Lutein Protects against Methotrexate-Induced and Reactive Oxygen Species-Mediated Apoptotic Cell Injury of IEC-6 Cells

Purpose High-dose chemotherapy using methotrexate (MTX) frequently induces side effects such as mucositis that leads to intestinal damage and diarrhea. Several natural compounds have been demonstrated of their effectiveness in protecting intestinal epithelial cells from these adverse effects. In this paper, we investigated the protection mechanism of lutein against MTX-induced damage in IEC-6 cells originating from the rat jejunum crypt. Methods The cell viability, induced-apoptosis, reactive oxygen species (ROS) generation, and mitochondrial membrane potential in IEC-6 cells under MTX treatment were examined in the presence or absence of lutein. Expression level of Bcl2, Bad and ROS scavenging enzymes (including SOD, catalase and Prdx1) were detected by quantitative RT-PCR. Results The cell viability of IEC-6 cells exposed to MTX was decreased in a dose- and time-dependent manner. MTX induces mitochondrial membrane potential loss, ROS generation and caspase 3 activation in IEC-6 cells. The cytotoxicity of MTX was reduced in IEC-6 cells by the 24 h pre-treatment of lutein. We found that pre-treatment of lutein significantly reduces MTX-induced ROS and apoptosis. The expression of SOD was up-regulated by the pre-treatment of lutein in the MTX-treated IEC-6 cells. These results indicated that lutein can protect IEC-6 cells from the chemo-drugs induced damage through increasing ROS scavenging ability. Conclusion The MTX-induced apoptosis of IEC-6 cells was shown to be repressed by the pre-treatment of lutein, which may represent a promising adjunct to conventional chemotherapy for preventing intestinal damages.


Introduction
Cancers are leading causes of mortality in both adults and children. Multiple drugs have been developed to treat cancers. An antifolate, methotrexate (MTX) is widely used to treat cancers and a number of other malignant and nonmalignant diseases. The effect of MTX is attributed to its inhibition of dihydrofolate reductase, purine synthesis or blocking DNA repair, but can cause an acute injury to the intestinal epithelium characterized by reduced mitosis in the crypts and shortened villi [1,2]. High-dose MTX treatment is often accompanied by adverse effects, such as severe enterocolitis, weight loss, anorexia, diarrhea and intestinal atrophy [3,4]. MTX induces damage to the villi of the small intestine, leading to decreased surface area of the small intestine. Shortened villi of the small intestine change the physical structure of brush-border membranes and compromise the components of the small intestinal mucosa such as proteins and lipids, and. The chemical and morphological changes in the small intestinal may be triggered by crypt cells injury. In addition to the morphological changes, MTX is known to be a source to decrease the intestinal absorption of nutrients and drugs [5,6]. MTX is also known as a pro-oxidant compound that causes depletion of the dihydrofolate pool. It directly affects the synthesis of thymidilate, suppresses DNA synthesis, inhibits epithelial proliferation, and induces apoptosis in the small intestinal crypts [7]. Therefore, MTX is widely used for cancer and also for rapidly-dividing cells of the intestinal crypt. Nevertheless, MTX raises an important complication for patients who are undergoing cancer chemotherapy. The clinical application of this drug is limited by its toxic dose-related side effects. Methotrexate has been shown to decrease viability and to induce apoptosis in the small intestine in rats [8]. In order to minimize the side effects in patients taking MTX as chemotherapeutic agent, it is important to reduce the damages and to stimulate tissue repair [9]. Growth factors such as insulin-like growth factor 1 and keratinocyte growth factor were reported to protect mice from gastrointestinal injury by stimulating growth of the damaged intestine [10,11]. Other studies using vitamin A [6,12], aged garlic extract (AGE) [8], apricot, beta-carotene [13], melatonin [14] and prostaglandins [15], showed protective effect on the MTX-induced damages to the small intestine in rats.
Lutein is one of the most commonly found carotenoids in deepyellow vegetables and fruits, including cooked spinach, lettuce, Broccoli, peas, lima beans, orange juice, celery, string beans, and squash [16]. The structure of lutein is similar to that of betacarotene. Lutein derivatives have various biologic properties such antioxidant, anti-inflammatory and anti-tumor potential. Furthermore, lutein has neuroprotective effects in the retina mainly through its antioxidant ability [17]. Studies also demonstrated that lutein prevents the ischemia reperfusion injury induced by free radical species in the rat small intestine [18]. Apricot and betacarotene that are structurally similar to lutein also protect the impairment of oxidative stress in the small intestine of rats from MTX-induced damages [13]. However, lutein was reported to suppress cancer cell growth by inducing apoptosis [19,20]. Thus, it has been suggested that lutein has different effects on tumor cells and normal intestinal cells.
IEC-6 is an immortalized epithelial cell line derived from neonatal rat ileum and has been extensively used as an in vitro intestinal model for the research of floate and its transport derivatives [21]. In the present study, we examined the effects of lutein to protect the intestine from MTX-induced cytotoxic injuries in an IEC-6 intestinal epithelial cell chemotherapy damage model. Cell culture IEC-6 cells (ATCC#CRL1592) were obtained from American Type Culture Collection (Rockville, MD). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Invitrogen), 2 mM GlutaMAX-I (Invitrogen), 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). Cells were incubated at 37uC in 5% CO 2 . The culture medium was changed every 2 days.

Cell viability assay
The effects of MTX, alone or in combination with lutein, in cell viability were determined by WST-1 reagent, according to the manufacturer's recommendations. In brief, 4610 3 cells were seeded on a 96-well plate and allowed to attach for 24 h. the cells were then treated with MTX, lutein, or MTX in the present of lutein. After the indicated time period, cells were incubated for 1 h with 10 ml of WST-1 reagent and the absorbance was measured using a microplate reader (PowerWave 6340, Bio-Tek Instruments, Inc., Winooski, USA) at 450 nm. In the pretreatment experiment, cells were pretreated 2 h or 24 h with lutein prior to MTX exposure and measured of cell viability.

Apoptosis assay
Apoptosis induction by MTX was assessed by (a) activation of caspase 3 activity; (b) detection of caspase 3 cleavage by Western blot.
1. Caspase 3/7 activity. Activation of caspase-3 was assayed using (Z-DEVD) 2 -R110 substrate as described [22]. Briefly, cells were plated in 96-well plates and allowed to attach by overnight incubation. The cells were then treated with DMSO (control) or the desired concentrations of MTX in the presence or absence of lutein for 24 h. Subsequently, the cells were directly lyzed by adding caspase 3 assay buffer containing (Z-DEVD) 2 -R110 substrates and incubated at 37uC for 1 hour. The fluorescent intensity of proteolytically released fluorophore R110 was then measured using a plate reader (Victor X2; PerkinElmer, Waltham, MA, USA) with an excitation of 485 nm and emission of 535 nm.
2. Detection of caspase 3 cleavage by Western blot. Cells subjected to the indicated treatment were harvested and lysed, and the protein concentration was determined by a BCA protein assay (Pierce, Rockford, IL, USA). Protein samples were separated by 14% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) before probing with antibodies (caspase-3 (active), Epitomics, Burlingame, CA; b-actin (as internal control), Sigma). Subsequent immunoblotting procedures were performed using a chemiluminescence procedure (Millipore) per the manufacturer's instructions. The intensity of immunoreactive bands was determined using GeneTools software

Detection of mitochondrial membrane potential
Changes in the mitochondrial membrane potential in cells were measured by flow cytometry using JC-1 as described [23]. Thirty minutes prior to cytometric analysis, JC-1 is added to the cells to a final concentration of 10 mM. Cells are then examined on a FL-1 (530 nm) versus FL-2 (585 nm) dot plot on a FACSCalibur flow cytometer (Becton Dickinson, BD.; NJ. USA). JC-1 has dual emission depending on the state of the mitochondrial membrane potential. It forms aggregates in cells with a high FL-2 fluorescence indicating a normal mitochondrial membrane potential. Loss of the mitochondrial membrane potential results in a reduction in FL-2 and with a concurrent gain in FL-1 fluorescence as the dye  shifts from an aggregate to monomeric state. Therefore, the retention of the dye in the cells can be monitored through the increase in FL-1 fluorescence.

ROS assay
Intracellular ROS generation in cells treated with MTX was measured by flow cytometry following staining with H 2 DCFDA as described [24]. Briefly, 2610 5 cells were plated in 6-well culture plates, allowed to attach overnight, and exposed to DMSO (control) or the desired concentration of MTX for the specific time intervals. The cells were stained with 10 mM H 2 DCFDA, and the fluorescence was measured using a FACSCalibur flow cytometer (BD), and the data were analyzed with CellQuest pro software (BD). In some experiments, cells were pretreated for 2 h or 24 h with Lutein prior to MTX exposure and analysis of ROS generation.

Quantitative RT-PCR
The expression levels of Bcl-2, Bad, superoxidase dismutase 1(SOD), catalase (CAT) and Peroxiredoxin 1 (Prdx1) were measured in MTX-treated cells with or without the pretreatment of lutein. Total RNA was isolated using TRIzol reagent (Invitrogen). RNA yields were measured using Nanodrop 2000 (Thermo Scientific, Rockford, IL, USA). First strand cDNA was synthesized from total RNA (2 mg) by First Strand cDNA Synthesis Kit (Thermo Scientific) using oligo dT primers. Q-PCR was performed in a 10 ml reaction that contained 0.5 ml of the cDNA and 16 KAPA SYBRH FAST Universal qPCR Master Mix (Kapa biosystems, Woburn, MA, USA), using the following PCR parameters: 95uC for 3 min followed by 40 cycles of 95uC for 10 s, 60uC for 30 s followed by melt curve: 65uC to 95uC increment 0.5uC for 5 s. The expression level of GAPDH was used as internal control. The relative expression was calculated using the comparative Ct and expressed as fold of control. The specific primer pairs used to amplify genes are listed in Table S1.

Statistical analysis
All experiments were performed in triplicate, and results are presented as the mean 6 S.D. Student's t-test was employed to compare control and experimental parameters with P#0.05 considered as statistically significant. SigmaPlot (vers. 10) was utilized for data analysis and graphical presentation.

Dose-and time-dependent cytotoxicity in IEC-6 cells upon MTX treatment
Most studies described a dose-and time-dependent effect of MTX on the inhibition of cell proliferation and the induction of apoptosis [25,26,27]. After exposing IEC-6 cells to different concentrations of MTX for 24 hours, we observed a clear effect made by the dose. As shown in Figure 1A

Induction of ROS and loss of mitochondrial membrane potential in IEC-6 cells treated with MTX
MTX was reported to induce ROS in the intestinal mucosa in rats [28]. We therefore detected the induction of ROS in IEC-6 cells treated with different dose of MTX. As shown in Figure 2A

The effect of lutein on IEC-6 cell viability and caspase 3 activation
Lutein is one of the naturally occurring carotenoids and has been proved to reduce H 2 O 2 induced ROS stress in cells [29,30,31]. Therefore, we hypothesized that lutein could suppress MTX-induced ROS and thus protect the damage of MTX in IEC-6 cells. To test this theory, we first investigated the effect of lutein on the viability of IEC-6 cells. As shown in Figure 3A, administration of lutein raging from 0.1 to 10 mM showed no effect on the viability of IEC-6 cells. And as expected, the caspase 3 activity was not altered by the administration of lutein in IEC-6 cells ( Figure 3B).

Pretreatment of lutein inhibits MTX-induced cell viability lost and apoptosis
To test whether lutein inhibits MTX-induced cell injury, we first detected the effects of lutein in cells treated with MTX. The cells were pretreated with lutein for 2 or 24 h prior to the administration of MTX, or co-treated with lutein and MTX. The cell viability was then detected. As shown in Figure 4A, cell viability was reduced to 62.163.3 and 52.664.1 % in cells treated with 1 or 10 mM of MTX. Co-treatment of 1, 2.5, 5 or 10 mM of lutein had no effect on the viability lost in MTX-treated cells. Two hours of lutein pre-treatment has little but no significant effect in cells treated with 1 mM of MTX. Cell viability was slightly increased in 10 mM MTX treated cells that pretreated with lutein for 2 h. The rescue of cell viability lost was observed in cells pretreated with lutein for 24 h then treated with 1 or 10 mM MTX. We next detected the activity of caspase 3 in MTX-treated cells with the lutein co-treatment or pre-treatment. The results were consistent with the cell viability, cells pre-treated with lutein for 24 h suppressed the MTX-induced activation of caspase 3 ( Figure 4B). These results were further confirmed by direct monitoring the expression levels of cleavage caspase 3 in MTXtreated cells with 24 h lutein pre-treatment using Western blot ( Figure 4C). The apoptotic index (expression ratio of Bad/Bcl2) also showed decreased apoptosis in MTX-treated cells with 24 h pre-treatment of lutein ( Figure 4D). The data suggests that lutein may protect cells against MTX-induced apoptosis.

Lutein suppresses MTX-induced ROS generation
We next evaluated the anti-oxidant activity of lutein in cells treated with MTX. We hypothesized that lutein suppresses MTXinduced ROS generation to inhibit apoptosis induction that causes lost cell viability. As shown in Figure 5, ROS generation in cells treated with MTX was suppressed by the pre-treatment of lutein. Pre-treatment of 5 and 10 mM of lutein suppressed the percentage of DCF-positive cells from 30.861.83% to 18.260.79% and 12.060.41%, respectively, in 100 mM MTX-treated IEC-6 cells.

Expression of ROS scavenging enzymes in MTX-treated IEC-6 cells with or without lutein pretreatment
To gain insight of the ability of lutein on suppressing ROS generation induced by MTX, we monitored the expression of ROS scavenging enzymes, including SOD, CAT and Prdx1. The expressions of these anti-oxidant enzymes were all decreased upon MTX treatment ( Figure 6). The expression of SOD was increased in cells pre-treated with lutein for 24 h. The expression level of SOD was even higher in lutein treated cells than the control cells. The expression of CAT was also up-regulated in cells pre-treated with lutein, but was lower than control. The expression of Prdx1 was reduced in MTX-treated cells and was not elevated with the pre-treatment of lutein.

Discussion
In the present study, we have shown that MTX treatment caused IEC-6 cell death in a time-and dose-dependent manner via apoptosis, as demonstrated by the loss of IEC-6 cells viability, the induced activation of caspase-3 activity and the increased cleavage caspase 3. The MTX-induced loss of viable IEC-6 cells was prevented by pre-treatment with lutein. Our data suggests that lutein suppresses MTX-induced ROS generation by activating the expression of ROS scavenging enzymes and therefore reduces MTX-induced apoptosis in IEC-6 cells. These results suggested that lutein may be useful for alleviating the adverse effects generated by the cancer chemotherapy with MTX.
Caspases are crucial mediators of programmed cell death. Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. Caspase-3 activity in IEC-6 cells exposed to MTX was significantly up-regulated. Aged garlic extract (5 mM) was shown to significantly reduce activation of caspase-3 induced by MTX [26]. Caspase-3, a key enzyme for execution of apoptosis in many instances, also plays a central role for MTX-induced apoptosis in IEC-6 cells.
The intrinsic apoptotic pathway is characterized by permeabilisation of the mitochondria and by release of cytochrome c into the cytoplasm. Cytochrome c then forms a multi-protein complex known as the 'apoptosome' and initiates activation of the caspase cascade through caspase 9. Active caspase 9 cleaves and activates downstream caspases-3, -6, and -8, leading to apoptosis [32]. Mitochondrial permeability is also related to the increased generation of reactive oxygen species (ROS), which plays a role in the degradation phase of apoptosis [33]. In our study, the MTX-treated IEC-6 cells were shown increased release of ROS with disrupted mitochondrial membrane potential and active caspase-3 that leads to apoptosis.
Recent reports demonstrated that MTX induces ROS generation and causes significant reduction in the antioxidant enzyme levels, sensitizing the cells to ROS [18,28,34,35,36,37]. Oxidative stress was reported to contribute to MTX-induced small intestinal toxicity in rats [38]. Administration of N-acetylcysteine (NAC) decreases the MTX-induced damage to the small intestine in rats [39]. Oncologists contended that antioxidants interfere with radiation and some chemotherapeutic treatments because those modalities kill by generating free radicals that are neutralized by antioxidants and that folic acid interferes with MTX. However, a previous study reviewing fifty human clinical randomized or observational trials involving 8,521 patients shown non-prescription antioxidants and other nutrients do not interfere with therapeutic modalities for cancer. Furthermore, they enhance the therapeutic outcomes by decreasing side effects and protect normal tissue [40]. We hereby showed that the pre-treatment of lutein suppressed the ROS induced by MTX and increased the expression of SOD and catalase in the intestinal epithelial cells. SOD and catalase are ROS scavenging enzymes, and have been demonstrated to protect small intestinal damages induced by ischemia reperfusion [41]. Absorption of lutein in human intestinal epithelium was demonstrated to be associated with the scavenger receptor class B member 1 (SR-BI) protein [42]. Furthermore, the bioaccessibility of lutein in small intestine is greater than bcarotene or lycopene [43]. Our results indicate that pre-treatment of lutein inhibited MTX-induced ROS generation and apoptosis in IEC-6 cells, and suggest that lutein may have a potential role in adjuvant cancer chemotherapy by reducing the intestinal damage of anti-cancer drugs. Table S1 QPCR primers used in this study.