Evaluation of potential photodynamic therapy agents and patient-relevant biomarker combinations for the selective targeting of cancer

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Date
2018-08-21
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Publisher
Virginia Tech
Abstract

Cancer, the second leading cause of death worldwide, is characterized by uncontrolled and abnormal cell growth. Even though researchers have made significant progress in its treatment over the past several decades, innovative therapeutic approaches that both improve patient survival and lessen the many debilitating side effects of conventional cancer treatments are vital. Accordingly, we first investigated the mechanism of interaction of a bimetallic complex, Ru(II)-Rh(III), with DNA. Non-covalent binding of Ru(II)-Rh(III) is strong and involves electrostatic and, potentially, groove binding interactions. Ru(II)-Rh(III) photobinds and photocleaves DNA through an O2-independent, metal-center mediated mechanism that could be beneficial in hypoxic tumors. Furthermore, the extent of covalent binding and cleavage of DNA, which inhibit PCR amplification, is dependent upon the strength of the non-covalent interactions. These results suggest that the toxicity of Ru(II)-Rh(III) could be selectively generated in tissues irradiated with light (e.g., a tumor). Secondly, we identified protein combinations selectively present in melanoma, which could be utilized in heteromultivalency. Heteromultivalent scaffolds display higher affinity towards cells that express a protein combination in comparison to those with only one of the proteins, which facilitates cell discrimination. Using an empirically-optimized threshold-based screening method and expression profiles of melanoma patients and normal tissues, we identified surface proteins and protein combinations that are selectively found in melanoma patients and not in normal tissues. After a preliminary validation process using the scientific literature, we used immunofluorescence to confirm differential expression of some of these combinations in established melanoma cell lines in comparison to immortalized keratinocytes controls. Finally, we investigated the resazurin assay, a method used for the evaluation of proliferation and cytotoxicity in more than 2,000 publications. We found that only ~14% of these utilized validated assay conditions, while ~40% failed to report essential analytical parameters needed for their replication. We evaluated assay parameters needed for accurate estimation of cell number in eight cell lines, and found that these are highly variable and independent of tissue type, growth kinetics, and energetic parameters. Furthermore, we obtained some insights into the biochemical reduction of resazurin and proposed minimum reporting standards, along with a sample protocol for assay validation.

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Keywords
photodynamic therapy, excited state reactivity, DNA, heteromultivalency, melanoma, cytotoxicity, resazurin
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