Therapeutic ROS targeting of GADD45γ in the induction of G2/M arrest in primary human colorectal cancer cell lines by cucurbitacin E.

Y-C Hsu,M-J Chen,T-Y Huang

doi:10.1038/cddis.2014.151

Abstract

Cucurbitacin E (CuE) or α-elaterin is a natural compound previously shown to be an antifeedant as well as a potent chemopreventive agent against several types of cancer. The present study investigated the anticancer effects of CuE on colorectal cancer (CRC) using primary cell lines isolated from five CRC patients in Taiwan, Specifically, we explored the anti-proliferation and cell cycle G2/M arrest induced by CuE in CRC cells. MPM-2 flow cytometry tests show that CuE-treated cells accumulated in metaphase (CuE 2.5–7.5 μM). Results further indicate that CuE produced G2/M arrest as well as the downregulation of CDC2 and cyclin B1 expression and dissociation. Both effects increased proportionally with the dose of CuE; however, the inhibition of proliferation, arrest of mitosis, production of reactive oxygen species (ROS), and loss of mitochondrial membrane potential (ΔΨm) were found to be dependent on the quantity of CuE used to treat the cancer cells. In addition, cell cycle arrest in treated cells coincided with the activation of the gene GADD45(α, β, γ). Incubation with CuE resulted in the binding of GADD45γ to CDC2, which suggests that the delay in CuE-induced mitosis is regulated by the overexpression of GADD45γ. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activities in established CRC.

Highlights

Cucurbitacin E (CuE) is an active compound,[7] which was previously shown to be a strong antifeedant with the ability to disrupt cell actin[8] and cell adhesion.[9]
These findings indicate that the increase in G2/M phase colorectal cancer (CRC) cells resulted from non-CuE-induced apoptosis of primary CRC cells, the downregulation of cell division cycle gene 2 (CDC2), and the dissociation of the cyclin B1/CDC2 complex following incubation with CuE
Our results indicated that the activity of GADD45g/CDC2 complex was significantly suppressed in cells incubated at a CuE concentration of 7.5 mM (Figure 5b) (y 1⁄4 7.8819x þ 86.851, R2 1⁄4 0.7695)

Summary

Introduction

Cucurbitacin E (CuE) is an active compound,[7] which was previously shown to be a strong antifeedant with the ability to disrupt cell actin[8] and cell adhesion.[9]. No significant increase was observed in the percentage of five CRC cell lines undergoing necrosis, apoptosis (Figure 2a), or caspase 3 activation at CuE concentrations of 2.5–7.5 mM (data not shown).

Results

Conclusion