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. There is little basic research on the effectiveness of alternative and complementary therapies in cancer, and the few studies that have been performed were limited in scope. Beside this, immunostimulation by natural products has been attempted in various animal models and in human cancer patients as an adjunct to chemotherapy [24
A high diluted complex treatment in macrophages has been shown to suppress previously elevated levels of tumor necrosis factor-α (TNF-α), increase the activity of NADPH oxidase and the expression of inducible nitric oxide synthase (iNOS), and induce differential gene expression [9
]. Many studies have demonstrated the role of different high diluted complexes in cancer therapy immunomodulation [13
]. Regarding M8 (Calcarea carbonica
and associations), our previous results showed that in vitro
treatment significantly increased macrophages/lymphocyte interaction and effectiveness against melanoma cells [17
]. Extending these previous data to the in vivo
immunotherapeutic effect of this compound, we now demonstrate that M8 therapy prevents tumor growth and metastasis.
Progression of melanoma and other malignant cancers involves cellular changes such as the loss of E-cadherin expression and the gain of CD74 expression, which confer cell motility and immunologic escape, respectively [29
]. However, while both molecules were analyzed in the present study, no differences were found after M8 treatment (data not shown). Despite this finding, scanning electron micrographs analysis clearly showed the loss of adhesion of B16F10 cells, demonstrated by a decreased cell number on the matrigel subtract, and the permanence of non-degraded matrigel obstructing the pores in the cells treated with M8, leading to a diminished number of exposed pores (Figure ).
Since interferon type I based immunostimulation has been studied as an effective immunotherapy for melanoma cases [31
], we further investigated the in vitro
effect of M8 on type I IFN activity using MxRage reporter cells [18
]. When these cells were exposed to M8, no agonist or antagonism capacity was observed (Additional file 1
: Supplementary Figure A). Moreover, NF-κB has been reported to increase tumourigenesis by promoting anti-apoptotic activity, chemotherapy resistance, the expression of positive cell cycle regulators and the expression of other survival factors [32
]. However, no agonistic or antagonistic NF-κB activation was found after M8 treatment in the HT29 reporter model (Additional file 1
: Supplementary Figure B).
On the other hand, lung metastasis regression was observed in vivo
after M8 treatment (Figure and ). In order to evaluate the mechanism of action of M8 in vivo
, the level of immune cells was analyzed. Previous studies have shown that this category of medicament increases blood lymphocytes such as TCD4, TCD8 and NK, which are associated with tumor regression in an albino Swiss mice/Sar-180 model [13
]. In a C57BL/6 mice/B16F10 model, there was no significant difference in lymphocyte level from blood and bone marrow (Figure and ). However, the difference between the results obtained with the C57BL/6 strain and albino Swiss strain may be because the first mouse model has an intrinsic and well established innate and Th1 response that limits its use immunological questions [33
]. On the other hand, it is the most used murine model to simulate metastatic melanoma because it reflects several characteristics of human metastatic melanoma, and because B16F10 cell inoculation is only compatible with this strain [34
]. An increase in the number of circulating basophils and neutrophils was observed after M8 treatment. Basophils and neutrophils have been recently appointed as promissory innate cells to be targeted for anti-cancer treatment because are the first cells able to detect tissue abnormalities as tumor necrosis or growth damages tissue [35
]. There was no significant difference in bone marrow leukocytes number, but there was a clear tendency for CD4 and CD11c markers (Figure ) to be close to the levels seen in normal mice, demonstrating a "normalization" of bone marrow cell production.
This study provides some information to clarify the mechanism by which M8 treatment affects melanoma metastasis in this model. We observed a decrease in perlecan expression and a direct inhibition of cancer cell adhesion and invasion (Figure and ). Perlecan is an important component of basement matrix and its expression correlates strongly with the expression of several metastatic, angiogenic and invasive factors in tumor cells, particularly melanoma cells. Tumor-derived perlecan is distributed throughout its matrix and creates a microenvironment that favors neovascularization, tumor growth and invasion [36
]. Perlecan has been identified as a potential therapeutic target for the treatment of metastatic cancer because it is a necessary molecule for tumor vessels' structural integrity and forms the major storage site of neovascularization factor FGF-2 [7
]. Despite this, the inhibition of adhesion of extracellular matrix molecules in melanoma cells causes cell rounding, loss of survival and apoptosis [39
]. This result is corroborated by the invasion assay, which showed a loss of adhesion of B16F10 cells to the Matrigel substrate because of a decrease in the ECM proteolysis ability of the cells (Figure ).