Abstract
Radioresistance in non-small cell lung cancer (NSCLC) is a major contributor to radiation treatment failure. The transmembrane protein, Caveolin-1 (Cav1), is found to be highly expressed in numerous radioresistant tumors. However, the role of Cav1 in radioresistant NSCLC is still unexplored. Hence, in this study, we sought to demonstrate that modulation of CAV1 expression in NSCLC cells correlates with radioresistance . We have developed CRISPR/Cas9 mediated Cav1 knockout to enhance radiosensitivity in a novel acquired resistant NSCLC cell line, suggesting a possible role of Cav1 in mediating radioresistance. A549 cells were treated with photon therapy on a weekly basis (4Gy/week) to a maximum dose of 80 Gy to develop cell line with acquired radioresistance, which was confirmed by clonogenic assay, MTS assay and by western blot (WB). Cav1 knockout cells were developed using the CRISPR/Cas9 system with specific guide RNA (gRNA) targeting Cav1. T7 Endonuclease Assay was done to assess Cas9-editing efficiency and WB was done to confirm protein knockout. Clonogenic and MTS assays were also done to compare cell survival. The linear quadratic model was fit to each dataset and used to characterize differences in radiosensitivity. Further, sensitive A549 cells were transduced with lentivirus to overexpress Cav1. Expression was confirmed by flow cytometry and WB, and GFP competition assay was performed to assess cell proliferation rate. Our acquired radioresistant A549 cell line demonstrated stable radioresistance at different doses of 2, 4, 6 and 8 Gy in photon therapy with WB showing increased Cav1 expression compared to the parental cell line. Using CRISPR/Cas9, Cav1 knockout cells showed no Cav1 expression by WB as well as increased radiosensitization at different doses. Radiosensitive A549 cells were transduced with lentivirus and cells with more than 90% GFP positive in Flow cytometry were expanded to over expressed Cav1 and confirmed radioresistance at different doses compared to parental cells. We successfully established an acquired radioresistant A549 cell line and demonstrated that altering the expression of Cav1 can modulate radioresistance, thereby supporting a possible role of Cav1 in radioresistance. Using CRISPR/Cas9 system, Cav1 knockout cells were developed confirming its radiosensitizing effect. Our findings justify that Cav-1 could be a novel prognostic biomarker to monitor tumor radioresistance in NSCLC patients undergoing radiation therapy.
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More From: International Journal of Radiation Oncology*Biology*Physics
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