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.
Background: Kidney cancer is one of the most common malignancies, of which the most aggressive subtype was kidney renal clear cell carcinoma (KIRC), accounting for 80% of them. A growing number of studies point to the involvement of competitive endogenous RNAs in tumor development. However, the role of ceRNA network involved in KIRC remains unclear. Thus, the aim of this study was to investigate the BAP1-associated prognostic ceRNA in KIRC. Methods: We downloaded the RNAseq data from TCGA along with the relevant clinical data. We screened the differentially expressed lncRNAs, miRNAs, mRNAs according to the expression of BAP1 and established a ceRNA network. Results: After comprehensive bioinformatics analysis, we identified the XIST-miR-10a-5p-SERPINE1 ceRNA axis. Next, we confirmed the prognostic role of miR-10a-5p/SERPINE1 in KIRC using survival analysis and Cox regression analysis. To investigate the abnormally high expression of SERPINE1, we performed methylation analysis of SERPINE1 and concluded that the methylation level of SERPINE1 in KIRC was significantly lower than that in normal tissues. Furthermore, to study the role of SERPINE1 in the immune microenvironment in KIRC, we performed immune cell infiltration analysis and found that SERPINE1 expression was positively correlated with the level of multiple immune cell infiltration (CD 4+ T cell, CD 8+ T cell, macrophages, dendritic cells, neutrophils). Conclusion: We constructed a ceRNA (XIST/has-miR-10a-5p/SERPINE1) that can be used as prognostic biomarker of KIRC. Furthermore, we found that miR-10a-5p/SERPINE1 were significantly associated with clinical features and were independent prognostic factors of KIRC.
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