Effects of photodynamic exposure using chlorine E6 on U251 glioblastoma cell line in vitro





laser irradiation, chlorine E6, human glioblastoma U251 cell culture, cytodestructive effects, mitotic index, cell viability


Objective: to study the effect of photodynamic exposure with the use of chlorine E6 in cell cultures of the standardized human glioblastoma (GB) cell line U251 under different modes of laser irradiation (LI) in vitro.

Materials and methods. Groups of cell cultures of the U251 line were formed, depending on conditions of cultivation and exogenous influence: 1) control – cultivated in a standard nutrient medium (MEM with L-glutamine, 1 mml sodium pyruvate, 10% fetal calf serum) and experimental: 2) cultivated under conditions of adding a photosensitizer chlorine E6 (1.0, 2.0 and 3.0 μg/ml); 3) cultured in a nutrient medium without adding chlorine E6 and subjected to LI (intensity in the range 0.4–0.6 W, dose in the range 25–90 J/cm2, continuous or pulse mode); 4) cultivated under the conditions of adding chlorine E6 and subsequent exposure to LI in the specified modes. Intravital dynamic observation with photo-registration (fluorescence and light microscopy, survey staining methods, intravital staining with a vital dye (0.2% trypan blue solution), morphometric studies (mitotic index, numerical density of viable cells) were carried out.

Results. Cell cultures of the human GB U251 line are characterized by the formation of peculiar intercellular connections (reticular histoarchitectonics) of tumor cells with high polymorphism and proliferation activity. Chlorine E6 is incorporated into the cytoplasm of U251 cells with preservation of fluorescence intensity for 72 hours (observation period). The fluorescence intensity of chlorine E6, incorporated by non-tumorally transformed cells of the rat fetal brain (E14-16), is much weaker. Under the influence of chlorine E6 (1.0, 2.0 and 3.0 μg/ml), cytodestructive processes in U251 cell culture increase in a dose-dependent manner with a progressive loss of viability and a decrease of mitotic index. After exposure to LI in the studied regimes the viability of U251 cells decreases in a dose-dependent manner already 1 h after exposure, with a further decrease after 24 h (the most significant (~30%) – at doses of LI 75–90 J/cm2 in the pulse mode). Under the combined exposure of chlorine E6 (2.0 μg/ml) and LI, the viability of U251 cells decreases in a dose-dependent manner already 1 hour after exposure (by 4.5–10.0 times), the most significant (~80%) – at doses of LI 75–90 J/cm2 in pulse mode. After 24 h of observation under all modes of combined exposure of chlorine E6 and LI, viable cells in U251 cultures were not detected.

Conclusions. Sufficient effectiveness of the cytodestructive effect of chlorine E6 (2.0 μg/ml, preincubation for 6–24 h) and the lowest studied dose of LI (25 J/cm2) in the pulse mode in the cell culture of human GB U251 line was established. The use of vital dye provides an opportunity to record cytotoxic effects in the culture of U251 tumor cells at an early stage (within 1 h after exposure to chlorine E6 and LI).


1. Low JT, Ostrom QT, Cioffi G, Neff C, Waite KA, Kruchko C, Barnholtz-Sloan JS. Primary brain and other central nervous system tumors in the United States (2014-2018): A summary of the CBTRUS statistical report for clinicians. Neurooncol Pract. 2022 Feb 22;9(3):165-182. [CrossRef] [PubMed] [PubMed Central]

2. Fedorenko Z, Michailovich Yu, Goulak L, Gorokh Ye, Ryzhov A, Soumkina O, Koutsenko L. CANCER IN UKRAINE, 2020 - 2021: Incidence, mortality, prevalence and other relevant statistics. Bulletin of the National Cancer Registry of Ukraine. 2022;23. http://www.ncru.inf.ua/publications/BULL_23/index_e.htm

3. Ostrom QT, Cioffi G, Waite K, Kruchko C, Barnholtz-Sloan JS. CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2014-2018. Neuro Oncol. 2021 Oct 5;23(12 Suppl 2):iii1-iii105. [CrossRef] [PubMed] [PubMed Central]

4. Dupont C, Vermandel M, Leroy HA, Quidet M, Lecomte F, Delhem N, Mordon S, Reyns N. INtraoperative photoDYnamic Therapy for GliOblastomas (INDYGO): Study Protocol for a Phase I Clinical Trial. Neurosurgery. 2019 Jun 1;84(6):E414-E419. [CrossRef] [PubMed]

5. Rozumenko AV, Kliuchka VM, Rozumenko VD, Fedorenko ZP. Survival rates in patients with the newly diagnosed glioblastoma: Data from the National Cancer Registry of Ukraine, 2008-2016. Ukrainian Neurosurgical Journal. 2018;(2):33-9. [CrossRef]

6. van Solinge TS, Nieland L, Chiocca EA, Broekman MLD. Advances in local therapy for glioblastoma - taking the fight to the tumour. Nat Rev Neurol. 2022 Apr;18(4):221-236. [CrossRef] [PubMed]

7. Mahmoudi K, Garvey KL, Bouras A, Cramer G, Stepp H, Jesu Raj JG, Bozec D, Busch TM, Hadjipanayis CG. 5-aminolevulinic acid photodynamic therapy for the treatment of high-grade gliomas. J Neurooncol. 2019 Feb;141(3):595-607. [CrossRef] [PubMed] [PubMed Central]

8. Muller PJ, Wilson BC. Photodynamic therapy of brain tumors--a work in progress. Lasers Surg Med. 2006 Jun;38(5):384-9. [CrossRef] [PubMed]

9. Dubey SK, Pradyuth SK, Saha RN, Singhvi G, Alexander A, Agrawal M, Shapiro BA, Puri A. Application of photodynamic therapy drugs for management of glioma. Journal of Porphyrins and Phthalocyanines. 2019 Dec;23(11n12):1216-28. [CrossRef]

10. Cramer SW, Chen CC. Photodynamic Therapy for the Treatment of Glioblastoma. Front Surg. 2020 Jan 21;6:81. [CrossRef] [PubMed] [PubMed Central]

11. Castano AP, Demidova TN, Hamblin MR. Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death. Photodiagnosis Photodyn Ther. 2005 Mar;2(1):1-23. [CrossRef] [PubMed] [PubMed Central]

12. Kaneko S, Fujimoto S, Yamaguchi H, Yamauchi T, Yoshimoto T, Tokuda K. Photodynamic Therapy of Malignant Gliomas. Prog Neurol Surg. 2018;32:1-13. [CrossRef] [PubMed]

13. Li F, Cheng Y, Lu J, Hu R, Wan Q, Feng H. Photodynamic therapy boosts anti-glioma immunity in mice: a dependence on the activities of T cells and complement C3. J Cell Biochem. 2011 Oct;112(10):3035-43. [CrossRef] [PubMed]

14. Velazquez FN, Miretti M, Baumgartner MT, Caputto BL, Tempesti TC, Prucca CG. Effectiveness of ZnPc and of an amine derivative to inactivate Glioblastoma cells by Photodynamic Therapy: an in vitro comparative study. Sci Rep. 2019 Feb 28;9(1):3010. [CrossRef] [PubMed] [PubMed Central]

15. Rozumenko V.D., Semenova V.M., Stayno L.P. Issledovanie effekta fotodinamicheskoy terapii v kul'turakh gliom golovnogo mozga eksperimental'nykh zhivotnykh i cheloveka. Aspekty primeneniya metoda kul'tivirovaniya tkaney v neyrobiologii i neyroonkologii. Kiev: Interservis; 2018. Russian.

16. Fontana LC, Pinto JG, Pereira AHC, Soares CP, Raniero LJ, Ferreira-Strixino J. Photodithazine photodynamic effect on viability of 9L/lacZ gliosarcoma cell line. Lasers Med Sci. 2017 Aug;32(6):1245-1252. [CrossRef] [PubMed]

17. Skandalakis GS, Bouras A, Rivera D, Rizea C, Raj JG, Bozec D, Hadjipanayis CG. Photodynamic Therapy of Diffuse Intrinsic Pontine Glioma in Combination with Radiation. Neurosurgery. 2020 Dec;67(Supplement_1):nyaa447_873. [CrossRef]

18. An YW, Liu HQ, Zhou ZQ, Wang JC, Jiang GY, Li ZW, Wang F, Jin HT. Sinoporphyrin sodium is a promising sensitizer for photodynamic and sonodynamic therapy in glioma. Oncol Rep. 2020 Oct;44(4):1596-1604. [CrossRef] [PubMed] [PubMed Central]

19. Zhang X, Guo M, Shen L, Hu S. Combination of photodynamic therapy and temozolomide on glioma in a rat C6 glioma model. Photodiagnosis Photodyn Ther. 2014 Dec;11(4):603-12. [CrossRef] [PubMed]

20. Sun W, Kajimoto Y, Inoue H, Miyatake S, Ishikawa T, Kuroiwa T. Gefitinib enhances the efficacy of photodynamic therapy using 5-aminolevulinic acid in malignant brain tumor cells. Photodiagnosis Photodyn Ther. 2013 Feb;10(1):42-50. [CrossRef] [PubMed]

21. Fisher C, Obaid G, Niu C, Foltz W, Goldstein A, Hasan T, Lilge L. Liposomal Lapatinib in Combination with Low-Dose Photodynamic Therapy for the Treatment of Glioma. J Clin Med. 2019 Dec 14;8(12):2214. [CrossRef] [PubMed] [PubMed Central]

22. Chernov MF, Muragaki Y, Kesari S, McCutcheon IE (eds): Intracranial Gliomas. Part III - Innovative Treatment Modalities. Prog Neurol Surg. Basel, Karger. 2018; 32:1-13. [CrossRef]

23. Schipmann S, Müther M, Stögbauer L, Zimmer S, Brokinkel B, Holling M, Grauer O, Suero Molina E, Warneke N, Stummer W. Combination of ALA-induced fluorescence-guided resection and intraoperative open photodynamic therapy for recurrent glioblastoma: case series on a promising dual strategy for local tumor control. J Neurosurg. 2020 Jan 24:1-11. [CrossRef] [PubMed]

24. Vermandel M, Dupont C, Lecomte F, Leroy HA, Tuleasca C, Mordon S, Hadjipanayis CG, Reyns N. Standardized intraoperative 5-ALA photodynamic therapy for newly diagnosed glioblastoma patients: a preliminary analysis of the INDYGO clinical trial. J Neurooncol. 2021 May;152(3):501-514. [CrossRef] [PubMed]

25. Lietke S, Schmutzer M, Schwartz C, Weller J, Siller S, Aumiller M, Heckl C, Forbrig R, Niyazi M, Egensperger R, Stepp H, Sroka R, Tonn JC, Rühm A, Thon N. Interstitial Photodynamic Therapy Using 5-ALA for Malignant Glioma Recurrences. Cancers (Basel). 2021 Apr 7;13(8):1767. [CrossRef] [PubMed] [PubMed Central]

26. Kobayashi T, Nitta M, Shimizu K, Saito T, Tsuzuki S, Fukui A, Koriyama S, Kuwano A, Komori T, Masui K, Maehara T, Kawamata T, Muragaki Y. Therapeutic Options for Recurrent Glioblastoma-Efficacy of Talaporfin Sodium Mediated Photodynamic Therapy. Pharmaceutics. 2022 Feb 2;14(2):353. [CrossRef] [PubMed] [PubMed Central]

27. Della Puppa A, Lombardi G, Rossetto M, Rustemi O, Berti F, Cecchin D, Gardiman MP, Rolma G, Persano L, Zagonel V, Scienza R. Outcome of patients affected by newly diagnosed glioblastoma undergoing surgery assisted by 5-aminolevulinic acid guided resection followed by BCNU wafers implantation: a 3-year follow-up. J Neurooncol. 2017 Jan;131(2):331-340. [CrossRef] [PubMed]

28. Zavadskaya ТS. Photodynamic therapy in the treatment of glioma. Experimental oncology. 2015; Dec;37(4):234-41. https://exp-oncology.com.ua/wp/wp-content/uploads/2015/12/2204.pdf?upload=

29. PubChem. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004-. PubChem Compound Summary for CID 5479494, Chlorin e6. https://pubchem.ncbi.nlm.nih.gov/compound/Chlorin-e6

30. Pedachenko E, Liubich L, Staino L, Egorova D. Dynamics of morphological changes in neural cell culture with a model of neurotrauma under the influence of conditioned media of the rat fetal brain neurogenic cells. Cell Organ Transpl. 2020; 8(2):177-186. [CrossRef]

31. U-251 MG (formerly known as U-373 MG) (ECACC 09063001). Culture Collections. UK Health Security Agency; 2023. https://www.culturecollections.org.uk/products/celllines/generalcell/detail.jsp?refId=09063001&collection=ecacc_gc

32. Royko NV, Filenko BN, Nikolenko DE, Mamay IA. [Pathology of mitoses in tumours of different localization]. World of Medicine and Biology. 2013; 38(2):217-219.



How to Cite

Rozumenko, V. D., Liubich, L. D., Staino, L. P., Egorova, D. M., Vaslovych, V. V., Rozumenko, A. V., Komarova, O. S., Dashchakovskyi, A. V., Kluchka, V. M., & Malysheva T. А. (2023). Effects of photodynamic exposure using chlorine E6 on U251 glioblastoma cell line in vitro. Ukrainian Neurosurgical Journal, 29(2), 11–21. https://doi.org/10.25305/unj.273699



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