Alpha-Mangostin Reverses Multidrug Resistance by Attenuating the

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Alpha-mangostin reverses multidrug resistance by attenuating the function of the multidrug resistance-linked ABCG2 transporter Chung-Pu Wu, Sung-Han Hsiao, Megumi Murakami, Yu-Jen Lu, Yan-Qing Li, Yang-Hui Huang, Tai-Ho Hung, Suresh V. Ambudkar, and Yu-Shan Wu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00334 • Publication Date (Web): 22 Jun 2017 Downloaded from http://pubs.acs.org on June 24, 2017

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Molecular Pharmaceutics

Alpha-mangostin reverses multidrug resistance by attenuating the function of the multidrug resistance-linked ABCG2 transporter

Chung-Pu Wu a,b,c,e,*, Sung-Han Hsiao a, Megumi Murakami g, Yu-Jen Lu e, Yan-Qing Li b, Yang-Hui Huang c,e, Tai-Ho Hung d,f, Suresh. V. Ambudkar g and Yu-Shan Wu h,**

Authors' Affiliations: a

Graduate Institute of Biomedical Sciences, b Department of Physiology and

Pharmacology, c Molecular Medicine Research Center, and d Department of Chinese Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan. e Department of Neurosurgery, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan. f Department of Obstetrics and Gynecology, Taipei Chang Gung Memorial Hospital, Taipei, Taiwan. g Laboratory of Cell Biology, CCR, NCI, NIH, Bethesda, United States. h Department of Chemistry, Tunghai University, Taichung, Taiwan.

* Corresponding author at: 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan. Phone: +886-3-2118800, ext. 3754. Fax: +886-3-2118700. E-mail address: [email protected]

**Corresponding author at: 181 Taichung Harbor Road Section 3, Taichung, Taiwan. Phone: +886-4-2359-0121, ext 32248. Fax: +886-4-2359-0426. E-mail address: [email protected]

Keywords: Multidrug resistance; ABCG2; α-Mangostin; modulator Running Title: α-Mangostin inhibits ABCG2-mediated transport Abbreviations: MDR, multidrug resistance; ABC, ATP-binding cassette; FCS, fetal

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calf serum; PBS, phosphate-buffered saline; CCK-8, Cell Counting Kit-8; IMDM, Iscove's Modified Dulbecco's Medium; MTT, 3-(4,5-dimethylthiazol-yl)-2,5-diphenyllapatinibrazolium bromide; Vi, sodium orthovanadate; IAAP, Iodoarylazidoprazosin.

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ABSTRACT

The ATP-binding cassette (ABC) drug transporter ABCG2 can actively efflux a wide variety of chemotherapeutic agents out of cancer cells and subsequently reduce the intracellular accumulation of these drugs. Therefore, the overexpression of ABCG2 often contributes to the development of multidrug resistance (MDR) in cancer cells, which is one of the major obstacles to successful cancer chemotherapy. Moreover, ABCG2 is highly expressed in various tissues including the intestine and blood-brain barrier (BBB), limiting the absorption and bioavailability of many therapeutic agents. For decades, the task of developing a highly effective synthetic inhibitor of ABCG2 has been hindered mostly by the intrinsic toxicity, the lack of specificity and complex pharmacokinetics. Alternatively, considering the wide range of diversity and relatively non-toxic nature of natural products, developing potential modulators of ABCG2 from natural sources is particularly valuable. α-Mangostin is a natural xanthone derived from the pericarps of mangosteen (Garcinia mangostana L.) with various pharmacological purposes, including suppressing angiogenesis and inducing cancer cell growth arrest. In this study, we demonstrated that at non-toxic concentrations, α-mangostin effectively and selectively inhibits ABCG2-mediated drug transport and reverses MDR in ABCG2-overexpressing MDR cancer cells. Direct interactions between α-mangostin and the ABCG2 drug-binding site(s) were confirmed by stimulation of ATPase activity and by inhibition of photolabeling of the substrate-binding site(s) of ABCG2 with [125I]iodoarylazidoprazosin. In summary, our findings show that α-mangostin has great potential to be further developed into a promising modulator of ABCG2 for reversing MDR and for its use in combination therapy for patients with MDR tumors.

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INTRODUCTION

The ATP-binding cassette (ABC) drug transporters can utilize ATP hydrolysis to actively transport a wide variety of therapeutic agents out of cancer cells, rendering chemotherapy ineffective and increase the risk of cancer relapse and death 1. Therefore, the development of multidrug resistance (MDR) in cancer is often associated with overexpression of an ABC transporter, most commonly ABCB1(P-glycoprotein/ MDR1) or ABCG2 (BCRP; MXR) 2, 3. Human ABCG2 is one of the latest discovered major ABC drug transporters to be involved in MDR phenotype 4, 5. ABCG2 is capable of effluxing and conferring resistance to most conventional anticancer agents, and some molecularly targeted drugs such as tyrosine kinase inhibitors 3, 6-8. Consequently, overexpression of ABCG2 is often linked to MDR in patients with advanced non-small cell lung cancer or leukemia 1, 6, 8, 9. Moreover, ABCG2 is localized at many important biological barriers such as the luminal membrane of brain capillaries and the blood-brain barrier (BBB), protecting cells or tissues from xenobiotics and harmful chemotherapeutics, thus affecting the overall drug bioavailability, distribution, metabolism and elimination 3, 10, 11. Therefore, modulating the function or expression of ABCG2 has great clinical significance.

One of the most effective ways to overcome MDR in ABCG2-overexpressing cancer cells at present is by directly inhibiting the function of ABCG2 12. The idea is to introduce a selective modulator at a non-toxic concentration to transiently inhibit the transport function of ABCG2 in a competitive manner, elevating the accumulation of conventional anticancer drug in MDR cancer cells 3. Unfortunately, many failed attempts using immunosuppressants, channel blockers and synthetic chemicals to modulate the function of ABCG2 were associated with the lack of specificity, high 4


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intrinsic toxicity and adverse drug-drug interactions 3, 13, 14. As a result, none of the MDR modulators from the first three generations has been approved to treat patients with MDR tumors 13, and the discovery of modulators originating from natural sources is underway 15. The advantage of utilizing compounds originating from natural sources is that they are generally low in toxicity, better tolerated in human body and offer the widest range of diversity and novel chemical scaffolds 15. Therefore, it is not surprising that natural products are being used by most cancer patients in combination with anticancer drugs for the potential benefits in chemoprevention and delaying cancer progression 16.

α-Mangostin (see Fig. 1 for structure) is one of the most studied antioxidant phytochemicals 17-21 and the most abundant xanthone derived from the pericarps of mangosteen (Garcinia mangostana L.) 22, a popular fruit in Southeast Asia. Many biological properties of α-mangostin have been reported, including cardioprotective, neuroprotective, antidiabetic, anti-inflammatory, antimicrobial and antiprotozoal activities 23, 24. In addition, the antitumor activity of α-mangostin has been extensively studied in in vitro and in vivo systems 25. α-Mangostin has been shown to suppress tumor progression through induction of cell cycle arrest 21, 26, 27, apoptosis 26, 28, 29 and autophagy 30, as well as inhibition of angiogenesis 31, 32, invasion and metastasis 27, 31-34

. Moreover, α-mangostin has been reported able to selectively target cancer cells

without having cytotoxic effect to normal cells 35, 36 by acting synergistically with numerous conventional chemotherapeutic agents, including doxorubicin 37 and 5-FU 38

.

In the present study, we demonstrated for the first time that α-mangostin interacts directly with MDR-linked ABC transporter ABCG2 and inhibits its transport function 5



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without affecting the protein expression level of ABCG2. More importantly, α-mangostin significantly re-sensitizes ABCG2-overexpressing MDR cancer cells to chemotherapeutic drugs.

EXPERIMENTAL SECTION

Chemicals. Dulbecco's Modified Eagle's medium (DMEM), RPMI medium, fetal calf serum (FCS), phosphate-buffered saline (PBS), trypsin-EDTA, penicillin, and streptomycin were purchased from Gibco, Invitrogen (CA, USA). Tariquidar was obtained from MedKoo Biosciences (Morrisville, NC). [125I]-Iodoarylazidoprazosin (IAAP) (2200 Ci/mmol) was from Perkin-Elmer Life Sciences. All other chemicals were purchased from Sigma-Aldrich Company (St. Louis, MO, USA).

Cell lines and culture conditions. pcDNA3.1-HEK293, ABCB1-transected MDR19-HEK293, ABCG2-transfected R482-HEK293, KB-3-1, KB-V-1, MCF-7 and MCF7-FLV1000 were cultured in DMEM, whereas S1 and S1-M1-80 cells were cultured in RPMI-1640 (Gibco, Invitrogen). All cell lines were maintained at 37°C in culture media containing 10% FCS, 2 mM L-glutamine and 100 units of penicillin/streptomycin/mL in 5% CO2 humidified air. HEK293 and HEK293 transfected lines were maintained in 2 mg/mL G418 39, KB-V-1 cells were maintained in media containing 1 mg/mL vinblastine 40 and MCF7-FLV1000 cells were cultured in the presence of 1 µg/mL flavopiridol 41, 42, whereas S1-M1-80 cells were cultured in 80 µM of mitoxantrone 39. Cells were placed in drug-free medium 7 days prior to assay.

Fluorescent drug accumulation assay. A FACSort flow cytometer equipped 6


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with Cell Quest software (Becton-Dickinson) was used to monitor the intracellular accumulation of fluorescent substrates as described previously 43. Briefly, cells were harvested and resuspended in Iscove's modified Dulbecco's medium (IMDM) supplemented with 5 % FCS. Calcein-AM or pheophorbide A (PhA) was added to 3 x 105 cells in 4 mL of IMDM in the presence or absence of α-mangostin or respective reference inhibitors to determine ABCB1-mediated calcein-AM efflux or ABCG2-mediated PhA efflux according to the method described by Gribar et al 44.

Cytotoxicity assay. Cell Counting Kit-8 (CCK) assay was used to determine the cytotoxicity of drugs in HEK293 and HEK293 transfected with ABCB1 (MDR19-HEK293) or ABCG2 (R482-HEK293), whereas MTT assay was used to determine the cytotoxicity of drugs in human cancer cell lines according to the method described by Ishiyama et al 45. For the reversal of cytotoxicity assays, a nontoxic concentration of α-mangostin or reference inhibitor of ABCB1 or ABCG2 was added into the cytotoxicity assay, and the extent of reversal was determined based on the calculated fold-reversal (F.R) values.

ATPase assay and photoaffinity labeling of ABCG2 with [125I]IAAP. The effect of α-mangostin on vanadate (Vi)-sensitive ATPase activity of ABCG2 was determined using crude membranes isolated from High-Five cells based on the endpoint Pi assay as described previously 46. Photoaffinity labeling assays were performed using membranes (50 - 75 µg protein/mL) prepared from ABCG2-overexpressing MCF7-FLV1000 cells as described previously 46. Membranes were first incubated with α-mangostin for 10 min at room temperature in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, before 3-6 nmol/L [125I]IAAP (2200 Ci/mmol) was added. The samples were then photocrosslinked with UV light (366 nm) and 7



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processed as described previously 39.

Immunoblotting. Antibodies C219 (1:1000), BXP-21 (1:500) and α-tubulin (1:2000) were used in Western blot immunoassay to detect ABCB1, ABCG2 and tubulin, respectively. The horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000) was used as the secondary antibody. Signals were detected as described previously 39.

Molecular modeling of ABCG2. For structure prediction, the three dimensional structure of ABCG2 was predicted using SWISS-MODEL, an automated protein homology-modeling server. The amino acid sequence of the protein was submitted to SWISS-MODEL server and templates were searched with BLAST and HHBlits against SWISS-MODEL template library. For each identified template, the template’s quality was predicted from features of the target-template alignment. The templates with the highest quality were then selected for model building 47-49. Models were built based on the target-template alignment using ProMod3. For ligand preparation and docking, the energy was minimized for both protein and ligand using Acclerys Discovery Studio 4.0 and docking was also performed by the same software.

Statistical analysis. Experimental data and IC50 values were obtained from at least three independent experiments, and presented as mean ± standard error of the mean (S.E.M) and mean ± standard deviation (SD), respectively. Two-sided Student’s t-test was used to determine the differences between any mean values, and results were considered statistically significant at P < 0.05.

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RESULTS

α-Mangostin inhibits ABCG2-mediated substrate transport. First, the effect of α-mangostin on the transport function of ABCB1and ABCG2 was determined as described in Experimental section. As shown in Fig. 2A, ABCB1-mediated efflux of fluorescent substrate calcein was unaffected by 5 µM α-mangostin in ABCB1-transfected MDR19-HEK293. In contrast, ABCG2-mediated efflux of fluorescent PhA, a known fluorescent substrate of ABCG2 43, was strongly inhibited by α-mangostin in ABCG2-transfected R482-HEK293 (Fig. 2B). Similarly, α-mangostin inhibited ABCG2-mediated PhA transport in ABCG2-overexpressing human S1-M1-80 colon cancer (Fig. 2C) and human MCF7-FLV1000 breast cancer (Fig. 2D) cell lines. As positive controls, 3 µM of tariquidar and 1 µM of Ko143 (dotted lines) were used in the assasy as specific reference inhibitors for ABCB1 and ABCG2, respectively. Of note, α-mangostin had no significant effect on the accumulation of fluorescent probes in any of the drug sensitive parental cells tested (Fig. 2A - 2D, left panels).

α-Mangostin reverses MDR in cells overexpressing ABCG2. Knowing that α-mangostin inhibits the function of ABCG2, we next examined the reversal effect of α-mangostin on ABCG2-mediated MDR in ABCG2-overexpressing cells. Without affecting the proliferation of drug sensitive cells (Fig. 3, left panels), α-mangostin restored the sensitivity of ABCG2-transfected R482-HEK293 cells to topotecan (Fig. 3A, right panel) and mitoxantrone (Fig. 3B, right panel), two well-established anticancer drug substrates of ABCG2, in a concentration-dependent manner. At a highest tested concentration of 2 µM, α-mangostin re-sensitized R482-HEK293 cells to topotecan and mitoxantrone by approximately 25-fold and 9-fold, respectively. 9



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Similarly, multiple non-toxic concentrations of α-mangostin were also tested in ABCG2-overexpressing S1-M1-80 cancer cells, and the reversal effect of α-mangostin on ABCG2-mediated MDR is summarized in Table 1. In contrast, α-mangostin was unable to re-sensitize ABCB1-overexpressing MDR19-HEK293 cells and KB-V-1 cancer cells to ABCB1 substrates 50 doxorubicin (Fig. 3C) and colchicine (Fig. 3D). The fold-reversal (F.R) value indicates the degree of cellular chemosensitivity restored by a particular reversing agent, which was calculated as described previously 51. Tariquidar and Ko143 were used as positive controls to reverse MDR conferred by ABCB1 or ABCG2, respectively 52. Our results indicate that α-mangostin is selective for ABCG2 relative to ABCB1.

ABCG2 does not confer resistance to α-mangostin in cancer cells. Knowing that α-mangostin interacts with ABCG2, we next examined whether overexpression of ABCG2 in cancer cells confers significant resistance to α-mangostin. The cytotoxicity of α-mangostin was determined in several drug-sensitive and ABCG2-overexpressing MDR cancer cell lines, and the calculated IC50 values are summarized in Table 2. The resistance factor (RF) value was calculated by dividing the IC50 value of α-mangostin in ABCG2-overexpressing MDR subline by the IC50 value of α-mangostin in the respective parental line, representing the extent of cellular resistance to α-mangostin caused by the overexpression of ABCG2. We did not observe significant differences in α-mangostin IC50 values in ABCG2-overexpressing cancer cell lines in respective to the drug-sensitive cancer cell lines. In addition, the cytotoxicity of α-mangostin was also determined in HEK293 cells and ABCG2-transfected R482-HEK293 cells to confirm our findings. We found that both cell lines were equally sensitive to α-mangostin (Table 2).

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α-Mangostin does not affect the protein expression of ABCG2. In addition to direct inhibition of ABCG2 transport function, drug-induced transient down-regulation of ABCG2 can potentially increase the drug sensitivity of ABCG2-overexpressing cancer cells to chemotherapeutics 53, 54. Therefore, we examined protein expression of ABCG2 after exposing human S1-M1-80 colon cancer cells to increasing concentrations of α-mangostin (0 - 3 µM) for 72 h and processed by immunoblotting as described in Experimental section. As shown in Fig. 4, α-mangostin does not significantly alter the protein expression of ABCG2 in S1-M1-80 cancer cells. Our results indicate that α-mangostin reverses ABCG2-mediated MDR by inhibiting the function of ABCG2, and not by downregulation of ABCG2.

Effect of α-mangostin on [125I]IAAP photoaffinity labeling and ATPase activity of ABCG2. To study the interaction of α-mangostin with the substrate-binding sites of ABCG2, we examined the effect of α-mangostin on photoaffinity labelling of ABCG2 with [125I]IAAP and vanadate (Vi)-sensitive ATPase activity of ABCG2. We found that 10 µM of α-mangostin inhibited the incorporation of [125I]IAAP into ABCG2 by approximately 60% (Fig. 5A), indicating direct binding of α-mangostin to the substrate-binding sites of ABCG2. Moreover, considering that substrate transport mediated by ABC transporter is coupled to ATP hydrolysis 55, 56, the effect of α-mangostin on Vi-sensitive ATPase activity of ABCG2 in membranes isolated from cells expressing ABCG2 was examined in order to gain insight into the interaction between α-mangostin and ABCG2. As shown in Fig. 5B, α-mangostin stimulated ABCG2 ATPase activity in a concentration-dependent manner, with approximately 2-fold maximum stimulation at 1 µM and with the concentration of 86 ± 16 nM (Fig. 5B, inset) for 50% stimulation. 11



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α-Mangostin docking analysis with molecular modeling of ABCG2. To better understand the binding of α-mangostin with ABCG2 transporter protein, docking study was performed using a homology model generated from currently available ABCG5 crystal structure as template 57, 58. The potential binding site for α-mangostin lies within the transmembrane domain (TMD) of ABCG2 and the binding was mostly stabilized by hydrophobic interactions with surrounding hydrophobic residues Pro485, Val442, Ala444, Val445, Val401, Ile399, Ile400 and Ala397. A hydrogen bond was formed between a phenolic hydroxyl group and the carbonyl moiety of Ala397 (Fig. 6).

DISCUSSION

ABCG2-mediated multidrug resistance to chemotherapeutic agents remains a substantial challenge to the effective treatment of cancer 59. Instead of developing novel synthetic compounds or using clinically active drugs at higher than intended concentrations as reversing agents, we and others have explored utilizing bioactive compounds originated from natural sources to overcome ABCG2-mediated MDR in cancer cells 15. Although many natural product modulators have been identified, most of them lack the combination of potency and selectivity 13, 15. Moreover, some of these modulators are toxic in nature, and therefore unsuitable for clinical use 15. For instance, fumitremorgin C (FTC) has one of the highest in vitro activity against ABCG2 60, however, the neurotoxic nature of FTC prevented it from clinical applications 61. The natural xanthone α-mangostin is one of the well-characterized bioactive natural compounds, known for its antiperoxidative, anti-inflammation and anticancer properties 21, 25, 62, 63.

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In the present study, we studied the potential modulatory effect of α-mangostin on ABCG2-mediated MDR in cancer cells. Several short-term drug accumulation assays were performed to evaluate the specificity and inhibitory activity of α-mangostin against drug efflux mediated by MDR-linked ABC drug transporters ABCB1 and ABCG2. We noticed that ABCG2-mediated drug efflux was significantly inhibited by α-mangostin. In contrast, the effect of α-mangostin on ABCB1-mediated drug efflux was extremely weak (Fig. 2). Our results indicate that α-mangostin selectively inhibits the function of ABCG2. Next, we explored whether inhibition of ABCG2 transport function by α-mangostin could lead to resensitization of ABCG2-overexpressing cells to chemotherapeutic drugs in the same manner as other natural product modulators, such as curcumin 64, resveratrol 65 and plumbagin 66. We found that at non-toxic concentrations, α-mangostin substantially re-sensitized ABCG2-overexpressing cells to topotecan and mitoxantrone. Interestingly, α-mangostin appears to be more effective in restoring the sensitivity of ABCG2-overexpressing cells to topotecan than mitoxantrone (Table 1). Of note, in spite of the weak inhibition ABCB1-mediated efflux by α-mangostin, there was still the possibility of α-mangostin reversing MDR in ABCB1-overexpressing cells. Therefore, we also examined the effect of α-mangostin on ABCB1-mediated resistance to doxorubicin and colchicine. As expected, α-mangostin was unable to restore chemosensitivity in ABCB1-overexpressing cells (Table 1). Considering the overlapping substrate specificity of ABCB1 and ABCG2 3, 67, it is not surprising that although many natural product modulators showed promising results in restoring the chemosensitivity of MDR cancer cells, most were not transporter-specific 15. For instance, curcumin is known to re-sensitize ABCG2-overexpressing cancer cells to chemotherapy, but it also re-sensitizes ABCB1-overexpressing cells in a same manner 68

. An alternative way to re-sensitize ABC transporter-overexpressing MDR cancer 13



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cells is to reduce transiently the expression of that particular ABC drug transporter 54, 69, 70

. We discovered that α-mangostin did not alter the protein expression of ABCG2

in ABCG2-overexpressing MDR cancer cells over a period of 72 h (Fig. 4), supporting the notion that α-mangostin re-sensitizes ABCG2-overexpressing MDR cancer cells to anticancer drugs by inhibiting the function of ABCG2. Next, we examined whether ABCG2 is likely to confer resistance to α-mangostin by comparing the intrinsic cytotoxicity of α-mangostin in multiple drug-sensitive cell lines and respective ABCG2-overexpressing MDR sublines. We discovered that ABCG2-overexpressing human colon, lung and breast cancer cells, as well as ABCG2-transfected cells were all equally sensitive to α-mangostin as their respective parental cells (Table 2). Again, these results show that α-mangostin behaves as a high-affinity competitive modulator of ABCG2 and not a transport substrate of ABCG2.

To confirm the direct interaction between α-mangostin and substrate-binding site(s) of ABCG2, photoaffinity labeling and ATPase assay of ABCG2 experiments were performed. [125I]IAAP is a transport substrate that binds directly to the substrate binding sites of ABCG2 71, and any substrate or inhibitor that binds to the same site will inhibit the photolabeling 39, 72. Our photoaffinity labeling results demonstrated direct and competitive binding of α-mangostin to the substrate-binding pocket(s) of ABCG2. Moreover, substrate transport by an ABC transporter is known to be coupled with the stimulation of ATPase activity 73, where rapid stimulation of ATP hydrolysis is associated with the presence of a substrate, while inhibition of ATP hydrolysis is associated with the presence of an inhibitor or substrate with a much lower transport rate 74. α-Mangostin produced a sharp stimulation at low concentrations (0.5-1 µM) and decreased stimulation of the ABCG2 ATPase activity at higher concentrations (2 14


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10 µM), suggesting that α-mangostin exhibits a high affinity for ABCG2. These results demonstrated that α-mangostin binds to the substrate-binding pocket(s) of ABCG2 and attenuates the transport function of ABCG2 in a competitive manner.

Since high-resolution crystal structure of human ABCG2 is still unavailable, an ABCG2 homology model was used instead to better understand the binding of α-mangostin with ABCG2. The docking data were in accordance with our experimental results that α-mangostin binds to the substrate binding pocket of ABCG2. In summary, our results indicate that α-mangostin directly and selectively inhibits the transport function of ABCG2 and re-sensitizes ABCG2-overexpressing MDR cancer cells to chemotherapeutic drugs without altering the expression level of ABCG2. Although studies showing favorable MDR reversal results do not necessarily translate into successful clinical outcomes 13, our data suggest that in combination with conventional anticancer agents, α-mangostin could improve oral bioavailability or treatment outcome in cancer patients.

CONFLICT OF INTEREST None.

ACKNOWLEDGMENTS This work was supported by the Chang Gung Medical Research Program CMRPD190653, CMRPD1D0153, CMRPD1G0111, BMRPC17 (CPW); the Ministry of Science and Technology of Taiwan MOST-105-2320-B-182-018 (CPW) and MOST-102-2113-M-029-005 (YSW); Taichung Veterans General Hospital TCVGH-T1057805 (YSW); the Ministry of Education EMRPD1G0121 (CPW) and the Intramural Research Program of the National Institutes of Health, National Cancer 15



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Institute, Center for Cancer Research (MM & SVA).

REFERENCES

1.

Wu, C. P.; Hsieh, C. H.; Wu, Y. S.

The emergence of drug transporter-mediated

multidrug resistance to cancer chemotherapy. Mol Pharm 2011, 8, (6), 1996-2011. 2. Gottesman, M. M. Mechanisms of cancer drug resistance. Annual review of medicine 2002, 53, 615-27. 3. Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M. Targeting multidrug resistance in cancer. Nature reviews 2006, 5, (3), 219-34. 4. Doyle, L. A.; Yang, W.; Abruzzo, L. V.; Krogmann, T.; Gao, Y.; Rishi, A. K.; Ross, D. D. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America 1998, 95, (26), 15665-70. 5. Allikmets, R.; Schriml, L. M.; Hutchinson, A.; Romano-Spica, V.; Dean, M. A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance. Cancer Res 1998, 58, (23), 5337-9. 6. Steinbach, D.; Sell, W.; Voigt, A.; Hermann, J.; Zintl, F.; Sauerbrey, A. BCRP gene expression is associated with a poor response to remission induction therapy in childhood acute myeloid leukemia. Leukemia 2002, 16, (8), 1443-7. 7. Dohse, M.; Scharenberg, C.; Shukla, S.; Robey, R. W.; Volkmann, T.; Deeken, J. F.; Brendel, C.; Ambudkar, S. V.; Neubauer, A.; Bates, S. E. Comparison of ATP-binding cassette transporter interactions with the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib. Drug metabolism and disposition: the biological fate of chemicals 2010, 38, (8), 1371-80. 8. Tang, C.; Schafranek, L.; Watkins, D. B.; Parker, W. T.; Moore, S.; Prime, J. A.; White, D. L.; Hughes, T. P.

Tyrosine kinase inhibitor resistance in chronic myeloid

leukemia cell lines: investigating resistance pathways. Leuk Lymphoma 2011, 52, (11), 2139-47. 9. Yoh, K.; Ishii, G.; Yokose, T.; Minegishi, Y.; Tsuta, K.; Goto, K.; Nishiwaki, Y.; Kodama, T.; Suga, M.; Ochiai, A. Breast cancer resistance protein impacts clinical outcome in platinum-based chemotherapy for advanced non-small cell lung cancer. Clin Cancer Res 2004, 10, (5), 1691-7. 10. Hori, S.; Ohtsuki, S.; Tachikawa, M.; Kimura, N.; Kondo, T.; Watanabe, M.; Nakashima, E.; Terasaki, T. Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s). J 16


Page 16 of 39

Page 17 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Neurochem 2004, 90, (3), 526-36. 11. Cooray, H. C.; Blackmore, C. G.; Maskell, L.; Barrand, M. A.

Localisation of

breast cancer resistance protein in microvessel endothelium of human brain. Neuroreport 2002, 13, (16), 2059-63. 12. Wu, C. P.; Calcagno, A. M.; Ambudkar, S. V. Reversal of ABC drug transporter-mediated multidrug resistance in cancer cells: evaluation of current strategies. Current molecular pharmacology 2008, 1, (2), 93-105. 13. Shukla, S.; Wu, C. P.; Ambudkar, S. V. Development of inhibitors of ATP-binding cassette drug transporters: present status and challenges. Expert Opin Drug Metab Toxicol 2008, 4, (2), 205-223. 14. Shukla, S.; Ohnuma, S.; Ambudkar, S. V.

Improving cancer chemotherapy with

modulators of ABC drug transporters. Curr Drug Targets 2011, 12, (5), 621-30. 15. Wu, C. P.; Ohnuma, S.; Ambudkar, S. V. Discovering natural product modulators to overcome multidrug resistance in cancer chemotherapy. Curr Pharm Biotechnol 2011, 12, (4), 609-20. 16. Dennis, T.; Fanous, M.; Mousa, S.

Natural products for chemopreventive and

adjunctive therapy in oncologic disease. Nutrition and cancer 2009, 61, (5), 587-97. 17. Devi Sampath, P.; Vijayaraghavan, K. Cardioprotective effect of alpha-mangostin, a xanthone derivative from mangosteen on tissue defense system against isoproterenol-induced myocardial infarction in rats. Journal of biochemical and molecular toxicology 2007, 21, (6), 336-9. 18. Sampath, P. D.; Kannan, V. Mitigation of mitochondrial dysfunction and regulation of eNOS expression during experimental myocardial necrosis by alpha-mangostin, a xanthonic derivative from Garcinia mangostana. Drug and chemical toxicology 2009, 32, (4), 344-52. 19. Sun, D.; Zhang, S.; Wei, Y.; Yin, L. Antioxidant activity of mangostin in cell-free system and its effect on K562 leukemia cell line in photodynamic therapy. Acta biochimica et biophysica Sinica 2009, 41, (12), 1033-43. 20. Buelna-Chontal, M.; Correa, F.; Hernandez-Resendiz, S.; Zazueta, C.; Pedraza-Chaverri, J. Protective effect of alpha-mangostin on cardiac reperfusion damage by attenuation of oxidative stress. Journal of medicinal food 2011, 14, (11), 1370-4. 21. Hafeez, B. B.; Mustafa, A.; Fischer, J. W.; Singh, A.; Zhong, W.; Shekhani, M. O.; Meske, L.; Havighurst, T.; Kim, K.; Verma, A. K. alpha-Mangostin: a dietary antioxidant derived from the pericarp of Garcinia mangostana L. inhibits pancreatic tumor growth in xenograft mouse model. Antioxidants & redox signaling 2014, 21, (5), 682-99. 22. Shan, T.; Ma, Q.; Guo, K.; Liu, J.; Li, W.; Wang, F.; Wu, E. Xanthones from 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 39

mangosteen extracts as natural chemopreventive agents: potential anticancer drugs. Current molecular medicine 2011, 11, (8), 666-77. 23. Reyes-Fermin, L. M.; Gonzalez-Reyes, S.; Tarco-Alvarez, N. G.; Hernandez-Nava, M.; Orozco-Ibarra, M.; Pedraza-Chaverri, J. Neuroprotective effect of alpha-mangostin and curcumin against iodoacetate-induced cell death. Nutritional neuroscience 2012, 15, (5), 34-41. 24. Al-Massarani, S. M.; El Gamal, A. A.; Al-Musayeib, N. M.; Mothana, R. A.; Basudan, O. A.; Al-Rehaily, A. J.; Farag, M.; Assaf, M. H.; El Tahir, K. H.; Maes, L. Phytochemical, antimicrobial and antiprotozoal evaluation of Garcinia mangostana pericarp and alpha-mangostin, its major xanthone derivative. Molecules 2013, 18, (9), 10599-608. 25. Zhang, K. J.; Gu, Q. L.; Yang, K.; Ming, X. J.; Wang, J. X.

Anticarcinogenic

Effects of alpha-Mangostin: A Review. Planta medica 2016. 26. Kurose, H.; Shibata, M. A.; Iinuma, M.; Otsuki, Y. Alterations in cell cycle and induction of apoptotic cell death in breast cancer cells treated with alpha-mangostin extracted from mangosteen pericarp. Journal of biomedicine & biotechnology 2012, 2012, 672428. 27. Xu, Q.; Ma, J.; Lei, J.; Duan, W.; Sheng, L.; Chen, X.; Hu, A.; Wang, Z.; Wu, Z.; Wu, E.; Ma, Q.; Li, X. alpha-Mangostin suppresses the viability and epithelial-mesenchymal transition of pancreatic cancer cells by downregulating the PI3K/Akt pathway. BioMed research international 2014, 2014, 546353. 28. Hsieh, S. C.; Huang, M. H.; Cheng, C. W.; Hung, J. H.; Yang, S. F.; Hsieh, Y. H. alpha-Mangostin induces mitochondrial dependent apoptosis in human hepatoma SK-Hep-1 cells through inhibition of p38 MAPK pathway. Apoptosis : an international journal on programmed cell death 2013, 18, (12), 1548-60. 29. Watanapokasin, R.; Jarinthanan, F.; Jerusalmi, A.; Suksamrarn, S.; Nakamura, Y.; Sukseree, S.; Uthaisang-Tanethpongtamb, W.; Ratananukul, P.; Sano, T. Potential of xanthones from tropical fruit mangosteen as anti-cancer agents: caspase-dependent apoptosis induction in vitro and in mice. Applied biochemistry and biotechnology 2010, 162, (4), 1080-94. 30. Chao, A. C.; Hsu, Y. L.; Liu, C. K.; Kuo, P. L.

alpha-Mangostin, a dietary

xanthone, induces autophagic cell death by activating the AMP-activated protein kinase pathway in glioblastoma cells. J Agric Food Chem 2011, 59, (5), 2086-96. 31. Shibata, M. A.; Iinuma, M.; Morimoto, J.; Kurose, H.; Akamatsu, K.; Okuno, Y.; Akao, Y.; Otsuki, Y. alpha-Mangostin extracted from the pericarp of the mangosteen (Garcinia mangostana Linn) reduces tumor growth and lymph node metastasis in an immunocompetent xenograft model of metastatic mammary cancer carrying a p53 mutation. BMC medicine 2011, 9, 69. 18


Page 19 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


32. Jittiporn, K.; Suwanpradid, J.; Patel, C.; Rojas, M.; Thirawarapan, S.; Moongkarndi, P.; Suvitayavat, W.; Caldwell, R. B.

Anti-angiogenic actions of the

mangosteen polyphenolic xanthone derivative alpha-mangostin. Microvascular research 2014, 93, 72-9. 33. Lei, J.; Huo, X.; Duan, W.; Xu, Q.; Li, R.; Ma, J.; Li, X.; Han, L.; Li, W.; Sun, H.; Wu, E.; Ma, Q.

alpha-Mangostin inhibits hypoxia-driven ROS-induced PSC

activation and pancreatic cancer cell invasion. Cancer Lett 2014, 347, (1), 129-38. 34. Wang, J. J.; Sanderson, B. J.; Zhang, W. Significant anti-invasive activities of alpha-mangostin from the mangosteen pericarp on two human skin cancer cell lines. Anticancer research 2012, 32, (9), 3805-16. 35. Matsumoto, K.; Akao, Y.; Kobayashi, E.; Ohguchi, K.; Ito, T.; Tanaka, T.; Iinuma, M.; Nozawa, Y.

Induction of apoptosis by xanthones from mangosteen in

human leukemia cell lines. Journal of natural products 2003, 66, (8), 1124-7. 36. Chen, J. J.; Long, Z. J.; Xu, D. F.; Xiao, R. Z.; Liu, L. L.; Xu, Z. F.; Qiu, S. X.; Lin, D. J.; Liu, Q. Inhibition of autophagy augments the anticancer activity of alpha-mangostin in chronic myeloid leukemia cells. Leuk Lymphoma 2014, 55, (3), 628-38. 37. Tangpong, J.; Miriyala, S.; Noel, T.; Sinthupibulyakit, C.; Jungsuwadee, P.; St Clair, D. K. Doxorubicin-induced central nervous system toxicity and protection by xanthone derivative of Garcinia mangostana. Neuroscience 2011, 175, 292-9. 38. Akao, Y.; Nakagawa, Y.; Iinuma, M.; Nozawa, Y. Anti-cancer effects of xanthones from pericarps of mangosteen. International journal of molecular sciences 2008, 9, (3), 355-70. 39. Wu, C. P.; Shukla, S.; Calcagno, A. M.; Hall, M. D.; Gottesman, M. M.; Ambudkar, S. V. Evidence for dual mode of action of a thiosemicarbazone, NSC73306: a potent substrate of the multidrug resistance linked ABCG2 transporter. Mol Cancer Ther 2007, 6, (12 Pt 1), 3287-3296. 40. Shen, D. W.; Fojo, A.; Chin, J. E.; Roninson, I. B.; Richert, N.; Pastan, I.; Gottesman, M. M.

Human multidrug-resistant cell lines: increased mdr1 expression

can precede gene amplification. Science 1986, 232, (4750), 643-645. 41. Honjo, Y.; Hrycyna, C. A.; Yan, Q. W.; Medina-Perez, W. Y.; Robey, R. W.; van de Laar, A.; Litman, T.; Dean, M.; Bates, S. E. Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells. Cancer Res 2001, 61, (18), 6635-6639. 42. Robey, R. W.; Medina-Perez, W. Y.; Nishiyama, K.; Lahusen, T.; Miyake, K.; Litman, T.; Senderowicz, A. M.; Ross, D. D.; Bates, S. E. Overexpression of the ATP-binding Cassette Half-Transporter, ABCG2 (MXR/BCRP/ABCP1), in Flavopiridol-resistant Human Breast Cancer Cells. Clin Cancer Res 2001, 7, (1), 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39

145-152. 43. Robey, R. W.; Steadman, K.; Polgar, O.; Morisaki, K.; Blayney, M.; Mistry, P.; Bates, S. E.

Pheophorbide a is a specific probe for ABCG2 function and inhibition.

Cancer Res 2004, 64, (4), 1242-1246. 44. Gribar, J. J.; Ramachandra, M.; Hrycyna, C. A.; Dey, S.; Ambudkar, S. V. Functional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system. J Membr Biol 2000, 173, (3), 203-14. 45. Ishiyama, M.; Tominaga, H.; Shiga, M.; Sasamoto, K.; Ohkura, Y.; Ueno, K.

A

combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull 1996, 19, (11), 1518-20. 46. Ambudkar, S. V.

Drug-stimulatable ATPase activity in crude membranes of

human MDR1-transfected mammalian cells. Methods Enzymol 1998, 292, 504-514. 47. Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006, 22, (2), 195-201. 48. Benkert, P.; Biasini, M.; Schwede, T.

Toward the estimation of the absolute

quality of individual protein structure models. Bioinformatics 2011, 27, (3), 343-50. 49. Biasini, M.; Bienert, S.; Waterhouse, A.; Arnold, K.; Studer, G.; Schmidt, T.; Kiefer, F.; Gallo Cassarino, T.; Bertoni, M.; Bordoli, L.; Schwede, T. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic acids research 2014, 42, (Web Server issue), W252-8. 50. Kartner, N.; Riordan, J. R.; Ling, V. Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines. Science 1983, 221, (4617), 1285-8. 51. Dai, C. L.; Tiwari, A. K.; Wu, C. P.; Su, X. D.; Wang, S. R.; Liu, D. G.; Ashby, C. R., Jr.; Huang, Y.; Robey, R. W.; Liang, Y. J.; Chen, L. M.; Shi, C. J.; Ambudkar, S. V.; Chen, Z. S.; Fu, L. W. Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2. Cancer Res 2008, 68, (19), 7905-7914. 52. Wu, C. P.; Hsiao, S. H.; Su, C. Y.; Luo, S. Y.; Li, Y. Q.; Huang, Y. H.; Hsieh, C. H.; Huang, C. W. Human ATP-Binding Cassette transporters ABCB1 and ABCG2 confer resistance to CUDC-101, a multi-acting inhibitor of histone deacetylase, epidermal growth factor receptor and human epidermal growth factor receptor 2. Biochem Pharmacol 2014, 92, (4), 567-76. 53. Cuestas, M. L.; Castillo, A. I.; Sosnik, A.; Mathet, V. L. Downregulation of mdr1 and abcg2 genes is a mechanism of inhibition of efflux pumps mediated by polymeric amphiphiles. Bioorg Med Chem Lett 2012, 22, (21), 6577-9. 20


Page 21 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


54. Natarajan, K.; Bhullar, J.; Shukla, S.; Burcu, M.; Chen, Z. S.; Ambudkar, S. V.; Baer, M. R.

The Pim kinase inhibitor SGI-1776 decreases cell surface expression of

P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent mechanisms. Biochem Pharmacol 2013, 85, (4), 514-24. 55. Ambudkar, S. V.; Dey, S.; Hrycyna, C. A.; Ramachandra, M.; Pastan, I.; Gottesman, M. M. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annual review of pharmacology and toxicology 1999, 39, 361-98. 56. Ambudkar, S. V.; Kimchi-Sarfaty, C.; Sauna, Z. E.; Gottesman, M. M. P-glycoprotein: from genomics to mechanism. Oncogene 2003, 22, (47), 7468-85. 57. Lee, J. Y.; Kinch, L. N.; Borek, D. M.; Wang, J.; Wang, J.; Urbatsch, I. L.; Xie, X. S.; Grishin, N. V.; Cohen, J. C.; Otwinowski, Z.; Hobbs, H. H.; Rosenbaum, D. M. Crystal structure of the human sterol transporter ABCG5/ABCG8. Nature 2016, 533, (7604), 561-4. 58. Laszlo, L.; Sarkadi, B.; Hegedus, T. Jump into a New Fold-A Homology Based Model for the ABCG2/BCRP Multidrug Transporter. PLoS One 2016, 11, (10), e0164426. 59. Noguchi, K.; Katayama, K.; Sugimoto, Y. Human ABC transporter ABCG2/BCRP expression in chemoresistance: basic and clinical perspectives for molecular cancer therapeutics. Pharmacogenomics and personalized medicine 2014, 7, 53-64. 60. Rabindran, S. K.; Ross, D. D.; Doyle, L. A.; Yang, W.; Greenberger, L. M. Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein. Cancer Res 2000, 60, (1), 47-50. 61. Allen, J. D.; van Loevezijn, A.; Lakhai, J. M.; van der Valk, M.; van Tellingen, O.; Reid, G.; Schellens, J. H.; Koomen, G. J.; Schinkel, A. H. Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. Mol Cancer Ther 2002, 1, (6), 417-25. 62. Jindarat, S. Xanthones from mangosteen (Garcinia mangostana): multi-targeting pharmacological properties. Journal of the Medical Association of Thailand = Chotmaihet thangphaet 2014, 97 Suppl 2, S196-201. 63. Xie, Z.; Sintara, M.; Chang, T.; Ou, B. Daily consumption of a mangosteen-based drink improves in vivo antioxidant and anti-inflammatory biomarkers in healthy adults: a randomized, double-blind, placebo-controlled clinical trial. Food science & nutrition 2015, 3, (4), 342-8. 64. Chearwae, W.; Shukla, S.; Limtrakul, P.; Ambudkar, S. V. 21


Modulation of the


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

function of the multidrug resistance-linked ATP-binding cassette transporter ABCG2 by the cancer chemopreventive agent curcumin. Mol Cancer Ther 2006, 5, (8), 1995-2006. 65. van de Wetering, K.; Burkon, A.; Feddema, W.; Bot, A.; de Jonge, H.; Somoza, V.; Borst, P. Intestinal breast cancer resistance protein (BCRP)/Bcrp1 and multidrug resistance protein 3 (MRP3)/Mrp3 are involved in the pharmacokinetics of resveratrol. Mol Pharmacol 2009, 75, (4), 876-85. 66. Shukla, S.; Wu, C. P.; Nandigama, K.; Ambudkar, S. V. The naphthoquinones, vitamin K3 and its structural analogue plumbagin, are substrates of the multidrug resistance linked ATP binding cassette drug transporter ABCG2. Mol Cancer Ther 2007, 6, (12 Pt 1), 3279-86. 67. Gottesman, M. M.; Fojo, T.; Bates, S. E.

Multidrug resistance in cancer: role of

ATP-dependent transporters. Nat Rev Cancer 2002, 2, (1), 48-58. 68. Chearwae, W.; Anuchapreeda, S.; Nandigama, K.; Ambudkar, S. V.; Limtrakul, P. Biochemical mechanism of modulation of human P-glycoprotein (ABCB1) by curcumin I, II, and III purified from Turmeric powder. Biochem Pharmacol 2004, 68, (10), 2043-52. 69. Ludwig, J. A.; Szakacs, G.; Martin, S. E.; Chu, B. F.; Cardarelli, C.; Sauna, Z. E.; Caplen, N. J.; Fales, H. M.; Ambudkar, S. V.; Weinstein, J. N.; Gottesman, M. M. Selective toxicity of NSC73306 in MDR1-positive cells as a new strategy to circumvent multidrug resistance in cancer. Cancer Res 2006, 66, (9), 4808-4815. 70. Su, F.; Bradley, W. D.; Wang, Q.; Yang, H.; Xu, L.; Higgins, B.; Kolinsky, K.; Packman, K.; Kim, M. J.; Trunzer, K.; Lee, R. J.; Schostack, K.; Carter, J.; Albert, T.; Germer, S.; Rosinski, J.; Martin, M.; Simcox, M. E.; Lestini, B.; Heimbrook, D.; Bollag, G. Resistance to selective BRAF inhibition can be mediated by modest upstream pathway activation. Cancer Res 2012, 72, (4), 969-978. 71. Shukla, S.; Robey, R. W.; Bates, S. E.; Ambudkar, S. V. The calcium channel blockers, 1,4-dihydropyridines, are substrates of the multidrug resistance-linked ABC drug transporter, ABCG2. Biochemistry 2006, 45, (29), 8940-51. 72. Sauna, Z. E.; Ambudkar, S. V. Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein. Proc Natl Acad Sci U S A 2000, 97, (6), 2515-2520. 73. Ambudkar, S. V.; Cardarelli, C. O.; Pashinsky, I.; Stein, W. D. Relation between the turnover number for vinblastine transport and for vinblastine-stimulated ATP hydrolysis by human P-glycoprotein. J Biol Chem 1997, 272, (34), 21160-21166. 74. Hegedus, C.; Ozvegy-Laczka, C.; Szakacs, G.; Sarkadi, B. Interaction of ABC multidrug transporters with anticancer protein kinase inhibitors: substrates and/or inhibitors? Curr Cancer Drug Targets 2009, 9, (3), 252-272. 22


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75. Wu, C. P.; Hsieh, Y. J.; Hsiao, S. H.; Su, C. Y.; Li, Y. Q.; Huang, Y. H.; Huang, C. W.; Hsieh, C. H.; Yu, J. S.; Wu, Y. S.

Human ATP-Binding Cassette Transporter

ABCG2 Confers Resistance to CUDC-907, a Dual Inhibitor of Histone Deacetylase and Phosphatidylinositol 3-Kinase. Mol Pharm 2016, 13, (3), 784-94.

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FIGURE LEGENDS

Figure 1. The chemical structure of α-mangostin.

Figure 2. The effect of α-mangostin on the fluorescent substrate transport mediated by ABCB1 or ABCG2. The accumulation of calcein-AM in parental HEK293 (A, left panel) and ABCB1-transfected MDR19-HEK293 cells (A, right panel), as well as the accumulation of pheophorbide A (PhA) in parental HEK293 (B, left panel) and ABCG2-transfected R482-HEK293 cells (B, right panel) or in S1 (C, left panel) and ABCG2-overexpressing S1-M1-80 (C, right panel) or in MCF-7 (D, left panel) and ABCG2-overexpressing MCF7-FLV1000 cancer cells (D, right panel), were measured in the absence (solid lines) or presence of 5 µM α-mangostin (shaded, solid lines) or a reference inhibitor (dotted lines) of ABCB1 (3 µM tariquidar) or ABCG2 (1 µM Ko143). Cells were analyzed immediately by flow cytometry as described in Experimental section. Representative histograms and immunoblots of ABCB1 (A, inset) or ABCG2 (B - D, inset) in total cell lysate protein (10 µg) from pcDNA-HEK293, MDR19-HEK293, R482-HEK293, S1, S1-M1-80, MCF7 and MCF7-FLV1000 cells are shown.

Figure 3. Effect of α-mangostin on reversing multidrug resistance mediated by ABCG2 or ABCB1. Cytotoxicity of topotecan (A) or mitoxantrone (B) in HEK293 cells (left panels) and ABCG2-transfected R482-HEK293 cells (right panels), as well as doxorubicin (C) or colchicine (D) in HEK293 cells (left panels) and ABCB1-transfected MDR19-HEK293 cells (right panels) was determined in the absence (open circles) or presence of α-mangostin at 0.5 µM (filled circles), 1.0 µM (open squares), 2.0 µM (filled squares) or 1 µM of reference inhibitor Ko143 or 24


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tariquidar (open triangles). Points: means from at least three independent experiments; bars: SEM.

Figure 4. Effect of α-mangostin on ABCG2 protein expression in human S1-M1-80 colon cancer cells. (A) Immunoblot detection of human ABCG2 and (B) quantification of total lysate protein (10 µg) from drug-resistant S1-M1-80 cells treated with increasing concentrations (0 - 3 µM) of α-mangostin (MG) for 72 h as described previously 75. α-Tubulin was used as loading control. Values are presented as mean ± SEM calculated from three independent experiments.

Figure 5. Effect of α-mangostin on photoaffinity labeling of ABCG2 with [125I]IAAP and vanadate (Vi)-sensitive ABCG2 ATPase activity. (A) Membrane protein (250 500 µg protein/mL) from MCF7-FLV1000 cells expressing ABCG2 was incubated in the presence or absence of 10 µM of α-mangostin at 21°C in 50 µmol/L Tris-HCL (pH 7.5), 150 mM NaCl. [125I]IAAP (2,200 Ci/mmol) at 3 to 6 nmol/L was added to the samples and incubated for 5 min under subdued light, then illuminated with an UV lamp (365 nm) for 10 min at room temperature. The labeled ABCG2 was processed and visualized as described previously 39. Representative autoradiogram (A, inset) and values represent means and standard deviations calculated from three independent experiments. (B) Membrane protein (50 - 100 µg protein/mL) from High-Five insect cells expressing ABCG2 was incubated at 37°C with 0 - 10 µM of α-mangostin and 0 - 1 µM of α-mangostin (B, inset) in the presence or absence of vanadate. The ABCG2 ATPase activity was measured as described previously 39. Points: mean from at least three independent experiments; bar: SEM.

Figure 6. Binding mode of α-mangostin in the homology model of ABCG2 by using 25



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Acclerys Discovery Studio 4.0 software as described in Experimental section. α-Mangostin is shown as ball and stick model with the atoms colored as carbon-dark gray, hydrogen-light gray, nitrogen-blue and oxygen-red. The same color scheme is used for interacting amino acid residues. Dotted green line indicates proposed hydrogen bond.

26


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Table 1. Effect of α-mangostin on ABCG2- and ABCB1-mediated multidrug resistance in transfected and drug-selected cell lines treatment

Mean IC50† ± SD and (FR‡)

concentration (µM)

pcDNA-HEK293

R482-HEKS293

[nM]

[nM]

-

17.90 ± 4.21 (1.0)

627.68 ± 135.29 (1.0)

+ α-mangostin

0.5

18.28 ± 4.46 (1.0)

252.47 ± 39.59** (2.5)

+ α-mangostin

1

19.18 ± 5.03 (0.9)

73.15 ± 8.21** (8.6)

+ α-mangostin

2

18.84 ± 4.47 (1.0)

25.21 ± 5.56** (24.9)

+ Ko143

1

19.06 ± 4.93

24.43 ± 7.46** (25.7)

mitoxantrone

-

1.93 ± 0.41 (1.0)

136.14 ± 27.99 (1.0)

+ α-mangostin

0.5

2.05 ± 0.44 (0.9)

84.53 ± 15.80* (1.6)

+ α-mangostin

1

2.20 ± 0.45 (0.9)

35.47 ± 4.73** (3.8)

+ α-mangostin

2

2.52 ± 0.48 (0.8)

15.62 ± 2.60** (8.7)

+ Ko143

1

1.72 ± 0.32 (1.1)

8.09 ± 1.88** (16.8)

topotecan

topotecan

S1

S1-M1-80

[nM]

[µM]

29.18 ± 7.29 (1.0)

7.60 ± 2.09 (1.0)

+ α-mangostin

0.5

27.69 ± 6.05 (1.1)

2.17 ± 0.41* (3.5)

+ α-mangostin

1

31.93 ± 6.99 (0.9)

0.77 ± 0.11** (9.9)

+ α-mangostin

2

25.75 ± 6.00 (1.1)

0.53 ± 0.14** (14.3)

+ Ko143

1

31.83 ± 7.82 (0.9)

0.40 ± 0.11** (19.0)

mitoxantrone

-

8.87 ± 1.91 (1.0)

89.98 ± 16.19 (1.0)

+ α-mangostin

0.5

11.15 ± 2.06 (0.8)

55.58 ± 13.69* (1.6)

27



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Page 28 of 39

+ α-mangostin

1

10.67 ± 1.94 (0.8)

23.86 ± 4.36** (3.8)

+ α-mangostin

2

8.45 ± 1.98 (1.0)

9.11 ± 2.04** (9.9)

+ Ko143

1

7.90 ± 1.84 (1.1)

1.04 ± 0.16*** (86.5)

pcDNA-HEK293

MDR19-HEKS293

[nM]

[nM]

doxorubicin

-

11.84 ± 2.57 (1.0)

179.08 ± 48.63 (1.0)

+ α-mangostin

2

10.99 ± 2.54 (1.1)

226.67 ± 56.13 (0.8)

+ tariquidar

1

6.34 ± 1.49* (1.9)

20.43 ± 5.13* (8.8)

16.22 ± 5.83 (1.0)

317.87 ± 89.54 (1.0)

colchicine + α-mangostin

2

13.67 ± 4.18 (1.2)

228.31 ± 59.00 (1.4)

+ tariquidar

1

9.28 ± 3.79 (1.7)

14.15 ± 4.47** (22.5)

KB-3-1

KB-V-1

[nM]

[µM]

doxorubicin

-

30.60 ± 7.95 (1.0)

1.91 ± 0.37 (1.0)

+ α-mangostin

2

16.90 ± 5.49 (1.8)

1.24 ± 0.33 (1.5)

+ tariquidar

1

26.13 ± 6.07 (1.2)

42.44 ± 10.48 [nM]*** (45.0)

31.67 ± 10.53 (1.0)

1.64 ± 0.15 (1.0)

colchicine + α-mangostin

2

29.88 ± 9.35 (1.1)

1.54 ± 0.19 (1.1)

+ tariquidar

1

30.89 ± 9.95 (1.0)

44.20 ± 14.59 [nM]*** (37.1)

Abbreviation: FR, fold-reversal. † IC50 values are mean ± SD calculated from dose-response curves obtained from three independent experiments using cytotoxicity assay as described in Experimental section. ‡ FR values were obtained by dividing IC50 values of cells treated with a given anticancer drug in the absence of a modulator by IC50 values of cells treated with the same anticancer drug in the presence of a modulator. *P < 0.05; **P < 0.01 ; ***P < 0.001 28


Page 29 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Cytotoxicity of α-mangostin in drug-sensitivity and ABCG2-overexpressing multidrug resistant cells Cell line

Cancer origin

Transporter

IC50 (nM) †

R.F‡

expressed S1

colon

-

10.78 ± 4.48

1.0

S1-M1-80

colon

ABCG2

15.43 ± 5.63

1.4

H460

lung

-

11.58 ± 5.14

1.0

H460-MX20

lung

ABCG2

10.42 ± 4.39

0.9

A549

lung

-

13.04 ± 4.54

1.0

A549-Bec150

lung

ABCG2

10.84 ± 4.18

0.8

MCF7

breast

-

12.58 ± 6.97

1.0

MCF7-FLV1000

breast

ABCG2

13.18 ± 4.09

1.0

MCF7-AdVp3000

breast

ABCG2

17.89 ± 4.06

1.4

pcDNA-HEK293

-

-

17.31 ± 6.94

1.0

R482-HEK293

-

ABCG2

20.37 ± 7.64

1.2

Abbreviation: RF, resistance factor. † IC50 values are mean ± SD calculated from dose-response curves obtained from three independent experiments using cytotoxicity assay as described in Experimental section. ‡ RF values were calculated by dividing IC50 values of ABCG2-overexpressing MDR cells by IC50 values of drug-sensitive parental cells. *P < 0.05; **P < 0.01 ; ***P < 0.001

29



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For Table of Contents Only

30


Page 30 of 39

Page 31Wu of 39et 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

al. Figure 1



Wu et al. Figure 2

Page 32 of 39

MDR19

MDR19-HEK293

Cell Number

pcDNA-HEK293

pcDNA

A

ABCB1 Tubulin

Fluorescence Intensity


pcDNA-HEK293

R482-HEK293

Control + α-Mangostin + Tariquidar

R482

pcDNA

B

Cell Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


ABCG2 Tubulin




Control + α-Mangostin + Ko143

Page 33Wu of 39et


S1-M1-80

Cell Number

S1

S1 S1-M1-80

C

ABCG2 Tubulin




MCF-7

MCF7-FLV1000

MCF-7 FLV1000

D

Cell Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

al. Figure 2

ABCG2 Tubulin






Wu et al., Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

A

B

C


Page 34 of 39

Page 35 of 39


Wu et al., Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

D



Wu et al., Figure 4

A

S1-M1-80 0 0.5 1.0 2.0 3.0

α-Mangostin [μM]

ABCG2 Tubulin

B Relative ABCG2 protein expression (% control)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


Page 36 of 39

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Wu et al., Figure 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

A

ABCG2

71kDa 55kDa

B



Wu et al., Figure 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


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1 2 3 4 5 6 7



Alpha-Mangostin Reverses Multidrug Resistance by Attenuating the

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