SciELO - Scientific Electronic Library Online

 
vol.17 número3Methylene blue degradation assessment by advanced oxidation methodsNickel (II) sorption from aqueous media by Agave salmiana as biosorbent índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

Links relacionados

  • No hay artículos similaresSimilares en SciELO

Compartir


Journal of applied research and technology

versión On-line ISSN 2448-6736versión impresa ISSN 1665-6423

J. appl. res. technol vol.17 no.3 Ciudad de México may./jun. 2019  Epub 16-Abr-2020

 

Articles

In vitro assessment of murine melanoma cells sensitivity to non-thermal atmospheric plasma

Jorge Humberto Serment Guerreroa  * 

Karina Giron Romeroa 

Régulo López-Callejasb 

Rosendo Peña-Eguiluzb 

a Laboratory of Molecular Biology, Department of Biology, Instituto Nacional de Investigaciones Nucleares, Carretera México Toluca S/N, 52750 Ocoyoacac, México.

b Plasma Physics Laboratory, Instituto Nacional de Investigaciones Nucleares, Carretera México Toluca S/N, 52750 Ocoyoacac, México.


Abstract

Melanoma is a dangerous skin cancer incidence of which has been increasing over the past years, so is important the search for new treatments. The purpose of the present work is to evaluate the relative sensitivity of a melanoma cell line to helium-generated non-thermal plasma. For that, three cell types were used (murine melanoma B16 cells, mouse embryo fibroblasts, and peripherical blood lymphocytes) and both cytotoxicity and genotoxicity were evaluated. The lethality produced by no-thermal plasma was higher in melanoma mouse cells compared with lymphocytes and fibroblasts. Accordingly, B16 cells showed higher levels of DNA fragmentation by this agent. Overall, the results suggest that non-thermal plasma has the potential to become a good alternative for treating skin cancer.

Keywords: Non-thermal plasma; melanoma; genotoxicity

1. INTRODUCTION

Melanoma is a very dangerous skin cancer due to its ability to metastasize and acquire chemo- or radioresistance (Vermeylen et al., 2016). The incidence of metastatic melanoma has been increasing over the past 35 years (Gray-Schopfer, Wellbrock, & C Marais, 2007), so is important, along with early detection, the search for new treatments. The B16 murine cell line has been used extensively as a model for the study of this type of cancer.

In physics, plasma is defined as a partially ionized gas containing free charge carriers (ions and electrons), active radicals, excited molecules and ultraviolet emissions (Kogelschatz, 2003). In the laboratory, thermal (quasi-equilibrium) and non-thermal (out of equilibrium) are produced at different pressures, depending on the required technological application. In the last years, non-thermal plasma has been used for surface (Nehra, Kumar, & Dwivedi, 2008; Laroussi, 1996; Lerouge, Wertheimer, & Yahia, 2001) and heat sensitive medical material sterilization (Fridman, 2008), wound healing and tissue sterilization (Kieft, Darios, Rooks, & Stoffels, 2005; Laroussi, 2009), blood coagulation (Fridman, 2008) skin treatments (Fridman et al., 2007; Fridman, 2008), bacteria inactivation (Gallagher et al., 2007; García-Alcantara et al., 2012; Laroussi, Richardson, & Dobbs, 2002; Martines et al., 2009; Moisan et al., 2001; Sladek, Stoffels, Walraven, Tielbeek & Koolhoven, 2004;) and functional and structural modification of cancer cells (Fridman, 2008; Stoffels, 2003; Zimheld, Zucker, Zimheld, Zucker, DiSanto, Berezney, & Etemadi, 2010).

It has been proposed that the reactive oxygen/nitrogen species (RONS) generated by plasma can disrupt cell membrane and attack several biomolecules, including DNA (Yan, Sherman, & Keidar, 2017). Indeed, experiments made with a bacterial strain defective in a protective system to oxidizing agents showed that it was more sensitive to plasma than a wild-type strain (Garcia-Alcantara et al., 2013). Accordingly, the effect of non-thermal plasma upon the genetic material has been demonstrated before, either in naked DNA (Morales-Ramirez et al., 2013) or in living cells (García-Alcantara et al., 2013). To date, several assays have been used to assess DNA damage, such as plasmid breakage, micronuclei induction or phosphorylated histone H2AX detection in a wide range of cell types (Alkawareek et al., 2014; Kim, Kim, & Lee, 2010; Kluge et al., 2016).

The purpose of the present work is to evaluate the sensitivity of the murine melanoma cell line B16 to a helium-generated plasma needle exposure.

2. METHODOLOGY

2.1 CELL CULTURES

Murine melanoma cells (B16) and 3T3 fibroblasts were grown at 37°C and 5% CO2 atmosphere, in minimal essential medium (MEM) supplemented with 10% FBS, 1% antibiotics (penicillin, streptomycin and amphotericin) and 0.3 mg/ml L-glutamine until subconfluent phase was reached. Cultures growth was monitored using an inverted microscope. Cells were harvested by trypsinization with a 0.01% trypsin-EDTA solution, washed twice with Hanks Balanced Saline Solution (HBSS) and further incubated for at least one hour in MEM at 37°C to recover prior plasma exposure Human lymphocytes were obtained from blood samples drawn by venipuncture from healthy donors, mixed 1:1 with HBSS, gently poured on top of an equal volume of Ficoll Hypaque and centrifugated for 10 minutes at 2,000 rpm. The inter-phase ring of nucleated cells was then collected and washed twice with HBSS, resuspended in RPMI-1640 supplemented with 10% FBS and placed again at 37°C and a 5% CO2 atmosphere for at least one hour.

2.2 PLASMA EXPOSURES

Plasma generator is based on a radiofrequency 13.56 MHz commercial power source linked to a homemade PI matching box, which in turns supplies a punctual non-thermal plasma reactor (Pérez et al., 2008). The last has a coaxial configuration built with an energized copper filament surrounded by an isolating ceramic, both linked to a cylindrical Nylamid SL frame with 8 cm of length. This structure has a gas inlet and a female micro RF connector. A 1 mm visible non-thermal plasma discharge is produced at the head of copper filament which is encircled by an external thin copper grounded electrode. When 8 W were applied from the RF power source to the load (mode load power), it was generated a power density along the radiofrequency cable and the reactor of 0.2416 W/cm2. The three cell types were resuspended in HBSS and then 200 µl aliquots were distributed in a 96-microwell plate and exposed to plasma generated by a flow of 0.7 LPM of helium electrically excited by means of a 13.56 MHz radiofrequency generator at a power of 8 W. The plasma reactor outlet was kept at a distance of 2 mm from the liquid surface.

2.3 CYTOTOXICITY

Cell death was evaluated according to the method of Strauss (1991). After treatments, cells were stained with a 1:1 fluorescein diacetate (80 µg/ml) and ethidium bromide (50 µg/ml) solution and observed afterward under a Hund Wetzlar fluorescence microscope with an excitation filter of 488 nm (blue light). Living cells are stained in green while dead cells are stained in red. A minimum of 100 cells per treatment were scored and all the experiments were repeated at least three times. Survival was calculated by dividing the number of living cells by the total number of cells.

2.4 GENOTOXICITY

Genotoxicity was evaluated using the comet assay (Tice et al., 2000). After plasma exposure, cell suspensions were mixed with an equal volume of 1% low melting point agarose (LMPA) and immediately 80μl aliquots were dispensed on top of fully frosted slides, let to solidify for 5 minutes and then immerse in cold lysis solution (2.5M NaCl, 100mM EDTA, 10mM Tris, 1% SDS, 10% DMSO, 1% TRITON X100, pH10) for at least an hour. Afterward, slides were transferred to an electrophoresis cell filled with electrophoresis solution (0.3M NaHO, 0.1 mM Na2EDTA) for 20 minutes to allow DNA unwinding and thereafter a current was applied (20 V, 300 mA, 20 minutes). Slides were rinsed twice with neutralizing buffer (0.4M Tris-HCl, pH 7.5), stained with 60 µl of a 20 µg/ml ethidium bromide solution and observed under an epifluorescence microscope. Images (at least 100 per dose) were scored by the Comet Assay IV Analyzer (Perceptive Instruments Inc.). All the experiments were repeated no less than three times.

3. RESULTS AND DISCUSSION

In the conditions stated above, the non-thermal plasma generator delivered the following energy density (Table 1):

Table 1 Energy delivered at different exposition times. 

Time (seconds) Equivalent Energy Density
(J/cm2)
15 3.62
30 7.25
45 10.87
60 14.50

The results show that the exposure to the non-thermal plasma produced both cytotoxicity and genotoxicity upon cells in a dose-dependent manner. Overall, the effect of plasma was higher in melanoma cells than in human lymphocytes or fibroblasts. The idea to use lymphocytes was to compare the effect of non-thermal plasma upon proliferative and no proliferative cells. We decided to use human lymphocytes because of the volume required and the ease to obtain them.

As shown in Figure 1, fibroblasts are less sensitive to plasma exposure compared with lymphocytes and B16 cells. Lymphocytes were slightly more sensitive than fibroblasts, with about 50% of survival at the higher dose, while B16 cells had the highest sensitivity to non-thermal plasma, showing a cellular death above 90% at the higher dose.

Fig. 1 Survival to non-thermal plasma exposure of the different cell types used. 

B16 cells suffer higher genetic damage compared with fibroblasts and lymphocytes as the energy deposited in the cells rises, as shown in Figures 2 and 3. Nevertheless, fibroblasts and lymphocytes show a similar behavior, while the DNA breakage in both is lower than the one observed in B16 cells. Overall, all three cell lines show an increase in DNA fragmentation as the dose rises.

Fig 2 DNA fragmentation in cells exposed to non-thermal plasma, assessed by the comet assay. A) Tail Moment; B) Tail Length. 

Fig. 3 Images of the different comets found according to the dose. A) 0; B) 15; C) 30; D) 45; E) 60. 

As stated above, when plasma is generated intramolecular collisions occur resulting in the emergence of ions and free radicals. The interaction with air results in a partial dissociation and ionization of O2, N2 and H2O, generating reactive oxygen species (ie. O3, H2O2, OH or even N), oxides of nitrogen (ie. NO, NO2, N2O, ONOO), nitrous and nitric acid (HNO3, HNO2) (Yan et al., 2017). All of these products can attack different parts of the cell, such as lipids of the membrane (Van der Paal et al., 2016), proteins in general, mitochondria, as well as genetic material (Kalghatg et al., 2011; Morales-Ramirez et al., 2013). Plasma also generates UV radiation (Stoffels, Sakiyama, & Grave, 2008), especially UVB, which can produce cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) upon DNA, which arrest the replication fork and hence could result in cellular death or apoptosis (You et al., 2001).

According to survival results, non-thermal plasma produces higher cytotoxicity on B16 cells compared to lymphocytes or fibroblasts, maybe because this cell line is in active replication and according to the Bergonié and Tribondeau law’s, cells with high metabolic rates or in constant replication are more sensitive. In fact, this is the reason why lymphocytes (which are in a G0 state) were included in the present work. Although fibroblasts, as well as B16 cells, are in constant replication, it is possible that the presence of peroxisomes could protect these cells to the action of RONS, resulting in a higher survival percentage (Brun et al., 2014). Indeed, it has been demonstrated that low levels of RONS increase the migration and proliferation of fibroblast as part of tissue regeneration processes (Gurtner, Werner, Barrandon, & Longaker 2008), promoting an increase in the number of intracellular peroxisomes.

Genetic damage on the three cell lines was evaluated by comet assay. Results show a proportional increase of DNA fragmentation as the dose increases. The results data indicate that CAP breaks the genetic material, most probably due to the RONs generation. Furthermore, DNA damage generated by UV radiation could result in additional breakage due to the repair mechanisms. Once again, B16 cells showed a more severe DNA fragmentation most probably because of their accelerated metabolism. Fibroblasts appear to be as resistant to the genotoxic effect of plasma as lymphocytes, although they are in active replication. Once more, it is possible that the presence of peroxisomes protects the cells from the RONS, resulting in a lower DNA fragmentation. Additionally, recent studies demonstrated that many melanoma cell lines are defective in cytoglobin (Fujita et al., 2014), a new member of the globin family that has been proposed to have a potential role in reactive oxygen species (ROS) detoxification (Fordel et al., 2006; Hodges, Innocent, Dhanda, & Graham, 2008; Nishi et al., 2011). Indeed, recent results show a strong correlation between the expression level of Cygb, the intracellular ROS concentration and the sensitivity of melanoma cell lines towards non-thermal plasma treatment (De Backer et al., 2018).

4. CONCLUSIONS

Exposition to non-thermal plasma causes cytotoxicity and genotoxicity on the B16 cell in a dose-dependent way. It was observed that the lethality produced by this kind of plasma was higher in melanoma mouse cells compared with lymphocytes and fibroblasts. Accordingly, B16 cells showed higher levels of DNA fragmentation by this agent. Although in the present work only cyto- and genotoxicity data are presented, is important to underline that actually there are differences in the effect produced by non-thermal plasma on normal and melanoma cells, which is a key melanoma cells, which is a key feature in the safety treatment of cancer patients. The results suggest that non-thermal plasma has the potential to become a good alternative for skin cancer treatment.

REFERENCES

Alkawareek, M. Y., Nidá, H. A., Higginbotham, S., Flynn, P. B., Algwari, Q. T., Gorman, S. P., ... & Gilmore, B. F. (2014). Plasmid DNA damage following exposure to atmospheric pressure nonthermal plasma: kinetics and influence of oxygen admixture.Plasma Medicine, 4(1-4), 211-219. [ Links ]

Brun, P., Pathak, S., Castagliuolo, I., Pal, G., Zuin, M., Cavazzana, R., & Martines, E. (2014). Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells. PLoS ONE, 9(8), 1-9. [ Links ]

De Backer, J., Razzokov, J., Hammerschmid, D., Mensch, C., Kumar, N., Bogaerts, A., & Dewilde, S. (2018). The role of cytoglobin in the plasma-treatment of melanoma. Clinical Plasma Medicine , 9, 14. [ Links ]

Fordel, E., Thijs, L., Martinet, W., Lenjou, M., Laufs, T., Van Bockstaele, D., ... & Dewilde, S. (2006). Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death. Neuroscience Letters, 410(2), 146-151. [ Links ]

Fridman, A., (2008). Plasma Chemistry. Ed. Cambridge University Press, New York, pp. 1-978. [ Links ]

Fridman, G., Shereshevsky, A., Jost, M. M., Brooks, A. D., Fridman, A., Gutsol, A., ... & Friedman, G. (2007). Floating electrode dielectric barrier discharge plasma in air, promoting apoptotic behavior in melanoma skin cancer cell lines. Plasma Chemistry and Plasma Processing, 27(2), 163-176. [ Links ]

Fujita, Y., Koinuma, S., De Velasco, M. A., Bolz, J., Togashi, Y., Terashima, M., ... & Nishio, K. (2014). Melanoma transition is frequently accompanied by a loss of cytoglobin expression in melanocytes: a novel expression site of cytoglobin. PloS One, 9(4). [ Links ]

Gallagher, M. J., Vaze, N., Gangoli, S., Vasilets, V. N., Gutsol, A. F., Milovanova, T. N., ... & Fridman, A. A. (2007). Rapid inactivation of airborne bacteria using atmospheric pressure dielectric barrier grating discharge. IEEE Transactions on Plasma Science, 35(5), 1501-1510. [ Links ]

García-Alcantara, E., López-Callejas, R., Peña-Eguiluz, R., Lagunas-Bernabé, S., Valencia-Alvarado, R., Mercado-Cabrera, A., ... & de la Piedad-Beneitez, A. (2012). Time effect and aliquot concentration in Streptococcus mutans elimination by plasma needle. In Journal of Physics: Conference Series (Vol. 370 , No. 1, p. 012018 ). IOP Publishing.Links ]

García-Alcantara, E. , Lopez-Callejas, R., Serment-Guerrero, J., Peña-Eguiluz, R. , Muñoz-Castro, A. E., Rodríguez-Méndez, B. G., ... & Barbabosa-Pliego, A. (2013). Toxicity and genotoxicity in HELA and E. coli cells caused by a helium plasma needle. Applied Physics Research, 5(5), 21-28. [ Links ]

Goore, J., Liu, B., & Drake, D. (2006). Gas flow dependence for plasma-needle disinfection of S. mutans bacteria. Journal of Physics D: Applied Physics, 39(16), 3479-3486. [ Links ]

Gray-Schopfer, V., Wellbrock C., & Marais, R. (2007). Melanoma biology and new targeted therapy. Nature, 445, 851-857. [ Links ]

Gurtner, G.C., Werner, S., Barrandon, Y. & Longaker, M.T., (2008). Wound repair and regeneration. Nature, 453 (7193), 314-321. [ Links ]

Hodges, N.J., Innocent, N., Dhanda, S., Graham, M. (2008) Cellular protection from oxidative DNA damage by over-expression of the novel globin cytoglobin in vitro. Mutagenesis, 23(4), 293-298. [ Links ]

Kalghatgi, S., Kelly, C. M., Cerchar, E., Torabi, B., Alekseev, O., Fridman, A. , ... & Azizkhan-Clifford, J. (2011). Effects of non-thermal plasma on mammalian cells. PLoS ONE , 6(1), 1-11. [ Links ]

Kieft, I.E., Darios, D., Rooks, A.J.M., & Stoffels, E. (2005). Plasma treatment of mammalian vascular cells: a quantitative description. IEEE Transactions on Plasma Science, 33(2), 771-775. [ Links ]

Kim, G. J., Kim, W., Kim, K. T., & Lee, J. K. (2010). DNA damage and mitochondria dysfunction in cell apoptosis induced by non-thermal air plasma. Applied Physics Letters, 96(2). [ Links ]

Kluge, S., Bekeschus, S., Bender, C., Benkhai, H., Sckell, A., Below, H., & Kramer, A. (2016). Investigating the mutagenicity of a cold argon-plasma jet in an HET-MN model. PLoS One, 11(9). [ Links ]

Kogelschatz, U. (2003). Dielectric-barrier Discharges: Their history, discharge physics, and industrial applications. Plasma Chemistry and Plasma Processing, 23(1), 1-46. [ Links ]

Laroussi, M. (1996). Sterilization of contaminated matter with an atmospheric pressure plasma. IEEE Transactions on Plasma Science, 24(3), 1188-1191. [ Links ]

Laroussi, M. (2009). Low-temperature plasmas for medicine. IEEE Transactions on Plasma Science, 37(6), 714-724. [ Links ]

Laroussi, M., Richardson, J.P., & Dobbs, F.C. (2002). Effects of nonequilibrium atmospheric pressure plasmas on the heterotrophic pathways of bacteria and on their cell morphology. Applied Physics Letters , 81(4), 773-774. [ Links ]

Lerouge, S., Wertheimer, M.R., & Yahia, H. (2001). Plasma sterilization: A review of parameters. mechanisms, and limitations. Plasmas and Polymers, 6(3), 175-188. [ Links ]

Martines, E. , Zuin, M. , Cavazzana, R. , Gazza, E., Serianni, G., Spagnolo, S., ... & Aragona, M. (2009). A novel plasma source for sterilization of living tissues. New Journal of Physics, 11(1), 115014. [ Links ]

Moisan, J., Barbeau, S., Moreau, J., Pelletier, M., Tabrizia, M., & Yahia, L.H. (2001). Low-temperature sterilization using gas plasmas: A review of the experiments and an analysis of the inactivation mechanisms. International Journal of Pharmaceutics, 226(1-2), 1-21. [ Links ]

Nehra, V., Kumar, A., & Dwivedi, H.K. (2008). Atmospheric non-thermal plasma sources. International Journal of Engineering, 2(1), 53-68. [ Links ]

Nishi, H., Inagi, R., Kawada, N., Yoshizato, K., Mimura, I., Fujita, T., & Nangaku, M. (2011). Cytoglobin, a novel member of the globin family, protects kidney fibroblasts against oxidative stress under ischemic conditions. American Journal of Pathology, 178:128-139. [ Links ]

Morales-Ramirez, P., Cruz-Vallejo, V., Peña-Eguiluz, R. , Lopez-Callejas, R. , Rodríguez-Méndez, B. G. , Valencia-Alvarado, R. , ... & Munoz-Castro, A. E. (2013). Assessing cellular DNA damage from a helium plasma needle. Radiation Research 179(6), 669-673. [ Links ]

Pérez‐Martínez, José A., Rosendo Peña‐Eguiluz, Régulo López‐Callejas, Antonio Mercado‐Cabrera, Raul Valencia Alvarado, Samuel R. Barocio, & Aníbal de la Piedad‐Beneitez (2008). Power Supply for Plasma Torches Based on a Class‐E Amplifier Configuration. Plasma Processes and Polymers, 5(6), 593-598. [ Links ]

Sladek, R.E.J., Stoffels, E. , Walraven, R., Tielbeek, P.J.A., & Koolhoven, R.A. (2004). Plasma treatment of dental cavities: a feasibility study. IEEE Transactions on Plasma Science, 32(4), 1540-1543. [ Links ]

Stoffels, E. (2003). Plasma needle: Treatment of living cells and tissues. American Physical Society, 56th Gaseous Electronics Conference, October 21-24, San Francisco, CA. [ Links ]

Stoffels, E. , Sakiyama, Y., & Grave, D.B: (2008). Cold atmospheric plasma: charged species and their interactions with cells and tissues. IEEE Transactions on Plasma Science, 36(4), 1441-1457. [ Links ]

Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., ... & Sasaki, Y. F. (2000). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Mutagenesis , 35(3), 206-221. [ Links ]

Van der Paal, Jonas, Erik C. Neyts, Christof CW Verlackt, & Annemie Bogaerts (2016). Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress. Chemical science, 7(1), 489-498. [ Links ]

Vermeylen, S., De Waele, J., Vanuytsel, S., De Backer, J. , Van der Paal, J., Ramakers, M., Leyssens, K., Marcq, E., Van Audenaerde, J., L.J. Smits, E., & Dewilde, S. (2016). Cold atmospheric plasma treatment of melanoma and glioblastoma cancer cells. Plasma Processes and Polymers, 13(12), 1195-1205. [ Links ]

Yan, D., Sherman, J.H., & Keidar, M. (2017). Cold atmospheric plasma, a novel promising anti-cancer treatment modality. Oncotarget, 8(9), 15977-15995. [ Links ]

You, Y. H., Lee, D. H., Yoon, J. H., Nakajima, S., Yasui, A., & Pfeifer, G. P. (2001). Cyclobutane pyrimidine dimers are responsible for the vast majority of mutations induced by UVB irradiation in mammalian cells. Journal of Biological Chemistry, 276(48), 44688-44694. [ Links ]

Zirnheld, J. L., Zucker, S. N., DiSanto, T. M., Berezney, R., & Etemadi, K. (2010). Nonthermal plasma needle: development and targeting of melanoma cells.IEEE Transactions on Plasma Science,38(4), 948-952. [ Links ]

Peer Review under the responsibility of Universidad Nacional Autónoma de México.

* Corresponding author. E-mail address: jorge.serment@inin.gob.mx (Jorge H. Serment Guerrero).

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License