RTA404, an Activator of Nrf2, Activates the Checkpoint Kinases and Induces Apoptosis through Intrinsic Apoptotic Pathway in Malignant Glioma

Tai-Hsin Tsai; Ann-Shung Lieu; Tzuu-Yuan Huang; Aij-Lie Kwan; Chih-Lung Lin; Yi-Chiang Hsu

doi:10.3390/jcm10214805

RTA404, an Activator of Nrf2, Activates the Checkpoint Kinases and Induces Apoptosis through Intrinsic Apoptotic Pathway in Malignant Glioma

J Clin Med. 2021 Oct 20;10(21):4805. doi: 10.3390/jcm10214805.

Authors

Tai-Hsin Tsai^{1

2

3}, Ann-Shung Lieu^{1

2

3}, Tzuu-Yuan Huang⁴, Aij-Lie Kwan^{1

2

3}, Chih-Lung Lin^{1

2

3}, Yi-Chiang Hsu⁵

Affiliations

¹ Division of Neurosurgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan.
² Department of Surgery, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
³ Graduate Institutes of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
⁴ Department of Neurosurgery, Changhua Christian Hospital, Changhua 500, Taiwan.
⁵ School of Medicine, I-Shou University, Kaohsiung 824, Taiwan.

Abstract

Background: Malignant glioma (MG) is an aggressive malignant brain tumor. Despite advances in multidisciplinary treatment, overall survival rates remain low. A trifluoroethyl amide derivative of 2-cyano-3-,12-dioxoolean-1,9-dien-28-oic acid (CDDO), CDDO-trifluoroethyl amide (CDDO-TFEA) is a nuclear erythroid 2-related factor 2/antioxidant response element pathway activator. RTA404 is used to inhibit proliferation and induce apoptosis in cancer cells. However, its effect on tumorigenesis in glioma is unclear. Methods: This in vitro study evaluated the effects of RTA404 on MG cells. We treated U87MG cell lines with RTA404 and performed assessments of apoptosis and cell cycle distributions. DNA content and apoptosis induction were subjected to flow cytometry analysis. The mitotic index was assessed based on MPM-2 expression. Protein expression was analyzed through Western blotting. Results: RTA404 significantly inhibited the cell viability and induced cell apoptosis on the U87MG cell line. The Annexin-FITC/PI assay revealed significant changes in the percentage of apoptotic cells. Treatment with RTA404 led to a significant reduction in the U87MG cells' mitochondrial membrane potential. A significant rise in the percentage of caspase-3 activity was detected in the treated cells. In addition, these results suggest that cells pass the G2 checkpoint without cell cycle arrest by RTA404 treatment in the MPM-2 staining. An analysis of CHK1, CHK2, and p-CHK2 expression suggested that the DNA damage checkpoint system seems also to be activated by RTA404 treatment in established U87MG cells. Therefore, RTA404 may not only activate the DNA damage checkpoint system, it may also exert apoptosis in established U87MG cells. Conclusions: RTA404 inhibits the cell viability of gliomas and induces cancer cell apoptosis through intrinsic apoptotic pathway in Malignant glioma. In addition, the DNA damage checkpoint system seems also to be activated by RTA404. Taken together, RTA404 activated the DNA damage checkpoint system and induced apoptosis through intrinsic apoptotic pathways in established U87MG cells.

Keywords: RTA404; apoptosis; checkpoint kinase; malignant glioma.