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Experimental Biology and Medicine 229:1017-1025 (2004)
© 2004 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Dietary Corn Oil Promotes Colon Cancer by Inhibiting Mitochondria-Dependent Apoptosis in Azoxymethane-Treated Rats

Bin Wu*, Ryuichi Iwakiri*, Akifumi Ootani*, Seiji Tsunada*, Takehiro Fujise*, Yasuhisa Sakata*, Hiroyuki Sakata*, Shuji Toda{dagger} and Kazuma Fujimoto*,1

* Departments of Internal Medicine, and {dagger} Pathology, Saga Medical School, 5-1-1 Nabeshima, Saga 849–8501, Japan

1 To whom requests for reprints should be addressed at Department of Internal Medicine, Saga Medical School, 5-1-1 Nabeshima, Saga 849–8501, Japan. E-mail: fujimoto{at}post.saga-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
How dietary corn oil is involved in colon carcinogenesis and cancer development is poorly understood. The aim of this study was to investigate whether long-term dietary corn oil promotes colon cancer by inhibiting the tumor suppressor gene p53-mediated mitochondria-dependent apoptosis in azoxymethane (AOM)-treated rats. Male Sprague-Dawley rats were injected with AOM or with saline and fed on a basal diet or basal diet supplemented with 10% corn oil for 48 weeks. Colonic aberrant crypt foci (ACF) and tumors, including adenomas and carcinomas, were examined. Colonic apoptosis and cell proliferation were evaluated. Wild type (wt) p53 was analyzed using reverse transcription–polymerase chain reaction (RT-PCR) and Western blotting. In addition, Bcl-2, Bcl-xL, Bax, and Bak localized in the mitochondria were detected. Long-term dietary corn oil increased ACF in AOM-treated rats at 12 weeks and promoted colon cancer invasion at 48 weeks. Cancer invasion was not observed in the AOM-treated rats without dietary corn oil, although colon adenomas and cancers were detected. Apoptosis was decreased and cell proliferation was increased in the AOM-treated rats with dietary corn oil, compared with the AOM-treated rats with dietary basal diet. In these rats, mitochondrial wt p53 was significantly inhibited through decreased mitochondrial localization of wt p53 and increased cytosolic p53, resulting in the upregulation of Bcl-2 and Bcl-xL and the downregulation of Bak in the mitochondria. Results suggest that long-term dietary corn oil promotes AOM-induced colon cancer development partly by inhibiting the tumor suppressor gene p53-mediated mitochondria-dependent apoptosis.

Key Words: polyunsaturated fatty acid • aberrant crypt foci • adenoma • Bcl-2 family protein • mitochondria


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Colon cancer is one of the leading causes of cancer death in both men and women in Western countries (1). Epidemiological studies have suggested a positive association between dietary fat and colon cancer (2, 3). Furthermore, animal model studies have shown that the colon tumor–promoting effect of a high-fat diet depends not only on the amount consumed but also on the fatty acid composition. A high-fat diet rich in corn oil (n-6 polyun-saturated fatty acid [n-6 PUFA]) promotes colon carcinogenesis, particularly in the postinitiation or promotional phases or both (3–5), whereas a diet rich in fish oil (n-3 PUFA) or olive oil (n-9 PUFA) decreases colon tumor incidence in both the initiation and postinitiation phases (4, 6).

Colon carcinogenesis is a multistep processor that requires considerable time for these chance events to accumulate. Several observations have confirmed the putative association between aberrant crypt foci (ACF) and colon cancer in animals (7–9) and have suggested that ACF might be a high risk factor for colon cancer in humans (10, 11). Quantification of the formation and growth of colonic ACF has been used as a short-term bioassay to evaluate the roles of nutritive components at a very early stage of colon carcinogenesis in animals and humans (7–11). It has been proposed that potentially tumorigenic clones might be eliminated through apoptotic targeting of damaged intestinal epithelial stem cells and that inhibition of this apoptosis causes aberrant cell survival and contributes to oncogenesis (12–14).

Wild type (wt) p53 is thought to function as a gene-transcription factor, and the active wt p53 can induce the expression of a large number of genes, many of which evoke either cell cycle arrest or apoptosis (15–17). Wild type p53 induces cell death through a multitude of molecular pathways involving transcription-dependent or independent functions or both (18, 19). It can mediate apoptosis via transcriptional activation of proapoptotic genes such as BH3 proteins (e.g., Noxa and Puma) and Bcl-2 family members (e.g., Bax and Bak) (20–22). In addition, it is a potent inhibitor of cell growth and tumor growth, and its inactivation or mutation or both is considered a prerequisite for tumor formation (23). Furthermore, evidence for transcription-independent wt p53–mediated apoptosis has been accumulated, and suggests that wt p53 has a direct apoptotic genetic role in the mitochondria (24).

Previous studies revealed that dietary corn oil rich in n-6 PUFA promoted colon cancer (3–5). However, how dietary corn oil is involved in colon carcinogenesis and cancer development is poorly understood. Mitochondria play a key role in cellular survival and death, and wt p53–mediated mitochondria-dependent apoptosis (24, 25), but it is unclear whether dietary corn oil inhibits wt p53–mediated mitochondria-dependent apoptosis during colon carcinogenesis and cancer development. Azoxymethane (AOM), a standard chemical carcinogen, was used in this study. AOM treatment increases ACF formation in rats, which leads to the induction of colon adenomas and cancers (26–28). The aim of the present study was to investigate whether long-term dietary corn oil promotes colon cancer development by inhibiting the tumor suppressor gene p53-mediated mitochondria-dependent apoptosis in AOM-treated rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Experimental Procedures.
Male Sprague-Dawley rats were used in this study. At 5 weeks of age, the rats were divided into four groups: (i) basal diet plus vehicle rats were fed on a basal diet (Clea, Tokyo, Japan) and given intraperitoneal injections of 1 ml physiological saline once a week for 2 weeks; (ii) corn oil plus vehicle rats were fed on a basal diet supplemented with 10% corn oil (Clea) consisting of 8.1% (w/w) n-6 PUFA in the total diet and given intraperitoneal injections of 1 ml physiological saline once a week for 2 weeks; (iii) basal diet plus AOM rats were fed on a basal diet and given intraperitoneal injections of AOM (Sigma, St. Louis, MO) dissolved in 1 ml physiological saline once a week for 2 weeks at a dose of 15 mg/kg body wt; and (iv) corn oil plus AOM rats were fed on a basal diet supplemented with 10% corn oil and given injections of AOM as described in Group 3. The basal diet included 8.9% moisture, 25.4% crude protein, 4.4% crude fat, 4.1% crude fiber, and 6.9% crude ash. The corn oil included 56.8% linoleic acid, 29.0% oleic acid, 10.5% palmitic acid, and 1.9% stearic acid. At 12 weeks, after the second injection of AOM, colonic ACF formation was analyzed, and at 12, 24, 36, and 48 weeks colon carcinogenesis and cancer development were investigated.

Collection of Intestinal Tissue Samples.
The entire colon was carefully removed and placed on ice-cold glass except for the cecum. Each segment was rinsed thoroughly with physiological saline and opened longitudinally on its antimesenteric border to expose the mucosa. The mucosa and tumors were carefully harvested, respectively.

DNA Fragmentation Assay.
At 48 weeks, the colonic mucosa and tumors of all four groups of rats were examined. The amount of fragmented DNA was determined as previously described (29). Tissues were homogenized in 10 volumes of a lysis buffer (pH 8.0) consisting of 5 mM Tris-HCl, 20 mM EDTA (Sigma), and 0.5% (w/v) t-octylphenoxypolyethoxyethanol (Triton X-100, Sigma). One-milliliter aliquots of each sample were centrifuged for 20 mins at 27,000 g to separate the intact chromatin (pellet) from the fragmented DNA (supernatant). The supernatant was decanted and saved, and the pellet was resuspended in 1 ml of Tris buffer (pH 8.0) consisting of 10 mM Tris-HCl and 1 mM EDTA. The pellet and supernatant fractions were assayed for DNA content using a diphenylamine reaction. The results were expressed as the percentage of fragmented DNA divided by the total DNA. Six rats were studied in each group.

DNA Ladders Assay.
At 48 weeks, the colonic mucosa (basal diet plus vehicle and corn oil plus vehicle rats) and the colon tumors were examined. The total DNAs were extracted sequentially using a phenol-chloroform-isoamyl alcohol mixture (25:24:1, v/v/v) to remove proteins and then purified as previously described (29). Resolving agarose gel electrophoresis was performed using a 1.5% gel containing 1.0 µg/ml ethidium bromide. DNA standards were included to identify the sizes of the DNA fragments. The DNA ladders were observed under ultraviolet fluorescent light.

ACF Analysis.
For ACF assessment, the colon was removed at 12 weeks, after the second injection of AOM, opened longitudinally, rinsed in ice-cold physiological saline, placed flat mucosal side up between wet filter papers, and fixed in 10% buffered formalin for 24 hrs. The colon was then stained with 0.2% methylene blue dissolved in the same formalin solution for 5 mins and rinsed in physiological saline. After staining, the entire colonic mucosa was observed using a stereoscopic microscope. Crypt multiplicity was determined as the number of crypts in each colon and was categorized as containing <4 or ≥4 aberrant crypts. Six rats were examined in each group.

Histological Analyses.
At 12 weeks, after the assessment as described above, the ACF were embedded in paraffin. At 24, 36, and 48 weeks, after the second injection of AOM, colon tumors over 5 mm were collected and fixed in 10% buffed formalin and embedded in paraffin. Four-micrometer sections were cut and stained with hematoxylin and eosin (H&E) and periodic acid-schiff (PAS) and subjected to histological analysis. Six rats were examined in each group at each time point.

Total RNA Extraction and Reverse Transcription–Polymerase Chain Reaction (RT-PCR).
At 48 weeks, after the second injection of AOM, the colonic mucosa (basal diet plus vehicle and corn oil plus vehicle rats) and the colon tumors were examined. Total RNA was extracted using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. RT-PCR specific for the rat wt p53 mRNA was amplified using a primer specific for the leader sequence of exon 6–7 of the wt p53 allele (5'-GGCTCCTCCCCAACATCTTATC-3') and the downstream primer (5'-TCTCCCAGGACAGGCACAAAC-3'). For the internal control, the expression of the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene in each sample was also quantified using the primer (5'-TCCACCACCCTGTTGCTGTA-3') and the downstream primer (5'-ACCACAGTCCATGCCATCAC-3'). RT-PCR was performed on 1 µg total RNA using a RT-PCR kit (Toyobo Co., Osaka, Japan). The PCR products were electrophoresed in 2% agarose gels and visualized under ultraviolet fluorescent light after staining with ethidium bromide. Densitometric assessment of the auto-radiogram bands was conducted using Image Gauge Version 3.12 (Fujifilm, Tokyo, Japan). Band intensity was quantified by measuring the absolute integrated optical intensity, which estimates the volume of the band in the lane profile. Six rats were studied in each group. The results were expressed as a ratio to G3PDH densitometry units.

Western Blotting Analysis.
At 48 weeks, after the second injection of AOM, the colonic mucosa (basal diet plus vehicle and corn oil plus vehicle rats) and the colonic mucosa and tumors were examined. Total proteins, cytosolic fractions, and mitochondrial fractions were purified as previously described (29–32). Equal quantities of protein were electrophoresed in a sodium dodecyl sulfate polyacrylamide gel and electroblotted onto a nitrocellulose membrane (Trans-Blot Bio-Rad; Hercules, CA). After blocking with phosphate-buffered saline containing 0.1% polyoxyethylene sorbitan monolaurate (Tween-20, Sigma) and 5% skimmed milk at 4°C overnight, the membrane was respectively incubated with rabbit polyclonal anti–proliferating cell nuclear antigen (PCNA) antibody (1:1000), mouse monoclonal anti–wt p53 antibody (1:1000), mouse monoclonal anti–Bcl-2 antibody (1:500), mouse monoclonal anti–Bcl-xL antibody (1:500), mouse monoclonal anti-Bax antibody (1:500), rabbit polyclonal anti-Bak antibody (1:1000), rabbit polyclonal anti–cytochrome-c antibody (1:1000), and rabbit polyclonal anti–caspase-3 antibody (1:1000, all from Santa Cruz Biotech, Santa Cruz, CA) for 1 hr. Antigen-antibody complexes were detected with horse-radish peroxidase–conjugated anti-mouse IgG or anti-rabbit IgG (1:1000, Santa Cruz Biotech). Detection of the chemiluminescence was carried out using Western blotting detection reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK). Densitometric assessment of the auto-radiogram bands was conducted using Image Gauge Version 3.12 (Fujifilm). Band intensity was quantified by measuring the absolute integrated optical intensity, which estimates the volume of the band in the lane profile. Six rats were studied in each group. The results were expressed as a ratio to ß-actin densitometry units.

Statistical Analysis.
Results are expressed as mean ± SD. Data were evaluated by analysis of variance (ANOVA) in which multiple comparisons were performed by the method of least significant difference. Colon cancer incidence is expressed as the percentage of animals with cancer, and the results were analyzed statistically using the chi-square test. Differences were considered significant if the probability of the difference occurring by chance was less than 5 in 100 (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Long-Term Dietary Corn Oil Promoted Colon Carcinogenesis and Cancer Invasion in AOM-Treated Rats.
At 12 weeks, after the second injection of AOM, the entire colonic mucosa was stained with 0.2% methylene blue to evaluate ACF formation using stereoscopic microscopy and they then were histologically examined. The ACF had dilated irregular luminal openings, thicker epithelial linings, and protrusions towards the lumen (data not shown). Few ACF were found in both the dietary basal diet and the dietary corn oil rats without AOM treatment. A large number of ACF (especially ≥4 ACF) were observed in the AOM-treated rats. Compared with AOM-treated rats with dietary basal diet, the number of total ACF and ≥4 ACF were about 2-fold higher in the AOM-treated rats with dietary corn oil as shown in Table 1Go. ANOVA revealed that corn oil (total ACF, F1,20 = 8.7; ≥4 ACF, F1,20 = 14.6), AOM (total ACF, F1,20 = 102.5; ≥4 ACF, F1,20 = 134.9), and interaction of the two factors (total ACF, F1,20 = 8.5; ≥4 ACF, F1,20 = 14.6) significantly induced ACF formation in the colonic mucosa (P < 0.01 in each).


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  		Table 1. Long-Term Dietary Corn Oil Ingestion Promoted Colonic Aberrant Crypt Foci (ACF) Formation in Azoxymethane (AOM)-Treated Rats at 12 Weeksa
 
Colon tumors were not observed in rats without AOM treatment throughout the study period. No tumors were observed in AOM-treated rats at 12 weeks. At 24 weeks, colon tumors were found in the AOM-treated rats, and all tumors were tubular adenomas (data not shown). At 36 and 48 weeks, the tumors showed well-differentiated adenocarcinomas with polypoid growth. The number of colon tumors was significantly increased in the corn oil plus AOM rats compared with the basal diet plus AOM rats as indicated in Figure 1Go (F1,46 = 12.2, P < 0.01). At 48 weeks, cancer invasion was only observed in the corn oil plus AOM rats (Fig. 2Go). As shown in Table 2Go, all AOM-treated rats fed on dietary corn oil (100%) had invasive colon cancers, but no invasive cancer was found in the AOM-treated rats with dietary basal diet. Histologically, poorly differentiated or undifferentiated adenocarcinomas were not found in the colons of these rats. These results suggest that long-term dietary corn oil might promote not only colon carcinogenesis but also cancer invasion.



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  		Figure 1. Long-term dietary corn oil ingestion promoted azoxymethane (AOM)-induced colon carcinogenesis and cancer development. The number of colon tumors is significantly increased in the AOM-treated rats with dietary corn oil compared with the AOM-treated rats with dietary basal diet. Values are the means ± SD, six rats were examined in each group at each time point. #P < 0.01 compared with the AOM-treated rats with basal diet.

 


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  		Figure 2. Histological findings of invasive colon cancers. A well-differentiated adenocarcinoma with cancer invasion was observed in the azoxymethane-treated rats with dietary corn oil at 48 weeks.

 

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  		Table 2. Long-Term Dietary Corn Oil Ingestion Promoted Colon Cancer Invasion in azoxymethane (AOM)-Treated Rats at 48 Weeks
 
Long-Term Dietary Corn Oil Inhibited Cell Apoptosis and Promoted Cell Proliferation in AOM-Induced Colonic Mucosa and Colon Cancer.
Apoptosis was evaluated using a DNA fragmentation assay (Fig. 3AGo). After AOM treatment, the percentage of fragmented DNA was significantly inhibited in the colonic mucosa and tumors (P < 0.01 in each). This inhibitory effect of AOM was significantly enhanced by dietary corn oil in the mucosa and tumors (P < 0.05 in each). The DNA ladders assay also showed that colon tumor apoptosis was significantly inhibited in the AOM-treated rats at 48 weeks, and the inhibitory effect was enhanced by dietary corn oil (Fig. 3BGo).



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  		Figure 3. Long-term dietary corn oil ingestion enhanced the inhibitory effect of azoxymethane (AOM) on apoptosis in colonic mucosa and cancer. At 48 weeks, the colonic mucosa (basal diet plus vehicle and corn oil plus vehicle rats) and the colonic mucosa and tumors (basal diet plus AOM rats and corn oil plus AOM rats) were examined. (A) The percentage of fragmented DNA. Values are means ± SD, six rats were studied in each group. *P < 0.01 compared with the vehicle-treated rats. #P < 0.05 compared with the AOM-treated rats with basal diet. (B) DNA ladders. Lanes 1–4, basal diet plus vehicle rat, corn oil plus vehicle rat, basal diet plus AOM rat, and corn oil plus AOM rat, respectively. Lanes 5 and 6 contain marker DNA.

 
Cell proliferation was evaluated by assessing PCNA as shown in Figure 4Go. After AOM treatment, the colonic mucosal and tumors PCNA significantly increased at 48 weeks (P < 0.01 in each). This effect of AOM was significantly enhanced by dietary corn oil in the mucosa and tumors (P < 0.01 in each).



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  		Figure 4. Long-term dietary corn oil ingestion promoted cell proliferation in azoxymethane (AOM)-induced colonic mucosa and cancer. At 48 weeks, the colonic mucosa (basal diet plus vehicle and corn oil plus vehicle rats) and the colonic mucosa and tumors (basal diet plus AOM and corn oil plus AOM rats) were examined. PCNA was evaluated in the total protein. Lanes 1–6: mucosa in basal diet plus vehicle rats, mucosa in corn oil plus vehicle rats, mucosa in basal diet plus AOM rats, mucosa in corn oil plus AOM rats, tumors in basal diet plus AOM rats, and tumors in corn oil plus AOM rats, respectively. Values are means ± SD, six rats were studied in each group. *P < 0.01 compared with the vehicle-treated rats. #P < 0.01 compared with the AOM-treated rats with basal diet.

 
Long-Term Dietary Corn Oil Enhanced the Inhibitory Effects of AOM on Mitochondrial wt p53.
The mRNA of wt p53 was confirmed by RT-PCR, and wt p53 protein expression was determined by Western blotting analysis. As shown in Figure 5Go, wt p53 mRNA and total protein were not inhibited by AOM treatment at 48 weeks, but cytosolic wt p53 significantly increased in AOM-treated rats, and this was enhanced by a corn oil diet. At 48 weeks, compared with the rats with vehicle treatment, mitochondrial wt p53 was decreased in the rats with AOM treatment, although total wt p53 was not inhibited. The reduction in mitochondrial wt p53 was significantly enhanced by dietary corn oil.



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  		Figure 5. Long-term dietary corn oil ingestion enhanced the inhibitory effect of azoxymethane (AOM) on mitochondrial wt p53 in colon cancer. At 48 weeks, the colonic mucosa (basal diet plus vehicle and corn oil plus vehicle rats) and tumors (basal diet plus AOM and corn oil plus AOM rats) were assessed. Total, cytosolic, and mitochondrial wt p53 were measured using Western blotting. Lanes 1–4, basal diet plus vehicle rats, corn oil plus vehicle rats, basal diet plus AOM rats, and corn oil plus AOM rats, respectively. (A) Wt p53 determined using RT-PCR and Western blotting. (B) The band quantified. Values are means ± SD, six rats were studied in each group. *P < 0.01 compared with the vehicle-treated rats. #P < 0.01 compared with the AOM-treated rats with basal diet.

 
Long-Term Dietary Corn Oil Enhanced the Inhibitory Effects of AOM on Mitochondrial Apoptotic Signaling.
We have shown that mitochondrial localization of wt p53 is inhibited by AOM treatment, and that dietary corn oil enhanced this inhibitory effect at 48 weeks. Furthermore, we investigated mitochondrial apoptotic signaling. The results are shown in Figure 6Go. Inhibition of mitochondrial wt p53 localization caused by AOM treatment resulted in upregulation of the mitochondrial antiapoptotic members, Bcl-2 and Bcl-xL. Long-term dietary corn oil promoted Bcl-xL accumulation in the mitochondria by enhancing the inhibitory effect of AOM on mitochondrial wt p53 localization. AOM treatment with a corn oil diet induced the downregulation of mitochondrial proapoptotic Bak members at 48 weeks but without any influence on Bax. Further, AOM treatment significantly inhibited procaspase-3 formation. The release of cytochrome-c from the mitochondria into the cytosol was decreased, resulting in the reduction of cleaved caspase-3 at 48 weeks in the AOM-induced tumors, and these effects were also enhanced by a corn oil diet. These results suggest that long-term dietary corn oil enhances the inhibitory effect of AOM on wt p53–mediated mitochondrial apoptotic signaling.



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  		Figure 6. Long-term dietary corn oil ingestion enhanced the inhibitory effects of azoxymethane (AOM) on mitochondrial apoptotic signaling in colon cancer. At 48 weeks, the colonic mucosa (basal diet plus vehicle and corn oil plus vehicle rats) and tumors (basal diet plus AOM and corn oil plus AOM rats) were assessed. Lanes 1–4, basal diet plus vehicle rats, corn oil plus vehicle rats, basal diet plus AOM rats, and corn oil plus AOM rats, respectively. (A) The image of Western blotting. (B and C) The band quantified. Values are means ± SD, six rats were studied in each group. *P < 0.01 compared with the vehicle-treated rats. #P < 0.01 compared with the AOM-treated rats with basal diet.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several observations have confirmed the putative association between ACF and cancer in the colon and AOM-induced ACF formation in animals (7–9). In this study, the results show that colonic ACF was about 2-fold higher in corn oil plus AOM rats compared with basal diet plus AOM rats and suggest that long-term dietary corn oil might be involved in the induction of colon cancer with ACF formation. Furthermore, our data demonstrate that long-term dietary corn oil promotes colon cancer invasion after AOM treatment. Previous studies have reported that corn oil rich in n-6 PUFA involved colon carcinogenesis occurring in the postinitiation or promotional phase or both (4, 5). The present results suggest that long-term dietary corn oil is involved in colon cancer in both ACF formation and cancer invasion. Tumor growth is determined not only by increased cell proliferation but also by decreased tumor-cell apoptosis (12, 13). Our results showed that AOM treatment significantly inhibited cell apoptosis and promoted cell proliferation leading to malignant cell growth in both the colonic mucosa and tumors, and this effect was significantly enhanced by long-term dietary corn oil.

Wild type p53 has multiple functions as a tumor suppressor, including cell cycle arrest in response to DNA damage, induction of apoptosis, and DNA repair (33–36). In its normal state, the tumor suppressor action of wt p53 is present in the nucleus and mediated by specific DNA binding and protein-protein interactions within the nucleus, although sometimes a secondary event occurs in which wt p53 is lost (37). Abnormal cytosolic wt p53 localization has been observed in a number of cancer cell lines, and this cytosolic wt p53 is stable and inactive (38). Our data indicate that an increase in cytosolic wt p53 localization occurs after AOM treatment leading to reduced wt p53 activity in colon cancer, although wt p53 mRNA and total proteins were not inhibited by AOM. Dietary corn oil enhanced this AOM-induced cytosolic wt p53 localization. These results suggest that long-term dietary corn oil might enhance colon cancer development after AOM treatment via wt p53 inactivation.

Cytosolic wt p53 is inactivated, but when it is localized in the mitochondria it can contribute toward apoptosis. Previous evidence has shown that wt p53 has an apoptotic role in mitochondria (39–41), but how mitochondrial wt p53 induces apoptosis has not been clearly demonstrated. Our data revealed that mitochondrial wt p53 induces apoptosis by regulating mitochondrial Bcl-2 family proteins. The Bcl-2 family proteins are central regulators of the mitochondria-dependent apoptotic pathway and can be subdivided into two classes: antiapoptotic members, such as Bcl-2 and Bcl-xL, which protect cells from apoptosis, and proapoptotic members, such as Bax and Bak, which trigger or sensitize cells for apoptosis. The ratio of antiapoptotic and proapoptotic Bcl-2 members determines the susceptibility of cells to death signals. Following multiple death stimuli, Bax and Bak induce mitochondrial dysfunction and cell death, while Bcl-2 and Bcl-xL prevent Bax- and Bak-mediated mitochondrial apoptosis (42). Bcl-2 family members are integral proteins located mainly on the outer membrane of mitochondria, and their overexpression prevents the efflux of cytochrome-c from the mitochondria and the initiation of apoptosis (30, 43). We previously demonstrated that the release of cytochrome-c from the mitochondria to the cytosol is important for apoptosis in intestinal mucosa (29, 44, 45). Cytosolic cytochrome-c forms a complex with Apaf-1 and caspase-9, resulting in the activation of caspase-9 and caspase-3 (46). Caspase-3 possesses a small prodomain and participates in the execution phase of apoptosis. In this study, the results show that long-term dietary corn oil enhanced the inhibitory effects of AOM on mitochondrial cytochrome-c release, procaspase-3 formation, and caspase-3 cleavage. This result suggests that inhibition of mitochondrial cytochrome-c release might be involved in inhibition of caspase-3 in both procaspase-3 synthesis and caspase-3 activation. Our results revealed that AOM treatment significantly inhibited the mitochondrial localization of wt p53, preventing both the mitochondrial cytochrome-c release and the caspase-3 activation partly through the regulation of Bcl-2 family members. Long-term dietary corn oil significantly enhanced this inhibitory effect, resulting in inhibited mitochondrial apoptotic signaling.

In conclusion, colon cancer invasion was developed in AOM-treated rats after long-term dietary corn oil ingestion, suggesting that dietary corn oil can enhance AOM-induced colon carcinogenesis and cancer development. The reason why cancer development was enhanced by long-term dietary corn oil in this model might be in part explained by the inhibition of the tumor suppressor gene p53-mediated mitochondria-dependent apoptosis. These results warrant further exploration including the effect of diet oil with other carcinogens and the effect of other diet oils on colon cancer development.


   Acknowledgments
 
We thank Hiroyoki Ideguchi for excellent technical assistance.


   Footnotes
 
This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, for scientific research 15590656 (K.F.), 15590659 (S.T.), and 14570478 (R.I.).

Received for publication April 29, 2004. Accepted for publication July 24, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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