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IMMUNEPOTENT CRP is a dialyzable leukocyte extract obtained from bovine spleen with immunomodulatory and antitumour properties; therefore, when administrated as an adjuvant therapy for cancer patients, it has increased their survival and quality of life. The bioavailability of any orally administered compound can be reduced due to gastrointestinal enzymes. In this study, we evaluated if IMMUNEPOTENT CRP is resistant to the treatment with different enzymes (proteases, nucleases, polysaccharide-degrading enzymes or lipase), using as parameters for biological activity measurement its in vitro antitumour and antioxidant properties and in vivo the antitumour effect of IMMUNEPOTENT CRP treated with proteinase K. In conclusion, we consider necessary to include the antioxidant and cytotoxic activity on the MCF-7 cancer cell line as parameters for the quantitative determination of biological activity or potency tests for batch release. Additionally, the results showed that different enzymatic treatments do not affect the antitumour and antioxidant activities of IMMUNEPOTENT CRP in vitro, suggesting that this product can be administered orally without any loss of biological activity. Furthermore, IMMUNEPOTENT CRP treatment with proteinase K increases the antitumour activity in vivo.

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Our main focus was to evaluate the efficacy of silver and sodium dichloroacetate as dual-function agents to be used in melanoma treatment. This strategy is designed to increase the activity of these two compounds that affect DNA integrity and the mitochondria at different levels. Furthermore, we evaluated if the cell death mechanism induced by our treatments was immunogenic cell death. To evaluate antitumor efficacy, we assessed tumor volume and production of tumor necrosis factor-α, nuclear factor κ B (both by ELISA), and nitric oxide levels (Nitrate/Nitrite colorimetric assay kit); for immunogenic cell death, we evaluated the release of danger-associated molecular patterns using immunohistochemistry and flow cytometry, as well as an in vivo challenge. Our results showed that the combination of colloidal silver and sodium dichloroacetate is more effective than each treatment alone and that the antitumor mechanism is not through immunogenic cell death. Furthermore, this study can broadly contribute to the development of dichloroacetate-loaded silver nanoparticles and to the design targeted pharmacological formulations to fight melanoma as well as other types of cancer.