Melanosomal localization is required for GIF-2115/2250 to inhibit melanogenesis in B16F10 melanoma cells.

OBJECTIVE: Tyrosinase inhibitors suppress melanogenesis in melanocytes. During a screening for tyrosinase inhibitors, however, we noticed some discrepancies in inhibitory efficacies between melanocytes and in vitro assays. The compound (S)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-indan-1-amine (GIF-2115) exerts antioxidative stress activity upon accumulation in late endosomes and lysosomes. GIF-2115 was also identified as a potent antimelanogenic reagent in B16F10 mouse melanoma cells. GIF-2115 inhibited the activity of mushroom tyrosinase and the lysates of B16F10 cells. However, structure-activity relationship studies indicated that GIF-2238, which lacks the benzene ring in the aminoindan structure of GIF-2115, inhibited tyrosinase activity in vitro but did not inhibit melanogenesis in B16F10 cells. The aim of the present study is to show the importance of the intracellular distribution of tyrosinase inhibitors in exerting their antimelanogenic activity in melanocytes. METHODS: The intracellular distribution of compounds was monitored by linking with the fluorescent group of 7-nitro-2,1,3-benzoxadiazole (NBD). To mislocalize GIF-2115 to mitochondria, the mitochondria-preferring fluoroprobe ATTO565 was used. RESULTS: We reconfirmed the localization of GIF-2250 (GIF-2115-NBD) not only to matured but also to early-stage melanosomes. Although GIF-2286 (GIF-2238-NBD) maintained tyrosinase inhibitory activity, it did not show specific intracellular localization. Moreover, when GIF-2115 was linked with ATTO565, the resultant compound GIF-2265 did not inhibit melanogenesis in B16F10 cells, despite its strong tyrosinase inhibitory activity. CONCLUSION: These results suggest that melanosomal localization is essential for the antimelanogenic activity of GIF-2115, and GIF-2115 derivatives may be new guides for drugs to endosomes and lysosomes as well as melanosomes.

OBJECTIF: Les inhibiteurs de la tyrosinase suppriment la mélanogenèse dans les mélanocytes. Lors d'un criblage d'inhibiteurs de la tyrosinase, cependant, nous avons remarqué des différences dans les efficacités inhibitrices entre les mélanocytes et les essais in vitro. Le composé (S)-N-{3-[4-(diméthylamino)phényl]propyl}-N-méthyl-indan-1-amine (GIF-2115) exerce une activité antioxydante en cas de stress lors de l'accumulation dans les endosomes tardifs et les lysosomes. GIF-2115 a également été identifié comme un puissant réactif antimélanogène dans les cellules de mélanome murin B16F10. GIF-2115 a inhibé l'activité de la tyrosinase de champignon et les lysats des cellules B16F10. Cependant, des études de relation structure-activité ont indiqué que GIF-2238, à qui il manque l'anneau benzénique dans la structure aminoindan de GIF-2115, inhibait l'activité de la tyrosinase in vitro mais n'inhibait pas la mélanogenèse dans les cellules B16F10. L'objectif de la présente étude est de montrer l'importance de la distribution intracellulaire des inhibiteurs de la tyrosinase dans l'exercice de leur activité antimélanogène dans les mélanocytes. MÉTHODES: La distribution intracellulaire des composés a été surveillée en les liant au groupe fluorescent de la 7-nitro-2,1,3-benzoxadiazole (NBD). Pour délocaliser GIF-2115 vers les mitochondries, le fluorophore ATTO565 préférant les mitochondries a été utilisé. RÉSULTATS: Nous avons confirmé la localisation de GIF-2250 (GIF-2115-NBD) non seulement dans les mélanosomes matures mais aussi dans les mélanosomes à un stade précoce. Bien que GIF-2286 (GIF-2238-NBD) ait maintenu une activité inhibitrice de la tyrosinase, il n'a pas montré de localisation intracellulaire spécifique. De plus, lorsque GIF-2115 a été lié à ATTO565, le composé résultant GIF-2265 n'a pas inhibé la mélanogenèse dans les cellules B16F10, malgré son activité inhibitrice de la tyrosinase forte. CONCLUSION: Ces résultats suggèrent que la localisation dans les mélanosomes est essentielle pour l'activité antimélanogène de GIF-2115, et que les dérivés de GIF-2115 peuvent être de nouveaux guides pour les médicaments vers les endosomes et les lysosomes ainsi que les mélanosomes.

Simple methods for measuring milk exosomes using fluorescent compound GIF-2250/2276.

Exosomes are small extracellular vesicles (EVs) found in culture supernatants, blood, and breast milk. The size of these nanocomplexes limits the methods of EV analyses. In this study, nitrobenzoxadiazole (NBD), a fluorophore, conjugated endosome-lysosome imager, GIF-2250 and its derivative, GIF-2276, were evaluated for exosome analyses. A correlation was established between GIF-2250 intensity and protein maker levels in bovine milk exosomes. We found that high-temperature sterilization milk may not contain intact exosomes. For precise analysis, we synthesized GIF-2276, which allows for the covalent attachment of NBD to the Lys residue of exosome proteins, and labeled milk exosomes were separated using a gel filtration system. GIF-2276 showed chromatographic peaks of milk exosomes containing >3 ng protein. The area (quantity) and retention time (size) of the exosome peaks were correlated to biological activity (NO synthesis suppression in RAW264.7 murine macrophages). Heat denaturation of purified milk-derived exosomes disrupted these indicators. Proteome analyses revealed GIF-2276-labeled immunomodulators, such as butyrophilin subfamily 1 member A1 and polymeric immunoglobulin receptor. The immunogenicity and quantity of these factors decreased by heat denaturation. When milk exosomes were purified from market-sourced milk we found that raw and low-temperature sterilization milk samples, contained exosomes (none in high-temperature sterilization milk). These results were also supported by transmission electron microscopy analyses. We also found that GIF-2276 could monitor exosome transportation into HEK293 cells. These results suggested that GIF-2250/2276 may be helpful to evaluate milk exosomes.

Intermittent inhibition of FYVE finger-containing phosphoinositide kinase induces melanosome degradation in B16F10 melanoma cells.

BACKGROUND: Melanosomes are lysosome-related organelles that contain melanogenic factors and synthesize melanin as they mature. FYVE finger-containing phosphoinositide kinase (PIKfyve) regulates late endosome and lysosome morphology, vesicle trafficking, and autophagy. In melanocytes, PIKfyve inhibition has been reported to induce hypopigmentation due to impairments in the metabolism of early-stage melanosomes. METHODS AND RESULTS: Here, we report a new type of melanosome metabolism: post-PIKfyve inhibition, which was found during the characterization of the endosome/lysosome fluoroprobe GIF-2250. In B16F10 mouse melanoma cells, GIF-2250 highlighted vesicles positive for lysosomal-associated membrane protein 1 (lysosome marker) and other endosome/lysosome markers (CD63 and Rab7/9). When cells were continuously treated with PIKfyve inhibitors, intracellular vacuoles formed, while GIF-2250 fluorescence signals diminished and were diffusely distributed in the vacuoles. After removal of the PIKfyve inhibitors, the GIF-2250 signal intensity was restored, and some GIF-2250-positive vesicles wrapped the melanosomes, which spun at high speed. In addition, intermittent PIKfyve inhibition caused melanin diffusion in the vacuoles and possible leakage into the cytoplasmic compartments, and melanosome degradation was detected by a transmission electron microscope. Melanosome degradation was accompanied by decreased levels of melanin synthesis enzymes and increased levels of the autophagosome maker LC3BII, which is also associated with early melanosomes. However, the protein levels of p62, which is degraded during autophagy, were increased, suggesting an impairment in autophagy flux during intermittent PIKfyve inhibition. Moreover, the autophagy inhibitor 3-methyladenine does not affect these protein levels, suggesting that the melanosome degradation by the intermittent inhibition of PIKfyve is not mediated by canonical autophagy. CONCLUSIONS: In conclusion, disturbance of PIKfyve activity induces melanosome degradation in a canonical autophagy-independent manner.

Increased expression of glucagon-like peptide-1 and cystic fibrosis transmembrane conductance regulator in the ileum and colon in mouse treated with metformin.

Metformin, an oral medication, is prescribed to patients with type 2 diabetes mellitus. Although the efficacy, safety, and low economic burden of metformin on patients have long been recognized, approximately 5% of the patients treated with this drug develop severe diarrhea and discontinue the treatment. We previously reported that 1,000 mg·kg-1·day-1 of metformin induced diarrhea in diabetic obese (db/db) mice and wood creosote (traditional medication for diarrhea) ameliorated the symptoms. In this study, we attempted to elucidate the molecular mechanisms by which metformin induces diarrhea. Cystic fibrosis transmembrane conductance regulator (CFTR) is a key ion (chloride) channel in cyclic adenosine monophosphate (cAMP)-induced diarrhea. Metformin treatment increased bile flow (bile acids and bilirubin) in the ileum of mice. In addition, the treatment was accompanied by an increase in mRNA and protein levels of CFTR in the mucosa of the ileum and colon in both wild-type (C57BL/6J) and db/db mice. Glucagon-like peptide-1 (GLP-1), as well as cholic acid, induces CFTR mRNA expression in human colon carcinoma Caco-2 cells through cAMP signaling. Although wood creosote (10 mg/kg) ameliorated diarrhea symptoms, it did not alter the mRNA levels of Glp-1 or Cftr. Similar to overeating, metformin upregulated GLP-1 and CFTR expression, which may have contributed to diarrhea symptoms in mice. Although we could not identify db/db mouse-specific factors associated with metformin-induced diarrhea, these factors may modulate colon function. Wood creosote may not interact with these factors but ameliorates diarrhea symptoms.

Visualization of mitophagy using LysoKK, a 7-nitro-2,1,3-benzoxadiazole-(arylpropyl)benzylamine derivative.

7-Nitro-2,1,3-benzoxadiazole (NBD) is an environmentally responsive fluorophore. We have reported that GIF2114 and GIF2115, anti-ferroptotic N,N-dimethylaniline-compounds, localize to lysosome when they are visualized by NBD. Here we show that the NBD fluorescence of GIF2259, a hybrid derivative of GIF2114 and GIF2115, was quenched in aqueous buffer. However, the fluorescence was recovered when GIF2259 was localized on lysosomes. Although the dimethylamine group of GIF2259 is not essential for the lysosome localization, it contributes to a high specific/nonspecific ratio of fluorescence. Under a normal condition, the lysosomal signal visualized by GIF2259 did not overlap with mitochondria, while, under starved or depolarization conditions, it overlapped with mitochondria, suggesting that GIF2259 could be used as a simple tool for monitoring lysosomal metabolism and mitochondrial turnover, that is mitophagy.