Elektroporasyon İşlemi Sırasında Oluşan Hücre Ölümünde Darbe Frekansının Önemi

Year 2021, , 887 - 890, 26.12.2021

Mehmet Eşref Alkış , Yusuf Alan

https://doi.org/10.18586/msufbd.1010048

Abstract

Tersinir elektroporasyon (EP), darbeli elektrik alanlarının uygulanmasıyla hücre zarının geçirgenliğinin arttırıldığı biyofiziksel bir yöntemdir. Kanser tedavisinde, EP tümör hücrelerinde antikanser ilaçlarının alımını arttırmak, dolayısıyla sitotoksik etkilerini yükseltmek için kullanılır, ancak hücre ölümüne de yol açabilmektedir. Bu çalışmanın amacı tersinir EP uygulamasında meydana gelen hücre ölümünde darbe frekansının etkisini incelemektir. Çalışmada HepG2 karaciğer kanser hücreleri kullanıldı. Hücreler 200 Hz, 1 kHz, 5 kHz, 10 kHz ve 20 kHz frekanslarında 70-1250 V/cm elektrik alana sahip, 100μs süreli 8 kare dalga elektrik darbesine maruz bırakıldı. Elektrik maruziyetinden sonra hücre canlılığı MTT tahlili ile belirlendi. Çalışmamızın MTT bulgularına göre, EP’de uygulanan darbe frekansı arttıkça HepG2 kanser hücrelerinde inhibisyon azalmaktadır. EP uygulanan HepG2 hücrelerinde en yüksek inhibisyon, 1000 V/cm elektrik alanında ve 200 Hz'lik frekansta meydana geldi. 10 kHz ve 20 kHz frekanslarında uygulanan düşük elektrik alanlar (70 V/cm, 250 V/cm) hücre canlılığında azalmaya neden olmadı. Düşük elektrik alanlarında 1 kHz frekansına göre 5 kHz frekansından daha fazla hücre ölümü gerçekleşse de, yüksek elektrik alanlarında 1 kHz frekansında daha yüksek oranda hücre ölümü tespit edildi. Sonuçlarımız, elektroporasyonda düşük frekanslardaki darbelerin daha fazla hücre inhibisyonuna neden olabildiğini, bu yüzden yüksek frekanslı darbelerin kullanımı daha uygun olabileceğini göstermektedir.

Keywords

Darbe Frekans, Elektrokemoterapi, Elektroporasyon, HepG2 Hücreleri, Hücre İnhibisyonu

The Importance Of Pulse Frequency in Cell Death During Electroporation Process

Abstract

Reversible electroporation (EP) is a biophysical method in which the permeability of the cell membrane is enhanced by the application of pulsed electric fields. In cancer treatment, EP is used to increase the uptake of anticancer drugs in tumor cells, thus increasing their cytotoxic effects, but it can also lead to cell death. The aim of this study is to examine the effect of pulse frequency on cell death in reversible EP applications. HepG2 liver cancer cells were used in the study. The cells were exposed to 8 square wave electrical pulses of 100 μs duration with an electric field of 70-1250 V/cm at frequencies of 200 Hz, 1kHz, 5kHz, 10kHz and 20kHz. Cell viability after electrical exposure was determined by MTT assay. According to the MTT findings of our study, inhibition in HepG2 cancer cells decreases as the pulse frequency applied in EP increases. The highest inhibition in EP applied HepG2 cells occurred at 1000 V/cm electric field and 200 Hz frequency. Low electric fields (70 V/cm, 250 V/cm) applied at 10 kHz and 20kHz frequencies did not cause a decrease in cell viability. Although more cell death occurred at a frequency of 5 kHz than at a frequency of 1kHz in low electric fields, a higher rate of cell death was detected at a frequency of 1kHz in high electric fields. Our results show that low-frequency pulses in electroporation can cause more cell inhibition, so the use of high-frequency pulses may be more appropriate.

Keywords

Cell Inhibition, Electrochemotherapy, Electroporation, HepG2 Cells, Pulse Frequency

References

• Kotnik T., Rems L., Tarek M., Miklavčič D. Membrane electroporation and electropermeabilization: Mechanisms and models, Annual Review of Biophysics, 48:1 63-91, 2019.
• Gehl J. Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research, Acta Physiologica Scandinavica, 177:4 437-447, 2003.
• Cemazar M., Sersa G. Recent Advances in Electrochemotherapy, Bioelectricity, 1:4 204-213, 2019.
• Luz J.C.D.S. da, Antunes F., Clavijo-Salomon M.A., Signori E., Tessarollo N.G., Strauss B.E. Clinical applications and immunological aspects of electroporation-based therapies, Vaccines, 9:7 727, 2021.
• Domanico R., Trapasso S., Santoro M., Pingitore D., Allegra E. Electrochemotherapy in combination with chemoradiotherapy in the treatment of oral carcinomas in advanced stages of disease: efficacy, safety, and clinical outcomes in a small number of selected cases, Drug Design, Development and Therapy, 9 1185-1191, 2015.
• Kunte C., Letulé V., Gehl J., Dahlstroem K., Curatolo P., Rotunno R. Electrochemotherapy in the treatment of metastatic malignant melanoma: a prospective cohort study by InspECT, The British Journal of Dermatology, 176:6 1475-1485, 2017.
• Esmaeili N., Friebe M. Electrochemotherapy: A review of current status, alternative IGP approaches, and future perspectives, Journal of Healthcare Engineering, 2019 2784516, 2019.
• Batista Napotnik T., Polajžer T., Miklavčič D. Cell death due to electroporation-A review, Bioelectrochemistry, 141 107871, 2021.
• Alkış M.E., Keleştemür Ü., Alan Y., Turan N., Buldurun K. Cobalt and ruthenium complexes with pyrimidine based schiff base: Synthesis, characterization, anticancer activities and electrochemotherapy efficiency, Journal of Molecular Structure, 1226 129402, 2021.
• Yadegari-Dehkordi S., Firoozabadi S.M., Forouzandeh Moghadam M., Shankayi Z. Role of endocytosis pathways in electropermeablization of MCF7 cells using low voltage and high frequency electrochemotherapy, Cell Journal, 23:4 445-450, 2021.
• Campana L.G., Miklavčič D., Bertino G., Marconato R., Valpione S., Imarisio I. Electrochemotherapy of superficial tumors - Current status: Basic principles, operating procedures, shared indications, and emerging applications, Seminars in Oncology, 46:2 173-191, 2019.
• Sersa G., Cemazar M., Snoj M. Electrochemotherapy of tumours, Current Oncology, 16:2 34-35, 2009.
• Fiorentzis M., Kalirai H., Katopodis P., Seitz B., Viestenz A., Coupland S.E. Electrochemotherapy with bleomycin and cisplatin enhances cytotoxicity in primary and metastatic uveal melanoma cell lines in vitro, Neoplasma, 65:2 210-215, 2018.
• Zimmermann C.E., Faesser H.A., Gassling V., Wiltfang J. The role of electrochemotherapy with intratumoral bleomycin for early tongue carcinoma, Acta Oto-Laryngologica, 141:4 424-431, 2021.
• Alki̇s M.E. Effects of electroporation on cytotoxicity of 4-aminopyrimidin-2-(1H)-one based ligand and its Cobalt (II) and Ruthenium (II) complexes in MCF-7 cancer cells, Dicle Medical Journal, 498-506, 2021.
• Sersa G., Miklavcic D., Cemazar M., Rudolf Z., Pucihar G., Snoj M. Electrochemotherapy in treatment of tumours. European Journal of Surgical Oncology: The Journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology, 34:2 232-240, 2008.
• Miklavcic D., Pucihar G., Pavlovec M., Ribaric S., Mali M., Macek-Lebar A., et al. The effect of high frequency electric pulses on muscle contractions and antitumor efficiency in vivo for a potential use in clinical electrochemotherapy, Bioelectrochemistry, 65:2 121-128, 2005.
• Pucihar G., Mir L. M., Miklavcic D. The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy, Bioelectrochemistry, 57:2 167-172, 2002.
• Cadossi R., Ronchetti M., Cadossi M. Locally enhanced chemotherapy by electroporation: clinical experiences and perspective of use of electrochemotherapy, Future Oncology, 10:5 877-890, 2014.
• Corovic S., Bester J., Miklavcic D. An e-learning application on electrochemotherapy, Biomedical Engineering Online, 8:1 26, 2009.
• Zupanic A., Ribaric S., Miklavcic D. Increasing the repetition frequency of electric pulse delivery reduces unpleasant sensations that occur in electrochemotherapy, Neoplasma, 54:3 246-250, 2007.
• Polajžer T., Dermol-Černe J., Reberšek M., O’Connor R., Miklavčič D. Cancellation effect is present in high-frequency reversible and irreversible electroporation, Bioelectrochemistry, 132:107442 107442, 2020.
• Shankayi Z., Firoozabadi S.M. Antitumor efficiency of electrochemotherapy by high and low frequencies and repetitive therapy in the treatment of invasive ductal carcinoma in BALB/c mice, Cell Journal, 14:2 110-115, 2012.
• Shankayi Z., Firoozabadi S.M.P., Hassan Z.S. Optimization of electric pulse amplitude and frequency in vitro for low voltage and high frequency electrochemotherapy, The Journal of Membrane Biology, 247:2 147-154, 2014.
• Fusco R., Di Bernardo E., D’Alessio V., Salati S., & Cadossi M. Reduction of muscle contraction and pain in electroporation-based treatments: An overview, World Journal of Clinical Oncology, 12:5 367-381, 2021.
• Klein N., Mercadal B., Stehling M., Ivorra A. In vitro study on the mechanisms of action of electrolytic electroporation (E2), Bioelectrochemistry, 133:107482 107482, 2020.
• Alkış M.E., Buldurun K., Turan N., Alan Y., Yılmaz Ü.K., Mantarcı A. Synthesis, characterization, antiproliferative of pyrimidine based ligand and its Ni(II) and Pd(II) complexes and effectiveness of electroporation, Journal of Biomolecular Structure Dynamics, 1-11, 2020.
• Alkış M.E., Turan N., Alan Y., Irtegun Kandemir S., Buldurun K. Effects of electroporation on anticancer activity of 5-FU and newly synthesized zinc(II) complex in chemotherapy-resistance human brain tumor cells, Medical Oncology, 38:11 129, 2021.
• Jakstys B., Jakutaviciute M., Uzdavinyte D., Satkauskiene I., Satkauskas S. Correlation between the loss of intracellular molecules and cell viability after cell electroporation, Bioelectrochemistry, 135:107550 107550, 2020.
• Saczko J., Kamińska I., Kotulska M., Bar J., Choromańska A., Rembiałkowska N. Combination of therapy with 5-fluorouracil and cisplatin with electroporation in human ovarian carcinoma model in vitro, Biomedecine Pharmacotherapie, 68:5 573-580, 2014.
• Shankayi Z., Firoozabadi S., Hassan Z. Comparison of low voltage amplitude electrochemotherapy with 1 Hz and 5 kHz frequency in volume reduction of mouse mammary tumor in Balb/c mice, Koomesh, 13:4 486-490, 2012.
• Vernhes M.C., Cabanes P.A., Teissie J. Chinese hamster ovary cells sensitivity to localized electrical stresses, Bioelectrochemistry and Bioenergetics, 48:1 17-25, 1999.