Induction of dsDNA Dispersion in SH-SY5Y Cells by Chloroquine and Doxorubicin
Introduction
Human Neuroblastoma (NB) is a pediatric tumor that begins in nerve tissues of the adrenal gland, and nerve tissues of the neck, chest, or pelvis. Drug resistance poses a significant challenge in cancer patients, hindering the effectiveness of treatments such as Doxorubicin (Doxo). The administration of Doxo involves a 24-hour treatment followed by a designated period of recovery for the patient. Doxo disrupts the tumor tissue environment, promoting increased cytotoxicity within cells, which ultimately induces cell death. It forms a complex with DNA through the G bases on both DNA strands. Autophagy inhibitors hinder or reduce the occurrence of autophagy in cells triggered by drugs or compounds. Within eukaryotic cells, Chloroquine (CQ), an antimalarial medication, strongly affects lysosomotropic and dsDNA activities, effectively inhibiting autophagy. When administered, CQ leads to an elevation in acidity within the lysosomes of cancer cells, which play a crucial role in breaking down and recycling cellular components. In this study, we investigated the impact of CQ on Doxo treatment in the SH-SY5Y cell line, focusing on the inhibition of autophagy and its effect on dsDNA dispersion. Following a 48-hour period, cells treated with Doxo and CQ exhibited reduced dsDNA dispersion compared to those treated with Doxo alone.
Methods
A cell viability assay was done by allowing the cells to reach confluency in DMEM supplemented with 3% FBS, 10 mM all-trans-retinoic acid (RA), and 1% P/S at 37°C and 5% CO2 for five days. These cells were treated with 0.2, 0.5, 1, 10, 20, and 40 µM Doxo, CQ, and 25, 50, 100 µM CQ and Doxo. Following treatment of cells for 24 and 48 h, a medium containing non-adherent and dead cells was removed and set aside. To examine the effect of the incubation time and concentration of Doxo and CQ, cell morphology was examined by phase contrast microscopy. We studied viability by determining the percentage of intact, round, and Trypan blue-stained cells at different times of exposure. Cells were counted in eight quadrants of a hemocytometer with a light microscope, with cells stained blue counted as dead, and results were expressed as a percentage of total cells using GraphPad Prism v.5.0. The dispersion of dsDNA was analyzed by treating SH-SY-5Y cells with 0.2µM Doxo, 25 µM, and 100 µM CQ for 48h, and co-stained with Lamin B1, dsDNA, and H3K9 dimethyl that were observed in the cytoplasm. The dispersion was calculated by dsDNA dispersed cell percentage total cell (dsDNA amount/dispersed cell number).
Results
The data demonstrate a dose-response relationship in cells exposed to both pre-treatment and post-treatment conditions. Cell death of SH-SY5Y cells, obtained from a metastatic bone tumor biopsy, was observed in a manner dependent on both the dosage and duration of exposure to Doxo. Hence, the induction of cell death in response to Doxo treatment exhibited a dependence on both the dose and duration, emphasizing the dose- and time-dependent nature of the phenomenon. The dispersion of dsDNA increased in Doxo treatments compared with Doxo and CQ together in all doses.
Conclusion
These findings align with the established mechanism of action of Doxo, which involves DNA, cell membrane, and mitochondrial damage, ultimately resulting in cell death. The inclusion of CQ did not intensify cell death or dsDNA dispersion, suggesting that it may not be the optimal co-treatment option with Doxo. Exploring alternative drugs that operate through autophagy could hold therapeutic potential.