Original Article

Study Of Dacryodes Edulis Pulp Oil On 3-Methylcholanthrene Induced Breast Cancer and Expression of Cyclooxygenase-2 And Peroxisome Proliferator-Activated Receptor-Gamma in Wistar Rats

[*] Uhunmwangho E S, Oyiborhoro O, Olafusi C O

Bioactive lipids in cancer and toxicology research laboratory, Department of Biochemistry, Faculty of Basic Medical Sciences, University of Medical Sciences, Ondo, Nigeria.

ABSTRACT

Background: Breast cancer is a major cause of death in women, and dietary fat has been implicated among the factors that influence its incidence. This study investigated the effect of Dacryodes edulis pulp oil on 3-methylcholanthrene (MCA) induced breast cancer and the expression of cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptor gamma (PPAR-ɣ) in female Wistar rats. Methods: The pulp oil was extracted with n-hexane using the Soxhlet apparatus and characterized by gas chromatography. Results: A total of 48 Wistar rats (4 weeks old) were used and they were divided into three groups. Group A animals were fed with a diet containing 10% Dacryodes edulis pulp oil for 14 days before MCA (250mg/kg) was intraperitoneally administered. Afterwards, they were fed with pulp oil for twelve weeks before sacrifice. Group B animals received MCA (250mg/kg) to induce breast cancer and were fed a diet containing no Dacryodes edulis pulp oil. Group C animals were fed a diet containing 10% Dacryodes edulis pulp oil only. COX-2 and PPAR-ɣ were significantly (P < 0.05) less and more expressed in pulp oil-fed animals respectively. The Tumour latency period was longer on animals fed with seed oil compared with those not fed a diet containing pulp oil. Conclusion: Results from gas chromatography analysis indicated that the omega-3 polyunsaturated fatty acids were incorporated into the mammary gland cell membrane which opposed the progression of carcinogenesis in animals treated with Dacryodes edulis pulp oil, this indicates that Dacryodes edulis pulp oil is able to oppose breast carcinogenesis induced by 3-methylcholanthrene.

Keywords:

Dacryodes edulis, 

3-methylcholanthrene,

Cyclooxygenase-2,

Carcinogenesis,

Pulp oil.

[*] Corresponding author; 

email: euhunmwangho@unimed.edu.ng; 

Tel: +2348033542957

INTRODUCTION

Dacryodes edulis is a diecious shade-loving species of non-flooded forests in the humid tropical zone. It 

is found in countries such as Nigeria, Cameroon, Malaysia, Liberia and Sierra Leone ¹. It is known as ‘elemi’ in Yoruba, ‘Ube’ in Igbo and "Atili" in Hausa. It belongs to the family ‘Burseraceae’ whose members are characterized by an ovary of 2-5 cells, prominent as in ducts in the bark, wood and intrastaminal disk².  It was shown to possess antimicrobial, antioxidant, cardiovascular, antidrapanocytary activities and also shows no signs of toxicity ³. It was also reported that Dacryodes edulis possess anti-proliferative, pro-aprotic effects against tumour cells, thus, being capable of breaking down multi-cellular tumours, regulating molecules, in apoptosis, signal transduction and cell cycle progression, hence, its anti-cancer activity ⁴ edulis is a versatile plant in African ethnomedicine as its various parts are employed to treat several diseases. The bark of the plant has long been used to cicatrize wounds in Gabon ^2,4. In the Democratic Republic of Congo, the plant is employed for the treatment of leprosy, tonsillitis and dysentery, anaemia, spitting blood, pains and stiffness and skin diseases ⁵. In Congo Brazzaville, the leaves are boiled with those of Lanata camara, and Cymbopogon citretus in water to form a decoction for treating malaria ⁶. The bark resin is used in Nigeria to treat parasitic-skin diseases and jiggers and when applied in lotions and creams, the resin smoothens and protects the skin. The aroma of the resin is liberated through burning, which is believed to ward off evil spirits in Nigeria⁷. The leaves are often crushed and the juice is released to treat generalized skin diseases such as scabies, ringworm, rash and wound, while the stems are employed as chewing sticks for oral hygiene ⁴. The essential oil of the plant showed a potent antibacterial effect against staphylococcus aureus, Bacillus aurens, Escherichia coli and proteus minibillis ^8-10. Dacryodes edulis has been reported to decrease the HDL cholesterol level in the serum of rats ¹¹. The genus Dacryodes consists of about 40 species ^8,9. Two varieties are recognized; Var. edulis and Var. parricarpa, whose conical fruits are smaller with the pulp. Var. edulis exhibit verticillate branching, while branching is slender and opposite in Var. parricarpa (Ajibesin, 2011). Breast cancer starts when cells in the breast begin to grow out of control¹³. These cells usually form a tumour that can often be seen on an x-ray or felt as a lump. The tumour is malignant (cancer) if the cells can grow into (invade) surrounding tissues or spread (metastasize) to distant areas of the body. Breast cancer occurs almost entirely in women, but men can get breast cancer too¹³. Around 5% of breast cancers and up to 25% of familial breast cancer cases are caused by a BRCA1 or BRCA2 mutation ¹⁴. A woman carrying a BRCA1 mutation has a 65 95% lifetime risk of breast cancer, and more than 90% of hereditary breast and ovarian cancers are thought to be due to a mutation in BRCA1 or BRCA2 ¹⁴. Everyone has BRCA1 and BRCA2 genes. Some people have an inherited mutation in one or both of these genes that increases the risk of breast cancer. BRCA1/2 mutations can be passed on from either parent and can affect the risk of cancers in both women and men. Breast cancer women who have a BRCA1 or BRCA2 gene mutation have an increased risk of breast cancer. Estimates of risk are different for BRCA1 carriers and BRCA2 carriers. BRCA1/2 mutations are thought to explain a large portion of hereditary breast cancers¹⁴. A person who has a BRCA1/2 mutation is sometimes called a BRCA1/2 carrier. The identification of the BRCA1 and BRCA2 genes will have vast significance in furthering our understanding of breast pathogenesis. These two genes are tumour suppressor genes shown to be involved not only in breast cancer but also in ovarian and prostate cancers. BRCA1 and BRCA2 are responsible for 80-90% of all familial breast cancer ¹⁴. This means therefore, there is a need to ascertain the role of Dacryodes edulis pulp oil influencing the range it exerts on lipid bilayers which influences the carcinogen-metabolizing enzymes by which it may exert anti-cancer effects. In this study, we shall investigate the effect of feeding Dacryodes edulis pulp oil on 3-methylcholanthrene-induced mammary cancer in female Wistar rats.

MATERIAL AND METHODS

The Study Location: Bioactive lipids in cancer and toxicology research laboratory, Department of Biochemistry, University of Medical Sciences, Ondo City, Ondo State, Nigeria.

Reagents/Chemicals: All reagents used were of analytical grade. Methanol (Sigma Chemicals Co, London), Chloroform (Sigma Chemicals Co., London), 3-methylcholanthrene, Sulphuric Acid Aldrich Chemical Company, USA.

Plant material (The sample collection): Fresh Dacryodes edulis fruits were obtained from farms in Laje town near Ondo City, Ondo State, Nigeria. The fruits were authenticated by a Taxonomist of the Botany Department, University of Medical Sciences, Ondo. The collected fruits were cleaned with a moist soft cotton wool and then the seeds carefully separated from the fruits and dried at 65⁰ C for 4 hrs. in an oven, crushed with a laboratory mortar and pestle and were kept in a well labelled airtight screw-capped bottles at 4⁰ C for extraction.

Extraction of oil from African walnuts: The Soxhlet extraction method was employed^15,16. 

Ethical approval: Ethical approval was granted by the University of Medical Sciences, Ondo, Ethics committee for the use of animals.    

Feeding the animals with a diet containing Dacryodes edulis oil 

Female Wistar rats (28 days old) were obtained from the animal house of the University of Medical Science, Ondo, and were housed in metal cages in a well-ventilated room and they were allowed access to water and ad libitum. The experimental diet comprised among others Dacryodes edulis pulp oil (10.0%). Overall, 48 female Wistar rats were used. The animals were randomly divided into three major groups of 16 animals each. Group 1 animals were fed for 12 weeks with a diet containing Dacryodes edulis pulp oil (10%) and the animals were administered 3-methylcholanthrene (250mg/kg body weight) intraperitoneally after 4 weeks of feeding. Group 2 were fed for 12 weeks with a diet containing Dacryodes edulis pulp oil (10%) only. Group 3 animals were fed for 12 weeks with a diet containing no Dacryodes edulis pulp oil, and were administered 3-methylcholanthrene (250mg/kg body weight) intraperitoneally after 4 weeks of feeding.  The animals were palpated weekly to determine the time of appearance of tumours and body weight.

At necropsy, mammary glands were exposed and tumours were excised. Tumour incidence, volume and weight were determined. Animals from each group were sacrificed at the end of 12 weeks, and the serum and tissues were collected for enzymes and biochemical analysis. Portions of mammary tissue from no tumour bearing and tumour tissue were preserved in RNA later for gene expression studies. Another portion of tumour tissue was fixed in formalin (10%) for histopathological studies.

Fatty Acid Determination: Fatty Acids were determined according to the method of Manni and Caron ¹⁷.

COX-2 and PPAR-γ gene expression: The liver samples were placed in triazole (a molecular grid RNA isolating reagent). The samples were homogenized and chloroform was added for homogenate gradient separation. This was followed by centrifugation at 15,000rpm for 15 minutes. After centrifugation, the upper phase (clear supernatant containing RNA) was aspirated into a new sterile eppendorf tube of 1.5ml. The clear supernatant was precipitated by adding isopropanol. This was followed by centrifugation at 15,000rpm for 5 minutes. RNA pellet was air-dried for 15 minutes and resuspended in nuclease-free water (30 microliters). RNA samples were quantified and absorbance was checked using a spectrophotometer. RNA samples were optimized using a PCR machine for 1 hour at 420C. The samples were amplified and gel electrophoresis was carried out at 70 volts, 500 milli amperes for 10 minutes, the samples were placed in a UV documentary for viewing the expression bands.

Statistical analysis: The values were expressed as mean ± SE. Kruskal-Wallis one-way analysis of variance (ANOVA) was used for the feed intake, body weight, tumour weight, tumour volume and COX-2 and PPAR-γ gene expression using Systat 7.0 software (Spss Inc., Chicago, USA). Statistical analysis of tumour incidence was done by Chi-square test using Systat 7.0 software. A difference with P<0.05 was considered statistically significant.

RESULTS

Table 1. Summarizes the data on incidence, latency period and weight and volume of tumours in the mammary glands. The incidence of tumours on a pulp oil diet (35.9%) was significantly (P<0.05) lower than on diet without Dacryodes edulis pulp oil (87.4%). The tumour latency period was 7 weeks in animals fed with pulp oil compared to 4 weeks in animals fed without pulp oil. The average size of the tumour was generally larger in animals that were not fed with Dacryodes edulis pulp oil than in animals fed with a diet containing Dacryodes edulis pulp oil. Similarly, average tumour volume was significantly (P<0.05) less in animals fed with a diet containing Dacryodes edulis pulp oil than in animals fed with a diet containing no pulp oil.

Table 1 Effect of feeding Dacryodes edulis pulp oil on mammary carcinogenesis in MCA-administered rats

Values are mean ± SE; *P<0.05 compared to Dacryodes edulis pulp oil group

Figure 1: Major fatty acids composition (%) in mammary tissue of animals fed with and without pulp oil diet. 

The major unsaturated FAs in mammary cells of animals fed diets containing Dacryodes edulis pulp oil were gamma linoleic acid, docosatrienoic acid, eicosadienoic acid, and linolenic acid. The concentration of the FAs from the Dacryodes edulis pulp oil-fed rats was significantly different (p ˂0.05) from animals not fed with Dacryodes edulis pulp oil. 

The effect of dietary fat on the expression of COX-2 and PPAR-γ was investigated in the normal mammary glands, Dacryodes edulis-treated animal mammary glands and tumour-bearing rats tissue. COX-2 was not expressed in normal mammary tissue but its expression was induced in response to MCA treatment. 

Figure 2. Expression of Cyclooxygenase-2 in the mammary tissues. Values are mean ± SEM. (* = P<0.05).

In MCA-treated rats, the expression of COX-2 was greater in tumour-bearing than in no tumour-bearing and Dacryodes edulis-treated rats’ tissues. Furthermore, the expression of COX-2 was greater in tumour tissues than in other tissues studied. In carcinogen-treated rats wherein no tumour appeared, the expression of PPAR-γ in Dacryodes edulis-only fed rats were almost of the same magnitude as observed in treated counterpart’s groups. 

DISCUSSION

Fewer tumour incidence, smaller tumour size and greater tumour latency period on animals administered with MCA pulp oil were suggestive of protection conferred by Dacryodes edulis pulp oil in mammary gland carcinogenesis. The role of Dacryodes edulis pulp oil in mammary carcinogenesis may be explained by their ability to modulate the pathway of prostaglandin synthesis. Mammary carcinogenesis is triggered by inappropriate induction and upregulation of COX-2. It was reported ¹⁸ that the expression of the normally silent COX-2 gene results in excess production of prostaglandin E2 and an increase in local estrogen biosynthesis by aromatase.

In this study, COX-2 was undetectable in normal mammary tissue, the group fed with Dacryodes edulis pulp oil only, and its expression induced by 3-MCA treatment was significantly higher in tumour tissue as compared to Dacryodes edulis pulp oil-treated mammary tissue. Cyclooxygenase-2 is an inflammation-associated enzyme involved in the pathogenesis of carcinogenesis. Inhibition of COX-2 and blockade of prostaglandin cascade may lead to the reduction of carcinogenesis ¹⁹ but overexpression of COX-2 initiates and promotes carcinogenesis. Figure 2.0 shows there was no significant difference (p˃0.05) between the group fed with Dacryodes edulis pulp oil only and the animals in the group administered with MCA but treated with Dacryodes edulis pulp oil. Hence, there was a significant difference (p˂0.05) in the expression of COX-2 between the groups fed with Dacryodes edulis pulp oil and the group which was induced with 3-Methylcholanthrene but was not treated with Dacryodes edulis pulp oil which implies that the use of Dacryodes edulis pulp oil has a high activity against COX-2 in groups fed with Dacryodes edulis pulp oil. This proves that Dacryodes edulis pulp oil extract was effective in suppressing the expression of COX-2 in groups that were fed with the pulp oil. This result is in agreement with Chinery et al²⁰ who conducted an experiment on cyclooxygenase-2 expression in colorectal cancer cells. Peroxisome proliferators activated receptor-γ (PPAR-γ), a key component in the regulation of growth and progression of mammary cancer, is expressed in normal as well as in malignant mammary epithelial cells and its activation by ligands induces cellular differentiation^20-22. The activation of PPAR-γ induces proapoptotic caspase-3 protein in human liver cancer cell lines and reduces the antiapoptotic Bcl-2 protein levels in human colon cancer cells ²³. An inverse relationship between COX-2 and PPAR-γ expression was observed in the present study and it was associated with decreased mammary tumour incidence in Dacryodes edulis pulp oil-fed rats compared to non-Dacryodes edulis pulp oil-fed ones. In the genesis of breast cancer, evidence suggests that induction of COX-2 and downregulation of PPAR-γ can be the key components ^24,25. Simultaneous targeting with COX-2 inhibitor (celecoxib) and PPAR-γ agonist [N - (9-fluorenyl-methoxycarbonyl)-L-leucine] has been reported to inhibit mammary gland carcinogenesis in rats ²⁴. Further, activation of PPAR-γ by cigiltazone (PPAR-γ ligand) decreases the COX-2 expression (Yang and Fruchth, 2001), and the inhibition of COX-2 induces PPAR-γ expression ²⁶. Feeding GLA during the pubescent mammary gland development period lowers the population and proliferating activity of the terminal end buds’ cells ²⁷, which are the target sites for the development of adenocarcinomas in response to a carcinogenic stimulus. In this present study, the feeding of animals with Dacryodes edulis pulp oil started during the pubescent period of mammary gland development might have resulted in decreased tumour incidence and progression to malignancy. The anticarcinogenic effect of GLA may be partly explained by its effect on COX-2. Gamma linolenic acid affects the COX-2 at the level of mRNA as well as protein in cultured macrophage cell line ²⁷. It represses AP-1-mediated activation of COX-2 transcription in MCF-7 breast cancer cells ²⁷. The treatment of colon cancer cells with CLA inhibits cell proliferation; increases expression of PPAR-γ and downregulates APC and c-myc proteins ^12,28. The higher tumour incidence and faster progression of MCA-induced mammary carcinogenesis in rats fed on Dacryodes edulis pulp oil compared to animals not fed with Dacryodes edulis pulp oil could be due, partly, to the high content of GLA, eicosadienoic acid, dihomotrienoic acid and linoleic acid in Dacryodes edulis pulp oil. 

CONCLUSION

The conclusion from this study is that Dacryodes edulis pulp oil opposed MCA-induced mammary carcinogenesis and the effect is mediated through decreased expression of COX-2 and increased expression of PPAR-γ. 

ACKNOWLEDGEMENT

The authors sincerely thank the cancer unit of the Biochemistry Department, University of Medical Sciences, Ondo, Nigeria, for supplying the reagents and chemicals used during this research.

CONFLICTS OF INTEREST

No conflict of interest among the authors. 

REFERENCES 

1. Adebayo–Tayo, B. C. and Ajibesin, K. K. Antimicrobial activity of Coula edulis. Res J Med Plant 2008, 2: 86-91 
2. Ajibesin, K. K. Dacryodes edulis (G. Don) H.J. Lam: A Review on its Medicinal, Phytochemical and Economical Properties. Research Journal of Medicinal Plants 2011, 5: 32-41.
3. Ajibesin, K. K., Ekpo, B. J., Bala, D. N., Essien, E. E. and Adesanya, S. A. Ethnobotanical survey of Akwa Ibom State of Nigeria. J Ethnopharmacol 2008, 115: 387-408 
4. Akin-Osanaiye, B. C. and Ahmad. A. A. Nutrient Composition Analysis of African Walnut (Tetracarpidium Conophorum) and Isolation of Microorganisms Responsible for its Spoilage. Direct Res J Agric Food Sci. 2018, 6 (7): 173-181   
5. Anonymous, Dacryodes edulis. Portabase, Plant Resources of Tropical Africa (PROTA) 2010.
6. AOAC, AOAC official method 994.10: Cholesterol in foods- Direct saponification – Gas chromatography method. Association of analytical chemists Arlington, VA 1996. 
7. Badawi, A. F. and Badr, M. Z. Expression of cyclooxygenase-2 and peroxisome proliferator-activated receptor-γ and levels of prostaglandin E2 and 15-deoxy-Δ12, 14 -prostaglandin J2 in human breast cancer and metastasis. Int J Cancer 2003, 103: 84-90 
8. Bozzo, F., Bocca, C., Colombatto, S. and Miglietta. A. Antiproliferative effect of conjugated linoleic acid in caco-2 cells: involvement of PPAR gamma and APC/beta-catenin pathways. Chem Biol Interact 2007, 169: 110–121. 
9. Degner, S. C., Kemp, M. Q., Bowden, G. T. and Romagnolo, D. F. Conjugated linoleic acid attenuates cyclooxygenase-2 transcriptional activity via an anti-AP-1 mechanism in MCF-7 breast cancer cells. J Nutr 2006, 136: 421-427 
10. Edu, E. A. and Akwaji, P. I. Pentaclethra macrophylla Benth. (African oil bean tree) and Parkia biglobosa Jacq (African locust bean tree): The declining giants of the rainforest of Nigeria (The Northern Cross River situation). World Scientific News 2017, 64, 127-138
11. Gavamukulya, Y., Abou-Elella, F., Wamunyokoli, F. and ElShemy, H. A. GC-MS Analysis of bioactive phytochemicals present in ethanolic extracts of leaves of Annona muricata: a further evidence for its medicinal diversity. Pharmacognosy Journal 20015, 7(5): 300-304. 
12. Gimble, J. M. Pighetti, G. M., Lerner M. R. et al. (1998). Expression of peroxisome proliferator activated receptor mRNA in normal and tumorigenic rodent mammary glands. Biochem Biophys Res Commun 1998, 253: 813-817. 
13. Harris, R. E., Robertson, F. M., Abou-Issa, H. M., Farrar, W. B. and Brueggemeier, R. Genetic induction and upregulation of cyclooxygenase (COX) and aromatase (CYP19): an extension of the dietary fat hypothesis of breast cancer. Med Hypotheses 1999, 52: 291–292.  
14. Ikhuoria, E. U. and Maliki, M. Characterization of Avocado pear (Paesea ignallin) and African pear (Dacryodes edulis) extracts. Afr J Trad Cam 2007, 2: 134-152 
15. Ikpa, C. B. C. and Maduka, T. O. D. Antimicrobial properties of methanol extract of Dacryodes edulis seed and determination of phytochemical composition using FTIR and GC-MS. Chemistry Africa 2020, doi.org/10.1007/s42250-020-00176-x
16. Koudou, J., Edou, P., Obame, L. C., Bassole, I. H. and Figueredo, G. Volatile components, antioxidant and antimicrobial properties of the essential oil of Dacryodes edulis from Gabon. J Applied science 2008,  8: 3532-3535 
17. Maduka, T. O. D., Ikpa, C. B. C. and Kalu, G. I. Physicochemical characterization and assessment of bioactive chemical compounds of Persea americana (Avocado) Seed. Nat Ayurvedic Med 2020, 4(1): 000229. 
18. Magaji, 1. H., Musa, A. A., Abubakar, H. Y. and Umar, A.T. Synthesis, Characterization and Antimicrobial Evaluation of Silver Nanoparticles Embedded Alkyd Resin Derived from Neem Seed Oil. Journal of Applied Chemistry 2018, 11(2):13-20
19. Manni, G. and Caron, F. Calibration and Determination of Volatile Fatty Acids in Waste Leachates by Gas Chromatography. Journal of chromatography 1995, 690: 237  
20. Nagawa, C., Böhmdorfer, S. and Rosenau, T. Chemical composition and anti-termitic activity of essential oil from Canarium schweinfurthii Engl. Industrial Crops and Products 2015, 71:75-79. 
21. Obonga, W. O., Omeje, E.O. Nnadi, C. O. and Ocheme, W.G. Phytochemical evaluation of extracts and GC-MS analysis of oil from Monodora myristica seed. Dhaka Univ J Pharm Sci 2019, 18(1): 69-73.
22. Okwu, D. E. and Nnamdi, D. A. Evaluation of the chemical composition of Dacryodes edulis and Raphia hookeri.  Maun and wendi exudates used in herbal medicine in south eastern Nigeria.  Afr J Trad Global 2008, 5:194-200. 
23. Oyen, L. P. A. and Lemens, R. H. M. Plant Resources of Tropical Africa, Wageneningen, Netherlands. 2002. 
24. Shiff, J. and Rigas, B. The role of cyclooxygenase inhibition in the antineoplastix effect of non-steroidal anti-inflammatory drugs (NSAIDS). Journal of Experimental Medicine 1999, 190(4): 445-450. 
25. Skol, D. S., Sasaski, M. M. and Onel, K. The genetics of breast cancer risk in the post-genome era: thoughts on study design to move past BRCA and towards clinical relevance. Breast cancer Res 2016, 18: 99. 
26. Sofowora, A. Medicinal plants and traditional medicine in Africa.  2nd Edn. Spectrum books Ltd. Ibadan, Nigeria 2008. 10-158. 
27. Toyoda, M. Takagi, H. Horiguchi, N. et al. A ligand for peroxisome proliferator activated receptor gamma inhibits cell growth and induces apoptosis in human liver cancer cells. Gut 2002, 50: 563-567. 
28. Uhunmwangho, E. S. and Omoregie, E. S. Changes in lipid profile and fatty acid composition during the development of African walnut (Tetracarpidium conophorum) seeds. World Applied Sciences Journal 2017, 35: 1174-1179. 
29. Yang, W. L. and Frucht, H. Activation of the PPAR pathway induces apoptosis and COX-2 inhibition in HT-29 human colon cancer cells. Carcinogenesis 2001, 22: 1379-1383. 
30. Yasui, Y., Hosokawa, M. and Sahara, T. et al. Bitter gourd seed fatty acid rich in 9c,11t,13 tconjugated linolenic acid induces apoptosis and up-regulates the GADD45, p53 and PPARgamma in human colon cancer Caco-2 cells. Prostaglandins Leukot Essent Fatty Acids 2005, 73: 113-119. 
31. Yee, L. D., Guo, Y. Bradbury, J., Suster, S., Clinton, S. K. and Seewaldt, V. L. The antiproliferative effects of PPAR-γ ligands in normal human mammary epithelial cells. Breast Cancer Res Treat 2003, 78: 179–192.