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Enhanced efficacy of bleomycin in bladder cancer cells by photochemical internalization.

ABSTRACT: Bleomycin is a cytotoxic chemotherapeutic agent widely used in cancer treatment. However, its efficacy in different cancers is low, possibly due to limited cellular internalization. In this study, a novel approach known as photochemical internalization (PCI) was explored to enhance bleomycin delivery in bladder cancer cells (human T24 and rat AY-27), as bladder cancer is a potential indication for use of PCI with bleomycin. The PCI technique was mediated by the amphiphilic photosensitizer disulfonated tetraphenyl chlorin (TPCS(2a)) and blue light (435?nm). Two additional strategies were explored to further enhance the cytotoxicity of bleomycin; a novel peptide drug ATX-101 which is known to impair DNA damage responses, and the protease inhibitor E-64 which may reduce bleomycin degradation by inhibition of bleomycin hydrolase. Our results demonstrate that the PCI technique enhances the bleomycin effect under appropriate conditions, and importantly we show that PCI-bleomycin treatment leads to increased levels of DNA damage supporting that the observed effect is due to increased bleomycin uptake. Impairing the DNA damage responses by ATX-101 further enhances the efficacy of the PCI-bleomycin treatment, while inhibiting the bleomycin hydrolase does not.

SUBMITTER: Baglo Y

PROVIDER: S-EPMC4101207 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Enhanced efficacy of bleomycin in bladder cancer cells by photochemical internalization.

Baglo Yan Y Hagen Lars L Høgset Anders A Drabløs Finn F Otterlei Marit M Gederaas Odrun A OA

BioMed research international 20140630

Bleomycin is a cytotoxic chemotherapeutic agent widely used in cancer treatment. However, its efficacy in different cancers is low, possibly due to limited cellular internalization. In this study, a novel approach known as photochemical internalization (PCI) was explored to enhance bleomycin delivery in bladder cancer cells (human T24 and rat AY-27), as bladder cancer is a potential indication for use of PCI with bleomycin. The PCI technique was mediated by the amphiphilic photosensitizer disulf ...[more]

PMID: 25101299

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Project description: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.

Project description:Development of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma.5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin.The 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS2a, altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population.Our findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models.

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Enhanced efficacy of bleomycin in bladder cancer cells by photochemical internalization.

Publications

Enhanced efficacy of bleomycin in bladder cancer cells by photochemical internalization.

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