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.
Highlights
Caspase-3, an important executioner in apoptosis, exists in cells as an inactive zymogen and can be activated by proteolysis through either the intrinsic mitochondria-mediated pathway or the extrinsic death-receptor-mediated pathway.[10]
We found that staurosporine, a well-known apoptotic trigger for many cell types,[17] could further activate caspase-3 in cells pretreated with Photofrin-Photodynamic therapy (PDT)
We examined the effect of Photofrin-PDT on the TRAIL or FasL-mediated apoptotic cell death in Jurkat T cells and found that PDT with 28 mg/ml of Photofrin could significantly attenuate the anti-Fas antibody- and TRAIL-mediated caspase-3 activation (Supplementary Figure 1)
Summary
Caspase-3, an important executioner in apoptosis, exists in cells as an inactive zymogen and can be activated by proteolysis through either the intrinsic mitochondria-mediated pathway or the extrinsic death-receptor-mediated pathway.[10]. Owing to its critical role in apoptosis, numerous reports have investigated the regulation of caspase-3 activity by different post-translational modifications, such as nitrosylation, glutathionylation and phosphorylation, all of which inhibited caspase-3 activity.[11,12,13] it is currently unknown whether caspase-3 could be affected and modified by other reactive oxygen species (ROS), such as the singlet oxygen elicited by clinically approved PDT. Some studies have shown that caspase-3 was activated by PDT, whereas others found that PDT-induced necrosis occurred without caspase-3 activation.[3,14,15] In this study, we detected significant inhibition of caspase-3 activation exposed to higher (but not lower) doses of Photofrin-PDT, and which could dose-dependently induce covalent modification of procaspase-3 in human cells. We observed that Met-44, one of the major modification sites, has an important role in the activation of procaspase-3
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