Abstract 1373: Insight into the mechanisms of anti-angiogenesis mediated by phosphaplatins
Authors: Yang Lu 
Published: August 2015

Phosphaplatins are a new class of monomeric Pt(II) and Pt(IV)- pyrophosphato complexes that contain amine ligands. These complexes containing 1, 2-cyclohexanediamine exhibit excellent in vitro and in vivo efficacy against both cisplatin- and carboplatin-sensitive and -resistant human cancer cells without exhibiting severe toxicities commonly associated with platinum therapies. We report here unique anti-angiogenic properties of phosphaplatins through several experiments in HUVEC and human ovarian cancer (A 2780) cells as described below.

Inhibition of vascularization. As cell migration is necessary for endothelial cells in angiogenesis and for cancer growth and metastasis, wound healing migration assay was performed. The data obtained from these experiments indicate that phosphaplatins strongly inhibited the migration of HUVECs while cisplatin did not. The ability of endothelial cells forming tube-like structures was also investigated. Remarkably, phosphaplatins resulted in a significantly reduced tubule length. These morphological changes suggested that phosphaplatins inhibited the HUVECs migration at low concentrations.

RNA and Protein expression of VEGFR-2 Inhibition. To understand the inhibitory effects of phosphaplatins on cell migration and tube formation, angiogenesis PCR array was applied to determine changes in gene expression before and after 24 hours treatment. VEGFR-2 and VEGF-A were decreased by 50 - 80% in their mRNA levels of A2780s. Protein expression by Western blot showed that VEGFR-2 was typically decreased after 12 hours during the treatment with phosphaplatins. These results are consistent with RT-PCR data suggesting that VEGFR-2 was down-regulated by phosphaplatins, which probably lead to anti-angiogenic property.

Kinase Inhibition and Enzyme kinetics. To investigate whether phosphaplatins directly inhibited the activation of VEGFR-2 or not, kinase inhibition assay was performed. These results also confirmed that phosphorylation of VEGFR-2 was inhibited, indicating that phosphaplatins are indeed inhibitors of VEGFR-2. These data also suggest that phosphaplatins might be directly bound to VEGFR-2.

LC-MS/MS and protein mapping. To understand the mode of interactions between phosphaplatins and VEGFR-2, comparison between treated and untreated VERGF-2 was subject to mass spectroscopy analysis after trypsin digestion. Data indicated deamidations in some amino acids, such as Glutamine 847, Asparagine 1216, Glutamine 1222, indicating that phosphaplatins establish ionic interactions or hydrogen binding with VEGFR-2, rather than covalent binding.

In summary, our results show phosphaplatins functions as an inhibitor of VEGFR-2, leading to the inhibition of angiogenesis and tumorigenesis. The data suggest a new mode of action for phosphaplatins which is not known for other platinum therapies.

Citation Format: Lu Yang. Insight into the mechanisms of anti-angiogenesis mediated by phosphaplatins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1373. doi:10.1158/1538-7445.AM2015-1373