Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity

Abstract

Verteporfin (VP) was first used in Photodynamic therapy, where a non-thermal laser light (689 nm) in the presence of oxygen activates the drug to produce highly reactive oxygen radicals, resulting in local cell and tissue damage. However, it has also been shown that Verteporfin can have non-photoactivated effects such as interference with the YAP-TEAD complex of the HIPPO pathway, resulting in growth inhibition of several neoplasias. More recently, it was proposed that, another non-light mediated effect of VP is the formation of cross-linked oligomers and high molecular weight protein complexes (HMWC) that are hypothesized to interfere with autophagy and cell growth. Here, in a series of experiments, using human uveal melanoma cells (MEL 270), human embryonic kidney cells (HEK) and breast cancer cells (MCF7) we showed that Verteporfin-induced HMWC require the presence of light. Furthermore, we showed that the mechanism of this cross-linking, which involves both singlet oxygen and radical generation, can occur very efficiently even after lysis of the cells, if the lysate is not protected from ambient light. This work offers a better understanding regarding VP's mechanisms of action and suggests caution when one studies the non-light mediated actions of this drug.

Figure 1. Light is needed for the formation of p62 (1A) Diap1 (1B) and Rock1(1C) oligomers in MEL 270, HEK-293 and MCF-7 cell lines.

Western-blot of protein extracts from cells treated with vehicle (veh.), low dose (LD, 1.25 μg/ml) or high dose (HD, 7.5 μg/ml) Verteporfin (VP) for 0 and 6 hours. Verteporfin treatment of cells was always performed in darkness. Right column demonstrates experiments performed in absolute darkness during all steps (treatment, lysis and electrophoresis). Left column demonstrates experiments performed without shielding from light subsequent to cell lysis. (i.e. cell lysis and electrophoresis performed in ambient light). When all steps were performed in absolute darkness no high molecular weight cross-linked oligomers were observed. Ambient light after cell lysis led to VP inducing cross-linked oligomers.

Figure 2. Cellular homogenates spiked with VP and incubated in ambient light exhibit formation of p62, Diap1, and Rock1 oligomers.

Western-blot of protein extracts from cells never exposed to VP in culture but incubated (6hrs, room temperature) with 7.5 μg/ml Verteporfin post lysis either in absolute darkness (incubation and electrophoresis) or in light (incubation and electrophoresis). When incubation and electrophoresis were performed in absolute darkness there was no formation of high molecular weight complexes.

Figure 3. Time course of high molecular weight complexes (HMWC) formation in cellular homogenates spiked with Verteporfin.

Western-blot of protein extracts from cells never exposed to VP in culture but incubated at room temperature with 7.5 μg/ml VP post lysis in ambient light (incubation and electrophoresis) for 0 h (5 min), 1 h, 3 h and 6 h. P62 dimers were noticed as early as 1 h for HEK, earlier for MEL 270 at 0 h (5 min). The same finding was observed for Diap1, Rock1 and HMWC in light as early as 1 h and in a cell dependent manner.

Figure 4. Presence of N-Acetyl Cysteine (NAC) or L-histidine in cellular homogenates decreases the formation of VP induced cross-linked oligomers and HMWC.

Western-blot analysis of p62 in protein extracts from cells never exposed to VP in culture. Cellular homogenates were pre-treated with N-Acetylecysteine- NAC (1 mM) or L-histidine (7.5 mM) 30 min before incubation with high dose (HD- 7.5 μg/ml) Verteporfin. Extracts pretreated with NAC or L-Histidine demonstrated decrease or no formation of VP induced cross-linked oligomers and HMWC compared to controls. (Multiple exposures are provided in Supplemental Information).

Figure 5. Increased expression of O-methionine in light treated cells with VP.

O-Met Western-blot of protein extracts from cells treated with vehicle (veh.), low dose (LD, 1.25 μg/ml) or high dose (HD, 7.5 μg/ml) of Verteporfin (VP) for 0 and 6 hours. Right column demonstrates cells that kept in absolute darkness during all steps (treatment, lysis and electrophoresis). Left column demonstrates cells that were kept in darkness only for the duration of treatment with subsequent steps of cell lysis and electrophoresis in ambient light. When all steps were performed in light we see an increase O-MET expression in a pattern that coincides to the one observed in HMWC formation of the aforementioned proteins. In absolute darkness, the increase in O-Met reactivity was abrogated.

Figure 6. Light activated VP mediates down - regulation of YAP and phospho -YAP (p-YAP) protein expression in MEL 270(4A), HEK 293 (4B) and MCF-7 (4C) perhaps by the formation of HMWC.

Western-blot of protein extracts from cells treated with vehicle (veh.), low dose (LD, 1.25 μg/ml) and high dose (HD, 7.5 μg/ml) of Verteporfin (VP) for 0 and 6 hours either in absolute darkness during all steps (treatment, lysis and electrophoresis) or in darkness only for the duration of treatment with subsequent steps of cell lysis and electrophoresis in ambient light. When all steps were performed in absolute darkness no difference in expression of YAP and p-YAP and there was no high molecular weight complexes. Notably there was no change in the expression of TEF1.

Figure 7. Absence of light from lysis and electrophoresis decreases the formation of protein oligomers induced by Verteporfin in MEL 270, HEK-293 and MCF-7 cell lines.

Western-blot of protein extracts from cells treated in ambient light with vehicle (veh.), low dose (LD, 1.25 μg/ml) and high dose (HD, 7.5 μg/ml) of Verteporfin (VP) for 0 and 6 hours. All the subsequent steps from lysis to electrophoresis were performed in complete darkness. Absence of light from lysis and electrophoresis decreases the formation of dimers and HMWC (compare to Fig. 1).

Figure 8. Effects of VP on cell growth (MEL 270, MCF-7, and HEK293) when shielded or not shielded form ambient light exposure.

MTT assay was performed on MEL 270 (6A, 6B, 6C), HEK293 (6D, 6E, 6F) and MCF-7(6 G, 6 H, 6I) after treatment with vehicle (control), low dose (LD, 1.25 μg/ml) or high dose (HD, 7.5 μg/ml) of Verteporfin (VP) for 0, 6 and 24 hours. Cell viability was significantly decreased in cells not shielded from ambient light, whereas toxicity was minimal in 0 h and 6 h in darkness and increased at 24 hours.