Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy

Abstract

BackgroundAccumulating 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.MethodsIn 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.ResultsPhotofrin-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.ConclusionsAltogether, 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.

Role of Bcl-2 Family Proteins in Photodynamic Therapy Mediated Cell Survival and Regulation

“…Pharmacological inhibitors like 3-Methyladenine, bafilomycin A1, obatoclax, clarithromycin, chloroquine and hydrochloroquine, etc., tend to target the inhibitory effect of cytoprotective autophagy to overcome therapy resistance [ 95 ]. Another study by Domagala et al also confirms that ATG5 gene knockout in HeLa and MCF-7 cells moderately increases the efficacy of photofrin-based PDT, thus suggesting involvement of the autophagy gene in PDT therapy [ 96 ]. This shows that autophagic genes need to be blocked or somehow be inhibited, in order to increase the efficiency of PDT therapeutic responses.…”

The Switch between Protective and Nonprotective Autophagy; Implications for Autophagy Inhibition as a Therapeutic Strategy in Cancer

“…Over the course of the last decade, one strategy that has been considered to have the potential to both enhance the response to cancer therapeutics and also to overcome drug and radiation resistance is that of autophagy inhibition. Preclinical studies of therapy-induced autophagy have generally shown autophagy to have a cytoprotective function [1][2][3][4]; cytoprotective autophagy has been demonstrated quite extensively in studies wherein autophagy inhibition promotes (apoptotic) cell death by cancer therapeutics. Consequently, it has been quite logical to infer that autophagy inhibition would result in an enhancement of therapeutic sensitivity in patients, paralleling the outcome in cell culture and tumor-bearing animal studies.…”

“…It must again be acknowledged that extensive data in the preclinical literature does largely support the concept of cytoprotective autophagy as a response to cancer therapeutics in the tumor cell. Specifically, studies have shown that either pharmacological inhibitors of autophagy (such as chloroquine, bafilomycin, or 3-methyladenine) or genetic inhibition of autophagy through the knockdown or silencing of autophagy-regulatory genes, often results in an enhanced tumor response to various therapeutic modalities, both in cell culture and in tumor bearing animal studies [1][2][3][4]. However, there is also currently clear evidence for what we have termed the "nonprotective' function of autophagy; here, neither pharmacological nor genetic autophagy inhibition produces a discernible influence on the therapeutic response [5][6][7][8][9][10].…”

Photodynamic therapy induces autophagy-mediated cell death in human colorectal cancer cells via activation of the ROS/JNK signaling pathway

“…In addition, it is critical for maintaining homeostasis and cell growth 28 . Evidence has shown that autophagy participates in tumor progression as well as a response to anticancer therapies 29 . It has also been shown that photodamage can lead to autophagy induction 29 , 30 .…”

“…Evidence has shown that autophagy participates in tumor progression as well as a response to anticancer therapies 29 . It has also been shown that photodamage can lead to autophagy induction 29 , 30 . However, autophagy might play dual roles in tumor suppression and promotion depending on the photosensitizers, cell types, and light dose used 31 .…”