Verteporfin-induced formation of protein oligomers is mediated by light and leads to cell toxicity.

Abstract

Purpose : Verteporfin (VP) is a photosensitizer with clinical uses, most notably in Photodynamic therapy (PDT) for neovascular AMD. Recently non-photo-activated effects of VP have been proposed, including inhibition of HIPPO pathway (Genes Dev 26, 1300-1305 2012, and Exp Eye Res 124, 67-73 2014) and induction of oligomers and high molecular weight complexes (HMWC) of proteins, especially ones with PB1 domain. (PLoS One 9, 2014 e114964 and J. Biol Chem 286, 7290-7300 2011.) Given the highly reactive nature of VP we wanted to further investigate some of the proposed mechanisms of non-light activated VP effects and to determine if some of these observed effects could be an artifact of incomplete shielding from light, such as light present in the experiments after cell lysis.

Methods : Incubation with low dose VP (1.25 μg/mL) or high dose VP (7.5 μg/mL) for 6 hours compared to no incubation, was carried out using three different cell lines (human uveal melanoma cells MEL 270, human breast cancer cells MCF7 and human embryonic kidney cells HEK), with four different combinations of darkness and light exposure: (i. light during both treatment and lysis, ii. darkness during treatment but light during lysis, iii. light during treatment but darkness during lysis and iv. darkness during both treatment and lysis.). In separate experiments, cell lysates never previously exposed to VP were spiked with doses of VP and incubated in dark or ambient light at room temperature. Formation of HMWC of p62, Diap1 and Rock1, YAP1, p-YAP and TEF1 were analyzed with Western blotting. Cell viability and toxicity were measured by means of MTT.

Results : Our study demonstrates that VP-induced HMWC formation requires light and is therefore not light-independent as previously suggested, and can take place after cell lysis. This oligomer formation was not restricted to PB1 domain-containing proteins but also occurs with proteins lacking the domain, and proteins with WW-containing domain. Both cancerous and non-cancerous cells were growth inhibited by VP exposure when not shielded from ambient light.

Conclusions : Our study suggests that VP-induced HMWC formation is a light-dependent event. These effects of ambient light-activated VP further strengthen the clinical recommendation of avoiding exposure to ambient light of healthy tissue after VP PDT treatment.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.