n-3 Polyunsaturated fatty acids enhance the antitumor effect of 5-fluorouracil by inhibiting bcl-2...

Research Article

n-3 Polyunsaturated fatty acids enhance the antitumor effect of5-fluorouracil by inhibiting bcl-2 and mutant-p53

Yao‐Zong Tan1*, Wen‐Ge Huang2*, Feng‐Ying Chen2, Jie Li2, Jing‐Ya Zhou1, Li‐JunWang1, Li Chen1

and Hui‐Lian Zhu1

1 Guangdong Provincial Key Laboratory of Food, Nutrition and Health, School of Public Health, Sun Yat‐SenUniversity, Guangzhou, China

2 Laboratory Animal Center of Sun Yat‐Sen University, Guangzhou, China

The study determined whether n‐3 polyunsaturated fatty acids (n‐3 PUFAs) enhance thechemotherapeutic ability of 5‐fluorouracil (5‐FU) to affect bcl‐2 and mutant‐p53 (mt‐p53). Thirty‐two BalbC/c mice bearing SW480 colorectal cancer (CRC) xenografts were randomly divided into fourgroups and fed with basic diet (5% of fat) and diets (20% of fat) with 0.69, 14.84, and 24.27% of n‐3PUFAs, while all mice received 5‐FU injections (35mg/kg) every 3 days. After 21 days’ treatment, tumorswere subjected to HE staining, TUNEL staining, and immunohistochemical analyses for bcl‐2 andmt‐p53, and serum n‐3 PUFAs composition were determined by GC‐FID. The proportion of serum n‐3PUFAs increased in the high (17.1%) and low (12.3%) n‐3 groups (vs. 3.1% in control). The high n‐3group showed slower tumor growth (Dvolume: 370.3mm3 vs. 556.9mm3, p< 0.05), larger tumornecrosis areas with more loosely arranged cells, and greater tumor cell apoptosis (positive rate: 9.4% vs.2.2%, p<0.05) compared to the control. Both Bcl‐2 and mt‐p53 expressions were inversely and dose‐dependently correlated to dietary n‐3 PUFAs (all p< 0.05). n‐3 PUFAs enhance the antitumor effect of5‐FU on SW480 xenografts probably through promoting tumor apoptosis via inhibiting bcl‐2 andmt‐p53.

Practical applications: n‐3 PUFAs have distinct anti‐cancer capacity and combination of n‐3 PUFAsand 5‐FU was considered as a potential approach for cancer chemotherapy, but the exact mechanism ofthe co‐anticancer effect of n‐3 PUFAs is still not clarified. Our present study demonstrated that n‐3PUFAs increase the apoptosis of CRC cells by inhibiting the expression of bcl‐2 and mt‐p53. Theseresults highlight the pro‐apoptosis effect of n‐3 PUFAs and reveal the possible mechanism of this effect.The main strategy of chemotherapy regimens is to enhance the pro‐apoptosis capacity of drugs againstmalignancy and n‐3 PUFAs, as a kind of dietary nutrient, are easy to be obtained and cause no toxicitycompared with other anti‐cancer drugs. In this context, n‐3 PUFAs may be developed as adjuvant anti‐cancer agent combining with 5‐FU or even other drugs.

Keywords: bcl‐2 / Colorectal cancer / 5‐Fluorouracil / Mutant‐p53 / n‐3 Polyunsaturated fatty acids

Received: June 3, 2013 / Revised: July 17, 2013 / Accepted: August 21, 2013

DOI: 10.1002/ejlt.201300017

1 Introduction

Colorectal cancer (CRC) is the second most prevalentmalignancy in western countries [1] and the fifth mostcommon malignancy in developing countries [2]. Therapeuti-cally, surgery is the primary treatment for potentially curablecancer, whereas chemotherapy and radiotherapy treatments areavailable for metastatic cancer [2]. 5‐Fluorouracil (5‐FU) has

*These authors contributed equally to this work.Color online: See the article online to view figures in colour.

Correspondence: Prof. Hui‐Lian Zhu, Guangdong Provincial KeyLaboratory of Food, Nutrition and Health, School of Public Health, Sun Yat‐Sen University, 74th Zhongshan Road II, Guangzhou 510080, ChinaE‐mail: [email protected]: þ86 20 87330446

Abbreviations: bcl‐2, B‐cell lymphoma 2; CRC, colorectal cancer; 5‐FU,5‐fluorouracil; HE, hematein & eosin; IHC, immunohistochemical; mt‐p53,mutant‐p53; n‐3 PUFAs, n‐3 polyunsaturated fatty acids; TUNEL,terminal‐deoxynucleoitidyl transferase‐mediated nick end labeling; VEGF,vascular endothelial growth factor

Eur. J. Lipid Sci. Technol. 2013, 115, 1483–1491 1483

� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

been used as themain chemotherapeutic protocol forCRC andother cancers for more than 50 years [3]. The chemotherapeu-tic effect of 5‐FU is attributed to inhibition of de novoDNA synthesis and altering the structure and function of RNAor DNA [4], leading to DNA damage, tumor cell apoptosisand, consequently, inhibition ofCRC [5].Despite this potentialfor effectiveness, the chemotherapeutic effects of 5‐FU can behampered by drug resistance or host toxicity [6–8].

Promoting apoptosis of tumor cells is a key strategy forcancer therapy. Multiple gene processes regulate tumor cellapoptosis and any of these factors may possibly influence thechemotherapeutic effect [9]. P53 is a tumor‐suppressingprotein which regulates a wide variety of target genesresponsible for cellular outcomes such as cell‐cycle arrest,apoptotic cell death, or nucleotide excision repair (NER)[10]. Mutation of the p53 gene is regarded as the mostcommon genetic change in the development of cancer andmutant type of p53 protein (mt‐p53) is detected at high levelsin most human cancer cells. Mt‐p53 not only loses its originalgrowth‐suppressive function but also gains the capacity forpromoting cancer cell growth, most likely by restoration ofcell cycle blocking, cell apoptosis, and NER [11]. B‐celllymphoma 2 (bcl‐2) is a tumor‐promoting protein, which isalso highly overexpressed in many cancers and enhances cellsurvival against programmed cell death (PCD) [12] throughinhibiting cell apoptosis caused by p53 or other factors [8, 13,14]. It is now well established that anticancer agents inducetumor cell apoptosis and that the promotion of apoptosisprograms increase cells’ sensitivity to chemotherapy. There-fore, inhibiting the expression of mt‐p53 and bcl‐2 mayenhance the apoptosis of tumor cells, and consequently exertadjuvant chemotherapy effects [15–18].

It is important to recognize that based on epidemiologicaldata [19–22], animalmodels, and cell culture studies [23–25],dietary fatty acids, especially saturated and unsaturated fattyacids, play a significant role in the pathogenesis of cancer. N‐3series polyunsaturated fatty acids (n‐3 PUFAs), includinglinolenic acid (LNA), docosapentanoic acid (DPA), eicosa-pentanoic acid (EPA), and docosahexanoic acid (DHA), havedistinct inhibitory effects on tumor growth, differentiation,and metastasis [26]. Calviello et al. [27] demonstrated that n‐3 PUFAs are able to inhibit the growth of human CRC cellseither in vitro or in vivo by inhibiting vascular endothelialgrowth factor (VEGF) expression through modulating thecyclooxygenase‐2/prostaglandin E2 (COX‐2/PGE2) path-way. A review by Dupertuis and co‐workers [28] also showsthat n‐3 PUFAs reduce the risk of CRC through modulatingthe gene expression of peroxisome proliferator activatedreceptor (PPAR), sterol regulatory element binding protein(SREBP), and nuclear factor‐kappa B (NF‐kB). In addition,Wynter and co‐workers [29] demonstrated that n‐3 PUFAsmay have potentiating effects on certain chemotherapyagents. Angela et al. [30] reported that the combination ofan n‐3 PUFAs‐containing lipid emulsion with 5‐FU shows anadditive inhibitory effect on CRC Caco‐2 cell line growth.

Calviello et al. [31] also found that DHA enhances thesusceptibility of CRC cells to the growth‐inhibitory action of5‐FU in vitro, probably due to suppression of the anti‐apoptotic proteins bcl‐2 and bcl‐XL. The mechanisms of thein vivo synergetic effect of n‐3 PUFAs and 5‐FU have stillnot been completely clarified.

In this study, we investigated the anticancer capacity ofn‐3 PUFAs combined with 5‐FU in a SW480 CRC xenograftmodel and determined whether n‐3 PUFAs enhance thechemotherapeutic effect of 5‐FU through the effect on bcl‐2and mt‐p53 expression.

2 Materials and methods

2.1 SW480 cell line and xenograft tumor model

Human SW480 cell line cultures were obtained from theLaboratory Animal Centre of Sun Yat‐Sen University. Cellswere cultured in RPMI1640 medium (Gibco Co., LTD.,USA) with 15% fetal bovine serum (Hangzhou SijiqingBiological Engineering Materials Co., LTD., Zhejiang,China) under standard conditions. After being passaged tothe third generation, cells were treated with trypsin, washedand re‐suspended in serum‐free RPMI1640 medium. Cellviability was �95%, as determined by trypan blue exclusionassay. Next, 2� 106 cells (1� 107/mL, 0.2mL) weresubcutaneously injected in the nuchal region of 4 BalbC/cnu/nu athymic mice (6‐wk‐old, female, obtained fromLaboratory Animal Centre of Sun Yat‐Sen University). Nudemice were kept in specific pathogen free (SPF) environmentand fed with basic rodent chow.Water and diet were providedad libitum. When the diameter of tumors reached �15mm,the nude mice were sacrificed. Tumors were excised,separated from their surrounding basement membranes,and cut into 27mm3 lumps for the grouping experiment.

2.2 Grouping experiment design of SW480 xenografts

Tumor lumps were subcutaneously transplanted in 32 femaleBALB/C nude mice. All nude mice were randomized intofour groups (n¼ 8) according to tumor size: basic group, n‐3control group, low n‐3 group, and high n‐3 group. Beforepreparation of each groups’ diet, the n‐3 PUFA content inbasic rodent chow, corn oil, and tuna oil (By‐HealthCorporation, China) were determined by gas chromatogra-phy with an FID detector. The compositions of n‐3 PUFAs(% of total fatty acids) were 1.84, 0.31, and 31.74% of thebasic rodent chow, corn oil, and tuna oil, respectively. Theformulas of each diet were as follows: (1) basic group: basicrodent chow (total fat content of 5%, containing 1.84% n‐3PUFAs); (2) n‐3 control group: rodent chowmixed with 15%corn oil (total fat content of 20%, containing 0.69% n‐3PUFAs); (3) low n‐3 group: rodent chowmixed with 9% tunaoil and 6% corn oil (total fat content of 20%, containing

1484 Y.‐Z. Tan et al. Eur. J. Lipid Sci. Technol. 2013, 115, 1483–1491


14.84% n‐3 PUFAs); and (4) high n‐3 group: rodent chowmixed with 15% tuna oil (total fat content of 20%, containing24.27% n‐3 PUFAs). All nude mice were kept in a SPFenvironment and received 5‐FU (SunRise Co., LTD., China)injection intraperitoneally (35mg/kg) every 3 days afterinitiation of the grouping experiment. Water and diet weregiven ad libitum throughout the experiment. Body weight andtumors’ long (a) and short (b) axes were measured every3 days, calculating tumor volume (V) as V¼ 0.5� a (mm)� b2 (mm2). After 21 days, all nude mice were sacrificed. Thetumors were stripped, weighed and then steeped in 4%paraformaldehyde for further analyses.

The animal experiment in this study was approved byExperimental Animals Ethics Committee of Sun Yat‐SenUniversity and was conducted following the universal higheststandard described by Workman et al. [32].

2.3 n‐3 PUFAs composition analysis

The n‐3 PUFAs composition in four diet samples of eachgroup and in serum specimens of each mice were analyzed bygas chromatography with flame ionization detector (GC‐

FID). Briefly, total dietary lipids were extracted withpetroleum ether using a Soxhlet extractor and the serumlipids with a 1:2 chloroform:methanol solution. The extractedlipids were then methylated by incubation with KOHmethanol (0.4mol/L) and the fatty acid methyl esters wereextracted with hexane prior to the gas–liquid chromatographyassay. A Finnigan Trace GC ultra (Thermo Fisher, USA),equipped with a capillary column (J and W DB‐FFAP,30m� 0.25mm), automatic injector and flame ionizationdetector, was used for the fatty acid separation and detection.Identification and quantitation of fatty acid were carried outby comparison of their retention times and peak area withthose of n‐3 PUFA standards (Sigma–Aldrich, USA), andvalues were expressed as g/100 g total fatty acids, respectively.

2.4 Histopathology analysis

The tumors steeped in 4% paraformaldehyde were used forparaffin sectioning. Briefly, the fixed tumors were trimmedinto an appropriate size and shape, washed with distilledwater, dehydrated in ethanol, cleared in xylene, and thenembedded in paraffin. Next, the paraffin blocks were cut on arotarymicrotome into 5mm sections andmounted onto slidesfor hematoxylin & eosin (HE), terminal‐deoxynucleoitidyltransferase‐mediated nick end labeling (TUNEL), andimmunohistochemical (IHC) staining.

After steeping in a xylene and ethanol (1:1) solution, thedeparaffinized slides were first stained with hematoxylin. Forstain separation, slides were washed with 0.5–1% hydro-chloric acid in 70% ethanol, followed by a 0.1–0.5% eosinsolution for counterstaining. Finally, the slides were dehy-drated in volumes of ascending concentrations of ethanol(70–100%), hyalinized by xylene and fixed by resinene.

2.5 TUNEL staining

The proportion of apoptotic cells in the tumor tissues wasdetermined using the DeadEnd™ Colorimetric TUNELSystem (Promega Co., LTD., USA). Briefly, the deparaffi-nized slides were incubated with 20mg/mL proteinase Ksolution. The end‐labeling reaction was conducted byterminal deoxynucleotidyl transferase (TdT) reaction mix(98% of equilibration buffer, 1% of biotinylated nucleotidemix and 1% of TdT enzyme), followed by incubation for60min at 37°C and then terminated by 2� SSC solution.Endogenous peroxidases were blocked with 0.3% hydrogenperoxide in PBS. Thereafter, slides were incubated withstreptavidin HRP solution for 30min at room temperature,and HRP activity was detected by DAB solution.

2.6 Immunohistochemical staining of bcl‐2 andmt‐p53

The IHC stainings of bcl‐2 and mt‐p53 were conducted byfollowing a two‐step method. Briefly, the deparaffinizedsections were treated with EDTA (pH 8.0, 1: 50 dilution) forantigen retrieval and then steeped in 3% hydrogen peroxidefor 10min. Thereafter, sections were incubated with primaryantibodies (mouse anti‐human IgG monoclonal antibody,1:50 dilution) raised against bcl‐2 (Shanghai Long IslandBiotech Corporation, China) or mt‐p53 (Fujian MaixinBiotech Corporation, China) at 37°C for 60min. Next,sections were treated with secondary antibody (Dako k5007)at 37°C for 40min. After cleaning with PBS, sections weretreated with DAB (1:50 dilution, Dako Envision K5007 colordeveloping system). Sections were next counterstained byhematoxylin, dehydrated in 95% ethanol and mounted byresinene.

The sections of HE, TUNEL, and IHC stainingwere analyzed using a Nikon Eclipse Ti‐E microscope withthe CFI60® optical system (Nikon Co., LTD., Japan) andImage‐Pro® Plus v. 6.0 software (Media Cybernetics Inc.,USA) by an experienced technician who was blinded tothe grouping protocol. Quantifications of apoptotic, bcl‐2‐positive and mt‐p53‐positive cells were performed bycounting the percentage of brown colored positive cellsamong 400 cells at five randomly selected fields undermicroscope.

2.7 Statistical analyses

All statistical analyses were performed with SPSS 13.0 (IBMInc., Armonk, NY, USA). Data are shown as the mean�SEM. The differences in body weight and tumor volumeamong the groups were compared using the analysis ofvariance for repeated measures. We compared the meandifferences of other data using post hoc multiple comparisontests (method: least significant difference) of one‐way analysisof variance, or using non‐parametric analyses in case of none‐

Eur. J. Lipid Sci. Technol. 2013, 115, 1483–1491 n‐3 Polyunsaturated fatty acids enhance the antitumor 1485


normal distribution. Pearson correlation was used to test thetrends of mean differences in tumor volume between groupsover time, and the association of the proportion of serum totaln‐3 PUFAs and the values of tumor volume, the percentagesof positive cells of bcl‐2, mt‐p 53, and TUNEL. Statisticallysignificant was defined as p<0.05.

3 Results

3.1 n‐3 PUFAs compositions of diets and serum

The fatty acids composition in diet and serum is shown inTable 1. The proportions of dietary total n‐3 PUFAs in thegroups of basic diet, n‐3 control, low n‐3, and high n‐3 dietswere 1.84, 0.69, 14.84, and 24.27%, respectively. The maincomposition of n‐3 PUFAs in diets were docosahexaenoicacid (DHA, C22:6), eicoapentaenoic acid (EPA, C20:5),alpha‐linolenic acid (ALA, C18:3), and docosapentaenoicacid (DPA, C22:5). Among them,DHA and EPA constitutedof 89, 64, 95, and 96% of the total n‐3 PUFAs in diets. Wefound that the pattern of serum n‐3 PUFAs were consistentwith those in diets. The proportional values of total n‐3PUFAs increased significantly in the basic group (7.0%), lown‐3 group (12.3%), and high n‐3 group (17.1%) as comparedwith the control group (3.1%) (all p<0.001).

3.2 n‐3 PUFAs inhibited the growth of SW480xenografts in nude mice

After subcutaneous implantation, tumors developed in allnude mice (Fig. 1A). The body weight curves are shown inFig. 1B. After transplantation, body weights showed a

downward trend but did not achieve significance amonggroups. Tumor growth curves based on mean tumor volumeof each group were shown in Fig. 1C and indicated thattumors in the high (D370.3mm3) and low n‐3 groups(D411.0mm3) grew slower than those in basic (D443.3mm3)and n‐3 control (D556.9mm3) groups, although only the highn‐3 group reached statistical significance using analysis ofvariance for repeated measures (p< 0.05). The meandecrease in tumor volume in high n‐3 group increasedsubstantially over time (r¼ 0.961, p< 0.001); similar trendwas also noted in the low n‐3 group (r¼0.693, p¼ 0.084) ascompared to the n‐3 control group. Correlation analysisshowed that the serum total n‐3 PUFAs was negativelycorrelated with tumor volume on Day 21 (r¼�0.66,p¼ 0.021) in the control, low n‐3 and high n‐3 groupscombined together. This result suggests that combining n‐3PUFAs supplements with 5‐FU has an extra inhibitory effecton the growth of SW480 CRC xenografts.

3.3 n‐3 PUFAs increased the necrosis of SW480xenograft cells

After all mice were sacrificed, the stripped tumors were cut,fixed, and stained with hematoxylin and eosin (HE). In thestained sections (Fig. 2A), we observed that necrotic areashad arisen in all tumor sections, but that the high n‐3 grouppresented larger necrosis areas, reaching nearly two‐thirds ofthe tumor sections. In the high power images (Fig. 2B, 400�magnification), we noticed that the cells in the high n‐3 grouparranged more loosely with pyknotic nuclei, vacuoles andapoptotic bodies. This suggests that apoptosis may play animportant role in the adjuvant anti‐tumor effect of n‐3 PUFAsto 5‐FU.

Table 1. The compositions of n‐3 PUFAs in diet and serum by group

Basic group n‐3 control group Low n‐3 group High n‐3 group

Diets (n¼ 3)Total n‐3 PUFA 1.84� 0.01 0.69� 0.03 14.84� 0.25 24.27� 0.75C18:3 (ALA) 0.07� 0.00 0.18� 0.02 0.14� 0.03 0.11� 0.04C20:5 (EPA) 1.05� 0.00 0.29� 0.00 3.35� 0.02 5.39� 0.02C22:5 (DPA) 0.14� 0.01 0.07� 0.01 0.55� 0.01 0.87� 0.01C22:6 (DHA) 0.58� 0.01 0.15� 0.00 10.80� 0.25 17.91� 0.35

Serum (n¼ 8)Total n‐3 PUFA 6.99� 1.30$ 3.10� 0.86 12.26� 2.25�,$ 17.13� 2.95�,$,#

C18:3 (ALA) 0.64� 0.28 0.22� 0.34 0.76� 0.06 1.17� 0.20C20:5 (EPA) 1.04� 0.30 0.05� 0.03 1.83� 0.51 2.44� 1.39C22:5 (DPA) 0.00� 0.00 0.00� 0.00 0.00� 0.00 0.00� 0.00C22:6 (DHA) 5.31� 0.91 2.83� 0.45 9.67� 1.55 13.51� 2.72

Values represent means�SD (g/100 g total fatty acids).�Compared to basic group, p< 0.001.$Compared to n‐3 control group, p< 0.001.#Compared to low n‐3 group, p< 0.001.



Figure 1. n‐3 PUFAs affect the growth of SW480 CRC xenografts in nudemice. (A) Tumors developed in all nudemice after transplantation.After 21 days, all nude mice were sacrificed and tumors were stripped. (B) Mean body weights (�SEM) of nude mice, receiving 5‐FUintraperitoneal injection (35mg/kg) in basic group, n‐3 control group, low n‐3 group and high n‐3 group after transplantation. (C) Mean tumorvolumes of each group (�SEM). Data are presented as growth curves. �Compared with basic group, p<0.05. $Compared with n‐3 controlgroup, p<0.05.

Figure 2. HE staining of xenografts. (A) Low power images (100�) showing the necrotic areas of the tumor tissue. (B) High power images(400�) showing the pyknotic nuclei (hollow arrows with solid line), vacuoles (solid arrows with dotted line), and apoptotic bodies (hollowarrows with dotted line).



3.4 n‐3 PUFAs promoted the apoptosis of SW480xenograft cells

To detect the apoptotic cells in xenograft sections in differentgroups, we next performed the TUNEL staining assay, withthe results shown in Fig. 3. We observed that the percentageof TUNEL‐positive cells of SW480 xenografts in high n‐3group (9.4%) and low n‐3 group (4.1%) were statisticallyhigher compared with n‐3 control group (2.2%, bothp< 0.002), showing a dose‐dependent effect in facilitatingthe apoptosis. We also observed that the percentage ofTUNEL‐positive cells in the high n‐3 group was significantlyhigher than in the low n‐3 group (p¼ 0.023) and basic group(3.3%, p¼ 0.038) (Fig. 3B). The serum n‐3 PUFAs waspositively correlated with the percentage of TUNEL‐positivecells (r¼ 0.94, p< 0.001).

3.5 n‐3 PUFAs inhibited the expression of bcl‐2 andmt‐p53

We next explored the possible mechanism of the pro‐apoptosis capacity of n‐3 PUFAs. For this purpose, IHCstaining was conducted to determine the expression of bcl‐2,an anti‐apoptosis protein, and mt‐p53, a tumor‐promotingprotein. The result of bcl‐2 staining (Fig. 4A and C) in thecytoplasm showed that bcl‐2 expression in the high n‐3 group(16.8%) was significantly lower than that in the low n‐3 group(22.0%, p¼ 0.039). The bcl‐2 expression in the high n‐3(16.8%, p< 0.001) and low n‐3 group (22.0%, p¼ 0.002)were both considerably lower than the n‐3 control group(32.7%). The mt‐p53 staining (Fig. 4B and D) showed that

the mt‐p53 displayed a negative dose‐dependent correlationto n‐3 PUFAs. Compared with n‐3 control group (71.6%),mt‐p53 expression, which was significant different betweeneach group (all p< 0.003), was reduced by 49.7% in high n‐3group and 15.6% in low n‐3 group. Serum n‐3 PUFAs wasnegatively associated with bcl‐2 (r¼�0.87, p¼ 0.002) andmt‐p53 (r¼�0.926, p< 0.001). These results suggest thatn‐3 PUFAs have inhibitory effect on bcl‐2 and mt‐p53 inCRC xenografts.

4 Discussion

The purpose of our present study was to investigate the co‐anticancer capacity of n‐3 PUFAs when combined with 5‐FUin a SW480 CRC xenograft model and to determine whethern‐3 PUFAs enhance the chemotherapeutic effect of 5‐FUthrough inhibition on bcl‐2 and mt‐p53. We found that n‐3PUFAs enhanced the inhibitory effect of 5‐FU on the growthof xenografts. The HE and TUNEL staining showed that n‐3PUFAs increased the apoptosis of xenograft cells, while IHCanalyses showed that n‐3 PUFAs reduced the expression ofbcl‐2 and mt‐p53. These results revealed that n‐3 PUFAsreduce the anti‐apoptosis capacity of tumor cells by inhibitingthe expression of bcl‐2 and mt‐p53, consequently enhancingthe chemotherapeutic effects of 5‐FU.

5‐FU is a general chemotherapeutic drug for CRCtherapy, but its therapeutic effect can be hampered by drugresistance or host toxicity.Many ancillary drugs and biologicalfactors have been developed to enhance the therapeuticefficacy of 5‐FU, including dietary fatty acids [33–35]. In

Figure 3. TUNEL staining of xenografts. (A) High‐power image of tumor sections (100�) showing dark brown stained nuclei (solid arrows).(B) The rate of positively stained cells (%) in each diet group. The percentage of positive cells was calculated by counting among 400 cellsat five randomly selected microscope fields. �Compared to basic group, p<0.01. $Compared to n‐3 control group, p< 0.01. #Comparedto low n‐3 group, p< 0.05.



many studies, n‐3 PUFAs were considered as anti‐cancerfactors. Bathen et al. [36] reported that feeding fish oil dietsuppresses the CRC xenografts growth in nude mice.Michaelet al. [29] reported the co‐anticancer effect of n‐3 PUFAscombining with 5‐FU in MAC16 colon xenograft bearingmice. Besides, Calviello et al. [31] demonstrated thatcombination of DHA and 5‐FU achieves a chemotherapeuticapproach with low toxicity in LS‐174, Colo 320 HSR, Colo205, and HT‐29 cell lines either. In consistent with thesestudies [29, 36], our study also showed that the combinationof n‐3 PUFAs and 5‐FU has fortified inhibitory effect onCRCxenografts growth. However, we observed n‐6 rich corn oiltended to promote the xenografts growth in mice (Fig. 1C) asnoted in other previous studies [37, 38].

Tumorigenesis is dependent on the complimentary effectsof rapid cell proliferation and diminished apoptosis, and both

play important roles in chemotherapeutic intervention [39].Angela and Jürgen [30] demonstrated that n‐3 PUFAs blockthe progression of cell cycle and induce apoptosis in the Caco‐2 cell line. By using HE and TUNEL staining, we observed inthe present study that n‐3 PUFAs enlarged the necrotic areaand promoted the apoptosis of xenograft tumor cells by overfourfold, compared with control (TUNEL positive rate of9.4% in high n‐3 group and 2.2% in n‐3 control group). Theenlarged necrotic area may be due to the increased apoptosisof xenografts as described by Kato et al. [40].

Further, our present study focused on the expression ofbcl‐2 and mt‐p53. The results show that n‐3 PUFAs have aninhibitory effect on the expression of bcl‐2 (16.8% in the highn‐3 group versus 32.7% in n‐3 control group) and mt‐p53(36.0% in high n‐3 group versus 71.6% in n‐3 control). Honget al. [41] showed that fish oil enhances apoptosis in colon

Figure 4. IHC staining of xenografts. (A and B) Positive rates (%) of bcl‐2 and mt‐p53, respectively. The percentage of positive cells wascounted among 400 cells at five randomly selected microscope fields. �Compared to basic group, p<0.01. $Compared to n‐3 control group,p<0.01. #Compared to low n‐3 group, p< 0.05. (C and D) Representative images of IHC staining of bcl‐2 (C, solid arrows) and mt‐p53(D, solid arrows with dotted line) at high magnification (400�).



tumors by the down‐regulation of bcl‐2. Bcl‐2 block the anti‐cancer progresses induced by 5‐FU by suppressing apoptosis.This anti‐apoptosis capacity of bcl‐2 may due to the pro‐inflammatory eicosanoid synthesis [41, 42]. However, n‐3PUFAs down‐regulate the synthesis of pro‐inflammatoryeicosanoids [28] and indirectly reduce the expression of bcl‐2. In addition, deletion, and/or mutation of wild‐type p53(wt‐p53) gene occurs in almost 70% of human CRC [43] andmt‐p53 causes a deletion of the apoptosis mechanism [11].Other researchers have shown that bcl‐2 blocks the apoptosispathway of wt‐p53 [13, 14]. Indeed, by inhibiting the bcl‐2andmt‐p53 expression, n‐3 PUFAs distinctly reduce the anti‐apoptosis capacity of CRC xenograft cells and consequentlyenhance the 5‐FU chemotherapeutic effect.

In conclusion, n‐3 PUFAs enhance the inhibitory effectsof 5‐FU on the growth of SW480 CRC xenografts. Thissynergistic effect is due to increasing tumor apoptosis byinhibition of bcl‐2 and mt‐p53.

The authors have declared no conflict of interest.

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