Selenized milk casein in the diet of BALB/c nude mice reduces growth of intramammary MCF-7 tumors

Abstract

Background: Dietary selenium has the potential to reduce growth of mammary tumors. Increasing the Se content of cows' milk proteins is a potentially effective means to increase Se intake in humans. We investigate the effects of selenized milk protein on human mammary tumor progression in immunodeficient BALB/c nude mice.

Methods: Four isonitrogenous diets with selenium levels of 0.16, 0.51, 0.85 and 1.15 ppm were formulated by mixing low- and high-selenium milk casein isolates with a rodent premix. MCF-7 cells were inoculated into the mammary fat pad of female BALB/c nude mice implanted with slow-release 17 β-estradiol pellets. Mice with palpable tumors were randomly assigned to one of the four diets for 10 weeks, during which time weekly tumor caliper measurements were conducted. Individual growth curves were fit with the Gompertz equation. Apoptotic cells and Bcl-2, Bax, and Cyclin D1 protein levels in tumors were determined.

Results: There was a linear decrease in mean tumor volume at 70 days with increasing Se intake (P < 0.05), where final tumor volume decreased 35% between 0.16 and 1.15 ppm Se. There was a linear decrease in mean predicted tumor volume at 56, 63 and 70 days, and the number of tumors with a final volume above 500 mm3, with increasing Se intake (P < 0.05). This tumor volume effect was associated with a decrease in the proportion of tumors with a maximum growth rate above 0.03 day-1. The predicted maximum volume of tumors (Vmax) and the number of tumors with a large Vmax, were not affected by Se-casein. Final tumor mass, Bcl-2, Bax, and Cyclin D1 protein levels in tumors were not significantly affected by Se-casein. There was a significantly higher number of apoptotic cells in high-Se tumors as compared to low-Se tumors.

Conclusions: Taken together, these results suggest that turnover of cells in the tumor, but not its nutrient supply, were affected by dairy Se. We have shown that 1.1 ppm dietary Se from selenized casein can effectively reduce tumor progression in an MCF-7 xenograft breast cancer model. These results show promise for selenized milk protein as an effective supplement during chemotherapy.

Figure 1

Proportion of large and fast-growing tumors within each treatment group. Proportion of tumors within each Se-casein treatment with a) final volume above 500 mm³, b) maximum growth rate above 0.03 d^-1, c) maximum volume (V_max) above 1600 mm³, d) inflection point after 28 days, and e) final growth rate above 0.025 d^-1 were subjected to an exact Cochran-Armitage test for linear effect of dietary Se level. P-values are shown as P_exact.

Figure 2

Tumor volume growth curves for individual mice in each treatment group. MCF-7 cells were xenografted into mammary fat pads of nude BALB/c mice implanted with slow-release estrogen pellets. Once tumor volumes reached 60 mm³ in volume, mice were assigned to dietary treatments of Se-casein at a) 0.16 ppm Se, b) 0.51 ppm Se, c) 0.85 ppm Se, and d) 1.15 ppm Se. Tumor volumes were estimated from caliper measurements once per week during treatment.

Figure 3

Final tumor volumes and masses. a) volume estimated from caliper measurements and b) mass measured after tumor excision, on day 70 of Se-casein treatment. Values are means ± SE for each dietary treatment group (n = 11, 10, 14 and 13, respectively). P-values represent linear effects of dietary Se level.

Figure 4

Mean tumor volumes predicted from fits of Gompertz equation to individual growth curves. Values are means for each Se-casein treatment (n = 11, 9, 14 and 13, respectively). Error bars represent pooled SE of the mean. Asterisks indicate significant linear effects of dietary Se from Se-casein (P< 0.05). 0.16 ppm Se (n=11), 0.51 ppm Se (n=9), 0.85 ppm Se (n=14), 1.15 ppm Se (n=13).

Figure 5

Tumor Se levels on a dry matter basis. MCF-7 tumors were excised from mice at day 70 of Se-casein treatment and subjected to Se analysis. Values are means ± SE for each treatment group (n = 11, 10, 14, and 13, respectively). The P-value represents the linear effect of dietary Se level.

Figure 6

Effect of Se-casein on apoptotic cell number. After 70 d on Se-casein treatment, the 4 largest MCF-7 tumors on each treatment were excised, embedded in wax, and sectioned at 5 μm onto microscope slides.The TUNEL assay was used to identify apoptotic cells and nuclei were counterstained with hematoxylin. a) mouse 13, 0.16 ppm Se. b) mouse 26, 0.51 ppm Se. c) mouse 3, 0.85 ppm Se. d) mouse 15. 1.15 ppm Se. Arrows indicate apoptotic cells. e) Apoptotic cell number was expressed as a percentage of total nuclei, counting 1500–2000 nuclei per sample. Values are means ± SE for each treatment group. The P-value represents the linear effect of dietary Se level.

Figure 7

Effects of selenium treatments on Bax, Bcl-2 and cyclin D1 protein levels. After 70 d of Se-casein treatment, tumors were excised from mice and subjected to western blot analysis for a) Bax, b) Bcl-2, c) Bax:Bcl-2 ratio and d) cyclin D1. Expression levels were normalized to β-tubulin. Values in bar graphs are means ± SE for each treatment group (n = 11, 10, 14 and 13, respectively). P-values represent linear effects of dietary Se level.