Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3

Abstract

Diverse death phenotypes of cancer cells can be induced by Photofrin-mediated photodynamic therapy (PDT), which has a decisive role in eliciting a tumor-specific immunity for long-term tumor control. However, the mechanism(s) underlying this diversity remain elusive. Caspase-3 is a critical factor in determining cell death phenotypes in many physiological settings. Here, we report that Photofrin-PDT can modify and inactivate procaspase-3 in cancer cells. In cells exposed to an external apoptotic trigger, high-dose Photofrin-PDT pretreatment blocked the proteolytic activation of procaspase-3 by its upstream caspase. We generated and purified recombinant procaspase-3-D3A (a mutant without autolysis/autoactivation activity) to explore the underlying mechanism(s). Photofrin could bind directly to procaspase-3-D3A, and Photofrin-PDT-triggered inactivation and modification of procaspase-3-D3A was seen in vitro. Mass spectrometry-based quantitative analysis for post-translational modifications using both 16O/18O- and 14N/15N-labeling strategies revealed that Photofrin-PDT triggered a significant oxidation of procaspase-3-D3A (mainly on Met-27, -39 and -44) in a Photofrin dose-dependent manner, whereas the active site Cys-163 remained largely unmodified. Site-directed mutagenesis experiments further showed that Met-44 has an important role in procaspase-3 activation. Collectively, our results reveal that Met oxidation is a novel mechanism for the Photofrin-PDT-mediated inactivation of procaspase-3, potentially explaining at least some of the complicated cell death phenotypes triggered by PDT.

Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach

“…This, together with our previous finding that Photofrin binds procaspase-3 and mediates its PDT-triggered Met oxidation and inactivation 29 , prompted us to search for additional Photofrin-binding proteins in human cells. We hypothesized that such proteins might be more susceptible to oxidative damage during Photofrin-mediated PDT.…”

31P NMR Evidence for Peroxide Intermediates in Lipid Emulsion Photooxidations: Phosphine Substituent Effects in Trapping

“…While 18 O and 15 N labeling have been exploited to elucidate mechanistic aspects of PDT, the natural 31 P abundance of phosphine traps has not been explored in depth yet, as done here in a PDT relevant project.…”

Interaction and oxidative damage of DVDMS to BSA: a study on the mechanism of photodynamic therapy-induced cell death

“…Numerous studies have demonstrated that PDT relies on the generation of ROS 12 13 14 15 . Our study also detected abundant intracellular and extracellular ROS generation after DVDMS-PDT treatment.…”

“…They exhibit remarkable photophysical properties, including high stability, water-solubility, high phototoxicity, and low dark toxicity 8 9 10 . Porfimer sodium (Photofrin II) was the first photosensitizer approved by the US Food and Drug Agency and has already been applied clinically to treat various cancers and non-cancer diseases 11 12 13 14 . However, patients treated with Photofrin II suffer from a skin photosensitivity, requiring protection from sunlight or bright lights exposure for up to 3 months 15 .…”

“…TOOKAD is not water soluble, so that it is administered in a Cremophor-based vehicle. Sinoporphyrin sodium (also called DVDMS) is the active ingredient of Photofrin II and gained independent intellectual property in China 12 18 19 . At a purity of above 98%, pharmacokinetic experiments showed that DVDMS preferentially accumulated in tumor tissues and metabolized quickly in normal tissues.…”