Discussion

Tumor cells use multiple mechanisms to escape detection and elimination by the immune system, prompting the development of chemotherapeutic drugs that harness both humoral and cellular immunity to target malignant cells. Innovative immunotherapy approaches include the use of immunotoxins to eliminate regulatory T cells (thereby allowing tumor-specific T cells to be activated), monoclonal antibodies to inhibit immunosuppressive cell signaling, and exvivo-expanded tumor-specific T cells in combination with chemotherapy [16]. There is little basic research on the effectiveness of homeopathic treatments in cancer, and the few studies that have been performed are limited in scope. One recent study examined the anti-tumor effect of the homeopathic agent, Lymphomyosot, in a B16F10 invivo model [17]. Previous results in mice showed that CHM treatment significantly reduced sarcoma size, infiltration of lymphoid cells, and occurrence of granulation tissue and fibrosis surrounding the tumor, and enhanced immune surveillance and promoted tumor regression [10].

The CHM that we have used here is neither toxic nor mutagenic. Our results show that it induces lymphocyte activation against B16F10 cells, primarily under conditions in which lymphocytes are stimulated by macrophages co-cultured (culture conditions M/Ly) in the presence of the CHM. These lymphocytes were activated by macrophage stimulation, even in the absence of cell contact. Electron microscopy is a powerful tool for gaining insight into the actions of homeopathic medicaments in cancer [18], providing images that show the reduction of melanoma cell density, membrane disruption, and altered morphology of tumor cells. The cytotoxic effects of macrophage-stimulated lymphocytes against B16F10 in a liquid culture system were also evident in the form of apoptotic cells detected by fluorescence and clearly seen in SEM images (Fig 6 and Fig 3H) and by melanoma cell density quantification (Fig 2). Although there was an apparent increase in apoptotic melanoma cells after M/Ly interaction, this difference was not significant, possibly reflecting the ability of melanoma cells to suppress apoptosis by over-expressing anti-apoptotic factors [19]. Our results showed that lymphocytes exerted a greater tumoricidal effect not only by inducing apoptosis but also by causing cell lyses. This is consistent with the fact that lymphocytes can kill foreign or tumor cells by other cytotoxic mechanisms, such as release of granzyme B or perforin [20]. The B16F10/M culture condition did not show increased effectiveness, probably because B16 melanoma cells are capable of inhibiting macrophage activation [21].

Macrophages and dendritic cells both serve important immunomodulatory functions, initiating a primary immune response and also activating lymphocytes. Macrophages can also regulate the intensity of the T-cell response to a pathogen [22,23]. Tumor progression in cancer may not simply result from the absence of an immune response, but rather from the inability of effector immune cells to control or destroy the tumor cells. The interaction between antigen-presenting cells (APCs), like dendritic cells and macrophages, and T cells is characterized by a bidirectional exchange of signals that can result in activation and maturation of effector T lymphocytes [24]. T lymphocytes have spontaneous anti-tumor activity and are capable of killing tumor cells, but the anti-tumor T cell can become inactive over time as a result of tumor-escape mechanisms [25]. Activation of T cells is dependent on APC-interaction mechanisms, and requires the interaction of MHC complexes with TCR, co-stimulatory molecules and/or mediators such as the cytokine, IL-2. Expression of the IL-2 receptor ?-chain (CD25) in activated lymphocytes confers greater effector capacity and cellular proliferation [26,27]. Our results confirm this, as evidenced by the fact that treatment with the CHM increased lymphocyte expression of CD25 and lymphocyte viability in conjunction with enhanced effectiveness against melanoma cells and showed to be important to keep the lymphocyte viability in co-culture without contact with macrophages.

Since this medication acts primarily through activation of macrophages [4,9], it is possible that, after being activated, macrophages modulate lymphocyte activation through membrane interactions or secretion of cytokines, such as IL-2, and thereby improve antitumor activity [28,29]. Other signaling pathways might also be involved in this process. Lymphocyte clustering on B16F10 was significantly increased in CHM-treated M/Ly co-cultures, indicating that macrophage stimulation promoted the interaction of lymphocytes with tumor cells. Many cancers and all melanomas express B7-H1 membrane proteins, which inhibit lymphocyte responses by directly inducing lymphocyte apoptosis. Pre-activated lymphocytes are better prepared to respond to this type of cancer defense [30]. Tumor cells can manipulate the immune microenvironment to their advantage, for example, by promoting TNF-? secretion. TNF-? is rarely cytotoxic to tumor cells and promotes cancer growth, invasion and metastasis, and controls the infiltration of antitumor lymphocytes [31]. This CHM decreased TNF-? production under pathological conditions, and exerted a positive immunomodulatory function by activating macrophages [4,9]. Individual lymphocytes appear not to be activated by the treatment, indicating the importance of immune cell interactions in general and macrophages in particular in CHM-mediated lymphocyte activation.

Conclusion

In summary, the present study allows us to conclude that this CHM indirectly activates lymphocytes through interaction with macrophages, even without direct cell-cell contact. The co-culture of macrophages and lymphocytes in the presence of the CHM promoted immunostimulation of lymphocytes, resulting in enhanced tumoricidal performance against a very aggressive lineage of melanoma cells. In addition, the lymphocytes activated by the treatment destroyed growing cancer cells more effectively than did control lymphocytes. Finally, these findings suggest that this medication is a promising combination-therapy candidate, which may enhance the efficacy of conventional medicines by improving the immune response or even induce dormancy in tumor cells.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

FSFG designed and performed all experiments and drafted the manuscript. APRA, BC, CCO, LFA and SMO collaborated on cell culture and microscopy experiments. LD and EST acquired and analyzed the microscopy data. JG performed the statistical analyses. DFB supervised all experiments and manuscript writing. All authors have read and approved the final manuscript.

Acknowledgements

We thank CAPES, CNPq, Fundação Araucária, and SETI-PR for financial support. We are especially grateful to Centro de Microscopia Eletrônica at Universidade Federal do Paraná – UFPR, and to Dr^a. Marta M. Cestari for their help with fluorescence microscopy.

References

1. Rigel DS, Carucci JA: Malignant melanoma: Prevention, early detection, and treatment in the 21st Century.

CA: Cancer Journal of Clinics 2000, 50:215-236. Publisher Full Text

2. Thompson JF, Scolyer RA, Kefford RF: Cutaneous melanoma.

Lancet 2005, 365:687-701. PubMed Abstract | Publisher Full Text

3. Milazzo S, Russell N, Ernst E: Efficacy of homeopathic therapy in cancer treatment.

European Journal of Cancer 2006, 42:282-289. PubMed Abstract | Publisher Full Text

4. Piemonte MR, Buchi DF: Analysis of IL-12, IFN-g and TNF-alpha production, ?5 ?1 integrins and actin filaments distribution in peritoneal mouse macrophages treated with homeopathic medicament.

Journal of Submicroscopical Cytology and Pathology 2002, 33(4):255-263.

5. Oliveira CC, Oliveira SM, Godoy LMF, Gabardo J, Buchi DF: Canova, a Brazilian medical formulation, alters oxidative metabolism of mice macrophages.

Journal of Infection 2006, 52:420-432. PubMed Abstract | Publisher Full Text

6. Lopes L, Godoy LMF, De Oliveira CC, Gabardo J, Schadeck RJG, Buchi DF: Phagocytosis, endosomal/lisosomal system and other cellular aspects of macrophage activation by Canova medication.

Micron 2006, 37:277-287. PubMed Abstract | Publisher Full Text

7. Pereira WKV, Lonardoni MVC, Grespan R, Caparroz-Assef SM, Cuman RKN, Bersani-Amado CA: Immunomodulatory effect of Canova medication on experimental Leishmania amazonensis infection.

Journal of Infection 2005, 51(2):157-164. PubMed Abstract | Publisher Full Text

8. Takahachi G, Maluf MLF, Svidzinski , Dalalio MMO, Bersani-Amado CA, Cuman RKN: In vivo and in vitro effects of the Canova medicine on experimental infection with Paracoccidioides brasiliensis in mice.

Indian Journal of Pharmacology 2006, 38(5):350-354.

9. Oliveira CC, de Oliveira SM, Goes VM, Probst CM, Krieger MA, Buchi DF: Gene expression profiling of macrophages following mice treatment with an immunomodulator medication.

Journal of Cellular Biochemistry 2008, 104:1364-1377. PubMed Abstract | Publisher Full Text

10. Seligmann IC, Lima PD, Cardoso PC, Khayat AS, Bahia MO, Buchi DF, Cabral IR, Burbano RR: The anticancer homeopathic composite “Canova Method” is not genotoxic for human lymphocytes in vitro.

Genetics and Molecular Research 2003, 2:223-228. PubMed Abstract | Publisher Full Text

11. Sato DYO, Wal R, De Oliveira CC, Cattaneo RII, Malvezzi M, Gabardo J, Buchi DF: Histopathological and immunophenotyping studies on normal and sarcoma 180-bearing mice treated with a Brazilian homeopathic medication.

Homeopathy 2005, 94(1):26-32. PubMed Abstract | Publisher Full Text

12. Abud APR, César B, Cavazzani LFM, De Oliveira CC, Gabardo J, Buchi DF: Activation of bone marrow cells treated with Canova in vitro.

Cell Biology International 2006, 30(10):808-816. PubMed Abstract | Publisher Full Text

13. Cesar B, Abud APR, De Oliveira CC, Cardoso F, Gremski W, Gabardo J, Buchi DF: Activation of mononuclear bone marrow cells treated in vitro with a complex homeopathic medication.

Micron 2008, 39(4):461-470. PubMed Abstract | Publisher Full Text

14. Buchi DF, de Souza W: Internalization of surface components during ingestion of Saccaromyces cerevisiae by macrophage.

Journal of Submicroscopical Cytology and Pathology 1992, 24(1):135-141.

15. Buchi DF, de Souza W: Internalization of surface components during Fc-receptor mediated phagocytosis by macrophages.

Cell Structure and Function 1993, 18:399-407. PubMed Abstract | Publisher Full Text

16. Dudley EM, Wunderlich JR, Robbins PF, Yang JC, et al.: Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes.

Science 2002, 298:850-854. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

17. Pascual CF, Vicente OV, Campos AM, Yañez GJ: In vivo treatment of melanoma (B16F10) with a homeopathic agent and with a cytokine (IFN-?).

Oncology Research 2006, 16(5):211-216. PubMed Abstract

18. Rahman A, Bukhsh K: Towards understanding molecular mechanisms of action of homeopathic drugs: An overview.

Molecular and Cellular Biochemistry 2003, 253:339-345. PubMed Abstract | Publisher Full Text

19. McGill GG, Horstmann M, Widlung HR, Du J, Motyckowa G, Nishimura EK, Lin Y, Ramaswamy S, Avery W, Ding H, Jordan SA, Jackson IJ, Korsmeyer SJ, Golub TR, Fisher DE: Bcl2 regulation by the melanocyte master regulator mitf modulates lineage survival and melanoma cell viability.

Cell 2002, 109:707-718. PubMed Abstract | Publisher Full Text

20. Griffioen M, Kessler JH, Borghi M, Soest RA, et al.: Detection and functional analysis of CD8+ TCells specific for PRAME: a target for T-cell therapy.

Clinical Cancer Research 2006, 12(10):3130-3136. PubMed Abstract | Publisher Full Text

21. Clarke JH, Cha JY, Walsh MD, Gamboni-Robertson F, Banerjee A, Reznikow LL, Dinarello CA, Harken AH, McCarter MD: Melanoma inhibits macrophage activation by suppressing toll-like receptor 4 signaling.

Journal of American College Surgeon 2005, 201(3):418-425. Publisher Full Text

22. Liu YJ, Kanzler H, Soumelis V, Gilliet M: (2001) Dentritic cell lineage, plasticity and cross-regulation.

Nature Immunology 2001, 2(7):585-589. PubMed Abstract | Publisher Full Text

23. Gordon S: Alternative activation on macrophages.

Nature Reviews – Immunology 2003, 3:23-35. PubMed Abstract | Publisher Full Text

24. Demeure CE, Wolfersc J, Martin-Garcia N, Gaulard P, Triebel FT: Lymphocytes infiltrating various tumor types express the MHC class II ligand lymphocyte activation gene-3 (LAG-3): role of LAG-3/MHC class II interactions in cell-cell contacts.

European Journal of Cancer 2001, 37:1709-1718. PubMed Abstract | Publisher Full Text

25. Boon T, Pierre G, Coulie-Benoit J, Eynde V, Bruggen P: Human T cell responses against melanoma.

Annual Review in Immunolology 2006, 24:175-208. Publisher Full Text

26. Liwski RS, Chase JC, Baldrige WH, Sadek I, et al.: Prolonged coestimulation is required for naïve T cell activation.

Immunology Letters 2006, 106:135-143. PubMed Abstract | Publisher Full Text

27. Lanzavecchia A, Sallusto F: Antigen decoding by T lymphocytes: From synapses to fate determination.

Nature Immunology 2001, 2:487-492. PubMed Abstract | Publisher Full Text

28. Fehniger TA, Cooper MA, Caligiuri MA: Interleukin-2 and interleukin- 15: immunotherapy for cancer.

Cytokine and Grown Factor Reviews 2002, 13(2):169-183. Publisher Full Text

29. Watford WT, Moriguchi M, Morinobu A, O’Shea JJ: The biology of IL-12: coordinating innate and adaptive immune responses.

Cytokine & Growth Factor Reviews 2003, 5:361-368. Publisher Full Text

30. Trapani JA: Tumor-mediated apoptosis of cancer-specific T lymphocytes – Reversing the “kiss of death”?

Cancer Cell 2002, 2(3):169-171. PubMed Abstract | Publisher Full Text

31. Szlosarek P, Charles KA, Balkwill FR: Tumor necrosis factor-? as a tumor promoter.

European Journal of Cancer 2006, 42:745-750. PubMed Abstract | Publisher Full Text