*Contributed equally
In the present study, the dietary effects of mead acid (MA; 5,8,11-eicosatrienoic acid) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer in female Sprague-Dawley rats were examined. The 2.4 and 4.8% MA diets were commenced when the rats were 6 weeks of age. DMBA was administered by a single oral ingestion when the rats were 7 weeks of age, and the rats were maintained on the respective diets until 19 weeks of age. Tumor weight, histopathology, cell kinetics, and the fatty acid composition in breast tissue and serum were examined. In the control (CTR) group, the DMBA-exposed rats were fed a basal diet (0% MA). The results revealed that there were no significant differences in tumor incidence, cell kinetics and in the N-6/N-3 ratio in breast tissue between the groups. Only the N-6/N-3 ratio of fatty acid composition in serum was significantly decreased in the 2.4% MA diet group. In previous studies, the 2.4% MA diet was shown to suppress
Breast cancer is the most frequent type of tumor occurring in women globally and its incidence has recently increased (
Mead acid (MA) is a 20:3 n-9 fatty acid (5,8,11-eicosatorienoic acid) that was characterized by Mead and Slaton (
The anticancer effects of MA were previously investigated against luminal type A breast cancer (
In addition to the present study, to the best of our knowledge, only one previous study has been conducted to date reporting that MA exerts an anticancer effect against breast cancer (
MA has been shown to exert various effects depending on the cancer type. However, the ability of MA to affect the prevention of carcinogenesis is not yet well understood. To further investigate the anticancer effects and mechanisms of MA, which could lead to new practical applications, such as a novel therapeutic agent, dietary habits and functional foods, additional studies with different models are required. In the present study, the anticancer effects of MA were investigated in a rat model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer, as previously described (
The experimental diets contained the same amounts of nutrients, but included different fatty acid compositions (
DMBA was obtained in powder form from Eastman Chemical. Prior to its use, DMBA was dissolved in sesame oil at 120˚C (DMBA 1,000 mg/50 ml sesame oil). A single dose of 80 mg/kg body weight was administered orally (
The study protocol and animal procedures were approved by the Animal Care and Use Committee of Kansai Medical University, Hirakata, Osaka, Japan (permit no. 13-060). Throughout the experiments, the animals were housed and treated in accordance with the Guidelines for Animal Experimentation of Kansai Medical University. In the present study, the following criteria for humane endpoints were also used (NIH guidelines for endpoints in animal study proposals): i) A tumor burden >10% of the animal body weight; ii) the tumor should not exceed 40 mm in any one dimension; iii) tumors that become ulcerated, necrotic or infected; iv) tumors that interfere with the eating ability or impair the ambulation of the animals.
In brief, 86 female Sprague-Dawley rats [Crl:CD(SD), 6 weeks old] were purchased from Charles River Laboratories Japan. They were housed in groups of 4 or 5 in plastic cages with paper bedding (Paper Clean; Japan SLC, Inc.) in a specific pathogen-free environment maintained at 22±2˚C and at 60±10% relative humidity with a 12-h light/dark cycle (lights on at 8:00 a.m. and lights off at 8:00 p.m.). In the experiment with 2.4% MA, the rats were randomly divided into 4 groups as follows: The CTR + sesame oil (n=5), CTR + DMBA (n=13), 2.4% MA + sesame oil (n=5) and 2.4% MA + DMBA (n=13) groups. In the experiment with 4.8% MA, the rats were divided into 4 groups as follows: CTR + sesame oil (n=10), 4.8% MA + sesame oil (n=10), CTR + DMBA (n=15) and 4.8% MA + DMBA (n=15) groups.
Fresh sterilized stocks of the pellet diet were provided to the animals twice a week starting at 6 weeks of age. The previous pellets were discarded to minimize the ingestion of oxidized fatty acids. The animals in the experimental groups received DMBA, whereas the animals in the CTR groups received sesame oil at 7 weeks of age and all animals remained on the same diets for the remaining duration of the experiments (until 19 weeks of age). The experimental diets and water were available freely. During the dosing period, the dose of the diet ingested, body weight and tumor volume were measured once a week. The tumor volume was calculated using the standard formula: Width2 x length x 0.5. The tumor volume measurement was used for monitoring of tumor incidence and growth. Prior to sacrifice, all rats were anesthetized with isoflurane (Wako Pure Chemical Industries, Ltd.). Before necropsy, the isoflurane was made to soak into paper and was put in closed chamber. Subsequently, rats were anesthetized in a pervasive chamber of isoflurane which was vaporized. A total of 4% of isoflurane was used for the induction of anesthesia and blood was sampled by inferior vena cava puncture. Subsequently, the animals were sacrificed by exsanguination and aortic transection. At necropsy, all organs were examined macroscopically and the breast tissue and tumors were examined histologically. The tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin and finally stained with hematoxylin and eosin (Wako Pure Chemical Industries, Ltd.). Cell kinetics were also assessed. The serum samples and the sections of the non-tumor breast tissues were used for fatty acid analysis. During the examination of the animals receiving the 4.8% MA diet, fatty acid analysis was not carried out.
The cell kinetics (cell proliferation activity and apoptosis) in the 6 largest DMBA-induced tumors were evaluated. The cell proliferative activity was evaluated using anti-Ki-67 antibody (cat. no. 418071, prediluted, clone SP6, Nichirei Biosciences). The incubation condition was 1 h at room temperature. The induction of apoptosis was evaluated by the anti-phospho-histone H2A.X (γ-H2A.X) antibody (cat. no. 2577S, x100, clone Ser139, 1:100; Cell Signaling Technology, Inc.), an immunomarker of the DNA damage response. The incubation condition was 1 h at room temperature. Immunohistochemical analysis was performed with the Histofine MAX-PO for rats kit (Nichirei Biosciences) according to the manufacturer's protocol. Each slide was scanned with a high-resolution digital scanner (NanoZoomer 2.0 Digital Pathology; Hamamatsu Photonics) to prepare the digital images (NDPI image). The NDPI image files were opened in color mode with the NDP.view software (Hamamatsu Photonics). The images were converted to the JPEG files (magnification, x400) in 5 randomly selected areas within each tumor and were analyzed by immunohistochemical staining, as previously described (
The fatty acid composition of the total phospholipid fraction of serum was determined and mammary gland samples were extracted using the method described in the study by Bligh and Dyer (
The values are expressed as the means ± standard error of the mean. The parameters body weight, tumor volume, tumor weight, fatty acid composition and the percentage of Ki-67-positive and γ-H2A.X-positive cells among the groups were analyzed using the Student's t-test. The incidence of breast cancer was analyzed using a χ2 test.
During the dosing period, the daily dose of food ingestion was compatible among the different groups. The parameter body weight did not reveal significant differences when the 2.4% MA diet was used, while in the 4.8% MA diet experimental protocol, the body weight in the group administered the 4.8% MA diet and exposed to DMBA was significantly decreased compared with that of the group administered the 4.8% MA diet and given sesame oil (
All mammary tumors were examined and confirmed histologically as mammary cancers. At the end of the experimental period, although the tumor incidence in both the 2.4 and 4.8% MA diet groups was lower than that noted in the CTR diet groups, the differences were not significant (
In the groups in which DMBA was not administered, the presence of breast tumors was not observed, in the presence of either the CTR or MA diet in both experimental settings. No conspicuous morphological differences were noted between the CTR diet and the MA diet groups. No lymph node metastasis was noted in any animal.
The percentages of Ki-67-positive cells and γ-H2A.X-positive cells from the CTR diet and MA diet groups were compared with regard to the ratio of proliferative cells and the apoptotic cell number. The proliferative cell number and apoptotic cell ratio are presented in
The different diet groups exhibited different fatty acid compositions in serum and mammary tissues, reflecting the content of the respective diets. Exposure to DMBA did not affect the fatty acid composition. The n-3, n-6 and n-9 (MA) fatty acid composition levels in the serum of the animals receiving the 2.4% MA diet were significantly increased compared with those noted in the CTR diet group, whereas the concentrations of OA, LA, AA and DHA were significantly decreased compared with those noted in the CTR + sesame oil group (
It is well known that there are a number of risk factors for breast cancer (e.g., an advanced age or viral infection) (
The present study investigated the concentration of fatty acids and its influence on breast cancer. Previous studies have examined the influence of fatty acid composition on breast carcinogenesis. The majority of previous studies have focused on n-3 PUFA and/or n-6 PUFA. The effects of n-9 PUFA were previously examined against breast cancer and the data indicated that MA inhibited the growth of luminal A type breast cancer by suppressing the expression of VEGFR. In addition, MA inhibited the growth of transplanted luminal A type breast cancer cells in nude mice and their metastasis to the lymph nodes (
The effects of MA on different cancer types vary greatly and only 4 studies have been previously published examining the association between MA and cancer cell progression (
In the present study, the MA diet did not suppress the incidence of breast cancer, although the Ki-67 labeling index was lower in the MA groups compared with that of the CTR diet group. The N-6/N-3 ratio in serum in the MA diet group indicated a significant decrease compared with that in the CTR diet group, whereas significant changes were not detected in the breast tissues in both groups. However, it has been previously reported that a lower ratio N-6/N-3 in the serum is associated with a lower incidence of breast cancer in humans (
In conclusion, the present study reported that the parameters tumor incidence, Ki-67 labeling index and γ-H2A.X-labeling index were not significantly affected by the specific MA diets in female Sprague-Dawley rats with DMBA-induced breast cancer. To further clarify the effects of MA on breast carcinogenesis, further investigations with different experimental breast cancer models are thus recommended.
The authors would like to thank Dr Robert R. Maronpot, Maronpot Consulting, LLC, for his excellent scientific advice and English grammar editing.
No funding was received.
All data generated or analyzed during this study are included in this published article or are available from the corresponding author on reasonable request.
YK, MYo, ATs and KY made substantial contributions to the conception and design of the study. YK, MYo, YM, TY, MYu, CK and ATa were involved in data acquisition, data analysis and interpretation. YK and KH were involved in fatty acid analysis. ATs and KY drafted the article or critically revised it for important intellectual content. All authors gave the final approval of the version to be published and all author agree to be accountable for all aspects of the work to ensure that questions regarding the accuracy or integrity of the work are appropriately investigated and resolved.
The study protocol and animal procedures were approved by the Animal Care and Use Committee of Kansai Medical University, Hirakata, Osaka, Japan (permit no. 13-060).
Not applicable.
The authors declare that they have no competing interests.
Changes in body weight of rats fed the CTR diet and 2.4% MA diet. (A) There was not significant difference in final body weight between the 2.4% MA diet group and CTR diet group at the end of the experiment. (B) DMBA treatment significantly decreased the body weight with the 4.8% MA diet. NS, not significant; CTR, control; MA, mead acid; DMBA, 7,12-dimethylbenz[a]anthracene.
Effects of MA on the incidence of breast tumors during the dosing period. (A) There were not significant differences between the 2.4% MA and CTR diet. (B) The 4.8% MA diet also did not significantly influence the tumor incidence. NS, not significant; CTR, control; MA, mead acid; DMBA, 7,12-dimethylbenz[a]anthracene.
The final average of DMBA-induced breast tumor weight in CTR diet group vs. MA diet group. (A) The 2.4% MA experimental group; (B) the 4.8% MA experimental group. No significant differences were observed between the CTR and MA diet (2.4% MA and 4.8% MA experimental groups, respectively). NS, not significant; CTR, control; MA, mead acid.
Immunohistochemical findings of (A) Ki-67 (right panel, CTR diet group; left panel, 2.4% MA diet group) and (B) γ-H2A.X (right panel, CTR diet group; left panel, 2.4% diet group) labeling index. From these findings, there were no distinct differences between CTR diet group and 2.4% MA diet group as regards cell kinetics and cell death. CTR, control; MA, mead acid.
Comparison of fatty acid composition in animals fed either the 2.4% MA diet or CTR diet for 13 weeks, and treated with or without DMBA. Fatty acid composition in (A) serum and (B) breast tissue. Fatty acid composition in serum and breast tissue reflected the differences in the contents of fatty acid induced by the different diets. *P<0.05. CTR, control; MA, mead acid; DMBA, 7,12-dimethylbenz[a]anthracene.
N-6/N-3 ratio in serum and breast tissue of animals fed the 2.4% MA diet or CTR diet, with or without DMBA treatment. (A) The MA diet significantly decreased the N-6/N-3 ratio in serum (P<0.05). (B) On the other hand, no significant differe nces were observed in breast tissue. NS, not significant; CTR, control; MA, mead acid; DMBA, 7,12-dimethylbenz[a]anthracene.
Composition of the experimental diets.
2.4% MA experimental group | 4.8% MA experimental group | |||
---|---|---|---|---|
Component | CTR diet | MA diet | CTR diet | MA diet |
Gasein | 20 | 20 | 20 | 20 |
DL-methionine | 0.3 | 0.3 | 0.3 | 0.3 |
Cornstarch | 43 | 43 | 38 | 38 |
α-Cornstarch | 12 | 12 | 12 | 12 |
Sucrose | 10 | 10 | 10 | 10 |
Cellulose | 5 | 5 | 5 | 5 |
AIN-76 mineral mix | 3.5 | 3.5 | 3.5 | 3.5 |
AIN-76 vitamin mix | 1 | 1 | 1 | 1 |
Choline bitartrate | 0.2 | 0.2 | 0.2 | 0.2 |
SUNTGM33 | 0 | 5 | 0 | 10 |
Olive oil | 5 | 0 | 10 | 0 |
Values are expressed in g/100 g diet. MA, mead acid; CTR, control.
Weight of DMBA-induced breast cancer (mg).
2.4% MA experimental group | 4.8% MA experimental group | |||
---|---|---|---|---|
CTR diet | MA diet | CTR diet | MA diet | |
Tumor weight | 1,323±251.4 | 1,019.3±178.6 | 941.3±231.4 | 1,235.6±208.4 |
Significance | NS | NS |
CTR, control; MA, mead acid; NS, not significant.
Cell kinetics of DMBA-induced breast cancer (%).
2.4% MA experimental group | 4.8% MA experimental group | |||
---|---|---|---|---|
Examination | CTR diet | MA diet | CTR diet | MA diet |
Ki-67 LI | 46.5±8.6 | 35.1±3.8 | 33.2±3.3 | 26.0±3.5 |
γH2A.X LI | 0.7±0.3 | 0.6±0.2 | 0.7±0.3 | 0.6±0.2 |
Significance | NS | NS |
CTR, control; MA, mead acid; LI, labeling index; NS, not significant.