Cellular Effects of Cyclodextrins: Studies on HeLa Cells
Abstract
:1. Introduction
2. Results
2.1. Cytotoxicity
2.2. Investigation of the Cellular Uptake of Cyclodextrins
2.3. NF-κB Pathway Activation Study
2.4. Autophagy Study
2.5. Investigation of Lysosomes
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Culture
4.3. Investigation of Cytotoxicity
4.4. Cellular Uptake of Fluorescently Labeled Cyclodextrin Derivatives on HeLa Cells
4.4.1. Fluorescence Microscopy
4.4.2. Flow Cytometry
4.5. Investigation of the NF-kB Pathway
4.6. Investigation of Autophagy
4.6.1. Fluorescence Microscopy
4.6.2. Microplate Reader
4.7. Investigation of the Lysosomes
4.7.1. Fluorescence Microscopy
4.7.2. Flow Cytometry
4.8. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Caco-2 Cell Line | HeLa Cell Line | |
---|---|---|
Cytotoxicity of cyclodextrins | 50 mM HPBCD and 10 mM RAMEB were cytotoxic (RTCA method) | |
Cellular internalization of fluorescently labeled cyclodextrins | Both fluorescein and rhodamine labeled HPBCD and RAMEB were localized in vesicles in the cytoplasm. (FM) | |
Normalized fluorescence intensity (FC, 50 µM): FITC-HPBCD: 35.63 ± 2.72 FITC-RAMEB: 4.96 ± 1.23 | Normalized fluorescence intensity (FC, 50 µM): FITC-HPBCD: 8.55 ± 2.26 FITC-RAMEB: 2.13 ± 0.33 | |
The internalization of cyclodextrins was significantly inhibited at 0 °C. (FC) | ||
Rottlerin significantly reduced the endocytosis both of FITC-HPBCD and FITC-RAMEB. (FC) | Rottlerin significantly increased the internalization of FITC-RAMEB. (FC) | |
Chlorpromazine significantly increased the internalization of the cyclodextrins. (FC) | Chlorpromazine had no significant effect on the cellular uptake of cyclodextrins. (FC) | |
Type of cyclodextrin endocytosis: fluid phase endocytosis predominates. | Type of endocytosis: its precise determination requires further experiments, presumably several simultaneous processes including clathrin-dependent endocytosis | |
Investigation of lysosomes | Cyclodextrin tested: Rho-HPBCD, Rho-RAMEB | Cyclodextrins tested: FITC-HPBCD, FITC-RAMEB |
Cyclodextrins entered the lysosomes, did not show more intense staining compared to the control and did not induce the formation of lysosomes. (FM, FC) | ||
Investigation of the NF-κB pathway | p65 translocation was not detected to the nucleus and induction of the NF-κB inflammatory pathway. (FM) | |
Investigation of the autophagy | Based on qualitative and quantitative studies, neither Rho-HPBCD nor Rho-RAMEB induced the formation of autophagosomes and autophagy. The effect of chloroquine (used as a positive control) was significantly different from that of the control sample. (FM, MR) |
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Rusznyák, Á.; Palicskó, M.; Malanga, M.; Fenyvesi, É.; Szente, L.; Váradi, J.; Bácskay, I.; Vecsernyés, M.; Réti-Nagy, K.S.; Vasvári, G.; et al. Cellular Effects of Cyclodextrins: Studies on HeLa Cells. Molecules 2022, 27, 1589. https://doi.org/10.3390/molecules27051589
Rusznyák Á, Palicskó M, Malanga M, Fenyvesi É, Szente L, Váradi J, Bácskay I, Vecsernyés M, Réti-Nagy KS, Vasvári G, et al. Cellular Effects of Cyclodextrins: Studies on HeLa Cells. Molecules. 2022; 27(5):1589. https://doi.org/10.3390/molecules27051589
Chicago/Turabian StyleRusznyák, Ágnes, Mercédesz Palicskó, Milo Malanga, Éva Fenyvesi, Lajos Szente, Judit Váradi, Ildikó Bácskay, Miklós Vecsernyés, Katalin Szászné Réti-Nagy, Gábor Vasvári, and et al. 2022. "Cellular Effects of Cyclodextrins: Studies on HeLa Cells" Molecules 27, no. 5: 1589. https://doi.org/10.3390/molecules27051589