We have recently shown that vascular endothelial growth factor-A (VEGFA), a major regulator of tumor vascularization, is essential for the organization of tumor cells into capillary-like structure (CLS), which is a hallmark of tumor vasculogenic mimicry (VM). Herein we further dissect the involvement of VEGFA and its downstream transducers, VEGF receptor 1 (VEGFR1), VEGFR2, and protein kinase C (PKC) in melanoma VM. The knockdown of VEGFR1 in three melanoma cell lines completely disrupts Matrigel-induced CLS formation, whereas inhibition of VEGFR2 kinase with a specific inhibitor, protein tyrosine kinase inhibitor II (PTKi-II), does not affect the process, indicating that VEGFR2 signaling is not involved in VEGFA-mediated melanoma VM. Furthermore, among tested PKC isoforms, only PKCα and δ are expressed in the melanoma cells during CLS formation. Pretreatment with selective PKCα and δ inhibitors blocked CLS formation. However, inhibition of PKCα, but not PKCδ, completely destroyed the previously formed CLS. Moreover, knockdown of PKCα, but not PKCδ, using small interfering RNAs abrogated CLS formation, suggesting that PKCα is the major contributory factor in melanoma VM. In-vivo experiments indicate that disruption of PKCα signaling significantly reduces the signs of VM in allografted B16/F10 melanoma. These findings may contribute to the development of new therapeutic agents that target melanoma VM.
Riboflavin (Rf) is a vitamin and endogenous photosensitizer capable to generate reactive oxygen species (ROS) under UV-blue irradiation and kill cancer cells, which are characterized by the enhanced uptake of Rf. We confirmed its phototoxicity on human breast adenocarcinoma cells SK-BR-3 preincubated with 30-μM Rf and irradiated with ultraviolet light, and proved that such Rf concentrations (60 μM) are attainable in vivo in tumour site by systemic intravascular injection. In order to extend the Rf photosensitization depth in cancer tissue to 6 mm in depth, we purpose-designed core/shell upconversion nanoparticles (UCNPs, NaYF4:Yb3+:Tm3+/NaYF4) capable to convert 2% of the deeply-penetrating excitation at 975 nm to ultraviolet-blue power. This power was expended to photosensitise Rf and kill SK-BR-3 cells preincubated with UCNPs and Rf, where the UCNP-Rf energy transfer was photon-mediated with ~14% Förster process contribution. SK-BR-3 xenograft regression in mice was observed for 50 days, following the Rf-UCNPs peritumoural injection and near-infrared light photodynamic treatment of the lesions.
Notch signaling plays an important role in tumor angiogenesis. Recent studies suggest that Notch signaling also regulates the progression of primary melanomas toward an aggressive phenotype. The aim of this study was to investigate the involvement of Notch signaling pathway in organization of tumor cells into capillary-like structures (CLS), the phenomenon also known as vasculogenic mimicry (VM). Here, we show that Notch signaling cascade was constitutively active in melanoma cell lines we used. Blocking Notch signaling with the γ-secretase inhibitors, DAPT, dibenzazepine or Jagged1 neutralizing antibody resulted in stabilization of CLS indicating that Notch signaling pathway attenuates melanoma VM. We further studied this phenomenon on melanomas grafted in nude mice. Compared to control, VM channels in DAPT-treated grafted melanoma became larger and more branched. DAPT-treated melanomas also exhibited an up-regulation of MMP-2 and VEGFR1, both known as VM mediators. Moreover, we did not observe necrosis in VM channels areas of DAPT-treated melanomas. These findings indicate that VM regulated by Notch signaling may present a novel target in melanoma therapy.
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