The proteome alterations associated with treatment with Photodynamic Therapy, endosomes after Bleomycine-mediated photochemical internalization on rat bladder cancer cells

ABSTRACT: Introduction: The photosensitizer, meso-tetraphenyl chlorine disulphonate, TPCS2a (Amphinex®) has been evaluated in a proteome analysis of rat bladder cancer cells (AY27) in combination with the chemotherapeutic drug Bleomycin (BML). Photochemical internalization (PCI) is a novel technology for cytosolic delivery of macromolecular therapeutics based on principles of photodynamic therapy (PDT). Since tumor cells have shown to release certain proteins into plasma membrane after PDT1, the we aimed at of this study was to quantifying the amount of endosomal proteins expressed following TPCS2a-PDT or BLM/TPCS2a-based PCI. Methods: AY27 cells were grown in isotopically labeled SILAC medium using the SILAC technology and further incubated with TPCS2a followed by BLM stimulation and blue light illumination (LumiSource, 13mW/cm2). Endosomal proteins were enriched by gradient-ultracentrifugation and measurements of BLM after endosomal uptake were performed by high pressure liquid chromatography (HPLC). Quantitative measurements of the pProteins were performed quantified by Orbitrap mass spectrometry (LC-MS/MS). These rRaw data files were analyzed using Max Quant v 1.5.5.12 thereby, mapping the spectra over rat canonical proteome with isoforms 3. The SILAC ratios hence obtained were log transformed and subjected to Student’s t-test in order to find identify differentially expressed proteins (DEPs) using Perseus platform4. Further these DEPs were mapped to Gene Ontology database to characterize the enrichment of ontological categories. Results: Both upregulated and downregulated DEPs were recorded from total cell extracts as well as from enriched endosomes after gradient-ultracentrifugation and further grouped according to their biological functions. Conclusions: The present work shows that SILAC analysis for in vitro PDT and PCI treatment is a promising technology approach for characterizing different processes related to e.g. cell death during PDT/PCI5 treatment based on quantified DEPs in these different conditions, respectively.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cell Culture, Urinary Bladder

DISEASE(S): Urinary Bladder Cancer

SUBMITTER: Animesh Sharma  

LAB HEAD: Odrun Gederaas

PROVIDER: PXD006915 | Pride | 2024-06-07

REPOSITORIES: Pride