GIF-2209, an Oxindole Derivative, Accelerates Melanogenesis and Melanosome Secretion via the Modification of Lysosomes in B16F10 Mouse Melanoma Cells

Melanogenesis and melanosome secretion are regulated by several mechanisms. In this study, we found that the oxindole derivative GIF-2209 accelerated melanogenesis associated with the discrimination in the expression and intracellular distributions of two melanogenic enzymes, tyrosinase (TYR) and tyrosinase-related protein-1 (TYRP-1). GIF-2209 upregulated the expression of TYR via a microphthalmia transcription factor (MITF)-independent mechanism, leading to high expression of protein. In contrast, GIF-2209 did not alter the mRNA levels of TYRP-1 and suppressed its protein levels. GIF-2209 induced the dissociation of TYR from TYRP-1 but did not alter the association between TYR and CD63, a melanosome and lysosome marker. The protein levels of CD63 were also upregulated by GIF-2209. GIF-2209 induced lysosome expansion and redistribution in all areas of the cytosol, accompanied by autophagy acceleration (upregulation of LC3BII protein levels and downregulation of p62 protein levels). In addition, GIF-2209 stimulated the secretion of melanosomes containing high levels of TYR, TYRP-1, and CD63 proteins. The GIF-2209 mediated melanosome secretion was sensitive to the lysosome inhibitor chloroquine. These results suggest that GIF-2209 may activate lysosomal functions with TYR gene expression, while it accelerates melanosome secretion, which finally leads to the depletion of intracellular melanogenic enzyme, especially TYRP-1 protein.

In vivo treatment with M8, a highly diluted tinctures complex, reduced the malignancy of a mouse melanoma model

Background: Cancer is a class of disease responsible for 13% of death cause worldwide. Among all types of cancers, one of the most aggressive and with the highest death rate is melanoma. It is highly metastatic and current treatments with chemotherapeutic drugs do not yield satisfactory results. Therefore, the interest on new therapeutics for cancer treatment has been increasing on research. Highly diluted tinctures (HDT) are intended to enhance immune system responses resulting in reduced frequency of various diseases, and often present no risk of serious side-effects due to its low toxicity. Previous results have demonstrated in vitro inhibition of invasion ability and in vivo anti-metastatic potential of B16F10 lung metastasis model after mice treatment with M8 inhalation. Aims: Now we have evaluated M8 effects on hyaluronic acid and its specific melanoma cell surface receptor (CD44) expression on lungs after inhalation by mice. Methodology: M8 compounds include Aconitum napellus 20dH, Arsenicum album 18dH, Asa foetida 20dH, Calcarea carbonica 16dH, Conium maculatum 17dH, Ipecacuanha 13dH, Phosphorus 20dH, Rhus toxicodendron 17H, Silicea 20dH, Sulphur 24dH, and Thuja occidentalis 19dH. B16F10 Melanoma cells were inoculated into C57B/L6 mouse lateral tail vein. Treatment started 24 hours after inoculation, and was repeated after each 12 hours during 14 days on an inhalation chamber that is adapted to little rodents. Mice were subjected to euthanasia by intraperitoneal injection of thiopental followed by decapitation. Lungs were surgically removed and analyzed under a stereomicroscope for the presence of metastatic foci. They were formaldehyde fixed, dehydrated and paraffin embedded. Histological sections were processed for hematoxilin/eosin (HE), Fontana-Masson and immunohistochemistry staining methods. Images were captured and blindly analysed by ImageJ (NIH) software. Results: HE and Fontana-Masson showed a reduction in number and size of metastatic nodules, as previously demonstrated. We have detected a reduction on hyaluronic acid as well as CD44 expression on mice lungs after M8 treatment. The high metastatic potential of melanoma is proportional to hyaluronic acid expression level, together with its specific cell surface receptor, the CD44. These results suggest that M8 treatment reduces malignancy of mouse melanoma through modulation of hyaluronic acid and CD44 expression, which play crucial roles in tumor invasion and growth. Conclusion: Even though further investigation are necessary to elucidate the mechanisms of action of M8 treatment there is an indication that these highly diluted tinctures could be a promising therapy to treat metastatic melanoma.

Novel Chemically Modified Curcumin (CMC) Derivatives Inhibit Tyrosinase Activity and Melanin Synthesis in B16F10 Mouse Melanoma Cells

Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its use, which has inspired synthesis of curcumin analogs. Here, we investigated four novel chemically modified curcumin (CMC) derivatives (CMC2.14, CMC2.5, CMC2.23 and CMC2.24) and compared them to the parent compound curcumin (PC) for inhibition of in vitro tyrosinase activity using two substrates for monophenolase and diphenolase activities of the enzyme and for diminution of cellular melanogenesis. Enzyme kinetics were analyzed using Lineweaver-Burk and Dixon plots and nonlinear curve-fitting to determine the mechanism for tyrosinase inhibition. Copper chelating activity, using pyrocatechol violet dye indicator assay, and antioxidant activity, using a DPPH radical scavenging assay, were also conducted. Next, the capacity of these derivatives to inhibit tyrosinase-catalyzed melanogenesis was studied in B16F10 mouse melanoma cells and the mechanisms of inhibition were elucidated. Inhibition mechanisms were studied by measuring intracellular tyrosinase activity, cell-free and intracellular α-glucosidase enzyme activity, and effects on MITF protein level and cAMP maturation factor. Our results showed that CMC2.24 showed the greatest efficacy as a tyrosinase inhibitor of all the CMCs and was better than PC as well as a popular tyrosinase inhibitor-kojic acid. Both CMC2.24 and CMC2.23 inhibited tyrosinase enzyme activity by a mixed mode of inhibition with a predominant competitive mode. In addition, CMC2.24 as well as CMC2.23 showed a comparable robust efficacy in inhibiting melanogenesis in cultured melanocytes. Furthermore, after removal of CMC2.24 or CMC2.23 from the medium, we could demonstrate a partial recovery of the suppressed intracellular tyrosinase activity in the melanocytes. Our results provide a proof-of-principle for the novel use of the CMCs that shows them to be far superior to the parent compound, curcumin, for skin depigmentation.