Selenium nanoparticle-enriched Lactobacillus brevis causes more efficient immune responses in vivo and reduces the liver metastasis in metastatic form of mouse breast cancer

Abstract

Background and the purpose of the study: Selenium enriched Lactobacillus has been reported as an immunostimulatory agent which can be used to increase the life span of cancer bearing animals. Lactic acid bacteria can reduce selenium ions to elemental selenium nanoparticles (SeNPs) and deposit them in intracellular spaces. In this strategy two known immunostimulators, lactic acid bacteria (LAB) and SeNPs, are concomitantly administered for enhancing of immune responses in cancer bearing mice.

Methods: Forty five female inbred BALB/c mice were divided into three groups of tests and control, each containing 15 mice. Test mice were orally administered with SeNP-enriched Lactobacillus brevis or Lactobacillus brevis alone for 3 weeks before tumor induction. After that the administration was followed in three cycles of seven days on/three days off. Control group received phosphate buffer saline (PBS) at same condition. During the study the tumor growth was monitored using caliper method. At the end of study the spleen cell culture was carried out for both NK cytotoxicity assay and cytokines measurement. Delayed type hypersensitivity (DTH) responses were also assayed after 72h of tumor antigen recall. Serum lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) levels were measured, the livers of mice were removed and prepared for histopathological analysis.

Results: High level of IFN-γ and IL-17 besides the significant raised in NK cytotoxicity and DTH responses were observed in SeNP-enriched L. brevis administered mice and the extended life span and decrease in the tumor metastasis to liver were also recorded in this group compared to the control mice or L.brevis alone administered mice.

Conclusion: Our results suggested that the better prognosis could be achieved by oral administration of SeNP-enriched L. brevis in highly metastatic breast cancer mice model.

Figure 1

Related tumor volume in tumor-bearing mice which received SeNP-enriched L. brevis, L. brevis alone and PBS buffer (control group). Tumor growth was measured twice a week and evaluated by caliper measurement of the tumor length. The tumor related volume which refers to the measurement of 15 tumors in each group was achieved by subtraction of first measurement from the latest. Asterisks indicate statistical significance (P ≤ 0.05 significance).

Figure 2

Foot pad induration in tumor-bearing mice which received SeNP-enriched L. brevis, L. brevis alone and PBS buffer (control group). Footpad induration was measured at 72 h after tumor antigen re-challenge using caliper measurements.

Figure 3

Inductions of IFN-γ (left illustration) and IL17 (right illustration) by administration of SeNP-enriched L. brevis in a spleen cell culture. The spleen cells were stimulated with tumor antigen for 72 h and then the levels of IFN-γ and IL-17 in the culture supernatants were determined by ELISA. Data represent the means ± standard deviations for triplicate cultures of eight animals per group * (P ≤ 0.05).

Figure 4

NK cell activity in spleen cell cultures measured by the LDH assay. A culture of 2.5 × 10⁶ cells/well from the splenocytes was added to K562 cells at a 1:100 ratio (Effector: Target ratio) and plates were gently centrifuged for five min at 50 × g, then cultured in RPMI1640 containing 10% FBS. The plates were incubated for seven h at 37°C in 5% CO₂ and the specific release of LDH was calculated as a percentage. A significant increase * * * (P ≤ 0.001) in the NK cytotoxicity was observed for the SeNP-enriched L. brevis group compared to the control group. The data are the means ± SD of triplicate cultures. SeNP-enriched L. brevis = selenium nanoparticle enriched Lactobacillus brevis.

Figure 5

Differences between the levels of ALP and LDH in the serum of mice, measured using the conventional IFCC method. Data are shown in the range of U/L.

Figure 6

The levels of tumor necrosis (left illustration) and liver metastasis (right illustration) cells were diagnosed in histopathological slides prepared from tissue samples removed mice which received SeNP-enriched L. brevis.

Figure 7

Survival rates of mice administered SeNP-enriched L. brevis and L. brevis alone when compared with control mice at the end of the study. A total of seven mice from each experimental group were kept under standard conditions until they died. The rate of death was registered every day and the obtained data were analyzed with a Kaplan-Meier test after the last death in both test and control groups. The lifespans of animals that received SeNP-enriched L. brevis or L. brevis alone were considerably increased compared to the lifespans of control mice.