Abstract 2063: GO-Chl induce necroptosis death in A549 lung cancer cells through p62/SQSTM1 mediated DNA damage response

Abstract

Abstract Autophagy is a highly regulated intracellular catabolic pathway for maintenance of cell homeostasis through the process of degrading proteins and organelles. Inhibition or modulation of autophagy may leads to accumulation of DNA damage and genomic instability. Recently, it has been reported that DNA damage or repair response by autophagy machinery can switch the cell signalling cascade from apoptosis to necroptosis. The synergistic effect of autophagy machinery with DNA damage pathways may provide a therapeutic window for next generation nanomedicine. Graphene oxide (GO) alters the autophagy response through toll like receptor signaling, lysosomal dysfunction, mitochondrial destabilization and NF- κB pathways. On the other hand, Chloroquine (Chl) an FDA approved drug inhibits the autophagy and has also shown anticancer potential. In the present study, we endeavored the conjugation of Chl onto GO nanosheets and evaluated the antiproliferative efficacy of GO-Chl nanoconjugate on human lung cancer A549 and normal lung BEAS-2B cell lines. Morphological properties of GO and GO-Chl have been analyzed through TEM, FESEM and AFM and formation of nearly monolayer highly exfoliated GO nanosheets has been observed. Further, structural, functional and optical properties of GO, Chl and GO-Chl have been investigated using Raman, FTIR and UV-Vis spectroscopy respectively. MTT assay has been performed for in- vitro cytotoxicity evaluation of GO, Chl and GO-Chl exposure on A549 and BEAS-2B cell lines and demonstrated that GO-Chl treatment exhibits significant cell death in A549 lung cancer cells. DCFDA assay reveals that GO-Chl exposure enhance generation of ROS. Flow cytometry based Propidium Iodide (PI) assay reveals the plasma membrane disruption leading to alteration in the cell cycle. Further, flow cytometry based annexin V/PI assay for cell cycle analysis indicate towards the halts of the cell cycle at G1 phase and possible DNA damage response. The integrity of A549 cells DNA was accesed using Comet assay. The results reveals a significant increase in Olive tail moment with increasing concentration of GO-Chl. Further, the autophagy response in A549 cells due to GO-Chl treatment is investigated through fluorescence microscopic analysis (MDC staining and GFP-LC3 plasmid), TEM observations and immunoblot analysis. Enhanced level of LC-3 I/II and Atg-5 markers signifies the autophagosomes formation and elevated expression of p62/SQSTM1 indicates the inhibition of autophagy at later stage. Recently, it was reported that enhanced level of p62/SQSTM1 may inhibit the DNA repair mechanism leading to decreased growth in-vitro and in-vivo. On the basis of results obtained, it is envisioned that GO-Chl nanoconjugate could be used as an effective cancer therapeutic agent, by targeting the DDR response through modualtion of autophagy. Note: This abstract was not presented at the meeting. Citation Format: Braham D. Arya, Surinder P. Singh. GO-Chl induce necroptosis death in A549 lung cancer cells through p62/SQSTM1 mediated DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2063.