ABCB1 cDNA in pBluescript KS (+) (Clone name: pMDRAl), human ABCC1 cDNA in pOTB7 (Clone name: RBbllBlO), and human ABCG2 cDNA in pOTB7 (Clone Name: IRAL039K21) were supplied by the Riken BRC DNA bank (Ibaraki, Japan). Fluman P-gp purified mouse monoclonal antibody (17F9) was ordered from BD Bioscience (San Jose, CA, USA), human MRP1 purified mouse monoclonal antibody (MRPm6) from Novus Biologicals (Littleton, CO, USA), human BCRP purified mouse monoclonal antibody (5D3) from Acris Antibodies (San Diego, CA, USA), and Alexa Fluor[R] 488-conjugated AffiniPure Goat Anti-Mouse IgG from Jackson ImmunoResearch (West Grove, PA, USA). All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).

Expression plasmids construction and cell line establishment

The human P-gp stable expression cells were constructed in our previous studies (Teng et al. 2015). For MRP1 stable expression cells, plasmid AfiCCJ/pKAlU5 cDNA was digested with Notl and EcoRI, and the ABCC7 cDNA fragment was ligated into pET-20b(+). The recombinant plasmid was digested with Notl and EcoRV, and the insert was further ligated into the mammalian expression vector pcDNA5/FRT (ABCCJ/pcDNA5). For BCRP stable expression cells, plasmid ABCG2/pOTB7 cDNA was digested with BamHI and Xhol, and the insert ABCG2 cDNA fragment was further ligated into the mammalian expression vector pcDNA5/FRT (ABCG2/pcDNA5). The sequences were confirmed by direct sequencing.

The constructed ABCC1IpcDNA5 or ABCG2/pcDNA5 were cotransfected with pOG44 (the FIp recombinase expression plasmid) into the FIp-In[TM]-293 cells. Stable cell lines were selected on the basis of hygromycin B resistance and parental FIp-In[TM]-293 cells were selected by zeocin. All cells were cultured in Dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum at 37[degrees]C, 95% humidity and 5% C[O.sub.2]. The protein expressions of P-gp, MRP1, and BCRP were confirmed by surface protein detection assay.

Parental and multi-drug resistant cancer cell line

Human cervical carcinoma cell line HeLaS3 and human nonsmall cell lung carcinoma cell line NCI-H460 were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). The multidrug resistant human cervical cancer cell line KB-vin was kindly provided by Dr. Kuo-Hsiung Lee (University of North Carolina, Chapel Hill, USA) and maintained with vincristine in a fixed period. MDR human non-small cell lung carcinoma cell line NCI-H460/MX20 was selected from NCI-H460 with gradually increased concentrations of mitoxantrone. All cells were cultured in RPMI-1640 containing 10% FBS at 37[degrees]C in a humidified atmosphere of 5% C[O.sub.2].

Surface protein detection assay and eFluxx-ID Green Dye assay

The surface expression of P-gp, MRP1, and BCRP in ABCB1/FlpIn[TM]-293, ABCC1/Flp-In[TM]-293, and ABCC2/Flp-In[TM]-293 was detected by specific antibody 17F9, MRPm6, and 5D3, respectively. The basic efflux function of ABCB1/Flp-In[TM]-293, ABCC1/Flp-In[TM]-293, and ABCG2IFlp-ln[TM]-293 was assessed by eFluxx-ID Green Dye assay (ENZO Life Sciences, Inc., Farmingdale, NY, USA). To be brief, on the day of the assay, 5 x [10.sup.5] cells/reaction were collected, washed with PBS and incubated with or without MDR inhibitors (verapamil for P-gp, NIK-571 for MRP1, and novobiocin for BCRP) in the presence of the eFluxx-ID[R] Green probes for 30 min at 37[degrees]C and then evaluated by FACS analysis immediately. Flow cytometry data were analyzed by calculating MDR Activity Factor (MAF) values using the following formula, where the [MFI.sub.inh] and [MFI.sub.0] were mean fluorescence intensity values measured in the presence and absence of inhibitor.

MAF = 100 x (MFlinh - MF10)/MFlinch

Cell viability assay

The effect of [beta]-carotene on the cell viability of Flp-In[TM]-293, ABCB1/Flp-In[TM]-293, ABCC1/Flp-In[TM]-293, and ABCG2/Flp-In[TM]-293 cells was determined by MTT assay as previously described (Teng et al. 2015). The influence of combinations of [beta]-carotene and chemotherapeutic agents on cell proliferation were evaluated in human cancer cell lines (HeLaS3, KB-vin, NCI-H460, and NCI-H460/MX20) and the established cell lines outlined above by SRB assay as previous studies (Nakagawa-Goto et al. 2015) with minor modifications. The drug incubation time for both assays was 72 h.

Calcein-AM uptake assay and mitoxantrone accumulation assay

The details for calcein-AM uptake assay were described previously (Teng et al. 2015). The calcein fluorescence generated within the cells was detected by BioTek Synergy HT Multi-Mode Microplate Reader at excitation wavelength 485 nm and emission wavelength 528 nm under the temperature of 37 [degrees]C every 3 min for 30 min.

For the mitoxantrone accumulation assay, 1 x [10.sup.6] cells/well were seeded in 6-well plates. After overnight incubation, cells were washed and pre-incubated with warm HBSS for 30 min, and subsequently with [beta]-carotene for 30 min. After pre-incubation, the medium was replaced with mitoxantrone and incubated for l h at 37 [degrees]C. The cells were collected after the treatment and intracellular mitoxantrone fluorescence was measured by FACS analysis with excitation wavelength 635 nm and emission wavelength 661 nm.

Real-time quantitative RT-PCR

ABCBl, ABCC1 and ABCG2 mRNA expression levels were quantified by real-time RT-PCR as described previously (Teng et al. 2015). The relative ABCBl, ABCC1 or ABCG2 mRNA expression levels were normalized to the amount of GAPDH in the same cDNA and evaluated by ABI Prism 7900 Sequence Detection System.

Rhodamine123 and doxorubicin efflux assay

The details for rhodamine123 and doxorubicin efflux assay were described previously (Teng et al. 2015). The fluorescence of rhodamine123 and doxorubicin was measured by BioTek Synergy HT Multi-Mode Microplate Reader (excitation/emission: 485/528 nm for rhodamine123, 485/590 nm for doxorubicin). Each experiment was performed at least three times, each in triplicate on different days.

Pgp-Glo[TM] assay and MDR1 shift assay

For the evaluation of [beta]-carotene's effect on P-gp ATPase activity, Pgp-Glo[TM] Assay System from Promega (Madison, WI, USA) was used, as previously described (Teng et al. 2015). Luminescence was measured using BioTek Synergy HT Multi-Mode Microplate Reader and data were presented as change in luminescence.

The conformation change of P-gp by [beta]-carotene was examined using MDR1 Shift Assay kit from EMD Millipore Co (Billerica, MA, USA), as previously described (Teng et al. 2015). The fluorescence of secondary antibody Alexa Fluor[R] 488-conjugated AffiniPure Goat Anti-Mouse IgG was evaluated by FACS analysis (BD FACSCanto System).

Data and statistical analysis

For evaluation of inhibitor potency, [IC.sub.50] values were determined from sigmoidal dose response curve. For kinetic studies, kinetic parameters were estimated by nonlinear regression using Scientist v2.01 (MicroMath Scientific Software, Salt Lake City, UT, U.S.A.). Statistical differences were evaluated by ANOVA followed post hoc analysis (Tukey's test) or the Student's t-test. The statistical significance was set at p value < 0.05.

Results

Expression and function of constructed models

The expression and function of human P-gp, MRP1, or BCRP in established cell lines were examined by specific surface antibody and eFluxx-ID Green Dye, respectively. The results of flow cytometry using the specific antibody of human P-gp, MRP1, or BCRP demonstrated that the individual cell line stably expressed the transfected transporter (Supplementary Fig. la, b, c). The transporter specific efflux function was evaluated by eFluxx-ID Green Dye assay and the results showed that the stable transfected cells exhibited individual transporter efflux function as indicated by the multidrug resistance activity factor (MAF) values (Supplementary Fig. 2a, b, c). The theoretical range of the MAF values was between 0 and 100. A MAF value < 20 suggested multidrug resistance negative, while MAF values > 25 was indicated as multidrug resistance positive specimens (Lebedeva et al. 20U).

In order to examine the overexpressed transporters in MDR cancer cell lines, real-time RT-PCR gene expression analysis was performed. The results revealed that, as compared to parental cell HeLaS3, the KB-vin MDR cell line expressed 1600 fold higher ABCBl gene, while the other two transporters, ABCC1 and ABCG2, remained equivalent levels in both cell lines (Fig. 4a). As for the NCI-H460/MX20 resistant cell line, it showed significant higher ABCBl and ABCG2 expression as compared to the NCI-H460 parental cell line (Fig. 4b).

Effects of [beta]-carotene on human P-gp, MRP1, and BCRP

A. Evaluation of cytotoxicity of [beta]-carotene.

The cytotoxicity of [beta]-carotene on Flp-In[TM]-293 (parental cell), ABCB1/Flp-In[TM]-293, ABCC1/Flp-In[TM]-293, and ABCG2/ Flp-In[TM]-293 were evaluated by MTT assay and the results demonstrated that [beta]-carotene showed no cytotoxicity toward these cell lines under 100 [micro]M.

B. Primary screen of the effect of [beta]-carotene on transport function of P-gp, MRP1, and BCRP.

The effect of [beta]-carotene on transport function of human P-gp was primarily screened by calcein-AM uptake assay. Hydrophobic P-gp substrate calcein-AM could be converted to hydrophilic fluorescent calcein by intracellular esterase. Therefore, the function of P-gp could be evaluated by measuring intracellular calcein fluorescence. [beta]-carotene significantly inhibited P-gp's efflux function in a concentration-dependent manner as indicated by the higher intracellular calcein fluorescence compared to the control group. Verapamil was used as a canonical P-gp inhibition positive control (Fig. 1a).

Calcein-AM is also a model substrate for MRP1. Hence, the effect of [beta]-carotene on transport function of human MRP1 was screened via calcein-AM uptake assay as well. The results showed that [beta]-carotene had no MRP1 inhibitory effect as compared to the positive control verapamil (Fig. 1b). The same conclusion can be drawn from the eFluxx-ID Green Dye assay (Supplementary Fig. 3a). Therefore, [beta]-carotene may have no influence on human MRP1 efflux function.

The effect of [beta]-carotene on transport function of human BCRP was evaluated by mitoxantrone accumulation assay as mitoxantrone is a standard BCRP substrate. As compared to control, [beta]-carotene showed weak inhibition potency toward human BCRP (Fig. 1c). Similar BCRP inhibition effect of [beta]-carotene was also revealed by eFluxx-ID Green Dye assay (Supplementary Fig. 3b). In addition, 5D3 shift assay was conducted to identify whether a compound is a substrate or inhibitor of BCRP by examining the epitope levels on extracellular loop 3 (ECL3) with BCRP specific surface antibody 5D3 (Stacy et al. 2013). Standard substrates hoechst33342 and mitoxantrone would change the ECL3 inward folding to outward folding as revealed by the right shift of the fluorescent peaks by flow cytometry. On the contrary, inhibitors like verapamil did not lead to the conformation change of BCRP and resulting in no movement of the fluorescent peak. The treatment of [beta]-carotene did not lead to shift of fluorescent peak either, which indicated that [beta]-carotene may possess inhibitory characteristic on BCRP efflux function (Supplementary Fig. 3c).

These results indicated that, among these three transporters, [beta]-carotene showed significant inhibitory effect on human P-gp efflux function and no or little influence on human MRP1 and BCRP.

C. Human P-gp inhibitory mechanisms of [beta]-carotene.

Whether treatment of [beta]-carotene influenced human P-gp expression was evaluated by real-time RT-PCR. After 48 h treatment of 100 [micro]M [beta]-carotene, the ABCB1 mRNA expression in ABCB1/Flp-In[TM]293 cells showed no difference as compared to the baseline (Supplementary Fig. 5a). In terms of the MDR cancer cells, treatment of [beta]-carotene 100 [micro]M for 72 h significantly elevated the ABCB1 mRNA expression in KB-vin cells (Supplementary Fig. 5b).

Inhibitory potency and mechanism of [beta]-carotene on P-gp were evaluated by standard fluorescent substrates rhodamine123 and doxorubicin efflux assay. The result of rhodamine123 efflux assay demonstrated that [beta]-carotene inhibited human P-gp via uncompetitive inhibition with an inhibitory IC50 around 25 [micro]M (25.72 [+ or -] 0.2 ([micro]M). The increase of [beta]-carotene concentration would lead to the decrease of both maximum rate ([V.sub.max]) and affinity (Km) of rhodamine 123 (Fig. 2a-c). In terms of doxorubicin efflux assay, [beta]-carotene was shown to inhibit P-gp via non-competitive inhibition with an inhibitory [IC.sub.50] around 17 [micro]M (16.81 [+ or -] 0.43 [micro]M). The increase of [beta]-carotene concentration would lead to the decrease of [V.sub.max] while the affinity of doxorubicin remained the same (Fig. 2a, d, e).

Whether the inhibition effect of [beta]-carotene on P-gp efflux function was related to influence of ATPase activity was further evaluated. The results of Pgp-Glo[TM] assay demonstrated that [beta]-carotene not only stimulated basal P-gp ATPase activity, but also increased verapamil-stimulated P-gp ATPase activity (Fig. 3). These results demonstrated that [beta]-carotene acted as an ATPase stimulator, like verapamil, and lead to the inhibition of P-gp efflux function.

The MDR1 shift assay was performed to further investigate whether [beta]-carotene would influence the conformation of P-gp. As compared to the positive control vinblastine, treatment of 100 [micro]M [beta]-carotene slightly changed P-gp's conformation as indicated by the increased binding of P-gp conformation-sensitive antibody UIC2. However, treatment of [beta]-carotene concentrations below 100 [micro]M showed no influence on P-gp's structure (Supplementary Fig. 4).

MDR reversal effects of [beta]-carotene

The MDR reversal ability of [beta]-carotene was evaluated by comparing the resistant fold of chemotherapeutic drugs only treatment with the resistant fold of the [beta]-carotene co-administrated with chemotherapeutic agent treatment. The resistant fold of doxorubicin treatment in ABCB1/Flp-In[TM]-293 cells was 1.86 fold higher than the resistant fold of combination treatment with 20 [micro]M [beta]-carotene (Table 1).

The reversal effects of [beta]-carotene were further examined in multidrug-resistant cancer cell lines. In KB-vin cells, the reversal potency of [beta]-carotene was most significant when combination with pachtaxel treatment (the resistant fold dropped by 81.2%), followed by combination with doxorubicin treatment (the resistant fold dropped by 45.4%) (Table 2). For NCI-H460/MX20 cells, the combination treatment of [beta]-carotene demonstrated significant reversal effect as well. The resistant fold decreased 70.5% as compared to the value without combination with [beta]-carotene treatment (Table 2).

Discussion

Multidrug-resistant (MDR) cancer is an unresolved problem in cancer treatment; however, natural products have been recognized as promising candidates for mitigating this concern. In the present study, [beta]-carotene was demonstrated to possess MDR reversal ability under noncytotoxic concentrations. It sensitized KB-vin and NCI-H460/MX20 resistant cancer cells to chemotherapeutic agents such as paclitaxel, doxorubicin, and mitoxantrone. [beta]-carotene inhibited P-gp by stimulating ATPase activity and inhibited P-gp transport of rhodamine123 and doxorubicin via uncompetitive and noncompetitive mechanisms, respectively. These results suggested that [beta]-carotene could be a promising candidate in combinatorial cancer treatment.

As a carotenoid, [beta]-carotene exists in various fruits and vegetables. Aside from its well-known antioxidant activities, studies have evaluated the effect of [beta]-carotene on cancer-related problems. Some observational studies have demonstrated that [beta]-carotene could prevent people from the risk of breast, head, and neck cancer (Eliassen et al. 2015; Leoncini et al. 2015), whereas another 18-year-period cancer preventive study found that [beta]-carotene supplements caused no reduction in incidences of cancer (Virtamo et al. 2014). In addition, it was shown that [beta]-carotene also exhibits the properties of inducing differentiation, and suppressing sternness and antimetastasis in cancer cells (Kim et al. 2014; Lee et al. 2013). Regarding drug efflux transporter inhibitors and MDR reversal agents, it has been reported that [beta]-carotene may possess P-gp inhibitory ability (Eid et al. 2012). Several secondary metabolites in plants, including [beta]-carotene, increased the intracellular accumulation of P-gp substrates, such as rhodamine123, calcein-AM, and doxorubicin, in the human colon cancer Caco-2 cell line (Eid et al. 2012). However, the influence of other efflux transporters, such as MRP1 or BCRP, could not be excluded. In the present study, by establishing cells expressing a specific transporter, we clarified the influence of [beta]-carotene on each transporter. In contrast to the well-known natural products curcuminoids which inhibit all three types of transporters (Chearwae et al. 2004; Limtrakul et al. 2007), the major efflux transporter that interacts with [beta]-carotene is P-gp. The binding of [beta]-carotene may stimulate the ATPase activity of P-gp, leading to inhibition of the P-gp efflux function. These results indicated that [beta]-carotene is a Class II compound, which could enhance ATPase activity in a dose-dependent manner (Ambudkar et al. 1999). The binding site of [beta]-carotene on AT-Pase could be investigated by observing the interaction with verapamil (a standard P-gp ATPase stimulator) (Ambudkar et al. 1999). For example, a well-known natural product named demethoxycurcumin (DMC) decreased the verapamil-stimulated ATPase activity when they were used in combination, indicating that DMC and verapamil may compete at the same binding site on P-gp ATPase (Teng et al. 2015). By contrast, the combined treatment of verapamil with [beta]-carotene showed that [beta]-carotene enhances the consuming volume of ATP compared with verapamil treatment alone, indicating that [beta]-carotene and verapamil may bind to different pockets on P-gp ATPase (Ambudkar et al. 1999).

The effect of [beta]-carotene in reversing MDR cancers was evaluated and it demonstrated different reversal potencies toward different MDR cell lines. The combination of [beta]-carotene with mitoxantrone exhibited significant reversal potency in the NCI-H460/MX20 MDR cancer cell line. Compared with parental NCI-H460 cells, resistant counterpart NCI-H460/MX20 cells significantly overexpressed not only the BCRP transporter but also the P-gp transporter (Fig. 4b). Therefore, the significant reversal potency of [beta]-carotene in NCI-H460/MX20 cells may result from the inhibitory effects of both transporters. This finding reflected that [beta]-carotene may have the potential to sensitize MDR cancer cells, which over-expressed both P-gp and BCRP. The effect of [beta]-carotene in reversing MDR cancer has also been shown in other studies (Eid et al. 2012; Molnar et al. 2004). A previous study, using the human colon cancer Caco-2 cell line, demonstrated that the [IC.sub.50] of chemotherapeutical agents was decreased during combination treatment with [beta]-carotene with agent-related reversal effects (Eid et al. 2012). In Caco-2 cells, combination treatment with p-carotene and 5-FU showed the strongest reversal effect (Eid et al. 2012). However, another study showed that [beta]-carotene has no reversal effect in human-MDR1-gene transfected mouse lymphoma cells (Molnar et al. 2004). Such discrepancies could be caused by several reasons including differences between cell lines, diverse assays used in each study, and uncertain expression types of transporters. The expressed transporters varied among different cancer cells, and the levels of expression may have been influenced by treatments as well.

There are three possible binding sites (modulation site, M, or polyspecific recognition sites, R or H) and four likely binding manners (nonsubstrates, transported substrates, nontransported substrates, and modulators) in P-gp, and there may be more than one binding situation for a single drug (Ferreira et al. 2013). For example, rhodamine123 could bind to both the M and R sites; therefore, the binding manner would be classified at the intersection of transported substrates and nontransported substrates (Ferreira et al. 2013). In the present study, the [beta]-carotene-P-gp interaction kinetics differed between the substrates. By using different P-gp substrates, we could demonstrate the possible P-gp-[beta]-carotene binding pocket through kinetic mechanism analyses. In addition, by using the P-gp conformation specific antibody UIC2, we showed that high concentrations of [beta]-carotene could have more binding opportunities than those of low concentrations, resulting in different conformation change patterns and therefore influencing the epitope levels detected by the UIC2 antibody. In addition to directly affecting P-gp efflux function, it was reported that [beta]-carotene modulated ABCB1 expression as an upstream pregnane X-receptor activator (Ruhl et al. 2004), whereas other studies have revealed that ABCB1 gene expression was downregulated by [beta]-carotene treatment (Eid et al. 2012). The present study showed that [beta]-carotene may increase ABCB1 gene expression in MDR cancer cells. In other words, [beta]-carotene may not be a suitable long-term daily supplement for advanced-stage cancer patients, although it possesses a MDR cancer reversal effect.

The strength of the present study was the utilization of cells that stably overexpress human P-gp, MRP1, and BCRP individually (ABCB1/FIp-In[TM]-293, ABCC?/FIp-ln[TM]-293, and ABCG2/FIp-In[TM]-293). These cell lines can prevent the involvement of other transporters; hence, the inhibitory potency and interaction kinetics of each transporter can be investigated separately. In addition, the evaluation of the reversal fold in various types of MDR cancer cells further confirmed the dual inhibition ability of [beta]-carotene. Nonetheless, there were still several weaknesses in this study. The low water solubility of [beta]-carotene would limit the bioavailability and the biotransformation processes would be another challenge. Several pharmaceutical modifications such as making finely dispersed powders or nanoparticles could be implemented to improve the absorption of [beta]-carotene and prevent it from being metabolized.

In conclusion, the present study demonstrated that [beta]-carotene significantly inhibits the function of human P-gp and stimulates the basal ATPase activity in a concentration-dependent manner. Furthermore, [beta]-carotene sensitizes KB-vin and NCI-F1460/MX20 MDR cancer cells to some conventional chemotherapeutic drugs. Additional in vivo studies regarding the MDR reversal effect of [beta]-carotene may provide additional evidence to support the use of combination treatment of [beta]-carotene and chemotherapeutic agents in clinical settings.

http://dx.doi.org/10.1016/j.phymed.2016.01.008

ARTICLE INFO

Article history:

Received 27 July 2015

Revised 8 January 2016

Accepted 13 January 2016

Conflict of interest

None.

Acknowledgments

Flow cytometry analyses were performed at the Medical Research Core Facilities Center, Office of Research & Development at China medical University, Taichung, Taiwan, R.O.C. We acknowledged Dr. Kazumitsu Ueda (Kyoto University) for providing the human MDR1 cDNA. This study was supported by grants from the Ministry of Science and Technology (MOST 103-2320-B-039-014) and China Medical University Hospital (DMR-103-106) in Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.01.008.

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Yu-Ning Teng (a), Ming-Jyh Sheu (a), Yow-Wen Hsieh (a,b), Ruey-Yun Wang (c), Yao-Chang Chiang (d), Chin-Chuan Hung (a,b),*

(a) Department of Pharmacy, College of Pharmacy, China Medical University, 91 Hsueh-Shih Road, Taichung, Taiwan 40402, R.O.C.

(b) Department of Pharmacy, China Medical University Hospital, 2 Yude Road, Taichung, 40447, Taiwan, R.O.C.

(c) Department of Public Health, China Medical University, 91 Hsueh-Shih Road, Taichung, Taiwan 40402, R.O.C.

(d) Center for Drug Abuse and Addiction, China Medical University, 91 Hsueh-Shih Road, Taichung, Taiwan 40402, R.O.C.

Abbreviations: MDR, multidrug resistance; ABC, ATP-binding cassette; P-gp, P-glycoprotein; MRP1, multidrug resistance protein 1; BCRP, breast cancer resistance protein.

* Corresponding author. Tel.: +886 4 22053366x5155; fax: +886 4 22078083.

E-mail address: cc0206hung@gmail.com (C.-C. Hung).

Table 1
Effects of [beta]-carotene on cytotoxicity [IC.sub.50] values of
doxorubicin in Flp-In[TM] 293 and ABCBl-Flp-In[TM]-293 cells.

Compound          [IC.sub.50] ([micro]M) (Resistant fold)

                  Flp-in[TM] 293         ABCB1/Flp-In[TM]-293

Doxorubicin       0.023 [+ or -] 0.001   0.235 [+ or -] 0.012 (10.22)

+10 [micro]M      0.020 [+ or -] 0.001   0.136 [+ or -] 0.011 (6.80) *
[beta]-carotene

+20 [micro]M      0.018 [+ or -] 0.001   0.099 [+ or -] 0.025 (5.50) *
[beta]-carotene

+10 [micro]M      0.015 [+ or -] 0.001   0.022 [+ or -] 0.002 (1.47) *
verapamil

* p < 0.05 as compared to doxorubicin treatment without
[beta]-carotene. The resistant fold was calculated by dividing the
individual [IC.sub.50] of ABCB1/Flp-In[TM]-293 by the [IC.sub.50] of
Flp-in[TM] 293.

Table 2
Effects of [beta]-carotene on cytotoxicity [IC.sub.50] values of
chemotherapeutic agents in HeLaS3, KB-vin, NCI-H460 and NC1-H460/MX20.

                                  [IC.sub.50] ([micro]M) (resistant
                                  fold)

Compound                          HeLaS3

[beta]-carotene                   79.270 [+ or -] 1.08
Paclitaxel                        0.00053 [+ or -] 0.00005
  + 50 [micro]M [beta]-carotene   0.00190 [+ or -] 0.00048
Doxorubicin                       0.50 [+ or -] 0.064
  + 50 [micro]M [beta]-carotene   0.46 [+ or -] 0.031
Etoposide                         4.83 [+ or -] 0.36
  + 50 [micro]M [beta]-carotene   3.82 [+ or -] 0.38
5-FU                              0.044 [+ or -] 0.003
  + 50 [micro]M [beta]-carotene   0.041 [+ or -] 0.008

                                  [IC.sub.50] ([micro]M) (resistant
                                  fold)

Compound                          KB-vin

[beta]-carotene                   185.56 [+ or -] 12.56
Paclitaxel                        1.80 [+ or -] 0.050 (3396.23)
  + 50 [micro]M [beta]-carotene   1.21 [+ or -]0.048 (636.84) *
Doxorubicin                       12.16 [+ or -] 0.001 (24.32)
  + 50 [micro]M [beta]-carotene   6.11 [+ or -] 0.41 (13.28) *
Etoposide                         13.63 [+ or -] 1.04 (2.82)
  + 50 [micro]M [beta]-carotene   17.90 [+ or -]0.35 (4.69)
5-FU                              0.092 [+ or -] 0.003 (2.09)
  + 50 [micro]M [beta]-carotene   0.079 [+ or -] 0.002 (1.93)

                                  [IC.sub.50] ([micro]M) (resistant
                                  fold)

Compound                          NCI-H460

[beta]-carotene                   > 200
Mitoxantrone                      0.090 [+ or -] 0.00085
+ 50 [micro]M [beta]-carotene     0.087 [+ or -] 0.00750

                                  [IC.sub.50] ([micro]M) (resistant
                                  fold)

Compound                          NCI-H460/MX20

[beta]-carotene                   > 200
Mitoxantrone                      13.23 [+ or -] 0.27 (147.00)
+ 50 [micro]M [beta]-carotene     3.77 [+ or -] 0.22 (43.33) *

* p < 0.05 as compared to chemotherapeutic agents treatment without
[beta]-carotene. The resistant fold was calculated by dividing the
individual [IC.sub.50] of  resistant cancer cell lines (KB-vin or
NCI-H460-MX20) by the [IC.sub.50] of parental cancer cell lines
(HeLaS3 or NCI-H460), respectively.
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Author:Teng, Yu-Ning; Sheu, Ming-Jyh; Hsieh, Yow-Wen; Wang, Ruey-Yun; Chiang, Yao-Chang; Hung, Chin-Chuan
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9TAIW
Date:Mar 15, 2016
Words:6226
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