Stimulation of lymphocyte anti-melanoma activity by co-cultured macrophages activated by complex homeopathic medication

Abstract

Background: Melanoma is the most aggressive form of skin cancer, and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have been uniformly disappointing. A Brazilian complex homeopathic medication (CHM), used as an immune modulator, has been recommended for patients with depressed immune systems. Previous studies in mice have demonstrated that the CHM activates macrophages, induces an increase in the number of leukocytes and improves the murine response against Sarcoma-180.

Methods: Here we studied the interaction of mouse lymph node lymphocytes, co-cultured in vitro with macrophages in the presence or absence of the CHM, with B16F10 melanoma cells.

Results: Lymphocytes co-cultured with macrophages in the presence of the CHM had greater anti-melanoma activity, reducing melanoma cell density and increasing the number of lysed tumor cells. There was also a higher proportion of activated (CD25+) lymphocytes with increased viability. Overall, lymphocytes activated by treatment destroyed growing cancer cells more effectively than control lymphocytes.

Conclusion: Co-culture of macrophages with lymphocytes in the presence of the CHM enhanced the anti-cancer performance of lymphocytes against a very aggressive lineage of melanoma cells. These results suggest that non-toxic therapies using CHMs are a promising alternative approach to the treatment of melanomas. In addition, they are attractive combination-therapy candidates, which may enhance the efficacy of conventional medicines by improving the immune response against tumor cells.

Figure 1

Microphotographs of control and treated cultures. The cells were fixed, stained with Giemsa, and observed under a light microscope. Left column: control culture conditions; right column: treated culture conditions. (A and B) B16F10 cells; (C and D) Mϕ/Ly; (E and F) B16F10/Ly; (G and H) B16F10/Mϕ; (I and J) B16F10/Ly-Mϕ. Original magnification: 40× objective for all figures. Black arrow: B16F10 cells; thin black arrow: macrophages.

Figure 2

Micrographs of B16F10 cell density after B16F10/Ly-Mϕ culture condition of control and treated cells respectively (A and B). Cell density was evaluated by cell area selection by ImageJ software and posterior pixel area quantification and comparison, showing a decrease of melanoma cell density on this culture condition (C). Original magnification: 40× objective for all figures.

Figure 3

Scanning electron micrographs of control and treated cultures. Left column: control culture condition; right column: treated culture condition. (A and B) B16F10 cells after 48 h in culture; (C and D) Mϕ/Ly co-culture; (E and G) untreated B16F10/Ly-Mϕ; (F and H) CHM-treated B16F10/Ly-Mϕ. White arrow: B16F10 cell; thin white arrow; macrophages; Open-headed arrow; lymphocytes.

Figure 4

CD25 expression and viability of lymphocytes after co-culture. (A and B) Lymphocytes co-cultured with macrophages for 24 h (treated or untreated) without cell contact (0.4 μm insert). In Ly/Mg* was observed a higher amount of unviable (7-AAD⁺) Ly cells in control group (Fig. A). Unviable cells were not included in CD25 analysis for to keep comparison between viable CD25+ cells; (C and D) melanoma cells co-cultured with lymphocytes previously co-cultured with macrophages. Percentage of CD25 and viability (7-AAD^low) subpopulations in lymphocytic cells are shown at the quadrant extremities.

Figure 5

Lymphocyte CD25 expression and viability (7-AAD^low) after macrophage co-culture. (A) Lymphocytes co-cultured with macrophages for 24 h (treated or untreated) without cell contact (0.4 μm insert). CD25 expression and viability significantly increased after treatment. In Ly/Mg* was observed a higher amount of unviable (7-AAD⁺) Ly cells in control group. Unviable cells were not included in CD25 analysis for to keep comparison between viable CD25+ cells. (B) Melanoma cells co-cultured with lymphocytes previously co-cultured with macrophages. Control lymphocyte CD25 expression and viability significantly decreased after 24 h. Y-axis: transformed mean.

Figure 6

Apoptotic cells detected by TUNEL assay and analyzed by a one-way ANOVA. Cell cluster formation was highly significant (P < 0.01), and apoptotic melanoma cell numbers were higher in treated cultures. Y-axis: transformed mean.