Song et al. Cell Death and Disease (2020) 11:938 https://doi.org/10.1038/s41419-020-03136-y Cell Death & Disease ARTICLE Open Access Photodynamic therapy induces autophagy- mediated cell death in human colorectal cancer cells via activation of the ROS/JNK signaling pathway Changfeng Song1,WenXu1,HongkunWu2,XiaotongWang1,QianyiGong1,ChangLiu2,JianwenLiu1 and Lin Zhou2 Abstract Evidence has shown that m-THPC and verteporfin (VP) are promising sensitizers in photodynamic therapy (PDT). In addition, autophagy can act as a tumor suppressor or a tumor promoter depending on the photosensitizer (PS) and the cancer cell type. However, the role of autophagy in m-THPC- and VP-mediated PDT in in vitro and in vivo models of human colorectal cancer (CRC) has not been reported. In this study, m-THPC-PDT or VP-PDT exhibited significant phototoxicity, inhibited proliferation, and induced the generation of large amounts of reactive oxygen species (ROS) in CRC cells. From immunoblotting, fluorescence image analysis, and transmission electron microscopy, we found extensive autophagic activation induced by ROS in cells. In addition, m-THPC-PDT or VP-PDT treatment significantly induced apoptosis in CRC cells. Interestingly, the inhibition of m-THPC-PDT-induced autophagy by knockdown of ATG5 or ATG7 substantially inhibited the apoptosis of CRC cells. Moreover, m-THPC- PDT treatment inhibited tumorigenesis of subcutaneous HCT116 xenografts. Meanwhile, antioxidant treatment 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; markedly inhibited autophagy and apoptosis induced by PDT in CRC cells by inactivating JNK signaling. In conclusion, inhibition of autophagy can remarkably alleviate PDT-mediated anticancer efficiency in CRC cells via inactivation of the ROS/JNK signaling pathway. Our study provides evidence for the therapeutic application of m- THPC and VP in CRC. Introduction for patients diagnosed with advanced-stage CRC is as low Colorectal cancer (CRC) is the third leading cause of as 13.1%3. Surgical resection is the main treatment cancer death globally, with a high incidence and mortality method for patients with early stage CRC, while che- rate1. CRC is stratified into two subgroups: early stage motherapy is regarded as the primary treatment option (stage I and II) and advanced-stage (stage III and IV)2. for patients with advanced-stage CRC2,4. Despite the The 5-year survival rate for patients diagnosed with early improvement in the treatment of CRC, the mortality rate stage CRC is approximately 90%, whereas the survival rate of CRC is still high. Thus, there is an urgent need to develop alternative treatments for CRC. Photodynamic therapy (PDT) is a minimally invasive, Correspondence: Jianwen Liu ([email protected])or effective cancer treatment modality that has emerged as Lin Zhou ([email protected]) 1 an alternative or additional approach to chemotherapy State Key Laboratory of Bioreactor Engineering and Shanghai Key Laboratory 5 of New Drug Design, School of Pharmacy, East China University of Science and and surgery . PDT has been clinically available and Technology, Shanghai 200237, P.R. China approved to treat some types of cancers, such as head and 2 Department of Laboratory Medicine, Changzheng Hospital, Naval Medical neck cancer, non-small cell lung cancer, prostate cancer, University, Shanghai 200003, P.R. China 6–9 These authors contributed equally: Changfeng Song, Wen Xu and colon cancer . PDT involves three primary Edited by G.M. Fimia © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Song et al. Cell Death and Disease (2020) 11:938 Page 2 of 14 components, namely a nontoxic photosensitizer (PS), a cancer cells to PDT33. In this study, we aimed to inves- light source, and oxygen10. During PDT, PSs absorb tigate whether m-THPC-PDT or VP-PDT treatment has visible light and convert energy to surrounding molecular an anticancer effect in CRC cells and explore the roles of oxygen and generate a range of highly reactive oxygen autophagy in PDT treatment in CRC. species (ROS), such as singlet oxygen, superoxide anions, and hydroxyl radicals11,12. High levels of ROS can rapidly Materials and methods cause significant toxicity, which eventually leads to cell Cell culture death via apoptosis, autophagy, and/or necrosis13,14. PSs Human colon cancer cell lines HCT116 and SW480 function as catalysts during the process of PDT15. Meta- were purchased from the American Type Culture Col- tetrahydroxyphenylchlorin (m-THPC) and verteporfin lection (ATCC, USA). HCT116 cells were cultured in (VP) are second-generation photosensitizers that exhibit RPMI 1640 medium (Thermo Fisher Scientific, Waltham, considerable photocytotoxicity to various tumor cells16,17. MA, USA) supplemented with 10% fetal bovine serum Emerging studies have found that m-THPC-PDT and VP- (Thermo Fisher Scientific) and 1% penicillin/streptomycin PDT could be promising therapeutic candidates for the (HyClone, Logan, UT, USA). SW480 cells were main- treatment of human cancers18,19. tained in Dulbecco’s modified Eagle’s medium (Thermo The role of a PS in the PDT process is similar to that of Fisher Scientific) supplemented with 10% fetal bovine chemical catalysts10. It can be excited by specific wave- serum (HyClone) and 1% penicillin/streptomycin. Cells lengths of light and absorb the energy of photons, con- were incubated in a humidified atmosphere of 5% CO2 at verting them from a stable ground state to a high-energy 37 °C. For starvation experiments, cells were washed three excited singlet state. Singlet oxygen generates free radicals times with phosphate-buffered saline (PBS), and once in the process of returning to the ground state, and free with serum-free media, and then incubated with modified radicals react with molecular oxygen to generate ROS20.A Earle’s Balanced Salt Solution medium (Sigma-Aldrich, St. variety of PSs exist in nature, but the PSs used for tumor Louis, MO, USA) containing 1.0 g/L glucose at 37 °C. treatment are demanding: they need to have the char- acteristics of high singlet oxygen yield, non-toxicity, rapid M-THPC-PDT and VP-PDT elimination from the body through metabolism, and easy The chemicals m-THPC and VP were purchased from accumulation in tumor tissues21,22. The PSs used in PDT MedChemExpress (Monmouth Junction, NJ, USA). They can be divided into porphyrin derivative PS, chlorophyll- were dissolved in DMSO as a 10 mmol/L stock solution derivative PS, and synthetic compound PS23. According to and stored at −80 °C, protected from light. Working the time of occurrence, it can be divided into first-gen- solutions of m-THPC were prepared fresh at various eration, second-generation, and third-generation PS24. concentrations (0, 0.375, 0.75, 1.5, 3.0, 6.0, and 12.0 μmol/ Choosing the right PS to treat a specific disease is parti- L) and working solutions of VP were prepared fresh at cularly critical. The properties of PS, such as charge and various concentrations (0, 0.375, 0.75, 1.5, 3.0, and polarity, are critical to their cellular localization, dis- 6.0 μmol/L), which were activated by a halogen lamp in tribution in the body, and therapeutic efficacy. Many PSs cell experiments, a laser power density of 10 mW/cm2 and selectively accumulate in specific organelles, such as late laser energy densities of 3 J/cm2. endosomes, lysosomes, mitochondria, or the endoplasmic reticulum25. In this case, light causes photo damage to Cell viability assay specific organelles. Therefore, determining the location of An MTT Cell Proliferation and Cytotoxicity Assay Kit the PS in the cell will provide a better understanding of (Beyotime Biotechnology, Shanghai, China) was used to the site of action of phototoxicity26. determine cell viability. HCT116 and SW480 cells (6 × Autophagy is a successive process of degrading and 103 cells per well) were seeded into 96-well plates and renewing cytoplasmic components27. In addition, it is incubated overnight at 37 °C. After that, HCT116 and critical for maintaining homeostasis and cell growth28. SW480 cells were treated with various concentrations of Evidence has shown that autophagy participates in tumor m-THPC (from 0 to 12.0 μmol/L) or VP (from 0 to progression as well as a response to anticancer thera- 6.0 μmol/L) for 8 h. Next, cells were irradiated with a pies29. It has also been shown that photodamage can lead light dose of 3 J/cm2 at a light power density of 10 mW/ to autophagy induction29,30. However, autophagy might cm2, and then incubated for 24 h without irradiation. play dual roles in tumor suppression and promotion Subsequently, cells were incubated with 20 μLof5mg/ depending on the photosensitizers, cell types, and light mL MTT for another 4 h. Subsequently, formazan dose used31. Xiong et al. reported that inhibition of crystals were dissolved using 150 μLofformazandis- autophagy increased photocytotoxicity of Photosan-II in solution reagent. Finally, the optical density value of CRC cells32,33. Meanwhile, Xue et al. indicated that each well at 570 nm was measured using a microplate knockdown of ATG7 enhanced the resistance of breast reader (BioTek, Winooski, VT, USA).