Effects of Zinc Phthalocyanine Photodynamic Therapy on Vital Structures and Processes in Hela Cells
<p>The dependence of tumoral and nontumoral cell viability on the concentrations of the zinc phthalocyanine photosensitizer. The dependence of the cellular viability on the concentration of the zinc phthalocyanine photosensitizer was determined by measuring the enzyme activity of living cells using the MTT test. The irradiation dose used was 5 Jcm<sup>−2</sup>. The control represents the irradiated cells without the photosensitizer (the negative control), and its value is set at 100%. Data are presented as ±SD from three independent measurements. The results were considered statistically significant when <span class="html-italic">p</span> < 0.05 and indicated by an asterisk symbol.</p> "> Figure 2
<p>ROS production and protein level changes after 5 Jcm<sup>−2</sup> ZnPc photodynamic therapy. The dependence of the HeLa cells’ ROS production on the concentration of the zinc phthalocyanine photosensitizer measured immediately after irradiation: (<b>A</b>) singlet oxygen production; (<b>B</b>) ROS production after type I of PDT reaction. The control represents the irradiated cells without the photosensitizer (the negative control). Data are presented as ±SD from three independent measurements. Results were considered statistically significant when <span class="html-italic">p</span> < 0.05 and indicated in the graphs by an asterisk symbol. (<b>C</b>) Changes in the proteins involved in oxidation stress reduction depending on incubation time after the therapy. The relative quantification was calculated as the proportion of protein in the control cell sample compared to the protein level in the treated cell sample. P32119 (UniProt identificator)—peroxiredoxin 2, P30044—peroxiredoxin 5, P30041—peroxiredoxin 6, P00441—superoxide dismutase [Cu-Zn], P10599—thioredoxin.</p> "> Figure 3
<p>Changes at the mitochondrial level after 5 Jcm<sup>-2</sup> ZnPc PDT. (<b>A</b>) Protein level changes depending on incubation time after the therapy (OPA1 (UniProt ID O60313—dynamin-like 120 kDa protein); VDAC (P21796—voltage-dependent anion-selective channel protein 1)). The relative quantification was calculated as the proportion of protein in the control cell sample compared to the protein level in the treated cell sample. (<b>B</b>) The change in HeLa cells’ mitochondria membrane potential using LC50 concentration of zinc phthalocyanine photosensitizer. The control represents the irradiated cells without a photosensitizer (the negative control). Data are presented as ±SD from three independent measurements. Results were considered statistically significant when <span class="html-italic">p</span> < 0.05 and indicated in the graphs by an asterisk symbol.</p> "> Figure 4
<p>Changes at the DNA level after 5 Jcm<sup>−2</sup> ZnPc PDT. (<b>A</b>) Changes in the protein level depending on the incubation period after the therapy. The relative quantification was calculated as the proportion of protein in the control cell sample compared to the protein level in the treated cell sample. Q16531—DNA damage-binding protein 1; P09874—poly [ADP-ribose] polymerase 1; P12004—proliferating cell nuclear antigen. (<b>B</b>) DNA damage evaluated 24 h after therapy by comet assay using LC50 concentration of zinc phthalocyanine photosensitizer. The control represents the irradiated cells without a photosensitizer (the negative control).). Data are presented as ±SD from three independent measurements. Results were considered statistically significant when <span class="html-italic">p</span> < 0.05 and indicated in the graphs by an asterisk symbol.</p> "> Figure 5
<p>The seven largest groups of the degraded proteins in terms of cell localization (G0 terms) identified in samples with 0, 4, and 24 h incubation time after in vitro 5 Jcm<sup>−2</sup> ZnPc PDT at the LC50 concentration on HeLa cells. Degraded proteins were identified by comparing the measured sample to the control cells without photosensitizer and irradiation. The proteins identified were sorted using David Bioinformatic Resources 6.8 when <span class="html-italic">p</span> < 0.0001 [<a href="#B44-ijms-25-10650" class="html-bibr">44</a>,<a href="#B45-ijms-25-10650" class="html-bibr">45</a>].</p> "> Figure 6
<p>The six largest groups of the degraded proteins in terms of molecular function (G0 terms) identified 24 h after in vitro 5 Jcm<sup>−2</sup> ZnPc PDT at the LC50 concentration on HeLa cells. Degraded proteins were identified by comparing the measured sample to the control cells without photosensitizer and irradiation and sorted using David Bioinformatic Resources 6.8 when <span class="html-italic">p</span> < 0.0001 [<a href="#B44-ijms-25-10650" class="html-bibr">44</a>,<a href="#B45-ijms-25-10650" class="html-bibr">45</a>].</p> "> Figure 7
<p>The dependence of tumoral HeLa and nontumoral BJ cells’ viability on the concentrations of the zinc phthalocyanine photosensitizer after sensitizer incorporation into the liposomes. The cellular viability was determined by measuring the enzyme activity of living cells using the MTT test. The irradiation dose used was 5 Jcm<sup>−2</sup>. The liposomal content was released from liposomes using ultrasound (3 Wcm<sup>−2</sup>, 60 s, pulse mode). The control represents the irradiated and sonicated cells without the photosensitizer (the negative control). Data are presented as ±SD from three independent measurements.</p> "> Figure 8
<p>The chemical structure of the zinc phthalocyanine (ZnPc) used in the study. The chemical formula of ZnPc: 2,3,9,10,16,17,23,24-Octakis[(2-(triethylammonio)ethyl)sulfanyl]phthalocyaninato]zinc(II) Octaiodide [<a href="#B52-ijms-25-10650" class="html-bibr">52</a>].</p> ">
Abstract
:1. Introduction
2. Results and Discussion
2.1. Cell Viability after 5 Jcm−2 ZnPc PDT Analysis
2.2. HeLa Cell Changes at the Oxidative Stress Level
2.3. HeLa Cell Changes at the Mitochondrial Level
2.4. Changes in HeLa Cells at the DNA Level
2.5. Cellular Localization and Molecular Function of Degraded Proteins
2.5.1. Cellular Localization
2.5.2. Molecular Function
2.6. Incorporation of the Sensitizer into the Liposomes
3. Materials and Methods
3.1. Photosensitive Substance and Cell Lines
3.2. Light Source and Exposure
3.3. MTT Viability Test
3.4. ROS Measurement
3.4.1. Type I Reaction Products’ Measurement
3.4.2. Type II Reaction Product (Singlet Oxygen) Measurement
3.5. Comet Assay
3.6. Protein Analysis
3.7. Mitochondrial Membrane Potential Measurement
3.8. Incorporation of the Sensitizer into the Liposomes
3.9. Statistical Analysis
4. Conclusions
5. Patents
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
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Cell Line | LC50 [µM] ZnPc |
---|---|
BJ | 0.09 ± 0.02 |
HaCat | 0.17 ± 0.03 |
MCF7 | 0.45 ± 0.17 |
HeLa | 0.03 ± 0.01 |
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Hosik, J.; Hosikova, B.; Binder, S.; Lenobel, R.; Kolarikova, M.; Malina, L.; Dilenko, H.; Langova, K.; Bajgar, R.; Kolarova, H. Effects of Zinc Phthalocyanine Photodynamic Therapy on Vital Structures and Processes in Hela Cells. Int. J. Mol. Sci. 2024, 25, 10650. https://doi.org/10.3390/ijms251910650
Hosik J, Hosikova B, Binder S, Lenobel R, Kolarikova M, Malina L, Dilenko H, Langova K, Bajgar R, Kolarova H. Effects of Zinc Phthalocyanine Photodynamic Therapy on Vital Structures and Processes in Hela Cells. International Journal of Molecular Sciences. 2024; 25(19):10650. https://doi.org/10.3390/ijms251910650
Chicago/Turabian StyleHosik, Jakub, Barbora Hosikova, Svatopluk Binder, Rene Lenobel, Marketa Kolarikova, Lukas Malina, Hanna Dilenko, Katerina Langova, Robert Bajgar, and Hana Kolarova. 2024. "Effects of Zinc Phthalocyanine Photodynamic Therapy on Vital Structures and Processes in Hela Cells" International Journal of Molecular Sciences 25, no. 19: 10650. https://doi.org/10.3390/ijms251910650