Tumor Growth Dynamics: Dietary Fish Oil Induced Inhibition of Human Breast Carcinoma Growth, a Phenomenon of Reduced Cellular DNA Synthesis or Increased Cell Loss?

Diets high in unsaturated fatty acids, especially those containing high levels of linoleic acid, e.g., corn oil, enhance mammary gland tumorigenesis in experimental animals. In contrast, diets high in long-chain polyunsaturated fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic (DHA), e.g. menhaden oil, appear to have a suppressive effect on this tumorigenic process. Many mechanisms have been proposed to explain the tumor inhibitory action exerted by menhaden oil and other fish oils, e.g., differences in prostaglandin metabolism, energy efficiency, alterations of the immune system, changes in lipid peroxidation, etc. Fundamental to a mechanistic understanding of this phenomenon, however, is an understanding as to whether or not the tumor inhibitory activities of dietary fish oil is mediated via an inhibition of tumor cell proliferation or mediated via an enhancement of tumor cell loss. Whether the amount of dietary fat or the type of fat effects mammary tumorigenic processes, via an effect on tumor cell proliferation or tumor cell loss, has not been clearly established. In the studies described in this communication, three methods were utilized to study tumor cell proliferation, i.e., H-thymidine autoradiographic analysis, 5-bromo 2'-deoxyuridine (Brdu) flow cytometric analysis, and proliferative cell nuclear antigen (PCNA) flow cytometric analysis. Two methods were used to study tumor cell loss, i.e., a determination of the IUrd tumor emission rate and a determination of a cell loss factor from the formulas of Steel and Begg. The tumor examined was the human breast carcinoma cell line MDAMB231 maintained in athymic nude mouse. No significant difference in cell proliferation between carcinomas of mice fed a high corn oil diet (20% w/w) and a diet high in fish oil (19% menhaden oil, 1% corn oil). In contrast, a significant (p<0.05) increase in the rate of IUrd emission rate and cell loss factor from the carcinomas in the fish oil fed mice compared to the corn oil fed mice was observed. In summary, the decreased tumor volume in the human breast carcinomas maintained in athymic nude mice fed a fish oil diet as compared to those fed a corn oil diet, appears to be due, at least in part, to an increased rate of carcinoma cell loss rather than a decreased rate of carcinoma cell proliferation. DOI : 10.14302/issn.2639-1716.jn-17-1495 Corresponding author: Michael J Gonzalez, University of Puerto Rico Medical Sciences Campus, Schools of Public Health and Pharmacy, San Juan PR. Email: michael.gonzalez5@upr.edu


Introduction :
The mechanism by which diets high in certain fats such as corn oil are capable of enhancing mammary tumorigenesis in rodents (1 ) and increase human breast carcinoma size in athymic nude mice (2) is unclear.
Moreover, the mechanism by which diets high in longchain polyunsaturated fatty acids (PUFA) such as fish oils, can effectively suppress mammary tumorigenesis in rodents (1) and inhibit human breast carcinoma growth in athymic nude mice (2,3) also remains to be determined. This raises a fundamental question in tumor biology that has not been rigorously examined.
How do dietary fats exert their enhancing or suppressive activity at a tumor growth kinetic (tumor cell loss vs tumor cell proliferation) level? Only a few research groups (4)(5)(6) have examined cell proliferation in tumors of animals fed high fat diets. One group (7) reports no difference in cell proliferation rates in transplantable mouse mammary tumors from animals fed diets composed of unsaturated vs saturated fats. In another study, Abraham et al. (8) hypothesized that the increase in tumor size induced by a corn oil diet compared to hydrogenated cottonseed oil or fish (menhaden) oil diet was due to a decrease in cell loss (cell death) as a result of the high corn oil diet impairing immune system activity.
In other studies (5,6,(9)(10), an increase in cell proliferation was observed by an increase in H 3thymidine incorporation into DNA of carcinogen-induced rat mammary tumors from rats fed a high corn oil diet compared to those fed lower levels of corn oil. They concluded that this increase in mammary tumor growth was due to an increase in carcinoma cell proliferation by providing diets high in corn oil. Our study was designed to determine if the growth of a human breast carcinoma cell line (MDA-MB231) in vivo (athymic nude mice), as a function of feeding high levels of either corn oil or fish oil (menhaden), is due to changes in carcinoma cell proliferation and/or changes in carcinoma cell loss. The knowledge of how dietary fats can affect mammary carcinoma growth dynamics is critical to a mechanistic understanding of nutritional tumorigenesis.  One hour prior to sacrifice, mice were injected i.p.

Assessment of Cell Loss in Human Breast Carcinomas
Cell loss is defined as the rate of loss of cells as a fraction of the rate at which cells are being added to the tumor volume by cell proliferation. Cell loss is an important factor in estimating the growth potential of a tumor (12). In order to facilitate the study of this phenomenon in our experimental model, we proceeded as follows.  (14).

Results
In Table 3  FO had a higher tumor volume than those fed 5% C0/15% FO but this difference did not reach a level of 5% significance. Also no significant difference was detected in tumor mean Brdu analysis between these three groups.
In   The method for the assessment of cell loss from growing tumors was originally described by Steel (12) and validated by Begg (13) Table 3, Study 7 in Table 4 and Study 8 in Table 5  Our results suggest that parameters other than cell proliferation may be the primary mechanism by which differences in tumor volume between dietary CO and FO fed animals is achieved. Studies 9,10,11 (Table   6)