Original Articles
Shanshan Qi, Lingyuan Guo, Shuzhen Yan, Robert J. Lee, Shuqin Yu, Shuanglin Chen. Hypocrellin A-based photodynamic action induces apoptosis in A549 cells through ROS-mediated mitochondrial signaling pathway[J]. Acta Pharmaceutica Sinica B, 2019, 9(2): 279-293

Hypocrellin A-based photodynamic action induces apoptosis in A549 cells through ROS-mediated mitochondrial signaling pathway
Shanshan Qia,b, Lingyuan Guoa, Shuzhen Yana, Robert J. Leeb, Shuqin Yuc, Shuanglin Chena
a Jiangsu Province Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China;
b College of Pharmacy, the Ohio State University, Columbus, OH 43210, USA;
c Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China
Over recent decades, many studies have reported that hypocrellin A (HA) can eliminate cancer cells with proper irradiation in several cancer cell lines. However, the precise molecular mechanism underlying its anticancer effect has not been fully defined. HA-mediated cytotoxicity and apoptosis in human lung adenocarcinoma A549 cells were evaluated after photodynamic therapy (PDT). A temporal quantitative proteomics approach by isobaric tag for relative and absolute quantitation (iTRAQ) 2D liquid chromatography with tandem mass spectrometric (LC-MS/MS) was introduced to help clarify molecular cytotoxic mechanisms and identify candidate targets of HA-induced apoptotic cell death. Specific caspase inhibitors were used to further elucidate the molecular pathway underlying apoptosis in PDT-treated A549 cells. Finally, down-stream apoptosis-related protein was evaluated. Apoptosis induced by HA was associated with cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and mitochondrial disruption, which were preceded by increased intracellular reactive oxygen species (ROS) generations. Further studies showed that PDT treatment with 0.08 mmol/L HA resulted in mitochondrial disruption, pronounced release of cytochrome c, and activation of caspase-3, -9, and -7. Together, HA may be a possible therapeutic agent directed toward mitochondria and a promising photodynamic anticancer candidate for further evaluation.
Key words:    Hypocrellin A    Photodynamic therapy    Reactive oxygen species    Proteomic    LC-MS/MS    iTRAQ   
Received: 2018-08-17     Revised: 2018-11-15
DOI: 10.1016/j.apsb.2018.12.004
Funds: This study was supported by the National Natural Science Foundation of China (Project No.81673214).The National Key Technology Research and National Key Technology Research and Development Program of the Ministry of Science and Technology of the People's Republic of China (Project No.2012BAD36B0502) and the Priority Academic Program Development of Jiangsu Higher Educational Institutions (China).
Corresponding author: Shuqin Yu, Shuanglin Chen     Email:yushuqin@njnu.edu.cn;chenshuanglin@njnu.edu.cn
Author description:
PDF(KB) Free
Shanshan Qi
Lingyuan Guo
Shuzhen Yan
Robert J. Lee
Shuqin Yu
Shuanglin Chen

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. Cancer J Clin 2011;61:69-90.
2. Monsuez JJ, Charniot JC, Vignat N, Artigou JY. Cardiac side-effects of cancer chemotherapy. Int J Cardiol 2010;144:3-15.
3. Dougherty TJ, Grindey GB, Fiel R, Weishaupt KR, Boyle DG. Photoradiation therapy. Ⅱ. Cure of animal tumors with hematoporphyrin and light. J Natl Cancer Inst 1975;55:115-21.
4. Allison RR, Sibata CH. Oncologic photodynamic therapy photosensitizers:a clinical review. Photodiagn Photodyn Ther 2010;7:61-75.
5. Wu HM, Lao XF, Wang QW, Lu RR, Shen CY, Zhang FX. The Shiraiachromes:novel fungal perylenequinone pigments from Shiraia bambusicola. J Nat Prod 1989;52:948-51.
6. Diwu Z. Novel therapeutic and diagnostic applications of hypocrellins and hypericins. Photochem Photobiol 1995;61:529-39.
7. Zhang J, Cao EH, Li JF, Zhang TC, Ma WJ. Photodynamic effects of hypocrellin A on three human malignant cell lines by inducing apoptotic cell death. J Photochem Photobiol, B 1998;43:106-11.
8. Fei XF, Jie C, Zheng KY, Wei W, Sun SJ, Li W. Apoptotic effects of hypocrellin A on HeLa cells. Chem Res Chin Univ 2006;22:772-5.
9. Kishi T, Tahara S, Taniguchi N, Tsuda M, Tanaka C, Takahashi S. New perylenequinones from Shiraia bambusicola. Planta Med 1991;57:376-9.
10. Qi SS, Lin X, Zhang MM, Yan SZ, Yu SQ, Chen SL. Preparation and evaluation of hypocrellin A loaded poly(lactic-co-glycolic acid) nanoparticles for photodynamic therapy. RSC Adv 2014;4:40085-94.
11. Rohlena J, Dong LF, Ralph SJ, Neuzil J. Anticancer drugs targeting the mitochondrial electron transport chain. Antioxid Redox Sign 2011;15:2951-74.
12. Dennis G, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC. DAVID:database for annotation, visualization, and integrated discovery. Genome Biol 2003;4(R60)1-11.
13. Szklarczyk D, Franceschini A, Kuhn M, Simonovic M, Roth A, Minguez P. The STRING database in 2011:functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res 2011;39:561-8.
14. Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M. KEGG:kyoto encyclopedia of genes and genomes. Nucleic Acids Res 1999;27:29-34.
15. Wang DF, Rong WT, Lu Y, Hou J, Qi SS, Xiao Q, et al. TPGS2k/PLGA nanoparticles for overcoming multidrug resistance by interfering mitochondria of human alveolar adenocarcinoma cells. ACS Appl Mater Interfaces 2015;7:3888-901.
16. Lu Y, Wang YY, Yang N, Zhang D, Zhang FY, Gao HT, et al. Food emulsifier polysorbate 80 increases intestinal absorption of di-(2-ethylhexyl) phthalate in rats. Toxicol Sci 2014;139:317-27.
17. Gao L, Fei J, Zhao J, Li H, Cui Y, Li J. Hypocrellin-loaded gold nanocages with high two-photon efficiency for photothermal/photodynamic cancer therapy in vitro. ACS Nano 2012;6:8030-40.
18. Soldani C, Scovassi AI. Poly(ADP-ribose) polymerase-1 cleavage during apoptosis:an update. Apoptosis 2002;7:321-8.
19. Gong JP, Traganos F, Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem 1994;218:314-9.
20. Zhang J, Jiang CS, Longob JP, Azevedo RB, Zhang H, Muehlmann LA. An updated overview on the development of new photosensitizers for anticancer photodynamic therapy. Acta Pharm Sin B 2018;8:137-46.
21. Crow JP. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro:implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1997;1:145-57.
22. Tian Y, Leung W, Yue K, Mak N. Cell death induced by MPPa-PDT in prostate carcinoma in vitro and in vivo. Biochem Biophys Res Commun 2006;348:413-20.
23. Dhanda S, Kaur S, Sandhir R. Preventive effect of N-acetyl-L-cysteine on oxidative stress and cognitive impairment in hepatic encephalopathy following bile duct ligation. Free Radic Biol Med 2013;56:204-15.
24. Cohen GM. Caspases:the executioners of apoptosis. Biochem J 1997;326:1-16.
25. Rohlena J, Dong LF, Ralph SJ, Neuzil J. Anticancer drugs targeting the mitochondrial electron transport chain. Antioxid redox Sign 2011;15:2951-74.
26. Lai SL, Wong PF, Lim TK, Lin Q, Mustafa MR. Cytotoxic mechanisms of panduratin A on A375 melanoma cells:a quantitative and temporal proteomics analysis. Proteomics 2015;15:1608-21.
27. Mo Y, Hou H, Li D, Liang Y, Chen D, Zhou Y. Mitochondrial protein targets of radiosensitisation by 1,8-dihydroxy-3-acetyl-6-methyl-9,10 anthraquinone on nasopharyngeal carcinoma cells. Eur J Pharmacol 2014;738:133-41.
28. Son YO, Heo JS, Kim TG, Jeon YM, Kim JG, Lee JC. Overexpression of JunB inhibits mitochondrial stress and cytotoxicity in human lymphoma cells exposed to chronic oxidative stress. BMB Rep 2010;43:57-61.
29. Kabbage M, Dickman MB. The BAG proteins:a ubiquitous family of chaperone regulators. Cell Mol Life Sci 2008;65:1390-402.
30. Deng J. How to unleash mitochondrial apoptotic blockades to kill cancers?. Acta Pharm Sin B 2017;7:18-26.
31. Xue L, Chiu S, Fiebig A, Andrews DW, Oleinick NL. Photodamage to multiple Bcl-xL isoforms by photodynamic therapy with the phthalocyanine photosensitizer Pc 4. Oncogene 2003;22:9197-204.
32. Gilmore TD. Introduction to NF-κB:players, pathways, perspectives. Oncogene 2006;25:6680-4.
33. Grosch S, Tegeder I, Niederberger E, Brautigam L, Geisslinger G. COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib. FASEB J 2001;15:2742-4.
34. Volanti C, Matroule JY, Piette J. Involvement of oxidative stress in NF-κB activation in endothelial cells treated by photodynamic therapy. Photochem Photobiol 2002;75:36-45.
35. Agostinis P, Buytaert E, Breyssens H, Hendrickx N. Regulatory pathways in photodynamic therapy induced apoptosis. Photochem Photobiol Sci 2004;3:721-9.
36. Ferrario A, Von TK, Wong S, Luna M, Gomer CJ. Cyclooxygenase-2 inhibitor treatment enhances photodynamic therapy-mediated tumor response. Cancer Res 2002;62:3956-61.
37. Denicourt C, Dowdy SF. Medicine. Targeting apoptotic pathways in cancer cells. Science 2004;305:1411-3.
38. Matroule JY, Hellin AC, Morliere P, Fabiano AS, Santus R, Merville MP, et al. Role of nuclear factor-κB in colon cancer cell apoptosis mediated by aminopyropheophorbide photosensitization. Photochem Photobiol 1999;70:540-8.
39. El-Sikhry HE, Miller GG, Madiyalakan MR, Seubert JM. Sonodynamic and photodynamic mechanisms of action of the novel hypocrellin sonosensitizer, SL017:mitochondrial cell death is attenuated by 11,12-epoxyeicosatrienoic acid. Investig New Drugs 2011;29:1328-36.
40. Fu NW, Chu YX. Photodynamic action of hypocrellin A on hepatoma cell mitochondria and microsomes. Acta Pharmacol Sin 1989;10:371-3.
41. Jiang Y, Leung AW, Xiang J, Xu C. LED light-activated hypocrellin B induces mitochondrial damage of ovarian cancer cells. Int J Photoenergy 2012;2012:1-5.
42. Zhao H, Yin R, Chen D, Ren J, Wang Y, Zhang J, et al. In vitro and in vivo antitumor activity of a novel hypocrellin B derivative for photodynamic therapy. Photodiagnosis Photodyn Ther 2014;11:204-12.
43. Kowaltowski AJ, Souza-Pinto NCD, Castilho RF, Vercesi AE. Mitochondria and reactive oxygen species. Free Radic Bio Med 2009;47:333-43.
44. Vaghy PL. Role of mitochondrial oxidative phosphorylation in the maintenance of intracellular pH. J Mol Cell Cardiol 1979;11:933-40.
45. Koppenol WH, Bounds PL, Dang CV. Otto Warburg's contributions to current concepts of cancer metabolism. Nat Rev Cancer 2011;11:325-7.
46. Cheng G, Zielonka J, Dranka BP, McAllister D, Mackinnon AC Jr, Joseph J, et al. Mitochondria-targeted drugs synergize with 2-deoxyglucose to trigger breast cancer cell death. Cancer Res 2012;72:2634-44.
47. Lu Z, Tao Y, Zhou Z, Zhang J, Li C, Ou LC, et al. Mitochondrial reactive oxygen species and nitric oxide-mediated cancer cell apoptosis in 2-butylamino-2-demethoxyhypocrellin B photodynamic treatment. Free Radic Biol Med 2006;41:1590-605.
48. Lam M, Oleinick NL, Nieminen AL. Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization. J Biol Chem 2001;276:47379-86.
49. Kim JS, Wang JH, Lemasters JJ. Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation:role of Ca2+-dependent mitochondrial formation of reactive oxygen species. Am J Physiol:Gastrointest Liver Physiol 2012;302:G723-31.
50. Zou H, Li YC, Liu HS, Wang XD. An APAF-1 center dot cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 1999;274:11549-56.