Published online Feb 11, 2022.
https://doi.org/10.4163/jnh.2022.55.1.47
Antiproliferative properties of luteolin against chemically induced colon cancer in mice fed on a high-fat diet and colorectal cancer cells grown in adipocyte-derived medium
Abstract
Purpose
Obesity and a high-fat diet (HFD) are risk factors for colorectal cancer. We have previously shown that luteolin (LUT) supplementation in HFD-fed mice markedly inhibits tumor development in chemically induced colon carcinogenesis. In this study, we evaluated the anticancer effect of LUT in the inhibition of cell proliferation in HFD-fed obese mice and HT-29 human colorectal adenocarcinoma cells grown in an adipocyte-derived medium.
Methods
C57BL/6 mice were fed a normal diet (ND, 11.69% fat out of total calories consumed, n = 10), HFD (40% fat out of total calories consumed, n = 10), HFD with 0.0025% LUT (n = 10), and HFD with 0.005% LUT (n = 10) and were subjected to azoxymethane-dextran sulfate sodium chemical colon carcinogenesis. All mice were fed the experimental diet for 11 weeks. 3T3-L1 preadipocytes and HT-29 cells were treated with various doses of LUT in an adipocyte-conditioned medium (Ad-CM).
Results
The weekly body weight changes in the LUT groups were similar to those in the HFD group; however, the survival rates of the LUT group were higher than those of the HFD group. Impaired crypt integrity of the colonic mucosa in the HFD group was observed to be restored in the LUT group. The colonic expression of proliferating cell nuclear antigen and insulin-like growth factor 1 (IGF-1) receptors were suppressed by the LUT supplementation in the HFD-fed mice. The LUT treatment (10, 20, and 40 μM) inhibited the proliferation and migration of HT-29 cells cultured in Ad-CM in a dose-dependent manner, as well as the differentiation of 3T3-L1 preadipocytes.
Conclusion
These results suggest that the anticancer effect of LUT is probably due to the inhibition of IGF-1 signaling and adipogenesis-related cell proliferation in colon cancer cells.
INTRODUCTION
The incidence rate of colorectal cancer (CRC) is mainly high in developed countries, and it has been reported that the incidence rate in advanced countries accounts for more than 63% of the total incidence rate [1, 2, 3]. These geographical differences appear to be closely related to the western culture of the region, including the diet. A high-fat diet (HFD) and excessive weight gain are major risk factors for CRC [2, 4, 5]. The World Cancer Research Fund and the American Institute for Cancer Research reported that increased body fat and abdominal obesity clearly increase the risk of colon cancer [1]. According to a recent epidemiological study, an increase in body mass index by 2 increases the risk of CRC by 7%, and an increase in waist circumference by 2 cm increases the risk by 4% [6]. In an animal model, when 40% of calories were fed as fat to ApcMin/+ mice, there was a 75% increase in colon polyps compared to that of the controls [7]. When obese mice were treated with azoxymethane (AOM) by supplying 60% of calories as fat, the occurrence of aberrant crypt foci, a precancerous lesion of CRC, increased compared to that of the control group [8]. Furthermore, in the colonic mucosa of HFD-induced obese mice, apoptosis was decreased and cell proliferation was increased, which promoted the development of CRC [9].
Luteolin (LUT; 3′,4′,5,7-tetrahydroxyflavone) is a flavonoid abundant in celery, parsley, broccoli, carrots, peppers, cabbage, apple peel, and chrysanthemum flowers [10]. It has a wide range of biological activities, including antioxidant, anti-obesity, and anti-cancer activities [10]. In an AOM-induced CRC model, LUT has been reported to inhibit cell proliferation by downregulating the Wnt signaling pathways and reducing proliferating cell nuclear antigen (PCNA) expression [11]. Oral administration of LUT has been shown to reduce tumor number in a 1,2-dimethylhydrazine-induced mouse CRC model [12]. Alternatively, recent studies have reported that LUT is effective in improving various symptoms of dietary obesity, such as increased thermogenesis of brown adipose tissue [13], decreased body weight and body fat mass [14], and suppressed adipocyte inflammatory response [15]. Previously, we showed that LUT supplementation markedly inhibited chemically-induced colon cancer by suppressing colonic inflammation in HFD-fed mice [16]. In this study, we investigated how LUT inhibits CRC in HFD-fed mice through in vivo and in vitro experiments with a focus on cell proliferation.
METHODS
Animal experiment
Colon carcinogenesis experiments were performed as previously reported [16]. Briefly, 5-week-old male C57BL/6 mice (Koatech Bio Inc., Busan, Korea) were divided into normal diet (ND), HFD (40% fat/kg diet), HFD with 0.0025% (w/w) LUT (HFD LL), HFD with 0.005% (w/w) LUT (HFD HL) groups (n = 10 for each group). LUT was purchased from LKT Laboratories (St. Paul, MN, USA). A single dose of 12.5 mg/kg body weight AOM (Sigma-Aldrich, St. Louis, MO, USA) was intraperitoneally injected to the mice at 6 weeks of age and dextran sulfate sodium (DSS; MP Biomedicals, Irvine, CA, USA) was provided to the mice in drinking water 1 week after the AOM injection [17]. Animals were given 2% DSS in drinking water for one cycle and 1% DSS for another cycle. Each cycle consisted of 5 days, and the cycles were separated by 16 days. The experimental diet (Table 1) was fed to mice after AOM initiation and continued for 11 weeks. The animal protocol used in this study was approved by the Animal Care and Use Committee at Catholic University of Daegu (IACUC-2017-067).
Table 1
Composition of experimental diets
Immunohistochemical analysis
Colonic tissue fixed in 10% formalin was dehydrated, embedded in paraffin, and sectioned. Sections were mounted on slides and blocked with 5% bovine serum albumin for 30 minutes. Sections were then incubated with primary antibody against PCNA (Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:500 overnight at 4°C. After washing with phosphate-buffered saline (PBS), the sections were incubated with peroxidase-conjugated secondary antibody (Biocare Medical Inc, Concord, CA, USA) for 30 minutes. After washing with PBS, the sections were incubated with 3, 3-diaminobenzidine solution for 10 minutes at room temperature in the dark. Finally, sections were washed with PBS and stained with hematoxylin and eosin according to standard protocols. The number of PCNA-positive cells in colonic epithelium was counted in 5 randomized tissue sections for each animal. The labeling index was calculated as the percentage of PCNA-positive cells to the number of 100 cells in the colonic epithelium.
Plasma insulin-like growth factor 1 (IGF-1) analysis
Plasma IGF-1 levels were measured using a mouse/rat IGF-1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
Protein extraction and immunoblot analysis
Frozen colorectal tissues were homogenized using a Mini-Bead Beater (Biospec Products, Bartlesville, OK, USA) in a cold radioimmunoprecipitation assay buffer (RIPA buffer, 0.1% SDS, 1% Triton X-100, 0.5% deoxycholate, 50 mM Tris [pH 7.5], 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10 μg/mL leupeptin, 10 μg/mL aprotinin). For the cell lysates, cells were washed with PBS, resuspended in mild-lysis buffer (10 mM Tris–HCl [pH 7.5], 100 mM NaCl, 1% NP-40, 50 mM NaF, 2 mM EDTA [pH 8.0], 1 mM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin). The lysates were centrifuged at 12,300 rpm for 10 minutes at 4°C and supernatants were assayed for protein concentration using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Cramlington, UK). Equal amounts of protein were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride (PVDF) membranes using an electrophoresis system. Non-specific binding was blocked by 5% non-fat dry milk in Tris-buffered saline-Tween (TBST) buffer for 1 hour. The membrane was probed with a primary antibody against PCNA and β-actin (Cell Signaling Technology) at 1:1,000 dilution for overnight at 4°C. After washing with TBST buffer 3 times, the membrane was incubated for 1 hour with horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (1:1,000). The protein levels were determined by using an Amersham™ ECL™ Prime Western Blotting Detection Reagent (GEHealthcare, Buckinghamshire, UK).
Cell culture
Mouse fibroblast 3T3-L1 cells and human colorectal adenocarcinoma HT-29 cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS) in a 5% CO2 incubator. 3T3-L1 preadipocytes were seeded at a density of 5 × 104 cells/60-mm dishes (Corning, NY, USA) in DMEM with 10% FBS. After reaching full confluence, cells were induced to differentiate for 2 days in MDI medium: DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St. Louis, MO, USA), 1 μM dexamethasone (Sigma-Aldrich), 1 μg/mL insulin (Sigma-Aldrich), 10% FBS and 1% PS. After 2 days, the culture medium was replaced with DMEM containing 1 μg/mL insulin, 10% FBS and 1% PS and cultured for 2 days. Thereafter, the medium was replaced with DMEM containing 10% FBS every 2 days for 4 days.
When 3T3-L1 adipocytes were differentiated, the cell culture medium was removed. Cell dishes were washed with PBS and replaced with DMEM containing 1% FBS and 1% PS for 24 hours. The supernatant was then collected in a sterile 50 mL conical tube and centrifuged at 1,000 rpm for 5 minutes to remove cell debris. The harvested supernatant was used as adipocyte-conditioned medium (Ad-CM) for subsequent in vitro studies.
Lipid accumulation quantification
Differentiated 3T3-L1 cells were washed 3 times with PBS and fixed overnight at room temperature using 10% formalin. The fixed cell dishes were then washed 3 times with PBS and stained with 1 mL of 60% Oil Red O working solution (saturated Oil Red-O solution in 6 parts of isopropyl alcohol and 4 parts of water) for 40 minutes. Cells were again washed 3 times with PBS and dried in air. Cells were visualized under a DM IL LED Microscope (Leica, Wetzlar, Germany) at 200× magnification. Oil Red O dye was dissolved in 100% isopropyl alcohol and absorbance was measured at 500 nm using a Thermo Scientific Multiskan GO spectrophotometer (Thermo Fisher Scientific, Vartaa, Finland). Relative values of lipid accumulation were expressed as a percentage of the control group considered 100%.
Cell viability assay
The viability of 3T3-L1 and HT-29 cells was assessed using the Quanti-MAX™ WST-8 Cell Viability Assay Kit (Biomax, Seoul, Korea). 3T3-L1 cells were seeded in a 48-well plate at a density of 5 × 104cells/well and treated with LUT at various concentrations of 5–40 μM for 48 hours. HT-29 cells were seeded in a 48-well plate at a density of 1 × 105cells/well and treated with LUT at various concentrations of 10–80 μM for 24 and 72 hours. After treatment, WST-8 reagent (10 µL) was added to each well and incubated for 1 hour before absorbance was measured at 450 nm using a Thermo Scientific Multiskan GO spectrophotometer. Relative values of cell viability were expressed as a percentage of control considered 100%.
Cell migration assay
HT-29 cells were seeded at a density of 3 × 104 cells/60 mm dish with DMEM containing 10% FBS until 80% confluence was reached. Using a 10 µL pipette tip, carefully scrape the cell monolayer and replace it with 1% FBS/DMEM medium with 50% Ad-CM. Images were captured at 100× magnification with a DM IL LED microscope. The analysis was repeated at least 3 times.
Statistical analysis
Data are presented as mean ± SE. All statistical analyzes were performed by the SPSS program (ver.19; IBM Corp., Chicago, IL, USA). These data were analyzed by one-way ANOVA and differences between experimental groups were assessed with Duncan’s multiple-range test. Cell viability assays were analyzed using unpaired Student’s t-test. The p-value < 0.05 indicates a significant difference.
RESULTS
Mouse survival and mucosal crypt integrity
We previously reported that LUT supplementation in HFD-fed mice significantly reduced colorectal tumor development in a dose-dependent manner compared to that of the HFD alone group [16]. The mouse mortality rate was also decreased by LUT supplementation. At the end of the experiment, 60% of the mice in the HFD group survived, 90% in the ND and HFD LL groups, and 100% in the HFD HL group (Fig. 1A). On the other hand, weekly body weights in the LUT groups were not different from that in the HFD group (Fig. 1B). Histological analysis of the colonic mucosa showed that crypt integrity was impaired in the HFD group, however, the crypts were restored in the LUT groups (Fig. 1C).
Fig. 1
ND, normal diet; HFD, high fat diet; HFD LL, HFD + 0.0025% luteolin; HFD HL, HFD + 0.005% luteolin. AOM, azoxymethane; DSS, dextran sulfate sodium.
LUT restores mucosal crypt integrity in mice fed a high-fat diet in a colon carcinogenesis model.
C57BL/6 mice were fed an experimental diet for 11 weeks and chemically induced colon carcinogenesis using AOM and DSS. (A) Survival rate of mouse. (B) Weekly changes of body weight. (C) H&E staining of large intestine (a: ND, b: HFD, c: HFD LL, and d: HFD HL). All sections were photographed at ×200. Each data are presented as mean ± SE. Means with different letters indicate significant differences between the groups at p < 0.05 assessed by ANOVA with Duncan’s multiple range test. Arrows indicate the treatment time of AOM and DSS.
Colonic mucosal cell proliferation
Immunohistochemical analysis of PCNA, a marker protein for cell proliferation, showed that PCNA staining of cells was 45.7%, 89.7%, 55.2%, and 49.0% in the ND, HFD, HFD LL, and HFD HL groups, respectively (Fig. 2A). The HFD group showed a 196% increase in PCNA labeling index compared with that of the ND group. The indices of the HFD LL and HFD HL groups decreased by 62% and 54%, respectively, compared with that of the HFD group. We analyzed insulin-like growth factor-1 receptor (IGF-1R) expression in colon tissue as another biomarker for proliferation. IGF-1R expression was significantly increased in the HFD group compared with that of the ND group, and IGF-1R expression in the HFD LL and HFD HL groups decreased to a level similar to that of the ND group (Fig. 2B). Plasma IGF-1 concentrations were also significantly lower in the LUT group than in the HFD group (Fig. 2C).
Fig. 2
ND, normal diet; HFD, high fat diet; HFD LL, HFD + 0.0025% luteolin; HFD HL, HFD + 0.005% luteolin; PCNA, proliferating cell nuclear antigens.
LUT reduces colonic cell proliferation in mice fed a high-fat diet in a colon carcinogenesis model.
(A) Colonic tissue sections were immunostained with antibodies to PCNA (a: ND, b: HFD, c: HFD LL, and d: HFD HL). All sections were photographed at ×200. The number of PCNA-positive cells in colonic epithelium was counted in 5 randomized tissue sections for each animal. The PCNA labeling index was calculated as the percentage of PCNA-positive cells to the number of 100 cells in the colonic epithelium. (B) Distal sections of the large intestine were immunoblotted with the relevant IGF-1R antibody. Photograph of chemiluminescent detection of the representative blots. The relative abundance of each band to β-actin was quantified. (C) Plasma IGF-1 was measured as described in Methods. Each data are presented as mean ± SE. Means with different letters indicate significant differences between the groups at p < 0.05 assessed by ANOVA with Duncan’s multiple range test.
Effect of LUT on 3T3-L1 preadipocyte differentiation
Colorectal tumor development was significantly increased in the HFD group, and LUT effectively reduced tumor multiplicity [16], therefore, we evaluated the effect of LUT on adipocyte differentiation. We observed a slight decrease (5%) in cell viability in 3T3-L1 preadipocytes treated with 40 μM LUT for 48 hours (Fig. 3A). Notably, LUT treatment reduced lipid accumulation in cells in a dose-dependent manner, while 3T3-L1 preadipocytes differentiated into adipocytes (Fig. 3B and C).
Fig. 3
LUT, luteolin.
LUT reduces differentiation and lipid accumulation in 3T3-L1 adipocytes.
(A) 3T3-L1 preadipocytes were treated with different concentrations of LUT for 48 hours. (B) The photographs of Oil Red O-stained adipocytes. (C) Stained oil droplets were dissolved in isopropylalcohol and quantified by reading the absorbance at 500 nm. Each data are presented as mean ± SE. Cell viability assays were analyzed using the unpaired Student’s t-test. p < 0.05 indicates a significant difference. Means with different letters indicate significant differences between the groups at p < 0.05 assessed by ANOVA with Duncan’s multiple range test.
Effect of LUT on proliferation of HT-29 cells cultured in adipocyte conditioned medium
Next, we determined if there was an interaction between colon cells and adipocytes. LUT treatment up to 40 μM did not affect the viability of HT-29 cells after 72 hours (Fig. 4A). However, at 80 μM, 30% and 60% cell death was achieved after 24 and 72 hours of treatment, respectively. HT-29 cells were treated with differentiated 3T3-L1 Ad-CM, and cell viability was analyzed. As shown in Fig. 4B, when more conditioned medium is added to the HT-29 cells, more cells are grown in culture. Interestingly, the addition of LUT-treated Ad-CM to HT-29 cells significantly reduced HT-29 cell viability in a LUT dose-dependent manner at Ad-CM volumes of both 25% and 50% (Fig. 4C). Furthermore, HT-29 cells grown in LUT-treated Ad-CM showed significantly reduced migration in a LUT dose-dependent manner (Fig. 4D). PCNA expression in HT-29 cells also increased by the addition of Ad-CM, however, the expression was decreased by LUT-treated Ad-CM in a dose-dependent manner within a given volume of Ad-CM (Fig. 4E).
Fig. 4
A25, 25% of Ad-CM; A50, 50% of Ad-CM; A100, 100% of Ad-CM; D, DMSO; L20, luteoiln 20 μM; L40, luteolin 40 μM; LUT, luteolin; DMEM, Dulbecco’s modified eagle’s medium; Ad-CM, adipocyte-conditioned medium; PCNA, proliferating cell nuclear antigen. *p < 0.05, ***p < 0.001 vs. control. Means with different letters indicate significant differences between the groups at p < 0.05 assessed by ANOVA with Duncan’s multiple range test.
LUT inhibits proliferation of HT-29 cells cultured in 3T3-L1 adipocyte conditioned medium.
(A) Viability of HT-29 cells cultured in DMEM with different concentrations of LUT for 24 and 72 hours. (B) Viability of HT-29 cells cultured in different volumes of Ad-CM for 48 hours. (C) HT-29 cells were incubated with LUT in 25% or 50% Ad-CM for 48 hours. (D) Migration of HT-29 cells cultured in Ad-CM with different LUT concentrations for 6 and 12 hours. (E) Protein expression of PCNA in HT-29 cells cultured in Ad-CM with different LUT concentrations. The relative abundance of each band to GAPDH was quantified. Each data are presented as mean ± SE. Mean values were analyzed using unpaired Student’s t-test.
DISCUSSION
The rapid increase in CRC in Korea is primarily due to westernized changes in diet such as high intake of fat, red meat, and processed meat and low intake of fiber-rich foods [18]. Previously, we reported that LUT is a potent anti-cancer flavonoid for CRC development in HFD-induced obese animals [16], and we identified that LUT inhibits colonic mucosal cell proliferation, possibly through inhibition of IGF-1 signaling and adipocyte-derived colon cell proliferation.
HFD is known to increase the risk of CRC. In the CRC xenograft mouse model, the HFD group fed 45% of total calories as fat had an increase in body weight, body fat mass, and tumor weight compared to that of the control group fed 13.5% fat [19]. Moreover, the expression of PCNA and COX-2 was significantly increased in the HFD group compared to that in the control group. When ApcMin/ + mice were fed 40% calories from fat, the HFD group showed an increase in body weight, body fat, and adipocyte size, and the number of colonic polyps increased by 75% compared to that of the control group [7]. However, LUT supplementation in HFD-fed mice reduced tumor multiplicity and incidence compared to that in controls [12, 16]. Contrary to the results of Kwon et al. [11], that is, the weight gain of mice supplemented with 0.0025% LUT to HFD was significantly reduced compared to that of the HFD group, the LUT and HFD groups in this study showed similar weight gain. In the AOM-DSS CRC model, LUT showed a high survival rate and anticancer efficacy by compensating for weight loss caused by DSS administration.
Increased bioavailability of insulin and IGF-1 has been proposed as a possible mechanism of obesity for CRC risk [20, 21]. Hyperinsulinemia in obesity leads to decreased hepatic synthesis of IGF binding protein 1 (IGFBP1) and IGFBP2, resulting in increased blood free IGF-1 levels. Indeed, a positive correlation was observed between IGF-1 level and body fat/waist circumference [22]. IGF-1 then binds to the IGF-1R and activates downstream signaling, including PI3K/Akt, which promotes cell proliferation and survival. IGF-1R expression has been shown to be upregulated in colon cancers in humans [23] and mice [24]. In HT-29 human colon cancer cells, LUT reduces IGF-II production and downregulates IGF-1R signaling [25]. It also inhibits IGF-1R signaling in prostate cancer cells [26]. In this study, we found that LUT supplementation in HFD-fed mice reduced the expression levels of plasma IGF-1 and colonic IGF-1R. This may explain the decreased colonic cell proliferation exhibited by mucosal thickness and PCNA expression.
Alternatively, various adipokines, cytokines, and chemokines produced by adipocytes are known to mediate inflammatory responses and proliferation that contribute to CRC development [5, 27]. In particular, leptin increases intestinal mucosal cell proliferation in human colon cells and enhances AOM-induced colonic precancerous lesions in mice [28, 29]. Leptin receptor overexpression has also been identified in intestinal cancers [24, 30]. The components of Ad-CM were not analyzed, however, it was confirmed that the addition of Ad-CM to HT-29 cells increased the proliferation of 3T3-L1 preadipocytes. When more Ad-CM was added, the greater the increase in cell proliferation. LUT treatment of 3T3-L1 preadipocytes effectively inhibited the proliferation and differentiation of Ad-CM-cultured HT-29 cells in a dose-dependent manner. LUT has been reported to inhibit triglyceride accumulation in adipocyte differentiation by downregulating the expression of the PPARγ and C/EBPα [31]. It will be interesting to investigate the interaction of the components of the LUT with the Ad-CM in future studies.
SUMMARY
In summary, CRC development was significantly inhibited in HFD-induced obese mice when LUT was included in the diet [16]. Our results suggest that LUT directly inhibits colonic cell proliferation by downregulating IGF-1 signaling in colon tissues and indirectly inhibiting adipokine-induced colon cancer cell proliferation by inhibiting adipocyte differentiation. In terms of gut health, it is important to develop a habit of eating foods rich in LUTs, such as celery, bell peppers, perilla leaves, broccoli, carrots, cabbage, and apple peel.
Funding:This work was supported by research grants from Daegu Catholic University in 2019.
Conflict of Interest:There are no financial or other issues that might lead to conflict of interest.
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