Multifunctional N-P-doped carbon dots for regulation of apoptosis and autophagy in B16F10 melanoma cancer cells and in vitro imaging applications

Abstract

Rationale: The present study reports the multifunctional anticancer activity against B16F10 melanoma cancer cells and the bioimaging ability of fluorescent nitrogen-phosphorous-doped carbon dots (NPCDs). Methods: The NPCDs were synthesized using a single-step, thermal treatment and were characterized by TEM, XPS, fluorescence and UV-Vis spectroscopy, and FTIR analysis. The anticancer efficacy of NPCDs was confirmed by using cell viability assay, morphological evaluation, fluorescent live-dead cell assay, mitochondrial potential assay, ROS production, RT-PCR, western-blot analysis, siRNA transfection, and cellular bioimaging ability. Results: The NPCDs inhibited the proliferation of B16F10 melanoma cancer cells after 24 h of treatment and induced apoptosis, as confirmed by the presence of fragmented nuclei, reduced mitochondrial membrane potential, and elevated levels of reactive oxygen species. The NPCDs treatment further elevated the levels of pro-apoptotic factors and down-regulated the level of Bcl2 (B-cell lymphoma 2) that weakened the mitochondrial membrane, and activated proteases such as caspases. Treatment with NPCDs also resulted in dose-dependent cell cycle arrest, as indicated by reduced cyclin-dependent kinase (CDK)-2, -4, and -6 protein levels and an enhanced level of p21. More importantly, the NPCDs induced the activation of autophagy by upregulating the protein expression levels of LC3-II and ATG-5 (autophagy-related-5) and by downregulating p62 level, validated by knockdown of ATG-5. Additionally, owing to their excellent luminescence property, these NPCDs were also applicable in cellular bioimaging, as evidenced by the microscopic fluorescence imaging of B16F10 melanoma cells. Conclusion: Based on these findings, we conclude that our newly synthesized NPCDs induced cell cycle arrest, autophagy, and apoptosis in B16F10 melanoma cells and presented good cellular bioimaging capability.