Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy
Abstract Background Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photof...
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| Format: | Article |
| Language: | English |
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BMC
2018-02-01
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| Series: | BMC Cancer |
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| Online Access: | http://link.springer.com/article/10.1186/s12885-018-4126-y |
| _version_ | 1818668662207283200 |
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| author | Antoni Domagala Joanna Stachura Magdalena Gabrysiak Angelika Muchowicz Radoslaw Zagozdzon Jakub Golab Malgorzata Firczuk |
| author_facet | Antoni Domagala Joanna Stachura Magdalena Gabrysiak Angelika Muchowicz Radoslaw Zagozdzon Jakub Golab Malgorzata Firczuk |
| author_sort | Antoni Domagala |
| collection | DOAJ |
| description | Abstract Background Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT. Methods In vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method. Results Photofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins. Conclusions Altogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy. |
| first_indexed | 2024-12-17T06:39:53Z |
| format | Article |
| id | doaj.art-16867412d425487fa07cdb3436c77e42 |
| institution | Directory Open Access Journal |
| issn | 1471-2407 |
| language | English |
| last_indexed | 2024-12-17T06:39:53Z |
| publishDate | 2018-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Cancer |
| spelling | doaj.art-16867412d425487fa07cdb3436c77e422022-12-21T21:59:53ZengBMCBMC Cancer1471-24072018-02-0118111010.1186/s12885-018-4126-yInhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapyAntoni Domagala0Joanna Stachura1Magdalena Gabrysiak2Angelika Muchowicz3Radoslaw Zagozdzon4Jakub Golab5Malgorzata Firczuk6Department of Immunology, Medical University of WarsawDepartment of Immunology, Medical University of WarsawDepartment of Immunology, Medical University of WarsawDepartment of Immunology, Medical University of WarsawDepartment of Immunology, Medical University of WarsawDepartment of Immunology, Medical University of WarsawDepartment of Immunology, Medical University of WarsawAbstract Background Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT. Methods In vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method. Results Photofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins. Conclusions Altogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy.http://link.springer.com/article/10.1186/s12885-018-4126-yAutophagyPhotodynamic therapyPhotofrinATG5CRISR/Cas-9 |
| spellingShingle | Antoni Domagala Joanna Stachura Magdalena Gabrysiak Angelika Muchowicz Radoslaw Zagozdzon Jakub Golab Malgorzata Firczuk Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy BMC Cancer Autophagy Photodynamic therapy Photofrin ATG5 CRISR/Cas-9 |
| title | Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy |
| title_full | Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy |
| title_fullStr | Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy |
| title_full_unstemmed | Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy |
| title_short | Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy |
| title_sort | inhibition of autophagy sensitizes cancer cells to photofrin based photodynamic therapy |
| topic | Autophagy Photodynamic therapy Photofrin ATG5 CRISR/Cas-9 |
| url | http://link.springer.com/article/10.1186/s12885-018-4126-y |
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