Notch 3 Affects Chemoresistance in Colorectal Cancer via DNA Base Excision Repair Enzymes

Abstract

Approximately 1.2 million cases of colorectal cancer (CRC) arise each year, and 40-50% of CRC patients will reach metastasis. The Notch pathway is known to be dysregulated in CRC, and its relationship with DNA repair mechanisms, which contribute to drug resistance, is currently being established. Previously, we observed a decrease in DNA base excision repair (BER) enzymes and drug resistance upon Notch 1 targeting. We have also observed that Notch 1 signaling is associated with promoting cancer stemness and epithelial to mesenchymal transition in CRC via upregulation of the Notch 3 receptor. Thus, we hypothesized that targeting Notch 3 will increase drug sensitivity in CRC via signaling effects on proteins associated with the DNA BER mechanism. Methods: In order to assess our hypothesis, the colon cancer cell line HCT 116 was transduced with a small hairpin messenger RNA construct that effectively knocked down the Notch 3 receptor, creating the Sh-N3 cell line. Culturing and Western blot analyses were conducted using standard methodology. Drug resistance was analyzed by treating cells with cisplatin or cytarabine, potent DNA damaging agents, and cytotoxicity was assessed. Microscopy was used to confirm effects of the DNA damaging agents. Proteins from the Notch 3 receptor targeted cell line treated with the DNA damaging agents were also studied. Results: Notch 3 targeted (Sh-N3) cells resulted in 1.5-fold lower plating efficiency compared to their counterpart controls (p<0.01). Western blot analysis showed that Notch 3 targeting led to a decrease in poly (ADP-ribose) polymerase (PARP1) expression by 34% in comparison to the parental cell line control (p<0.05), while apurinic/apyrmidinic endonuclease (APE1) expression was decreased by 47% (p<0.05). Sh-N3 cells treated with 20 [micro]g/mL of cisplatin for 48 hours showed a 2-fold increase in cell death compared to the controls (p<0.001). Additionally, cytotoxicity in Notch 3 null cells treated with 0.64 [micro]g/mL of cytarabine at 48 hours displayed a 1.7-fold increase compared to the controls (p<0.001). Microscopic observations confirmed these cytotoxicity results. Conclusions: This study further reinforces the importance of Notch signaling in drug resistance, and highlights the potential use of Notch 3 inhibition in conjunction with DNA BER protein inhibitors to effectively target chemoresistant CRC cells.