Nilotinib induction of melanogenesis via reactive oxygen species-dependent JNK activation in B16F0 mouse melanoma cells.

Nilotinib (AMN), a second-generation tyrosine kinase inhibitor, induces apoptosis in various cancer cells, and our recent study showed that AMN effectively reduced the viability of human ovarian cancer cells via mitochondrion-dependent apoptosis. The effect of AMN in the melanogenesis of melanoma cells is still unclear. In the present study, we found that the addition of AMN but not imatinib (STI) significantly increased the darkness of B16F0 melanoma cells, and the absorptive value increased with the concentration of AMN. A decrease in the viability of B16F0 cells by AMN was detected in a concentration-dependent manner, accompanied by increased DNA ladders, hypodiploid cells and cleavage of the caspase-3 protein. An in vitro tyrosinase (TYR) activity assay showed that increased TYR activity by AMN was detected in a concentration-dependent manner; however, induction of TYR activity by STI at a concentration of 40 µmol/L was observed. Increased intracellular peroxide by AMN was detected in B16F0 cells, and application of the antioxidant, N-acetylcysteine (NAC), significantly reduced AMN-induced peroxide production which also reduced the darkness of B16F0 cells. Additionally, AMN induced c-Jun N-terminal kinase (JNK) protein phosphorylation in B16F0 cells, which was inhibited by the addition of NAC. AMN-induced melanogenesis of B16F0 cells was significantly inhibited by the addition of NAC and the JNK inhibitor, SP600125 (SP). Data of Western blotting showed that increased protein levels of melanogenesis-related enzymes of tyrosinase-related protein-1 (TRP1), TRP2 and TYR were observed in AMN-treated B16F0 cells which were inhibited by the addition of NAC and SP. Evidence is provided supporting AMN effectively inducing the melanogenesis of B16F0 melanoma cells via reactive oxygen species-dependent JNK activation.

Imatinib mesylate induction of ROS-dependent apoptosis in melanoma B16F0 cells.

BACKGROUND: Imatinib mesylate (STI571), a protein tyrosine kinase inhibitor, was shown to reduce the viability of several cancer cell lines via apoptosis induction; however, the role of reactive oxygen species (ROS) in STI571-induced melanoma cell apoptosis is still undefined. OBJECTIVE: In this study, we investigated the contribution of ROS to STI571-induced apoptosis in melanoma B16F0 cells, and the apoptotic mechanism elicited by STI571 was illustrated. METHODS: Using an in vitro cell culture system, the effects of STI571 on ROS production, cell cycle progression, caspase activation, and mitochondrial functions were examined via Western blotting, a flow cytometric analysis, an enzyme activity assay, and a DNA integrity assay. In pharmacological studies, the ROS scavenger, N-acetyl cysteine (NAC), the NADPH oxidase inhibitor, dipheylene iodide (DPI), and mitogen-activated protein kinase (MAPK) inhibitors (PD98059, SP600125, and SB203580) were applied to investigate the mechanism. RESULTS: STI571 reduced the viability of melanoma cells B16F0, but not human skin fibroblasts WS1, via apoptosis induction. Besides, apoptosis induced by STI571 was inhibited by the addition of NAC and DPI, and an increase in the intracellular peroxide level by STI571 was identified in melanoma B16F0 cells. Activation of caspases 3 and 9 enzyme activities accompanied by disrupting the mitochondria membrane potential in according with stimulating JNK and p38 protein phosphorylation was identified in STI571-treated B16F0 cells. STI571-mediated a ROS-dependent apoptosis potentiated by JNK inhibitor SP600125 was first identified in melanoma B16F0 cells. CONCLUSION: Our results support the idea that ROS-dependent apoptosis in STI571-treated melanoma cells B16F0. The combination of a JNK inhibitor with STI571 for treating melanomas is suggested for further in vivo studies.

The wrinkles soothing effect on the middle and lower face by intradermal injection of botulinum toxin type A.

BACKGROUND: Botulinum toxin type A (BTA) has been used on facial expressive muscles to reduce wrinkles. We assumed that intradermal injection with BTA on the middle and lower face can produce face-lifting and wrinkles-soothing effect by promoting collagen synthesis. METHODS: Nine volunteers were included to undergo intradermal injections of a total dose of 20~25 U BTA into one-half of the face, and normal saline into the other half as control. Clinical photographs were taken every 4 weeks for 16 weeks, and evaluated by a doctor blinded to the assignments. Patients graded the results of wrinkles elimination on each follow-up. Skin biopsies were taken before the treatment and 8 weeks after the injection. The specimens were assessed by a pathologist using hematoxylin and eosin stain, elastin stain, Masson trichrome stain and immunohistochemical stain with type I procollagen. RESULTS: By photographic documentation, there was no significant face-lifting effect. However, there was statistical significance in wrinkles reduction on the BTA sides compared to pre-treatment. Subjectively, six subjects noticed better wrinkles soothing effect on the BTA sides. This effect was noted as early as 4 weeks after injection, and lasted for a minimum 8 weeks. The histologic examination revealed slight neocollagen synthesis by Masson trichrome stain on both sides. CONCLUSIONS: Based on this pilot study, the face-lifting effect of intradermal injection with BTA was not conclusive. Interestingly, BTA showed moderate but significant wrinkles-soothing effect without obvious side effects on the lower face.