This work presents results on the efficiency of newly designed zinc phthalocyanine-mediated photodynamic therapy of both tumoral and nontumoral cell models using the MTT assay. Further detailed examinations of mechanistic and cell biological effects were focused on the HELA cervical cancer cell model. Here, ROS production, changes in the mitochondrial membrane potential, the determination of genotoxicity, and protein changes determined by capillary chromatography and tandem mass spectrometry with ESI were analyzed. The results showed that, in vitro, 5 Jcm-2 ZnPc PDT caused a significant increase in reactive oxygen species. Still, except for superoxide dismutase, the levels of proteins involved in cell response to oxidative stress did not increase significantly. Furthermore, this therapy damaged mitochondrial membranes, which was proven by a more than 70% voltage-dependent channel protein 1 level decrease and by a 65% mitochondrial membrane potential change 24 h post-therapy. DNA impairment was assessed by an increased level of DNA fragmentation, which might be related to the decreased level of DDB1 (decrease in levels of more than 20% 24 h post-therapy), a protein responsible for maintaining genomic integrity and triggering the DNA repair pathways. Considering these results and the low effective concentration (LC50 = 30 nM), the therapy used is a potentially very promising antitumoral treatment.

Department of Medical Biophysics Faculty of Medicine and Dentistry Palacky University 77900 Olomouc Czech Republic

Laboratory of Growth Regulators Faculty of Science Palacky University and Institute of Experimental Botany of the Czech Academy of Sciences 77900 Olomouc Czech Republic

See more in PubMed

Correia J.H., Rodrigues J.A., Pimenta S., Dong T. Photodynamic Therapy Review: Principles, Photosensitizers, Applications, and Future Directions. Pharmaceutics. 2021;13:1332. doi: 10.3390/pharmaceutics13091332. PubMed DOI PMC

Kwiatkowaski S., Knap B., Przystupski D., Saczko J., Kedzierska E., Knap-Czop K., Kotlińska J., Michel O., Kotowski K., Kulbacka J. Photodynamic therapy—Mechanisms, photosensitizers and combinations. Biomed Pharmacother. 2018;106:1098–1107. doi: 10.1016/j.biopha.2018.07.049. PubMed DOI

Abrahamse H., Hamblin M.R. New photosensitizers for photodynamic therapy. Biomechem. J. 2016;473:347–364. doi: 10.1042/BJ20150942. PubMed DOI PMC

Mroz P., Bhaumik J., Dogutan D.K., Aly Z., Kamal Z., Khalid L., Kee H.L., Bocian D.F., Holten D., Lindsey J.S., et al. Imidazole metalloporphyrins as photosensitizers for photodynamic therapy: Role of molecular charge, central metal and hydroxyl radical production. Cancer Lett. 2009;282:63–76. doi: 10.1016/j.canlet.2009.02.054. PubMed DOI PMC

Ochsner M. Photophysical and photobiological processes in the photodynamic therapy of tumours. J. Photochem. Photobiol. B Biol. 1997;39:1–18. doi: 10.1016/S1011-1344(96)07428-3. PubMed DOI

Alvarez N., Sevilla A. Current Advances in Photodynamic Therapy (PDT) and the Future Potential of PDT-Combinatorial Cancer Therapies. Int. J. Mol. Sci. 2024;25:1023. doi: 10.3390/ijms25021023. PubMed DOI PMC

Petrat F., Pindiur S., Kirsch M., de Groot H. NAD(P)H, a Primary Target of 1O2 in Mitochondria of Intact Cells. J. Biol. Chem. 2003;278:3298–3307. doi: 10.1074/jbc.M204230200. PubMed DOI

Petrat F., Pindiur S., Kirsch M., de Groot H. “Mitochondrial” photochemical drugs do not release toxic amounts of 1O2 within the mitochondrial matrix space. Arch. Biochem. Biophys. 2003;412:207–215. doi: 10.1016/S0003-9861(03)00063-8. PubMed DOI

Castano A.P., Demidova T.N., Hamblin M.R. Mechanisms in photodynamic therapy: Part one—Photosensitizers, photochemistry and cellular localization. Photodiagnosis Photodyn. Ther. 2004;1:279–293. doi: 10.1016/S1572-1000(05)00007-4. PubMed DOI PMC

Ma J., Jiang L. Photogeneration of singlet oxygen (1O2) and free radicals (Sen−, O−2) bytetra-brominated hypocrellin B derivative. Free. Radic. Res. 2001;35:767–777. doi: 10.1080/10715760100301271. PubMed DOI

Nowis D., Golab J. Photodynamic Therapy and Oxidative Stress. In: Hamblin M.R., Mróz P., editors. Advances in Photodynamic Therapy—Basic, Translational, and Clinical. Artech House; New York City, NY, USA: 2008. p. 559.

Davies M.J. Singlet oxygen-mediated damage to proteins and its consequences. Biochem. Biophys. Res. Commun. 2003;305:761–770. doi: 10.1016/S0006-291X(03)00817-9. PubMed DOI

Diwanji N., Bergmann A. An unexpected friend—ROS in apoptosis-induced compensatory proliferation: Implications for regeneration and cancer. Semin. Cell Dev. Biol. 2018;80:74–82. doi: 10.1016/j.semcdb.2017.07.004. PubMed DOI PMC

Cai H., Meng Z., Yu F. The involvement of ROS-regulated programmed cell death in hepatocellular carcinoma. Crit. Rev. Oncol. 2024;197:104361. doi: 10.1016/j.critrevonc.2024.104361. PubMed DOI

Xing L., Tang Y., Li L., Tao X. ROS in hepatocellular carcinoma: What we know. Arch. Biochem. Biophys. 2023;744:109699. doi: 10.1016/j.abb.2023.109699. PubMed DOI

Gao Y., Huang D., Huang S., Li H., Xia B. Rational design of ROS generation nanosystems to regulate innate immunity of macrophages, dendrtical and natural killing cells for immunotherapy. Int. Immunopharmacol. 2024;139:112695. doi: 10.1016/j.intimp.2024.112695. PubMed DOI

Shah R., Ibis B., Kashyap M., Boussiotis V.A. The role of ROS in tumor infiltrating immune cells and cancer immunotherapy. Metabolism. 2024;151:155747. doi: 10.1016/j.metabol.2023.155747. PubMed DOI PMC

Bilski P., Motten A.G., Bilska M., Chignell C.F. The photooxidation of diethylhydroxylamine by rose bengal in micellar and nonmicellar aqueous solutions. Photochem. Photobiol. 1993;58:11–18. doi: 10.1111/j.1751-1097.1993.tb04896.x. PubMed DOI

Xu D.D., Leong M.M.L., Wong F.-L., Lam H.M., Hoeven R., Yang Y., Lv Q. Photodynamic therapy on prostate cancer cells involve mitochondria membrane proteins. Photodiagnosis Photodyn. Ther. 2020;31:101933. doi: 10.1016/j.pdpdt.2020.101933. PubMed DOI

Xue L.-Y., Chiu S.-M., Oleinick N.L. Photochemical destruction of the Bcl-2 oncoprotein during photodynamic therapy with the phthalocyanine photosensitizer Pc 4. Oncogene. 2001;20:3420–3427. doi: 10.1038/sj.onc.1204441. PubMed DOI

Jori G. Far-red-absorbing photosensitizers: Their use in the photodynamic therapy of tumours. J. Photochem. Photobiol. A Chem. 1992;62:371–378. doi: 10.1016/1010-6030(92)85065-3. DOI

Wilson B.C. Photodynamic therapy: Light delivery and dosage for second-generation photosensitizers. Ciba Found Symp. 1989;146:60–73; discussion 73–77. doi: 10.1002/9780470513842.ch5. PubMed DOI

Liu J.-Y., Li J., Yuan X., Wang W.-M., Xue J.-P. In vitro photodynamic activities of zinc(II) phthalocyanines substituted with pyridine moieties. Photodiagnosis Photodyn. Ther. 2016;13:341–343. doi: 10.1016/j.pdpdt.2015.07.003. PubMed DOI

Hsi R.A., Rosenthal D.I., Glatstein E. Photodynamic therapy in the treatment of cancer. Drugs. 1999;54:725–734. doi: 10.2165/00003495-199957050-00005. PubMed DOI

Wyss P., Schwarz V., Dobler-Girdziunaite D., Hornung R., Walt H., Degen A., Fehr M. Photodynamic therapy of locoregional breast cancer recurrences using a chlorin-type photosensitizer. Int. J. Cancer. 2001;93:720–724. doi: 10.1002/ijc.1400. PubMed DOI

Shanazarov N.A., Zare A., Mussin N.M., Albayev R.K., Kaliyev A.A., Iztleuov Y.M., Smailova S.B., Tamadon A. Photodynamic therapy of cervical cancer: A scoping review on the efficacy of various molecules. Ther. Adv. Chronic Dis. 2024;15:20406223241233206. doi: 10.1177/20406223241233206. PubMed DOI PMC

Fisher A.B., Vasquez-Medina J.P., Dodia C., Sorokina E.M., Tao J.-Q., Feinstein S.I. Peroxiredoxin 6 phospholipid hydroperoxidase activity in the repair of peroxidized cell membranes. Redox Biol. 2018;14:41–46. doi: 10.1016/j.redox.2017.08.008. PubMed DOI PMC

Li H., Benipal B., Zhou S., Dodia C., Chatterjee S., Tao J.-Q., Sorokina E.M., Raabe T., Feinstein S.I., Fisher A.B. Critical role of peroxiredoxin 6 in the repair of peroxidized cell membranes following oxidative stress. Free. Radic. Biol. Med. 2015;87:356–365. doi: 10.1016/j.freeradbiomed.2015.06.009. PubMed DOI PMC

Lien Y.-C., Feinstein S.I., Dodia C., Fisher A.B. The Roles of Peroxidase and Phospholipase A2Activities of Peroxiredoxin 6 in Protecting Pulmonary Microvascular Endothelial Cells Against Peroxidative Stress. Antioxid. Redox Signal. 2012;16:440–451. doi: 10.1089/ars.2011.3950. PubMed DOI PMC

Hanschmann E.-M., Godoy J.R., Berndt C., Hudemann C., Lillig C.H. Thioredoxins, Glutaredoxins, and Peroxiredoxins—Molecular Mechanisms and Health Significance: From Cofactors to Antioxidants to Redox Signaling. Antioxid. Redox Signal. 2013;19:1539–1605. doi: 10.1089/ars.2012.4599. PubMed DOI PMC

Greetham D., Vickerstaff J., Shenton D., Perrone G.G., Dawes I.W., Grant C.M. Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae. BMC Biochem. 2010;11:3. doi: 10.1186/1471-2091-11-3. PubMed DOI PMC

Pillay S.C., Hofmeyr J.H., Rohwer J.M. The logic of kinetic regulation in the thioredoxin system. BMC Syst. Biol. 2011;5:15. doi: 10.1186/1752-0509-5-15. PubMed DOI PMC

Porta C., Moroni M., Guallini P., Torri C., Marzatico F. Antioxidant enzymatic system and free radicals pathway in two different human cancer cell lines. Anticancer Res. 1996;16:2741–2747. PubMed

Gille J.J., Wortelboer H.M., Joenje H. Effect of normobaric hyperoxia on antioxidant defenses of HeLa and CHO cells. Free. Radic. Biol. Med. 1988;4:85–91. doi: 10.1016/0891-5849(88)90068-8. PubMed DOI

Ehlers R.A., Hernandez A., Bloemendal L., Ethridge R.T., Farrow B., Evers B. Mitochondrial DNA damage and altered membrane potential (ΔΨ) in pancreatic acinar cells induced by reactive oxygen species. Surgery. 1999;126:148–155. doi: 10.1016/S0039-6060(99)70148-0. PubMed DOI

Postiglione I., Chiaviello A., Barra F., Roscetto E., Soriano A.A., Catania M.R., Palumbo G., Pierantoni G.M. Mitochondrial Malfunctioning, Proteasome Arrest and Apoptosis in Cancer Cells by Focused Intracellular Generation of Oxygen Radicals. Int. J. Mol. Sci. 2015;16:20375–20391. doi: 10.3390/ijms160920375. PubMed DOI PMC

Jeong S.-Y., Seol D.-W. The role of mitochondria in apoptosis. BMB Rep. 2008;41:11–22. doi: 10.5483/BMBRep.2008.41.1.011. PubMed DOI

Hsieh Y., Wu C., Chang C., Yu J. Subcellular localization of Photofrin® determines the death phenotype of human epidermoid carcinoma A431 cells triggered by photodynamic therapy: When plasma membranes are the main targets. J. Cell. Physiol. 2003;194:363–375. doi: 10.1002/jcp.10273. PubMed DOI

Liu W., Vives-Bauza C., Acín-Peréz R., Yamamoto A., Tan Y., Li Y., Magrané J., Stavarache M.A., Shaffer S., Chang S., et al. PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson’s Disease. PLoS ONE. 2009;4:e4597. doi: 10.1371/journal.pone.0004597. PubMed DOI PMC

Lovejoy C.A., Lock K., Yenamandra A., Cortez D. DDB1 Maintains Genome Integrity through Regulation of Cdt1. Mol. Cell. Biol. 2006;26:7977–7990. doi: 10.1128/MCB.00819-06. PubMed DOI PMC

Hu J., McCall C.M., Ohta T., Xiong Y. Targeted ubiquitination of CDT1 by the DDB1–CUL4A–ROC1 ligase in response to DNA damage. Nat. Cell Biol. 2004;6:1003–1009. doi: 10.1038/ncb1172. PubMed DOI

Dougherty T.J., Gomer C.J., Henderson B.W., Jori G., Kessel D., Korbelik M., Moan J., Peng Q. Photodynamic Therapy. JNCI J. Natl. Cancer Inst. 1998;90:889–905. doi: 10.1093/jnci/90.12.889. PubMed DOI PMC

Wood S.R., Holroyd J.A., Brown S.B. The Subcellular Localization of Zn(ll) Phthalocyanines and Their Redistribution on Exposure to Light. Photochem. Photobiol. 1997;65:397–402. doi: 10.1111/j.1751-1097.1997.tb08577.x. PubMed DOI

Huang D.W., Sherman B.T., Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009;4:44–57. doi: 10.1038/nprot.2008.211. PubMed DOI

Huang D.W., Sherman B.T., Lempicki R.A. Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37:1–13. doi: 10.1093/nar/gkn923. PubMed DOI PMC

Saraste A. Morphologic criteria and detection of apoptosis. Henz. 1999;24:189–195. doi: 10.1007/BF03044961. PubMed DOI

Alberts B., Bray D., Johnson A., Lewis J., Raff M., Roberts K., Walter P. Základy Buněčné Biologie. 2nd ed. Espero Publishing, s.r.o.; Prague, Czech Republic: 1998. p. 740.

Heider T., Kanwan I.L., Pandey V., Jain P., Soni V. Targeted Nanomedicine for Breast Cancer Therapy. Elsevier; Amsterdam, The Netherlands: 2022. Tumor microenvironment-mediated targeted drug delivery to breast cancer cells; pp. 305–334.

Bi H., Xue J., Jiang H., Gao S., Yang D., Fang Y., Shi K. Current developments in drug delivery with thermosensitive liposomes. Asian J. Pharm. Sci. 2018;14:365–379. doi: 10.1016/j.ajps.2018.07.006. PubMed DOI PMC

Akram N., Afzaal M., Saeed F., Shah Y.A., Faisal Z., Asghar A., Ateeq H., Nayik G.A., Wani S.H., Hussain M., et al. Liposomes: A promising delivery system for active ingredients in food and nutrition. Int. J. Food Prop. 2023;26:2476–2492. doi: 10.1080/10942912.2023.2247578. DOI

de Matos M.B.C., Deckers R., van Elburg B., Lajoinie G., de Miranda B.S., Versluis M., Schiffelers R., Kok R.J. Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization. Front. Pharmacol. 2019;10:1463. doi: 10.3389/fphar.2019.01463. PubMed DOI PMC

Cidlina A. Diploma Thesis. Faculty of Pharmacy in Hradec Kralove; Charles University in Prague, Hradec Kralove, Czech Republic: 2011. Syntéza Kationických Ftalocyaninů.72p

Petřík I. Bachelor’s Thesis. Faculty of Science; Palacky University in Olomouc, Olomouc, Czech Republic: 2013. Analýza Proteinových Fosforylací Proteomickými Metodami.62p

Boersema P.J., Raijmakers R., Lemeer S., Mohammed S., Heck A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 2009;4:484–494. doi: 10.1038/nprot.2009.21. PubMed DOI