Anti-neoplastic and immunomodulatory potency of co-treatment based on bovine lactoferrin and/or muramyl dipeptide in tumor-bearing mice

Abstract

The current study investigates anti-neoplastic and immunomodulatory activities of co-treatment based on bovine lactoferrin (bLF) and/or muramyl dipeptide (MDP) with or without cisplatin (Cis) in tumor-bearing mice. In the present study, bLF (100 mg/kg; orally) and MDP (0.5 mg/kg; subcutaneously) was administered alone or together. MDP or bLF was co-treated with Cis (1 mg/kg; intraperitoneally) in mice-bearing Ehrlich solid carcinoma. Tumor size, tumor mass proliferation, apoptosis using immunohistochemistry, the alteration in spleen cell proliferation, phenotype using flow cytometry and white blood cells total and differential counts were detected. Treatment with Cis or (bLF and MDP) significantly reduced tumor size, upregulated the pro-apoptotic p53 expression and downregulated the anti-apoptotic Bcl-2 and proliferative marker PCNA expression compared to non-treated tumor-bearing animals. Moreover, co-treatment of MDP and Cis significantly potentiated the reduction of the tumor size, downregulated the Bcl-2 and PCNA expression and upregulated the p53 expression compared to Cis-treated animals. While bLF and Cis co-treatment positively controlled PCNA and p53 expression compared to tumor-bearing animals, it significantly potentiated the reduction of the tumor size and downregulated the Bcl-2 expression compared to Cis-treated animals. Co-treatment of (bLF and MDP), (bLF and Cis) or (MDP and Cis) increased the spleen cell proliferation and altered the immunological profile of the CD3⁺CD4⁺, CD3⁺CD8⁺, CD3⁺CD4⁺CD69⁺, CD3⁺CD8⁺CD69⁺ and CD11b⁺Ly6G⁺ cells to achieve better immune response against tumor. In conclusion, co-treatments based on bLF and/or MDP are promising therapies against cancer, through their potency to control proliferation, enhance apoptosis and improve the immune status against tumor cells.

Keywords: apoptosis; cisplatin; immunity; lactoferrin; muramyl dipeptide; tumor.

Graphical Abstract

Figure 1

Effect of co-treatment of bLF, MDP or each agent in combination with chemotherapeutic agent cisplatin (Cis) on tumor growth in EST-bearing mice. Data are expressed as the mean ± SD, n = 7. Statistical difference was calculated with an ANOVA and follow-up test (LSD). ^*P < 0.05 indicates significant difference compared to EST group.

Figure 2

Representative light micrographs of the proliferation marker PCNA protein expression in tumor mass after co-treatment with bLF and MDP. On day 21, thin sections in the right thigh muscles were prepared for immunohistochemical examination. The brown stain referred to positive immunohistochemical reaction; blue stain is hematoxylin counter satin (×400), n = 1.

Figure 3

Representative light micrographs of the pro-apoptotic p53 protein expression in tumor mass after co-treatment with bLF and MDP. On day 21, thin sections in the right thigh muscles were prepared for immunohistochemical examination. The brown stain referred to positive immunohistochemical reaction; blue stain is hematoxylin counter satin (×400), n = 1.

Figure 4

Representative light micrographs of the anti-apoptotic Bcl-2 protein expression in tumor mass after co-treatment with bLF and MDP. On day 21, thin sections in the right thigh muscles were prepared for immunohistochemical examination. The brown stain referred to positive immunohistochemical reaction; blue stain is hematoxylin counter satin (×400), n = 1.

Figure 5

Effect of co-treatment of bLF, MDP or each agent in combination with cisplatin on splenocyte proliferation rate in EST-bearing mice. On day 21, spleen was collected and 5 × 10⁷ spleen cells were labeled with 1 μM CFSE. The data obtained from four individual mice per group. Asterisk denotes P < 0.05 statistically compared with EST control group. Hash denotes P < 0.05 statistically compared with Cis-treated group.

Figure 6

Flow cytometric histogram plots of splenocytes phenotyping in EST-bearing mice showing the effect of co-treatment of bLF, MDP or each agent in combination with cisplatin. On day 21, spleens were collected and 1 × 10⁶ spleen cells were stained with the indicated surface marker and subjected to the flow cytometer. Numbers correspond to the percentage of double-positive fluorescence-labeled cells, n = 1.