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

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Azeem Khan (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

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.

Additional Information

Publication

Thesis

Language: English

Date: 2017

Keywords

Notch 3, PARP1, APE1, Notch Pathway

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