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In vitro and in vivo anticancer properties of a Calcarea carbonica derivative complex (M8) treatment in a murine melanoma model
© Guimarães et al; licensee BioMed Central Ltd. 2010
Received: 23 July 2009
Accepted: 25 March 2010
Published: 25 March 2010
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 had only marginal success. Previous studies in mice demonstrated that a high diluted complex derived from Calcarea carbonica (M8) stimulated the tumoricidal response of activated lymphocytes against B16F10 melanoma cells in vitro.
Here we describe the in vitro inhibition of invasion and the in vivo anti-metastatic potential after M8 treatment by inhalation in the B16F10 lung metastasis model.
We found that M8 has at least two functions, acting as both an inhibitor of cancer cell adhesion and invasion and as a perlecan expression antagonist, which are strongly correlated with several metastatic, angiogenic and invasive factors in melanoma tumors.
The findings suggest that this medication is a promising non-toxic therapy candidate by improving the immune response against tumor cells or even induce direct dormancy in malignancies.
Melanoma represents a significant worldwide public health risk and, from the standpoint of incidence, is the fastest growing of all cancer types. Malignnant melanoma is the most aggressive form of skin cancer, with a mortality rate that has risen about 2% annually since 1960. Although early stage melanoma can be cured surgically, once melanoma metastasizes to major organs (stage IV), it is almost always incurable . There are few chemotherapeutic agents available for treating metastatic melanoma, and those that have been used have all yielded unsatisfactory results. No single chemotherapeutic agent currently offers a response rate greater than 25%, and treatment is invariably accompanied by significant side effects, including myelosuppression, nausea and emesis [2–4].
Malignant transformation could be associated with general enzymatic changes leading to increased proteolytic and fibrinolytic activity in tumor cells. The significance of angiogenesis in tumor development and metastasis is well established, and it was previously confirmed that a significant correlation exists between tumor angiogenesis and the ability of a melanoma to metastasize . It has been observed that heparan sulfate proteoglycan expression, especially perlecan expression, is a prerequisite for melanoma tumor growth and metastasis [6, 7].
Immunotherapy remains the subject of intense investigation in both adjuvant and advanced disease settings, and attempts are being made to target melanoma defense mechanisms that blunt the effectiveness of host immune responses . Many natural compounds have been extensively studied to determine a possible anti-tumor effect. Our previous studies have demonstrated that a high diluted natural complex activates macrophages both in vivo and in vitro [9–11]. Moreover, that natural complex was neither toxic nor mutagenic . Similarly, an improvement of the immune response of treated mice has been demonstrated in studies with Sarcoma-180, including a significant infiltration of lymphoid cells, granulated tissue, fibrosis development around the tumor, sarcoma size reduction and an increase in the number of circulating leukocytes, such as B, NK and CD4+ cells . These results suggest that the medication has a direct or indirect action on hematopoiesis. A subsequent microscopic study of bone marrow cells showed that monocytic lineage (CD11b+) and stromal cells (adherent cells) were activated by treatment [14–16]. A previous study with an in vitro model showed that a specific high diluted complex derived from Calcarea carbonica and associations (here defined as M8 in the Materials and Methods section) activated lymphocytes even without direct contact with macrophages. A co-culture with macrophages and lymphocytes in the presence of M8 promoted stimulation of lymphocytes, resulting in an enhanced tumoricidal performance against a very aggressive lineage of melanoma cells .
We previously reported that M8 showed a promising capacity to stimulate immune cells against melanoma cells in vitro. In the present work we aimed to study the effects of M8 in vivo using a melanoma metastasis mouse model. We assessed blood and bone marrow cells immunophenothyping, tumor histopathology, immunohistochemistry, and ultrastructural analysis.
High diluted natural complexes represent a new form of immunomodulatory therapy and follow Hahnemann's ancient homeopathic techniques for dilution. Mother tinctures were purchased from authorized agencies sanctioned by the Brazilian Health Ministry. These agencies assure the quality (endotoxin free) and physico-chemical composition of their products. Starting from the original mother tincture -- an ethanolic extract in this case -- several dynamizations/succussion (shaking) and serial dilutions in distilled water were performed. The medication used to treat in vitro and in vivo models was a complex matrix obtained from Calcarea carbonica CH5 with associations, comprised of a 10%-20% concentration of each compound obtained after vigorous shaking. In addition to Calcarea carbonica, the final solution contained Aconitum napellus, Arsenicum album, Asa foetida, Conium maculatum, Ipecacuanha, Phosphorus, Rhus tox, Silicea, Sulphur, and Thuya occidentalis, all in decimal dilutions of Hahnemann (dH) in distilled water. The resulting aqueous solution was colorless and odorless, and had 1% alcohol concentration. This complex as well its vehicle (hydroalcoholic solution) used as control were sterilized by filtration through 0.22 μm MILLEX GV Durapore PVDF membranes (Millipore, Billerica, MA, USA), maintained at room temperature and vigorously shaken (succussed) immediately before each treatment. Since all experiments were performed in a double blind and randomized manner, the initial code applied for this studied complex (M8) remains in the Results/Discussion sections of this article.
For in vivo experiments, 2-3 month old male C57BL/6 mice were purchased from Central Animal House of the Federal University of Paraná (UFPR). All recommendations of the National Law (No. 6.638, November, 5, 1979) for scientific management of animals were followed and the Institutional Animal Care Committee of UFPR approved all related practices. All experiments were performed at least three times in quadruplicate and data analysis was performed in a double-blinded manner. Experiments were carried out at Laboratório de Pesquisa em Células Neoplásicas e Inflamatórias, UFPR, Brazil, and at Cell Biology Unit, IPMon, Uruguay.
Murine melanoma cells (B16F10 - ATCC: CRL-6475; mouse melanoma cell), were used for this study. Cells were maintained in DMEM containing 10% FBS, 1 U/mL penicillin, 1 μg/mL streptomycin, and 2.5 μg/mL amphotericin at 37°C in a humidified 5%CO2 atmosphere.
MxRage 7 cells
Transformed murine embryonic fibroblasts consist of an indicator cell line in which Cre-recombinase is driven by the IFN-inducible Mx1 promoter that, when activated, deletes a stop cassette upstream of the eGFP coding region, resulting in the expression of eGFP. The percentage of eGFP expressing cells, determined by fluorescence activated cell sorting (FACS), accurately correlates to the amount of type I IFN added to the culture . This cell line was maintained at 32°C in a humidified 5% CO2 atmosphere, using DMEM (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (GIBCO).
Human colon-rectal cancer cells (ATCC: HTB-38) were stably transfected with the pNF-κB-hrGFP Plasmid from the PathDetect Signal Transduction Pathway cis-Reporting Systems Kit (Stratagene) Briefly, subconfluent HT29 cells were transfected with pNF-κB-hrGFP plasmid using Lipofectamine 2000 (Invitrogen) and selected with hygromycin. After two weeks, cells were stimulated for 24 h with a pro-inflammatory cocktail (25 ng/mL TNF-α, 1.25 ng/mL IL-1β and 3.75 ng/mL IFN-γ) and GFP positive cells were sorted with a MoFlo cell sorter (Dako, Carpinteria, CA). This sorted cell line was maintained in RPMI (GIBCO) containing 10% FBS, 1 U/mL penicillin, 1 μg/mL streptomycin at 37°C in a humidified 5% CO2 atmosphere.
All in vitro experiments were performed at 37°C in a humidified 5% CO2 atmosphere for 48 h. All treatments were administered to log-phase growing cells and the different assayed conditions were divided into: a) cells without treatment (control culture conditions, named "Control"); b) cells treated with vehicle (1% hydroalcoholic solution, named "Vehicle"); or c) cells treated with M8 (named "M8"). An initial dose of 20% of M8 treatment was administered to the cells and, after 24 h, a reinforcement dose of 1% was administered according to a previous standard treatment protocol [10, 16].
Experiments on in vitro reporter cells (MxRAGE 7 and HT29-pNF-κB-hrGFP)
The reporter cell lines MxRAGE 7 and HT29-pNF-κB-hrGFP are routinely used at CBU (IPMon) to screen natural or synthetic compounds that interfere with type I IFN signaling pathway and/or modulate NF-κB activity. For the IFN assay, exponentially growing MxRage 7 cells were seeded in a 96-well plate and grown for 24 h at 32°C. The cells were then cultured for 48 h in presence or absence of fixed amounts of murine IFN-α11, with or without the vehicle or M8. Finally, cells were harvested using trypsin, resuspended in PBS and stained with 7-AAD to identify dead cells. Ten thousand events were acquired and analyzed on a CyAn™ ADP Flow Cytometer (Dako, Carpinteria, CA) using Summit v4.3 software. For the NF-κB activation assay, exponentially growing HT29 pNF-κB-hrGFP cells were treated cultured for 24 h in absence or presence of 3 ng/mL TNF-α, with or without the vehicle or M8 complex, and the percentage of positive GFP cells and the viability were determined using CyAn™ ADP Flow Cytometer (Dako, Carpinteria, CA) and Summit v4.3 software.
In vitro B16F10 cell experiments
Invasion activity of melanoma cells was assayed in a transwell cell culture chamber as previously described , with some modifications. Briefly, polyvinyl-pyrrolidone-free polycarbonate inserts with an 8.0 μm pore size (Corning-Costar, Cambridge, MA, USA) were pre-coated with 5 μg of fibronectin on the reverse side and dried at room temperature. Matrigel (containing laminin, collagen type IV, heparan sulfate proteoglycan and entactin from BD Pharmigen, San Diego, CA, USA) was diluted to 500 μg/mL with cold PBS, applied to the upper surface of the filter (5 μg/filter), and dried at room temperature. Log-phase growing B16F10 cultures were harvested by trypsin treatment, washed twice and re-suspended to give a final concentration of 2.0 × 106 cel/mL in serum free DMEM medium supplemented with 0.1% bovine serum albumin (BSA, Sigma-Aldrich Chemical Co., St. Louis, MO, USA). Cell suspensions (100 μl) were added to the upper compartment of the insert chamber and incubated for 6 h at 37°C in a humidified 5% CO2 atmosphere in the presence or absence (control) of M8 or its vehicle. Cells derived from the different assayed conditions were processed for scanning electron microscopy (SEM). Cell-containing filters were fixed with 2.5% glutaraldehyde (0.1 M cacodylate buffer, pH 7.2), washed with PBS and post-fixed in 1% OsO4 for 30 min in the dark at room temperature. After washing, the cells were dehydrated using increasing ethanol concentrations. Filter membranes were CO2 critical point dehydrated, metalized and observed using a JEOL JSM-6360 LV SEM.
Cultured B16F10 cells were analyzed by flow cytometry for cadherin, CD74 and 7-Amino-actinomycin D (7-AAD) as viability markers and propidium iodide (PI) as a cell cycle marker. Log-phase growing B16F10 cultures were harvested by trypsin digestion, washed twice and re-suspended in PBS containing 1% FBS. The harvested cells (106) were incubated with anti-CD74/FITC (BD Pharmingen, San Diego, CA, USA) in PBS containing 1% FBS for 30 min and washed three times with PBS. For intracellular CD74 and cadherin, a simultaneous incubation was performed with anti-CD74/FITC (BD Pharmingen) and anti-cadherin/FITC (Vector Labs, Burlingame, CA, USA) antibodies using 0.01% saponin in the incubation buffer. Cells were also stained with 7-AAD (BD Pharmingen) for 5 min to estimate the number of dead cells. Cell cycle was determined as previously described . Log-phase growing B16F10 cultures were detached by trypsin treatment, washed twice with PBS and fixed in chilled 70% ethanol. After centrifugation, the fixed cell pellet was treated with RNAse at a concentration of 50 μg/mL (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and stained with 50 μg/mL propidium iodide (Sigma-Aldrich) for 10 min at room temperature. For all flow cytometry experiments, ten thousand events were acquired on a FACSCalibur using the CellQuest software (Becton-Dickinson), and data were analyzed using WinMDI 2.9 software.
B16F10 lung metastasis and in vivo treatment
Blood leukocytes and bone marrow cell analysis
For immunophenotyping analyses, blood aliquots containing 106 leucokytes were incubated for 10 min with PharM Lyse (BD) for red blood cells lysis, followed by an 30 min incubation with anti-CD3/FITC, anti-CD4/PE, anti-CD8/PE, anti-CD11b, anti-CD16/32 (Fc block), anti-CD19/PE, anti-CD45 and anti-Pan-NK/PE. All antibodies were monoclonal antibodies (MAbs) from BD Pharmingen and were diluted in PBS with 1% FBS. After the MAbs binding, cells were washed three times with PBS and analyzed by flow cytometry using a FACSClibur (BD). For each sample, ten thousand events were acquired using the CellQuest software (BD) and data analysis was performed using WinMDI 2.9 software. FITC and PE isotype controls (BD Pharmingen) were used to calibrate the cytometer before the experimental sample acquisition. For bone marrow cells, the protocol was performed as previously described . Briefly, femurs were dissected and cleaned. Epiphyses were removed and the marrow was flushed with DMEM (GIBCO). Cells (106) were fixed with 1% paraformaldehyde, washed, and incubated for 40 min with biotinylated antibodies from a Mouse Lineage Panel Kit (BD, Pharmingen): anti-CD3, anti-B220, anti-Ly6G, anti-TER119, anti-CD11b, anti-CD11c. Samples were then washed with PBS and incubated with 0.5 μg/mL of streptavidin-PE (BD, Pharmingen) in PBS for 30 min and subsequently, cells were acquired and analyzed for blood samples as described. Blood aliquots were also stained with May-Gründ-Giemsa (InstantProv Kit, NewProv, Brazil), allowing for differential counting of basophils, neutrophils, eosinophils and monocytes by light microscopy.
Lung histopathology, immunohistochemistry, and ultrastructural pathology
After blood sampling, lungs were dissected, washed once with PBS and fixed by immersion in 4% paraformaldehyde in PBS for 1 hr. After washing with PBS, lungs were dehydrated using increasing ethanol concentrations, xylene and embedded in paraffin. For histopathology analysis, 5 μm sections of lungs were deposited on silane coated slides. Sections were deparaffinized in xylene, hydrated in alcohol, and incubated in 3% H2O2 in methanol to block endogenous peroxidase activity. A Retrievagen A (pH 6.0) kit (BD Pharmingen) was used for antigen unmasking, nonspecific binding was blocked by 1% BSA-PBS solution and possible aldehyde groups were blocked by 50 mM glycine-PBS. Anti-CD11c, anti-GR-1 (BD) and anti-perlecan proteoglycan (Affinity Bio-reagents), as well as the isotype controls (BD Pharmingen), were diluted 1:50 in 0.1% BSA/PBS and incubated on slides in a humidified chamber for 2 hr. After washing with PBS, the respective secondary biotinylated antibody (BD Pharmingen) was added at the same dilution and incubated for 1 hr more, then washed, followed by an incubation step with streptavidin-HSP (BD Pharmingen) for 1 hr. After washing with PBS, MAbs binding was revealed with a DAB Substrate kit (BD Pharmingen). Slides were stained with Giemsa (EMS) and rapidly dehydrated in ethanol, xylene and then coverslips were mounted with entellan. Sections were scanned with a Nikon Eclipse E200 microscope and the areas of the highest immunostaining were imaged at (12.5 × 100)× magnification. CD11c and GR-1 positive spots were counted in 10 chosen fields with highest density. Perlecan expression in tumor zones was quantified by color area measurement by ImageJ software (NIH) to distinguish DAB stain from melanin, which turns color from brown to dark green after Giemsa stain . For conventional histopathology analysis, a periodic acid-Schiff (PAS) stain was performed on the same samples and sections were scanned by a microscope at (12.5 × 40)× or 100× magnification.
For transmission electron microscopy, a rapid protocol was performed as previously described . Briefly, small pieces of lung tissue (1 mm3) containing melanoma nodules were fixed for 20-30 min with Karnovsky's fixative (2% glutaraldehyde, 4% paraformaldehyde, 5 mM CaCl2, in 0.1 M cacodylate buffer, pH 7.2 - 7.4); they were then washed with the same buffer and post-fixed with 1% osmium tetroxide, 1 mM CaCl2, 0.8% potassium ferricyanide in 0.1 M cacodylate buffer (pH 7.4) for 15 minutes and then rinsed twice for 1 min with the same buffer. The lung pieces were dehydrated with acetone 50%, 70%, 90% and 100% (2×) for 3 minutes each. The tissues were transferred to bean capsules containing 90% acetone and infiltrated in epoxy resin (Epon)/acetone solution (1:1) for 2 hr, then in pure Epon for 4 hr and, lastly, embedded in a new Epon solution overnight. Polymerization was carried out for 48 hr at 60°C. Ultra-thin sections were stained with aqueous uranyl acetate for 15 min and with lead citrate for 2 min. Samples were visualized with a Jeol JEM 1011 transmission electron microscope. A GATAN CCD camera and GATAN digital micrograph software were used to obtain the digital images.
Micrographs and statistics analysis
Micrographs obtained from immunohistochemistry and electron microscopy were analyzed by ImageJ software (NIH) to obtain mean/pixel values from the specific threshold of transwell insert pores or IHC chromogen. Data obtained from assays were transformed to conform to a normal distribution using the equation transformed data = . Statistical significance of transformed data was determined using a one-way analysis of variance (ANOVA), followed by Tukey post test. Statistical significance is presented as either P < 0.05 (*) or P < 0.01 (**). Data are representative of three independent experiments performed in triplicate.
M8 in vitro effects on B16F10 cells
In vivoevaluation of M8 treatment in lung metastasis
Peripheral blood and bone marrow cell subpopulation analysis after M8 treatment of mice with lung metastasis
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 [24–26].
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 [9–11]. Many studies have demonstrated the role of different high diluted complexes in cancer therapy immunomodulation [13, 17, 27, 28]. Regarding M8 (Calcarea carbonica and associations), our previous results showed that in vitro treatment significantly increased macrophages/lymphocyte interaction and effectiveness against melanoma cells . 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, 30]. 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 2).
Since interferon type I based immunostimulation has been studied as an effective immunotherapy for melanoma cases , we further investigated the in vitro effect of M8 on type I IFN activity using MxRage reporter cells . 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 . 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 3 and 4). 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 . In a C57BL/6 mice/B16F10 model, there was no significant difference in lymphocyte level from blood and bone marrow (Figure 6 and 7). 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 . 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 . 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 . There was no significant difference in bone marrow leukocytes number, but there was a clear tendency for CD4 and CD11c markers (Figure 7) 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 2 and 9). 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, 37]. 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, 38]. Despite this, the inhibition of adhesion of extracellular matrix molecules in melanoma cells causes cell rounding, loss of survival and apoptosis . 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 2).
Taken together, these results may explain the possible tumor cell dormancy and decrease in tumor nodule number and volume caused by M8 treatment. These findings suggest that M8 is a promising therapy that may improve the innate immune response against tumor cells, decreasing the cell adhesion or invasion of malignant cells or even inducing direct dormancy in malignancies. Further studies are necessary to clarify the precise and detailed mechanism of M8 treatment in anti-tumor effects in melanoma and its use as combination-therapy candidate used in addition with conventional medicines.
We thank CNPq, Fundação Araucária, and SETI-PR for financial support. We are especially grateful to Centro de Microscopia Eletrônica at UFPR and Laboratório Central de Microscopia Eletrônica at UFSC, and to UATE at IPMon, Uruguay.
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