1016 Preclinical report
Luteolin potentiates the sensitivity of colorectal cancer cell lines to oxaliplatin through the PPARγ/OCTN2 pathway Qiang Qua,b, Jian Qub, Yong Guoc, Bo-Ting Zhoua and Hong-Hao Zhoub Oxaliplatin is a chemotherapeutic agent used in the treatment of colorectal cancers. However, the mechanism controlling the cellular uptake and efflux of oxaliplatin is not completely understood. Organic cation/carnitine transporter 2 (OCTN2) is a member of the solute carrier superfamily and is a determinant of oxaliplatin uptake. OCTN2 is regulated by peroxisome proliferator-activated receptor γ (PPARγ) binding to the PPAR-response element within the first intron. Luteolin is a naturally occurring flavonoid and an agonist of PPARγ. Thus, we hypothesize that luteolin-mediated OCTN2 expression and activity potentiate the sensitivity of cancer cells to oxaliplatin. In this study, luteolin increased mRNA and protein expression of OCTN2 in a time-dependent and dose-dependent manner in colorectal cancer SW480 cells. This induction was attenuated by PPARγ antagonist GW9662 as well as by PPARγ knockdown, suggesting that the induction by luteolin is dependent on PPARγ. In uptake studies, luteolin increased the binding affinity of OCTN2 toward oxaliplatin and enhanced intracellular concentration of oxaliplatin. This finding is likely because of the increase of PDZ domain containing 1 (PDZK1) and PDZ domain containing 3
Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second most frequent cause of cancer-related death worldwide [1]. Oxaliplatin (trans-l1,2-diaminocyclohexane oxaliplatinum) is a thirdgeneration platinum chemotherapeutic that is used in the treatment of stage II, stage III, and stage IV CRC [2,3]. The cytotoxicity of oxaliplatin to cancer cells arises primarily from covalent binding to DNA, leading to a series of biochemical cascades, eventually cell death [4]. Studies aiming to identify such mechanisms have focused mainly on the influence of oxaliplatin on target genes and pathways involved in cell growth and apoptosis. However, the mechanism controlling the cellular uptake and efflux of oxaliplatin has not been investigated extensively, which could also be critical for increasing the therapeutic effect as well as reducing adverse reactions. Organic cation/carnitine transporter 2 (OCTN2) (SLC22A5) is an organic cation transporter of the solute carrier superfamily and is also well known as a sodiumSupplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.anti-cancerdrugs.com). 0959-4973 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
(PDZK2), which are known to facilitate the expression of OCTN2 on the cell surface and/or enhance transporter activity. Moreover, cell viability and cell apoptosis assays showed that luteolin increased oxaliplatin uptake and intracellular accumulation through OCTN2. Thus, our study showed that luteolin increased the sensitivity of colorectal cancer SW480 cells to oxaliplatin, likely through the PPARγ/OCTN2 pathway. Anti-Cancer Drugs 25:1016–1027 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Anti-Cancer Drugs 2014, 25:1016–1027 Keywords: colorectal cancer, luteolin, OCTN2, oxaliplatin, PPARγ a Department of Pharmacy, bInstitute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics and cDepartment of Surgery, Xiangya Hospital, Central South University, Changsha, China
Correspondence to Hong-Hao Zhou, PhD, Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Xiangya Hospital, Central South University, Changsha 410078, China Tel: + 86 731 848 05380; fax: + 86 731 823 54476; e-mail: [email protected] Received 14 November 2013 Revised form accepted 2 April 2014
dependent carnitine transporter [5]. OCTN2 is highly expressed in various tissues, such as the kidney, intestine, skeletal muscle, heart, colon, and brain [6,7]. A recent study showed that oxaliplatin accumulation and cytotoxicity were markedly increased in OCTN1-transfected and OCTN2-transfected cells, indicating that oxaliplatin was an excellent substrate of these transporters [8,9]. PDZ domain containing 1 (PDZK1) and PDZ domain containing 3 (PDZK2) are functional regulators of OCTN2 that modulate its expression and activity [10,11]. Peroxisome proliferator-activated receptor γ (PPARγ) regulates OCTN2 expression by binding to PPARresponse elements in the first intron as a heterodimer with the retinoid X receptor α (RXRα) [12]. Ligandactivated PPARγ has been investigated as a tumor suppressor with some favorable results, but remains controversial [13]. Although thiazolidinediones, the most widely used synthetic ligands for PPARγ, were used in preclinical studies as anticancer adjuvants against CRC cells, their uses may be limited because of predictable side effects on lipid and glucose metabolism in vivo [14]. Many herb products such as potent agonists of PPARγ have been investigated intensively for CRC prevention and therapeutic properties [15–17]. DOI: 10.1097/CAD.0000000000000125
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Luteolin and sensitivity of cancer cells Qu et al. 1017
Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a bioflavonoid, abundant in celery, green pepper, parsley, perilla leaf, and chamomile tea [18,19]. In 1,2-dimethyl hydrazineinduced and azoxymethane-induced colon cancer models, luteolin decreased the incidence of colon cancer, number of tumors per rat, and ameliorated the activities of the antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase [20,21]. Although the anticancer effects of luteolin have been investigated intensively, the mechanism and applicability of luteolin as possible adjuvants in combination with chemotherapeutics in CRC therapy remain less explained. A previous report suggested that luteolin showed agonist activity of PPARγ [22]. Thus, we hypothesized that luteolin probably acts as an activator of the PPARγ/OCTN2 pathway involved in oxaliplatin uptake. In the present study, we investigated the induction role of luteolin in PPARγ-activated OCTN2 expression and transport capability. We report that luteolin potentiates the sensitivity of SW480 cells to oxaliplatin by enhancing the uptake ability of OCTN2.
Materials and methods
(Thermo Scientific) following the instructions. The halfmaximal inhibitory concentration (IC50) was calculated using the SPSS 13.0 software (SPSS Inc., Chicago, Illinois, USA). Uptake experiment and high-performance liquid chromatography
Uptake studies of oxaliplatin were carried out in luteolinpretreated SW480 cells and quantified by highperformance liquid chromatography analysis. SW480 cells were pretreated with luteolin (5, 10, or 20 μmol/l) for 48 h and seeded in six-well plates at a density of 1 × 106/well. Cells were washed twice with PBS and incubated with 1 ml uptake buffer containing various concentrations of oxaliplatin (0, 1, 3, 10, 33, 100, or 300 μmol/l) for 30 min. Uptake of oxaliplatin was terminated by aspiration of uptake buffer. Cells were washed twice with PBS and lysed in 200 μl of 1% Triton X-100 solution for 6 h. Cell lysate (150 μl/well) was collected for the quantification of oxaliplatin by high-performance liquid chromatography as described elsewhere [8,9,23]. Cellular protein content was quantified using the BCA protein assay (Thermo Scientific). Vmax and Km were estimated by fitting data to the Michaelis–Menten equation using GraphPad Prism5 software (GraphPad, San Diego, California, USA).
Chemicals and reagents
GW9662, pioglitazone, oxaliplatin, and luteolin were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). TRIzol reagent was from Takara (Dalian, China). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, California, USA). All primary antibodies were purchased from Abcam Labs (Cambridge, Massachusetts, USA). Cell culture, treatments, and cell viability assay
CRC cell lines SW480 and LS174T were obtained from ATCC (Manassas, Virginia, USA). For cell treatments, SW480 or LS174T cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Scientific, Rockford, Illinois, USA) supplemented with 10% fetal bovine serum in six-well plates. A volume of 2 ml of medium containing various concentrations of luteolin or pioglitazone was added and incubated for 24, 48, or 72 h at 37°C. In the antagonist assay, SW480 cells were exposed to GW9662 (5, 10, or 20 μmol/l) for 1 h and then incubated with 20 μmol/l luteolin for 48 h. For cell viability assays, SW480 cells were pretreated with luteolin (5, 10, or 20 μmol/l) for 48 h and then seeded in a 96-well plate at 104 cells/well. Cells were treated with oxaliplatin at various concentrations for 48 h. As the vehicle control, dimethyl sulfoxide (0.1%) was added to control cells. Cell viability was determined with an MTS cell proliferating assay kit (Promega, Madison, Wisconsin, USA) using a Multiskan Ascent 354 Microplate Reader
Quantitative RT-PCR
After cell treatment, total RNA was extracted using TRIzol reagent and the concentration was measured. cDNA was synthesized from 2 μg of total RNA using the first cDNA synthesis kit (Takara). Quantitative RT-PCR reactions were carried out using the SYBR Green kit (Bio-Rad, Hercules, California, USA) with the primers listed in supplementary Table 1 (Supplemental digital content 1, http://links.lww.com/ACD/A66) and under the following conditions: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s (40 cycles), followed by a final incubation at 72°C for 2 min. The relative mRNA levels of PDZK1, PDZK2, PPARγ, RXRα, and OCTN2 were normalized to β-actin. The analysis was carried out using the 2 DDCt method [24]. Western blotting
After cell treatment, SW480 cells were lysed and the protein concentration was measured using the BCA protein assay (Thermo Scientific). The proteins were subjected to SDS-PAGE and electrophoretically transferred to a PVDF membrane. The membranes were incubated with anti-OCTN2 (1 : 400; Abcam Lab), anti-PPARγ (1 : 400), anti-RXRα (1 : 500), anti-PDZK1 (1 : 400), anti-PDZK2 (1 : 400), and anti-β-actin (1 : 1000) primary antibodies, and subsequently incubated with Dylight 800-labeled rabbit or mouse polyclonal antibody (1 : 7500; KPL Inc., Gaithersburg, Maryland,
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1018 Anti-Cancer Drugs 2014, Vol 25 No 9
Fig. 1
Relative OCTN2 mRNA levels (fold of control)
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Influence of luteolin (Lut) treatment on organic cation/carnitine transporter 2 (OCTN2) mRNA and protein levels. SW480 cells were treated with pioglitazone (Piog) and luteolin at various concentrations for 24, 48, or 72 h, and then total RNA and proteins were extracted and purified. Quantitative RT-PCR and western blotting were performed and analyzed. The level of β-actin was used as an internal control. Values are means ± SD of three separate experiments. *P < 0.05, statistically significant differences from the 0.1% dimethyl sulfoxide-treated group (control).
USA). The signal was detected and ratios of target protein against β-actin were calculated using the Odyssey Infrared Imaging System (LI-COR Biosciences, Cambridge, UK).
Knockdown of PPARγ by shRNA
To silence PPARγ expression, small hairpin RNA duplexes (shRNA) targeting the sequence AAAGCAA AGGCGAGGGCGATCTT of human PPARγ (sh-PPARγ)
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Luteolin and sensitivity of cancer cells Qu et al. 1019
Fig. 2
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Influence of luteolin (Lut) treatment on peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα) mRNA and protein levels. The methods of cell treatment, total RNA, and protein extraction, quantitative RT-PCR, and western blotting were analyzed. Values are means ± SD of three separate experiments. *P < 0.05, statistically significant differences from the 0.1% dimethyl sulfoxide-treated group (control).
[25] were synthesized and then inserted into the pGPH1-GFP/neo vector (GenePharma, Shanghai, China) for small interfering RNA production. SW480 cells were transfected by pGPH1-sh-PPARγ and pGPH1-sh-negative control using Lipofectamine 2000. Transfected cells were
sustained in 600 μg/ml kanamycin medium for 14 days and cell clones were selected by kanamycin resistance. PPARγ-knockdown and control cells were seeded in sixwell plates and exposed to 20 μmol/l luteolin or 0.1% dimethyl sulfoxide (vehicle control). Total cell proteins
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1020 Anti-Cancer Drugs 2014, Vol 25 No 9
Fig. 3
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Oxaliplatin (OXA) O
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Effects of luteolin (Lut) on the transport of oxaliplatin (OXA) by organic cation/carnitine transporter 2 (OCTN2) in SW480 cells. (a) The structure of oxaliplatin. (b) Chromatograms of the standard solution containing 100 ng/ml oxaliplatin. (c) Concentration-dependent oxaliplatin transport by OCTN2. SW480 cells were pretreated with luteolin at 5, 10, or 20 μmol/l for 48 h, and then exposed to oxaliplatin (0, 1, 3, 10, 33, 100, or 300 μmol/l) for an additional 30 min. Cells were harvested and oxaliplatin concentrations were determined by high-performance liquid chromatography. Values are means ± SD of three determinations. Results are expressed as pmol/l of oxaliplatin uptake/min/mg of total cell protein. DMSO, dimethyl sulfoxide.
were extracted and protein expression was determined by western blotting. Annexin V/propidium iodide double staining
SW480 cells were seeded in six-well plates and incubated for 48 h with DMEM containing positive control 5 μmol/l pioglitazone or luteolin at a final concentration of 5, 10, or 20 μmol/l, respectively. Cells were washed with PBS and continually exposed to 5 μmol/l oxaliplatin for 24 h. Each group was collected and cell apoptosis was analyzed by flow cytometry using the Annexin V-FITC staining kit (BD LSR II flow cytometer; BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instruction. Statistical analysis
For all results, statistical comparisons were performed with one-way analysis of variance, followed by a post-hoc test. A value of P less than 0.05 was considered statistically significant.
Results Luteolin activated PPARγ/RXRα and upregulated OCTN2 in SW480 cell lines
To investigate whether the upexpression of OCTN2 is the result of activating PPARγ/RXRα, SW480 cells were
exposed to pioglitazone (0–10 μmol/l) and luteolin (0–40 μmol/l) for 24, 48, or 72 h. As shown in Fig. 1a and b, pioglitazone, a PPARγ agonist, was used as a positive control, and it significantly increased the expression of OCTN2 at 24–48 h. Quantitative RT-PCR and protein analysis showed that luteolin markedly elevated the mRNA and protein levels of OCTN2 in a dose-dependent and time-dependent manner (Fig. 1a and c). As can be seen in Fig. 2, luteolin at 10–40 μmol/l elevated the mRNA and protein of RXRα compared with the control group at 48 and 72 h. However, protein and mRNA levels of PPARγ did not change (Fig. 2). Because of the treatment with higher concentrations of luteolin and the longer time of incubation, the cellular growth may be inhibited and the expression of OCNT2 probably was lower. Luteolin potentiates oxaliplatin uptake in SW480 cells
To examine the hypothesis that luteolin may enhance the transporting ability of OCTN2, uptake studies of oxaliplatin were performed and kinetic analyses were performed after SW480 cells were pretreated with luteolin for 48 h. With increasing concentrations of luteolin, Vmax was increased, but Km was decreased (Fig. 3 and Table 1). These results suggest that luteolin
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Luteolin and sensitivity of cancer cells Qu et al. 1021
Table 1 Michaelis–Menten kinetics for the effect of different concentrations of luteolin on oxaliplatin uptake in SW480 cells Treatments 0.01% DMSO + oxaliplatin 5 μmol/l luteolin + oxaliplatin 10 μmol/l luteolin + oxaliplatin 20 μmol/l luteolin + oxaliplatin
Km (μmol/l)
Vmax (pmol/mg protein/min)
31.12 ± 2.04 30.90 ± 3.30 22.18 ± 1.67* 19.91 ± 1.71*
21.66 ± 0.41 23.18 ± 0.71 25.44 ± 0.51* 29.46 ± 0.66*
Data are mean ± SD of three independent experiments. DMSO, dimethyl sulfoxide. *P < 0.05, significant differences from the 0.1% DMSO group.
increases the affinity of OCTN2 as well as the maximum transporting activity of OCTN2 to oxaliplatin. Luteolin modulates PDZK1 and PDZK2 expression in SW480 cell lines
We further measured the expression of the OCTN2 regulatory proteins, PDZK1 and PDZK2. In Fig. 4, the mRNA and protein levels of PDZK1 and PDZK2 were increased in a time-dependent and dose-dependent manner. Considering that luteolin at 20 μmol/l resulted in the highest increments in mRNA and protein expression for OCTN2, PDZK1, and PDZK2, this concentration and a 48 h period of treatment were used in the subsequent experiments. Luteolin-induced expression is dependent on PPARγ
To confirm the significant role of PPARγ in the expression of OCTN2, we pre-exposed SW480 cells to PPARγ antagonist GW9662 for 1 h, and then added 20 μmol/l luteolin. We found that GW9662 effectively prevented the induction effect of 20 μmol/l luteolin on PDZK1, PDZK2, RXRα, and OCTN2 in a concentrationdependent manner, but not on PPARγ expression (Fig. 5). It is possible that GW9662 abrogates the induction of luteolin by blocking the binding to PPARγ, but not decreasing PPARγ expression. To further gain more evidence that the above gene expression induced by luteolin is dependent on PPARγ, we used shRNA to knock down the expression of PPARγ in SW480 cells. As shown in Fig. 6, PPARγ expression in knockdown cells was markedly decreased to 37% of the control cells. Despite treatment with 20 μmol/l luteolin, PPARγ expression was only 61% of that of the control cells. Compared with the expression induced by 20 μmol/ l luteolin in control cells, protein levels of PDZK1, PDZK2, and OCTN2 in PPARγ knockdown cells were only 65.2, 58.7, and 69.9%, respectively. These results suggest that the effect of luteolin on target genes is dependent on PPARγ. Luteolin contributes toward the sensitivity of SW480 cells to oxaliplatin
To test whether luteolin potentiates oxaliplatin-induced cell apoptosis, cell viability was measured using an MTS assay. As shown in Fig. 7a, SW480 cells pretreated with
luteolin were more sensitive to oxaliplatin-induced apoptosis compared with cells treated with oxaliplatin alone. With increasing luteolin concentrations, the IC50 values of oxaliplatin in luteolin-pretreated cells decreased to 68.5, 37.1, and 31.1%, lower than the treatment of oxaliplatin alone. After pretreatment with luteolin (5, 10, or 20 μmol/l) for 48 h, SW480 cells were continually incubated with 5 μmol/l oxaliplatin for 24 h. The percentage of cell apoptosis was determined by flow cytometry. In Fig. 7b and c, SW480 cell apoptosis rates were increased after exposure to luteolin. The apoptosis rates of luteolin-pretreated groups (5, 10, or 20 μmol/l luteolin + 5 μmol/l oxaliplatin) were increased 1.7-, 1.8-, and 2.5-fold compared with the group of 5 μmol/l oxaliplatin alone. A similar result was observed in the positive group of 5 μmol/l pioglitazone. Compared with pretreatment with luteolin only (no oxaliplatin), the apoptosis rates were elevated after oxaliplatin exposure.
Discussion The aim of this work was to identify whether luteolin is an agonist of PPARγ and therefore plays an essential role in sensitizing SW480 cells to oxaliplatin through the PPARγ/OCTN2 pathway. In the present study, we showed that luteolin, by activating PPARγ, not only elevated the expression of OCTN2, PDZK1, and PDZK2 at both protein and mRNA levels but also increased the binding affinity of OCTN2 toward oxaliplatin. To the best of our knowledge, this is the first report to indicate that luteolin increases the sensitivity of SW480 cells to oxaliplatin, probably because of increased uptake and intracellular accumulation of oxaliplatin through OCTN2. Luteolin is effective as an antiproliferative agent for cancer prevention and therapy with multiple mechanisms [18,26]. The luteolin-induced PPARγ/ligand binding domain assay showed a dose-dependent curve with an EC50 of 15.6 μmol/l [22]. The PPARγ target gene GLUT4 was activated by luteolin to a similar degree as rosiglitazone [22]. Another study also found that 20 μmol/l luteolin activated the expression of adiponectin, leptin, GLUT4, and PPARγ2 in 3T3-L1 adipocytes and primary mouse adipose cells [27]. A previous EMSA assay showed that the first intron of human OCTN2 gene has a conserved noncanonical PPAR-responsive element [12]. A PPARγ agonist rosiglitazone could increase both mRNA and protein of OCTN2 in mouse colon epithelium [12]. These findings led us to postulate that luteolin probably induces OCTN2 through activation of PPARγ. In our studies, luteolin markedly increased both mRNA and protein of OCTN2 in a dose-dependent and timedependent manner in SW480 cells as well as in other colon cancer cells LS174T (Supplementary Fig. 1, Supplemental digital content 2, http://links.lww.com/ACD/ A67). The in-vitro and in-vivo PPAR-null models showed
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1022 Anti-Cancer Drugs 2014, Vol 25 No 9
Fig. 4
PDZK1
Relative PDZK1 mRNA levels (fold of control)
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Influence of luteolin (Lut) treatment on PDZ domain containing 1 (PDZK1) and PDZ domain containing 3 (PDZK2) mRNA and protein levels. Cell treatment, total RNA, and protein extraction, quantitative RT-PCR, and western blotting were performed as described under the Materials and methods section. Values are means ± SD of three separate experiments. *P < 0.05, statistically significant differences from the 0.1% dimethyl sulfoxide-treated group (control).
that only PPARγ transcriptionally regulated OCTN2 expression in human and mouse colon tissues, rather than other PPAR isoforms [12]. Previous studies suggested that luteolin inhibited the production of tumor necrosis factor α [27,28], which was reported to reduce RXRα
expression [29]. It is possible that luteolin indirectly upregulates RXRα through repression of tumor necrosis factor α production in SW480 cells. In sum, luteolin effectively activated PPARγ/RXRα and triggered the expression of OCTN2.
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Luteolin and sensitivity of cancer cells Qu et al. 1023
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Influence of the antagonist GW9662 on the induction effect of luteolin in SW480 cells. SW480 cells were coincubated with GW9662 at various concentrations and 20 μmol/l luteolin (Lut) for 48 h. Total proteins were extracted and western blotting was performed as described under the Materials and methods section. The value of the control group (0.1% dimethyl sulfoxide) was set as 1. *P < 0.05, statistically significant differences from the treatment group of 20 μmol/l luteolin alone. OCTN2, organic cation/carnitine transporter 2; PDZK1, PDZ domain containing 1; PDZK2, PDZ domain containing 3; RXRα, retinoid X receptor α.
Interestingly, animals overexpressing colon PPARγ showed not only a significant increase in OCTN2 expression, but the plasma concentration of carnitine [12], which is the classic substrate of OCTN2 [5]. This implies that the ligand-activating or overexpression of PPARγ facilitates the ability of OCTN2 transport. In our uptake studies, luteolin enhanced the capacity of OCTN2 to transport oxaliplatin in SW480 cells, which not only increased Vmax but also decreased Km in comparison with the control group. Thus, we focused on whether luteolin affected OCTN2 regulatory proteins, PDZK1 and PDZK2. PDZK1 is a functional regulator through direct interaction with the carboxyl terminus of OCTN2 to enhance the transport capacity, without influencing cell-surface expression of OCTN2 [10]. We speculated that luteolin may activate PPARγ, resulting in upregulation of PDZK1 through PPAR-response element in SW480 cells, the same binding region as PPARα in HepG2 cells [30]. PDZK2, another PDZ domain protein, is also increased in SW480 cells and, when colocalized with OCTN2, contributes toward the stabilization of OCTN2 expression on the cell surface [11]. Previous studies have presented an ‘intracellular pool’ model,
which involves dynamic regulation of a reservoir of OCTN2 [11]. Thus, we propose that luteolin probably indirectly modulates PDZK1 and PDZK2 and triggers the regulatory network, which not only enriches the expression level of OCTN2 but also increases the delivering capacity of OCTN2 from the ‘intracellular poor’ to the cell surface. To further identify whether luteolin-induced OCTN2 expression depends on PPARγ, we utilized a PPARγ antagonist GW9662 to block PPARγ. GW9662 might act through other PPARγ-independent pathways, but has been used to specifically block PPARγ activation as the primary action in many studies [31]. With increasing concentrations of GW9662, the proteins of PDZK1, PDZK2, and OCTN2 decreased in SW480 cells. Moreover, we found that knockdown of PPARγ can counteract the induction effect of luteolin to some extent. Thus, these findings strongly emphasize that luteolin-mediated OCTN2 expression in SW480 cells is dependent on PPARγ. Several reports showed that the decreased uptake of OCTN2 substrates caused by single nucleotide
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1024 Anti-Cancer Drugs 2014, Vol 25 No 9
(b)
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Influence of peroxisome proliferator-activated receptor γ (PPARγ) knockdown on the induction effect of luteolin in SW480 cells. SW480 cells were transfected with the pGPH1-sh-PPARγ vector and pGPH1-sh-negative control (NC), and sustained in G418 medium. Green fluorescent protein indicated the expression of sh-PPAR or sh-NC. Transfected SW480 cells were treated with luteolin (Lut) or 0.1% dimethyl sulfoxide (DMSO), then total proteins were extracted and expression was determined by western blotting as described under the Materials and methods section. SW480 cells transfected with sh-NC and incubated with 0.1% DMSO was used as the control group. Values are means ± SD of three separate experiments. *P < 0.05, statistically significant differences from the sh-NC group of 0.1% DMSO. #P < 0.05, statistically significant differences from the sh-NC group of either 0.1% DMSO or luteolin treatment. OCTN2, organic cation/carnitine transporter 2; PDZK1, PDZ domain containing 1; PDZK2, PDZ domain containing 3; PPARγ, peroxisome proliferator-activated receptor γ; RXRα, retinoid X receptor α.
polymorphisms lead to carnitine deficiency and reduced effectiveness of imatinib [32,33]. It is possible that the PPARγ agonist can activate OCTN2 expression and restore the uptake capacity of OCNT2. In our previous study, application of decitabine to HepG2 and LS174T cells reversed the hypermethylation status of the OCTN2 promoter and increased OCTN2 expression, enhancing cellular uptake of oxaliplatin [34]. In this study, cell viability tests and cell apoptosis rates showed that the sensitivity of SW480 cells to oxaliplatin is associated with luteolin pretreatment and that this process is likely because of the enhanced absorption of oxaliplatin by luteolin-induced OCTN2. At high concentrations (>50 μmol/l), luteolin leads to cell toxicity [22]; however, because of the low concentrations and short exposure
time we used, it was found that luteolin increased the sensitivity of SW480 cells to oxaliplatin mainly through enhanced OCTN2 transport. Many reporters have shown that the bioavailability of flavonoids was relatively low in human plasma after a realistic consumption, but up to 15 μmol/l of luteolin has been detected in plasma [35,36]. This implies that this concentration can activate PPARγ and induce OCTN2 expression. More interestingly, because of the high expression of PPARγ in tumors and colon cancer cell lines [37,38], it provides a favorable target for luteolin to induce OCTN2 expression. We also determined the effects on other normal cells such as HEK293. After the treatment of luteolin, we found that the alterations of
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Luteolin and sensitivity of cancer cells Qu et al. 1025
Fig. 7
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Control OXA (5 μmol/l)+Piog (0 μmol/l) Piog (5 μmol/l) OXA (5 μmol/l)+Piog (5 μmol/l)
0 Pretreatment of luteolin inhibits SW480 cells viability and enhances oxaliplatin-induced apoptosis of SW480 cells. (a) Influence of oxaliplatin (OXA) on the viability of SW480 cells after luteolin (Lut) pretreatment. SW480 cells were pretreated with luteolin (5, 10, or 20 μmol/l) for 48 h, and then exposed to oxaliplatin at various concentrations for 48 h. Cell viability was determined and half-maximal inhibitory concentration (IC50) values were calculated. *P < 0.05, statistically significant differences from the treatment group of luteolin alone. (b) Analysis of apoptosis of SW480 cells. After pretreatment with luteolin (5, 10, or 20 μmol/l) for 48 h, SW480 cells were incubated continually with 5 μmol/l oxaliplatin for 24 h. Cell apoptosis was analyzed by flow cytometry using the Annexin V-FITC staining kit. (c) SW480 cell apoptosis rate. The percentage of apoptotic cells is represented in a bar graph from three independent experiments. #P < 0.05, statistically significant differences from the control group in groups of luteolin treatment. *P < 0.05, statistically significant differences from the treatment group of luteolin alone before 5 μmol/l oxaliplatin exposure. §P < 0.05, statistically significant differences from the treatment group of 5 μmol/l oxaliplatin group alone. PI, propidium iodide.
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1026 Anti-Cancer Drugs 2014, Vol 25 No 9
OCTN2 mRNA were very minor (data not shown). A future pharmacokinetic study of luteolin in vivo will be useful to help determine a suitable dose range of luteolin that will effectively induce OCTN2 expression as well as enhance the effect of oxaliplatin, without markedly increasing toxicity in other tissues. Because of the effects of luteolin on OCTN2, we can also reduce the dosage of oxaliplatin in vivo without causing more side effects in normal tissues.
10
In conclusion, the present study elucidated the mechanism by which luteolin potentiated the sensitivity of colon cancer SW480 cells to oxaliplatin through the PPARγ/OCTN2 pathway. The effects of luteolin on the uptake of oxaliplatin are not only because of the increases in OCTN2, PDZK1, and PDZK2 expression but also because of the binding affinity of OCTN2 toward oxaliplatin. Our discovery that a PPARγ agonist luteolin modulates the expression and activity of OCTN2 has significant implications for cancer therapy, especially in terms of the effect of oxaliplatin against CRC.
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Acknowledgements
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This work was supported by the State Scholarship Fund from China Scholarship Council (File No. 201206370103), the National Scientific Foundation of China (No. 81273595), the Scientific Foundation of Hunan (No. 11K073, 10JJ4020), the ‘863’ Project (No. 2012AA02A518), NCET-10-0843, and the Research Innovation Foundation of Graduate Student in Hunan province, P.R.C. (CX2011B056).
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Conflicts of interest
There are no conflicts of interest.
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Luteolin potentiates the sensitivity of colorectal cancer cell lines to oxaliplatin through the PPARγ/OCTN2 pathway Qiang Qua,b, Jian Qub, Yong Guoc, Bo-Ting Zhoua and Hong-Hao Zhoub Oxaliplatin is a chemotherapeutic agent used in the treatment of colorectal cancers. However, the mechanism controlling the cellular uptake and efflux of oxaliplatin is not completely understood. Organic cation/carnitine transporter 2 (OCTN2) is a member of the solute carrier superfamily and is a determinant of oxaliplatin uptake. OCTN2 is regulated by peroxisome proliferator-activated receptor γ (PPARγ) binding to the PPAR-response element within the first intron. Luteolin is a naturally occurring flavonoid and an agonist of PPARγ. Thus, we hypothesize that luteolin-mediated OCTN2 expression and activity potentiate the sensitivity of cancer cells to oxaliplatin. In this study, luteolin increased mRNA and protein expression of OCTN2 in a time-dependent and dose-dependent manner in colorectal cancer SW480 cells. This induction was attenuated by PPARγ antagonist GW9662 as well as by PPARγ knockdown, suggesting that the induction by luteolin is dependent on PPARγ. In uptake studies, luteolin increased the binding affinity of OCTN2 toward oxaliplatin and enhanced intracellular concentration of oxaliplatin. This finding is likely because of the increase of PDZ domain containing 1 (PDZK1) and PDZ domain containing 3
Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second most frequent cause of cancer-related death worldwide [1]. Oxaliplatin (trans-l1,2-diaminocyclohexane oxaliplatinum) is a thirdgeneration platinum chemotherapeutic that is used in the treatment of stage II, stage III, and stage IV CRC [2,3]. The cytotoxicity of oxaliplatin to cancer cells arises primarily from covalent binding to DNA, leading to a series of biochemical cascades, eventually cell death [4]. Studies aiming to identify such mechanisms have focused mainly on the influence of oxaliplatin on target genes and pathways involved in cell growth and apoptosis. However, the mechanism controlling the cellular uptake and efflux of oxaliplatin has not been investigated extensively, which could also be critical for increasing the therapeutic effect as well as reducing adverse reactions. Organic cation/carnitine transporter 2 (OCTN2) (SLC22A5) is an organic cation transporter of the solute carrier superfamily and is also well known as a sodiumSupplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.anti-cancerdrugs.com). 0959-4973 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
(PDZK2), which are known to facilitate the expression of OCTN2 on the cell surface and/or enhance transporter activity. Moreover, cell viability and cell apoptosis assays showed that luteolin increased oxaliplatin uptake and intracellular accumulation through OCTN2. Thus, our study showed that luteolin increased the sensitivity of colorectal cancer SW480 cells to oxaliplatin, likely through the PPARγ/OCTN2 pathway. Anti-Cancer Drugs 25:1016–1027 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Anti-Cancer Drugs 2014, 25:1016–1027 Keywords: colorectal cancer, luteolin, OCTN2, oxaliplatin, PPARγ a Department of Pharmacy, bInstitute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics and cDepartment of Surgery, Xiangya Hospital, Central South University, Changsha, China
Correspondence to Hong-Hao Zhou, PhD, Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Xiangya Hospital, Central South University, Changsha 410078, China Tel: + 86 731 848 05380; fax: + 86 731 823 54476; e-mail: [email protected] Received 14 November 2013 Revised form accepted 2 April 2014
dependent carnitine transporter [5]. OCTN2 is highly expressed in various tissues, such as the kidney, intestine, skeletal muscle, heart, colon, and brain [6,7]. A recent study showed that oxaliplatin accumulation and cytotoxicity were markedly increased in OCTN1-transfected and OCTN2-transfected cells, indicating that oxaliplatin was an excellent substrate of these transporters [8,9]. PDZ domain containing 1 (PDZK1) and PDZ domain containing 3 (PDZK2) are functional regulators of OCTN2 that modulate its expression and activity [10,11]. Peroxisome proliferator-activated receptor γ (PPARγ) regulates OCTN2 expression by binding to PPARresponse elements in the first intron as a heterodimer with the retinoid X receptor α (RXRα) [12]. Ligandactivated PPARγ has been investigated as a tumor suppressor with some favorable results, but remains controversial [13]. Although thiazolidinediones, the most widely used synthetic ligands for PPARγ, were used in preclinical studies as anticancer adjuvants against CRC cells, their uses may be limited because of predictable side effects on lipid and glucose metabolism in vivo [14]. Many herb products such as potent agonists of PPARγ have been investigated intensively for CRC prevention and therapeutic properties [15–17]. DOI: 10.1097/CAD.0000000000000125
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Luteolin and sensitivity of cancer cells Qu et al. 1017
Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a bioflavonoid, abundant in celery, green pepper, parsley, perilla leaf, and chamomile tea [18,19]. In 1,2-dimethyl hydrazineinduced and azoxymethane-induced colon cancer models, luteolin decreased the incidence of colon cancer, number of tumors per rat, and ameliorated the activities of the antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase [20,21]. Although the anticancer effects of luteolin have been investigated intensively, the mechanism and applicability of luteolin as possible adjuvants in combination with chemotherapeutics in CRC therapy remain less explained. A previous report suggested that luteolin showed agonist activity of PPARγ [22]. Thus, we hypothesized that luteolin probably acts as an activator of the PPARγ/OCTN2 pathway involved in oxaliplatin uptake. In the present study, we investigated the induction role of luteolin in PPARγ-activated OCTN2 expression and transport capability. We report that luteolin potentiates the sensitivity of SW480 cells to oxaliplatin by enhancing the uptake ability of OCTN2.
Materials and methods
(Thermo Scientific) following the instructions. The halfmaximal inhibitory concentration (IC50) was calculated using the SPSS 13.0 software (SPSS Inc., Chicago, Illinois, USA). Uptake experiment and high-performance liquid chromatography
Uptake studies of oxaliplatin were carried out in luteolinpretreated SW480 cells and quantified by highperformance liquid chromatography analysis. SW480 cells were pretreated with luteolin (5, 10, or 20 μmol/l) for 48 h and seeded in six-well plates at a density of 1 × 106/well. Cells were washed twice with PBS and incubated with 1 ml uptake buffer containing various concentrations of oxaliplatin (0, 1, 3, 10, 33, 100, or 300 μmol/l) for 30 min. Uptake of oxaliplatin was terminated by aspiration of uptake buffer. Cells were washed twice with PBS and lysed in 200 μl of 1% Triton X-100 solution for 6 h. Cell lysate (150 μl/well) was collected for the quantification of oxaliplatin by high-performance liquid chromatography as described elsewhere [8,9,23]. Cellular protein content was quantified using the BCA protein assay (Thermo Scientific). Vmax and Km were estimated by fitting data to the Michaelis–Menten equation using GraphPad Prism5 software (GraphPad, San Diego, California, USA).
Chemicals and reagents
GW9662, pioglitazone, oxaliplatin, and luteolin were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). TRIzol reagent was from Takara (Dalian, China). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, California, USA). All primary antibodies were purchased from Abcam Labs (Cambridge, Massachusetts, USA). Cell culture, treatments, and cell viability assay
CRC cell lines SW480 and LS174T were obtained from ATCC (Manassas, Virginia, USA). For cell treatments, SW480 or LS174T cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Scientific, Rockford, Illinois, USA) supplemented with 10% fetal bovine serum in six-well plates. A volume of 2 ml of medium containing various concentrations of luteolin or pioglitazone was added and incubated for 24, 48, or 72 h at 37°C. In the antagonist assay, SW480 cells were exposed to GW9662 (5, 10, or 20 μmol/l) for 1 h and then incubated with 20 μmol/l luteolin for 48 h. For cell viability assays, SW480 cells were pretreated with luteolin (5, 10, or 20 μmol/l) for 48 h and then seeded in a 96-well plate at 104 cells/well. Cells were treated with oxaliplatin at various concentrations for 48 h. As the vehicle control, dimethyl sulfoxide (0.1%) was added to control cells. Cell viability was determined with an MTS cell proliferating assay kit (Promega, Madison, Wisconsin, USA) using a Multiskan Ascent 354 Microplate Reader
Quantitative RT-PCR
After cell treatment, total RNA was extracted using TRIzol reagent and the concentration was measured. cDNA was synthesized from 2 μg of total RNA using the first cDNA synthesis kit (Takara). Quantitative RT-PCR reactions were carried out using the SYBR Green kit (Bio-Rad, Hercules, California, USA) with the primers listed in supplementary Table 1 (Supplemental digital content 1, http://links.lww.com/ACD/A66) and under the following conditions: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s (40 cycles), followed by a final incubation at 72°C for 2 min. The relative mRNA levels of PDZK1, PDZK2, PPARγ, RXRα, and OCTN2 were normalized to β-actin. The analysis was carried out using the 2 DDCt method [24]. Western blotting
After cell treatment, SW480 cells were lysed and the protein concentration was measured using the BCA protein assay (Thermo Scientific). The proteins were subjected to SDS-PAGE and electrophoretically transferred to a PVDF membrane. The membranes were incubated with anti-OCTN2 (1 : 400; Abcam Lab), anti-PPARγ (1 : 400), anti-RXRα (1 : 500), anti-PDZK1 (1 : 400), anti-PDZK2 (1 : 400), and anti-β-actin (1 : 1000) primary antibodies, and subsequently incubated with Dylight 800-labeled rabbit or mouse polyclonal antibody (1 : 7500; KPL Inc., Gaithersburg, Maryland,
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1018 Anti-Cancer Drugs 2014, Vol 25 No 9
Fig. 1
Relative OCTN2 mRNA levels (fold of control)
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2.5 5.0 7.5 10.0 Piog concentration (μmol/l)
0
(b)
5 10 15 20 25 30 35 40 Lut concentration (μmol/l)
4.3
∗
Piog (μmol/l) 1.25
2.5
5
3.8
10 OCTN2
24 h β-Actin
OCTN2 48 h
OCTN2 protein levels (fold of control)
Control
∗
3.3 ∗
2.8 2.3
∗
∗
1.8
∗
∗
∗ 48 h
1.3
β-Actin
24 h
0.8 0.0
(c)
2.5 5.0 7.5 Piog concentration (μmol/l)
10.0
Lut (μmol/l) Control
5
10
20
40
OCTN2 48 h β-Actin
OCTN2
3.2 OCTN2 protein levels (fold of control)
β-Actin
72 h 48 h 24 h ∗
3.5
OCTN2 24 h
2.9
∗
∗
2.6
∗
2.3 ∗
2.0 1.7
∗ ∗
1.4
∗ ∗
1.1
∗
∗
0.8
72 h β-Actin
0
5
10 15 20 25 30 35 40 Lut concentration(μmol/l)
Influence of luteolin (Lut) treatment on organic cation/carnitine transporter 2 (OCTN2) mRNA and protein levels. SW480 cells were treated with pioglitazone (Piog) and luteolin at various concentrations for 24, 48, or 72 h, and then total RNA and proteins were extracted and purified. Quantitative RT-PCR and western blotting were performed and analyzed. The level of β-actin was used as an internal control. Values are means ± SD of three separate experiments. *P < 0.05, statistically significant differences from the 0.1% dimethyl sulfoxide-treated group (control).
USA). The signal was detected and ratios of target protein against β-actin were calculated using the Odyssey Infrared Imaging System (LI-COR Biosciences, Cambridge, UK).
Knockdown of PPARγ by shRNA
To silence PPARγ expression, small hairpin RNA duplexes (shRNA) targeting the sequence AAAGCAA AGGCGAGGGCGATCTT of human PPARγ (sh-PPARγ)
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Luteolin and sensitivity of cancer cells Qu et al. 1019
Fig. 2
(a)
PPARγ
RXRα
1.6 72 h 48 h 24 h
1.6
Relative RXRα mRNA levels (fold of control)
Relative PPARY mRNA levels (fold of control)
1.8
1.4 1.2 1.0 0.8
∗ ∗
1.4
∗
∗
∗
∗
∗
1.2
∗
72 h 48 h 24 h
1.0
0.8 0
5
10 15 20 25 30 35 40
0
5
Lut concentration (μmol/l)
10 15 20 25 30 35 40 Lut concentration (μmol/l)
(b)
5
10
20
40
24 h
48 h
PPARγ protein levels (fold of control)
Control
PPARγ
1.6
Lut (μmol/l)
PPARγ
72 h
72 h 48 h 24 h
1.4
1.2
1.0
0.8 0
5
10 15 20 25 30 35 40 Lut concentration (μmol/l)
5
10
20
40
24 h
48 h
RXRα protein levels (fold of control)
Control
RXRα
1.6
Lut (μmol/l)
RXRα
1.4
∗
∗
∗
∗
∗ 1.2
1.0
72 h 48 h 24 h
72 h 0.8 0
5
10 15 20 25 30 35 40 Lut concentration (μmol/l)
Influence of luteolin (Lut) treatment on peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα) mRNA and protein levels. The methods of cell treatment, total RNA, and protein extraction, quantitative RT-PCR, and western blotting were analyzed. Values are means ± SD of three separate experiments. *P < 0.05, statistically significant differences from the 0.1% dimethyl sulfoxide-treated group (control).
[25] were synthesized and then inserted into the pGPH1-GFP/neo vector (GenePharma, Shanghai, China) for small interfering RNA production. SW480 cells were transfected by pGPH1-sh-PPARγ and pGPH1-sh-negative control using Lipofectamine 2000. Transfected cells were
sustained in 600 μg/ml kanamycin medium for 14 days and cell clones were selected by kanamycin resistance. PPARγ-knockdown and control cells were seeded in sixwell plates and exposed to 20 μmol/l luteolin or 0.1% dimethyl sulfoxide (vehicle control). Total cell proteins
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1020 Anti-Cancer Drugs 2014, Vol 25 No 9
Fig. 3
(a)
(c)
Oxaliplatin (OXA) O
O
OXA (pmol/mg protein/min)
NH2 Pt
O
NH2
O
(b) 0.50 mAU 2.842
0.45
30 OXA+ 20 μmol/l Lut OXA+ 10 μmol/l Lut 20
OXA+ 5 μmol/l Lut OXA+ 0.1% DMSO
10
0 0
0.40 0.35
OXA (pmol/mg protein/min)
0.30 0.25
0.20 0.15
0.10 0.05
0.00 -0.05
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
min
50 100 150 200 250 300 OXA concentration ( μmol/l)
10 OXA+ 20 μmol/l Lut
8
OXA+ 10 μmol/l Lut 6
OXA+ 5 μmol/l Lut
4
OXA+ 0.1% DMSO
2 0 10 4 8 6 2 OXA concentration ( μmol/l)
0
Effects of luteolin (Lut) on the transport of oxaliplatin (OXA) by organic cation/carnitine transporter 2 (OCTN2) in SW480 cells. (a) The structure of oxaliplatin. (b) Chromatograms of the standard solution containing 100 ng/ml oxaliplatin. (c) Concentration-dependent oxaliplatin transport by OCTN2. SW480 cells were pretreated with luteolin at 5, 10, or 20 μmol/l for 48 h, and then exposed to oxaliplatin (0, 1, 3, 10, 33, 100, or 300 μmol/l) for an additional 30 min. Cells were harvested and oxaliplatin concentrations were determined by high-performance liquid chromatography. Values are means ± SD of three determinations. Results are expressed as pmol/l of oxaliplatin uptake/min/mg of total cell protein. DMSO, dimethyl sulfoxide.
were extracted and protein expression was determined by western blotting. Annexin V/propidium iodide double staining
SW480 cells were seeded in six-well plates and incubated for 48 h with DMEM containing positive control 5 μmol/l pioglitazone or luteolin at a final concentration of 5, 10, or 20 μmol/l, respectively. Cells were washed with PBS and continually exposed to 5 μmol/l oxaliplatin for 24 h. Each group was collected and cell apoptosis was analyzed by flow cytometry using the Annexin V-FITC staining kit (BD LSR II flow cytometer; BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instruction. Statistical analysis
For all results, statistical comparisons were performed with one-way analysis of variance, followed by a post-hoc test. A value of P less than 0.05 was considered statistically significant.
Results Luteolin activated PPARγ/RXRα and upregulated OCTN2 in SW480 cell lines
To investigate whether the upexpression of OCTN2 is the result of activating PPARγ/RXRα, SW480 cells were
exposed to pioglitazone (0–10 μmol/l) and luteolin (0–40 μmol/l) for 24, 48, or 72 h. As shown in Fig. 1a and b, pioglitazone, a PPARγ agonist, was used as a positive control, and it significantly increased the expression of OCTN2 at 24–48 h. Quantitative RT-PCR and protein analysis showed that luteolin markedly elevated the mRNA and protein levels of OCTN2 in a dose-dependent and time-dependent manner (Fig. 1a and c). As can be seen in Fig. 2, luteolin at 10–40 μmol/l elevated the mRNA and protein of RXRα compared with the control group at 48 and 72 h. However, protein and mRNA levels of PPARγ did not change (Fig. 2). Because of the treatment with higher concentrations of luteolin and the longer time of incubation, the cellular growth may be inhibited and the expression of OCNT2 probably was lower. Luteolin potentiates oxaliplatin uptake in SW480 cells
To examine the hypothesis that luteolin may enhance the transporting ability of OCTN2, uptake studies of oxaliplatin were performed and kinetic analyses were performed after SW480 cells were pretreated with luteolin for 48 h. With increasing concentrations of luteolin, Vmax was increased, but Km was decreased (Fig. 3 and Table 1). These results suggest that luteolin
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Luteolin and sensitivity of cancer cells Qu et al. 1021
Table 1 Michaelis–Menten kinetics for the effect of different concentrations of luteolin on oxaliplatin uptake in SW480 cells Treatments 0.01% DMSO + oxaliplatin 5 μmol/l luteolin + oxaliplatin 10 μmol/l luteolin + oxaliplatin 20 μmol/l luteolin + oxaliplatin
Km (μmol/l)
Vmax (pmol/mg protein/min)
31.12 ± 2.04 30.90 ± 3.30 22.18 ± 1.67* 19.91 ± 1.71*
21.66 ± 0.41 23.18 ± 0.71 25.44 ± 0.51* 29.46 ± 0.66*
Data are mean ± SD of three independent experiments. DMSO, dimethyl sulfoxide. *P < 0.05, significant differences from the 0.1% DMSO group.
increases the affinity of OCTN2 as well as the maximum transporting activity of OCTN2 to oxaliplatin. Luteolin modulates PDZK1 and PDZK2 expression in SW480 cell lines
We further measured the expression of the OCTN2 regulatory proteins, PDZK1 and PDZK2. In Fig. 4, the mRNA and protein levels of PDZK1 and PDZK2 were increased in a time-dependent and dose-dependent manner. Considering that luteolin at 20 μmol/l resulted in the highest increments in mRNA and protein expression for OCTN2, PDZK1, and PDZK2, this concentration and a 48 h period of treatment were used in the subsequent experiments. Luteolin-induced expression is dependent on PPARγ
To confirm the significant role of PPARγ in the expression of OCTN2, we pre-exposed SW480 cells to PPARγ antagonist GW9662 for 1 h, and then added 20 μmol/l luteolin. We found that GW9662 effectively prevented the induction effect of 20 μmol/l luteolin on PDZK1, PDZK2, RXRα, and OCTN2 in a concentrationdependent manner, but not on PPARγ expression (Fig. 5). It is possible that GW9662 abrogates the induction of luteolin by blocking the binding to PPARγ, but not decreasing PPARγ expression. To further gain more evidence that the above gene expression induced by luteolin is dependent on PPARγ, we used shRNA to knock down the expression of PPARγ in SW480 cells. As shown in Fig. 6, PPARγ expression in knockdown cells was markedly decreased to 37% of the control cells. Despite treatment with 20 μmol/l luteolin, PPARγ expression was only 61% of that of the control cells. Compared with the expression induced by 20 μmol/ l luteolin in control cells, protein levels of PDZK1, PDZK2, and OCTN2 in PPARγ knockdown cells were only 65.2, 58.7, and 69.9%, respectively. These results suggest that the effect of luteolin on target genes is dependent on PPARγ. Luteolin contributes toward the sensitivity of SW480 cells to oxaliplatin
To test whether luteolin potentiates oxaliplatin-induced cell apoptosis, cell viability was measured using an MTS assay. As shown in Fig. 7a, SW480 cells pretreated with
luteolin were more sensitive to oxaliplatin-induced apoptosis compared with cells treated with oxaliplatin alone. With increasing luteolin concentrations, the IC50 values of oxaliplatin in luteolin-pretreated cells decreased to 68.5, 37.1, and 31.1%, lower than the treatment of oxaliplatin alone. After pretreatment with luteolin (5, 10, or 20 μmol/l) for 48 h, SW480 cells were continually incubated with 5 μmol/l oxaliplatin for 24 h. The percentage of cell apoptosis was determined by flow cytometry. In Fig. 7b and c, SW480 cell apoptosis rates were increased after exposure to luteolin. The apoptosis rates of luteolin-pretreated groups (5, 10, or 20 μmol/l luteolin + 5 μmol/l oxaliplatin) were increased 1.7-, 1.8-, and 2.5-fold compared with the group of 5 μmol/l oxaliplatin alone. A similar result was observed in the positive group of 5 μmol/l pioglitazone. Compared with pretreatment with luteolin only (no oxaliplatin), the apoptosis rates were elevated after oxaliplatin exposure.
Discussion The aim of this work was to identify whether luteolin is an agonist of PPARγ and therefore plays an essential role in sensitizing SW480 cells to oxaliplatin through the PPARγ/OCTN2 pathway. In the present study, we showed that luteolin, by activating PPARγ, not only elevated the expression of OCTN2, PDZK1, and PDZK2 at both protein and mRNA levels but also increased the binding affinity of OCTN2 toward oxaliplatin. To the best of our knowledge, this is the first report to indicate that luteolin increases the sensitivity of SW480 cells to oxaliplatin, probably because of increased uptake and intracellular accumulation of oxaliplatin through OCTN2. Luteolin is effective as an antiproliferative agent for cancer prevention and therapy with multiple mechanisms [18,26]. The luteolin-induced PPARγ/ligand binding domain assay showed a dose-dependent curve with an EC50 of 15.6 μmol/l [22]. The PPARγ target gene GLUT4 was activated by luteolin to a similar degree as rosiglitazone [22]. Another study also found that 20 μmol/l luteolin activated the expression of adiponectin, leptin, GLUT4, and PPARγ2 in 3T3-L1 adipocytes and primary mouse adipose cells [27]. A previous EMSA assay showed that the first intron of human OCTN2 gene has a conserved noncanonical PPAR-responsive element [12]. A PPARγ agonist rosiglitazone could increase both mRNA and protein of OCTN2 in mouse colon epithelium [12]. These findings led us to postulate that luteolin probably induces OCTN2 through activation of PPARγ. In our studies, luteolin markedly increased both mRNA and protein of OCTN2 in a dose-dependent and timedependent manner in SW480 cells as well as in other colon cancer cells LS174T (Supplementary Fig. 1, Supplemental digital content 2, http://links.lww.com/ACD/ A67). The in-vitro and in-vivo PPAR-null models showed
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1022 Anti-Cancer Drugs 2014, Vol 25 No 9
Fig. 4
PDZK1
Relative PDZK1 mRNA levels (fold of control)
1.6 ∗
∗ 1.2
∗
∗
∗
1.4
∗
∗ ∗
∗
PDZK2
1.6
∗
∗
72 h
1.0
48 h 24 h
Relative PDZK2 mRNA levels (fold of control)
(a)
0.8
∗
∗ ∗
1.4
∗ ∗
∗ 1.2
∗
∗
∗ 72 h
1.0
48 h 24 h 0.8
0
5
10 15 20 25 30 35 40 Lut concentration (μmol/l)
0
(b)
5
10 15 20 25 30 35 40 Lut concentration (μmol/l) PDZK1
2.2 5
10
20
40
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48 h
PDZK1 protein expression (fold of control)
Lut (μmol/l)
PDZK1 Control
72 h
∗
2.0 1.8
∗
1.6
∗
∗ ∗
∗
∗
1.4 1.2
72 h
1.0
48 h 24 h
0.8 0
5
10 15 20 25 30 35 40 Lut concentration (μmol/l) PDZK2
2.2 5
10
20
40
24 h
48 h
PDZK2 protein expression (fold of control)
Lut (μmol/l)
PDZK2 Control
∗
∗
∗
2.0
∗
∗
1.8 ∗
1.6
∗ ∗
∗
∗
1.4 ∗
1.2 ∗
1.0
72 h
∗ 72 h 48 h 24 h
0.8 0
5
10 15 20 25 30 35 40 Lut concentration (μmol/l)
Influence of luteolin (Lut) treatment on PDZ domain containing 1 (PDZK1) and PDZ domain containing 3 (PDZK2) mRNA and protein levels. Cell treatment, total RNA, and protein extraction, quantitative RT-PCR, and western blotting were performed as described under the Materials and methods section. Values are means ± SD of three separate experiments. *P < 0.05, statistically significant differences from the 0.1% dimethyl sulfoxide-treated group (control).
that only PPARγ transcriptionally regulated OCTN2 expression in human and mouse colon tissues, rather than other PPAR isoforms [12]. Previous studies suggested that luteolin inhibited the production of tumor necrosis factor α [27,28], which was reported to reduce RXRα
expression [29]. It is possible that luteolin indirectly upregulates RXRα through repression of tumor necrosis factor α production in SW480 cells. In sum, luteolin effectively activated PPARγ/RXRα and triggered the expression of OCTN2.
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Luteolin and sensitivity of cancer cells Qu et al. 1023
0
5
10
20
Lut (μmol/l)
0
20
20
20
20
OCTN2
1.5 1.0
∗
∗
∗
0.5 0.0
PPARγ 1.5 1.2 0.9 0.6 0.3 0.0
GW9662 (μmol/l)
0
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5
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∗
∗
RXRα 1.6 1.2
∗
∗
∗
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∗
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PDZK1
1.5 1.2
∗
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0
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Lut (μmol/l)
0
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20
20
20
PDZK2 protein expression (% of control)
RXRα protein expression (% of control)
PDZK1
∗
2.0
PPARγ
RXRα
PPARγ protein expression (% of control)
0
OCTN2 2.5
Normalized PDZK1 protein levels (fold of control)
GW9662 (μmol/l)
OCTN2 protein expression (% of control)
Fig. 5
1.6
PDZK2
β-Actin
0
1.2
PDZK2
∗
∗
∗
∗
0.8 0.4
0.0 GW9662 (μmol/l) 0
0
5
10
20
Lut (μmol/l)
20
20
20
20
0
Influence of the antagonist GW9662 on the induction effect of luteolin in SW480 cells. SW480 cells were coincubated with GW9662 at various concentrations and 20 μmol/l luteolin (Lut) for 48 h. Total proteins were extracted and western blotting was performed as described under the Materials and methods section. The value of the control group (0.1% dimethyl sulfoxide) was set as 1. *P < 0.05, statistically significant differences from the treatment group of 20 μmol/l luteolin alone. OCTN2, organic cation/carnitine transporter 2; PDZK1, PDZ domain containing 1; PDZK2, PDZ domain containing 3; RXRα, retinoid X receptor α.
Interestingly, animals overexpressing colon PPARγ showed not only a significant increase in OCTN2 expression, but the plasma concentration of carnitine [12], which is the classic substrate of OCTN2 [5]. This implies that the ligand-activating or overexpression of PPARγ facilitates the ability of OCTN2 transport. In our uptake studies, luteolin enhanced the capacity of OCTN2 to transport oxaliplatin in SW480 cells, which not only increased Vmax but also decreased Km in comparison with the control group. Thus, we focused on whether luteolin affected OCTN2 regulatory proteins, PDZK1 and PDZK2. PDZK1 is a functional regulator through direct interaction with the carboxyl terminus of OCTN2 to enhance the transport capacity, without influencing cell-surface expression of OCTN2 [10]. We speculated that luteolin may activate PPARγ, resulting in upregulation of PDZK1 through PPAR-response element in SW480 cells, the same binding region as PPARα in HepG2 cells [30]. PDZK2, another PDZ domain protein, is also increased in SW480 cells and, when colocalized with OCTN2, contributes toward the stabilization of OCTN2 expression on the cell surface [11]. Previous studies have presented an ‘intracellular pool’ model,
which involves dynamic regulation of a reservoir of OCTN2 [11]. Thus, we propose that luteolin probably indirectly modulates PDZK1 and PDZK2 and triggers the regulatory network, which not only enriches the expression level of OCTN2 but also increases the delivering capacity of OCTN2 from the ‘intracellular poor’ to the cell surface. To further identify whether luteolin-induced OCTN2 expression depends on PPARγ, we utilized a PPARγ antagonist GW9662 to block PPARγ. GW9662 might act through other PPARγ-independent pathways, but has been used to specifically block PPARγ activation as the primary action in many studies [31]. With increasing concentrations of GW9662, the proteins of PDZK1, PDZK2, and OCTN2 decreased in SW480 cells. Moreover, we found that knockdown of PPARγ can counteract the induction effect of luteolin to some extent. Thus, these findings strongly emphasize that luteolin-mediated OCTN2 expression in SW480 cells is dependent on PPARγ. Several reports showed that the decreased uptake of OCTN2 substrates caused by single nucleotide
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1024 Anti-Cancer Drugs 2014, Vol 25 No 9
(b)
Control sh-NC
PDZK1
PDZK2
PDZK1 1.8 1.5
sh-PPARγ
#
1.2 0.9 0.6 0.3 0.0
Lut (20 μmol/l)
Control
Lut (20 μmol/l)
sh-PPARγ sh-NC
∗
sh-NC sh-PPARγ
PPARγ 1.5 1.2
sh-NC sh-PPARγ
0.9 0.6
∗#
∗#
0.3 0.0 Control
Lut (20 μmol/l)
PDZK2 protein expression (% of control)
sh-PPARγ
PPARγ protein expression (% of control)
sh-NC
RXRα protein expression (% of control)
(a)
PDZK1 protein expression (% of control)
Fig. 6
PDZK2 1.6
∗
sh-NC sh-PPARγ
1.2
#
0.8 0.4 0.0 Control
Lut (20 μmol/l)
RXRα 1.5
sh-NC sh-PPARγ
1.2
∗
0.9 0.6 0.3 0.0 Control
Lut (20 μmol/l)
RXRα
OCTN2
β-Actin
OCTN2 protein expression (% of control)
PPARγ OCTN2 2.0 1.6
sh-NC sh-PPARγ
∗ #
1.2 0.8 0.4 0.0 Control
Lut (20 μmol/l)
Influence of peroxisome proliferator-activated receptor γ (PPARγ) knockdown on the induction effect of luteolin in SW480 cells. SW480 cells were transfected with the pGPH1-sh-PPARγ vector and pGPH1-sh-negative control (NC), and sustained in G418 medium. Green fluorescent protein indicated the expression of sh-PPAR or sh-NC. Transfected SW480 cells were treated with luteolin (Lut) or 0.1% dimethyl sulfoxide (DMSO), then total proteins were extracted and expression was determined by western blotting as described under the Materials and methods section. SW480 cells transfected with sh-NC and incubated with 0.1% DMSO was used as the control group. Values are means ± SD of three separate experiments. *P < 0.05, statistically significant differences from the sh-NC group of 0.1% DMSO. #P < 0.05, statistically significant differences from the sh-NC group of either 0.1% DMSO or luteolin treatment. OCTN2, organic cation/carnitine transporter 2; PDZK1, PDZ domain containing 1; PDZK2, PDZ domain containing 3; PPARγ, peroxisome proliferator-activated receptor γ; RXRα, retinoid X receptor α.
polymorphisms lead to carnitine deficiency and reduced effectiveness of imatinib [32,33]. It is possible that the PPARγ agonist can activate OCTN2 expression and restore the uptake capacity of OCNT2. In our previous study, application of decitabine to HepG2 and LS174T cells reversed the hypermethylation status of the OCTN2 promoter and increased OCTN2 expression, enhancing cellular uptake of oxaliplatin [34]. In this study, cell viability tests and cell apoptosis rates showed that the sensitivity of SW480 cells to oxaliplatin is associated with luteolin pretreatment and that this process is likely because of the enhanced absorption of oxaliplatin by luteolin-induced OCTN2. At high concentrations (>50 μmol/l), luteolin leads to cell toxicity [22]; however, because of the low concentrations and short exposure
time we used, it was found that luteolin increased the sensitivity of SW480 cells to oxaliplatin mainly through enhanced OCTN2 transport. Many reporters have shown that the bioavailability of flavonoids was relatively low in human plasma after a realistic consumption, but up to 15 μmol/l of luteolin has been detected in plasma [35,36]. This implies that this concentration can activate PPARγ and induce OCTN2 expression. More interestingly, because of the high expression of PPARγ in tumors and colon cancer cell lines [37,38], it provides a favorable target for luteolin to induce OCTN2 expression. We also determined the effects on other normal cells such as HEK293. After the treatment of luteolin, we found that the alterations of
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Luteolin and sensitivity of cancer cells Qu et al. 1025
Fig. 7
(b)
OXA OXA+ 5 μmol/l Lut OXA+ 10 μmol/l Lut OXA+ 20 μmol/l Lut
120 100 80 60 40 20 0 100 0.1 10 1 Oxaliplatin concentration (μmol/l)
PI
R3
R4
0.24
R2
0.11 R5
R4
5 μmol/l OXA R2
R3
R2
8.12
0.01
0.00
∗
4
R5
R3
R5
10 μmol/l Lut R2
0.94
7.49
R4
R3
20 μmol/l Lut R2
4.84
R3
8.74
8.99 R5
R4
6.14
R5
R4
R5
5 μmol/l OXA + 5 μmol/l Lut
5 μmol/l OXA + 10 μmol/l Lut
5 μmol/l OXA + 20 μmol/l Lut
R2
R2
R2
R3
16.98
R4
R5
∗
2
R4
R5
R3
23.58
19.92
R4
R5
R3
35.50
30.09
32.81
38.12
24.91 R4
R3
∗
6
5 μmol/l Lut R2
5 μmol/l OXA + 5 μmol/l Piog
R3
8
OXA OXA+ 5 μmol/l Lut OXA+ 10 μmol/l Lut OXA+ 20 μmol/l Lut
0
5 μmol/l Piog
Control R2
10 IC50 ( μmol/l)
% cell viability
(a)
33.33 R4
R5
Annexin V (c)
∗
Cell apoptosis (%)
80
40 20
Cell apoptosis (%)
0 60 40 20
∗
∗
60 ∗ #
#
#
Control OXA (5 μmol/l)+Lut (0 μmol/l) Lut (5 μmol/l) OXA (5 μmol/l)+Lut (5 μmol/l) Lut (10 μmol/l) OXA (5 μmol/l)+Lut (10 μmol/l) Lut (20 μmol/l) OXA (5 μmol/l)+Lut (20 μmol/l)
Control OXA (5 μmol/l)+Piog (0 μmol/l) Piog (5 μmol/l) OXA (5 μmol/l)+Piog (5 μmol/l)
0 Pretreatment of luteolin inhibits SW480 cells viability and enhances oxaliplatin-induced apoptosis of SW480 cells. (a) Influence of oxaliplatin (OXA) on the viability of SW480 cells after luteolin (Lut) pretreatment. SW480 cells were pretreated with luteolin (5, 10, or 20 μmol/l) for 48 h, and then exposed to oxaliplatin at various concentrations for 48 h. Cell viability was determined and half-maximal inhibitory concentration (IC50) values were calculated. *P < 0.05, statistically significant differences from the treatment group of luteolin alone. (b) Analysis of apoptosis of SW480 cells. After pretreatment with luteolin (5, 10, or 20 μmol/l) for 48 h, SW480 cells were incubated continually with 5 μmol/l oxaliplatin for 24 h. Cell apoptosis was analyzed by flow cytometry using the Annexin V-FITC staining kit. (c) SW480 cell apoptosis rate. The percentage of apoptotic cells is represented in a bar graph from three independent experiments. #P < 0.05, statistically significant differences from the control group in groups of luteolin treatment. *P < 0.05, statistically significant differences from the treatment group of luteolin alone before 5 μmol/l oxaliplatin exposure. §P < 0.05, statistically significant differences from the treatment group of 5 μmol/l oxaliplatin group alone. PI, propidium iodide.
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1026 Anti-Cancer Drugs 2014, Vol 25 No 9
OCTN2 mRNA were very minor (data not shown). A future pharmacokinetic study of luteolin in vivo will be useful to help determine a suitable dose range of luteolin that will effectively induce OCTN2 expression as well as enhance the effect of oxaliplatin, without markedly increasing toxicity in other tissues. Because of the effects of luteolin on OCTN2, we can also reduce the dosage of oxaliplatin in vivo without causing more side effects in normal tissues.
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In conclusion, the present study elucidated the mechanism by which luteolin potentiated the sensitivity of colon cancer SW480 cells to oxaliplatin through the PPARγ/OCTN2 pathway. The effects of luteolin on the uptake of oxaliplatin are not only because of the increases in OCTN2, PDZK1, and PDZK2 expression but also because of the binding affinity of OCTN2 toward oxaliplatin. Our discovery that a PPARγ agonist luteolin modulates the expression and activity of OCTN2 has significant implications for cancer therapy, especially in terms of the effect of oxaliplatin against CRC.
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Acknowledgements
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This work was supported by the State Scholarship Fund from China Scholarship Council (File No. 201206370103), the National Scientific Foundation of China (No. 81273595), the Scientific Foundation of Hunan (No. 11K073, 10JJ4020), the ‘863’ Project (No. 2012AA02A518), NCET-10-0843, and the Research Innovation Foundation of Graduate Student in Hunan province, P.R.C. (CX2011B056).
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Conflicts of interest
There are no conflicts of interest.
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