Cucurbitacin B induces rapid depletion of the G-actin pool through reactive oxygen species-dependent actin aggregation in melanoma cells

Abstract

Cucurbitacin B (CuB), a triterpenoid compound isolated from Cucurbitaceae plants, has been reported as a promising anti-cancer agent, yet its action mechanism is still controversial. In this study, we explored the potential mechanism of CuB in murine B16F10 melanoma cells. Anti-proliferation and anti-invasion effects were assessed in cultured cells, and in vivo anti-tumor activity was evaluated in a murine subcutaneous melanoma model. Flow cytometry was adopted to analyze cell cycle distribution and reactive oxygen species (ROS) levels. Actin levels were determined by western blot analysis, and the profiles of differential expressed proteins were identified by a quantitative proteomic approach. The results showed that CuB exerted inhibitory effects on cell proliferation, colony formation, as well as migration and invasion potential of the melanoma cells. The growth of subcutaneous melanoma was significantly inhibited in mice treated with CuB when compared with control group. Furthermore, CuB treatment caused rapid cell membrane blebbing and deformation, and induced G 2 /M-phase arrest and formation of multiploid cells. Notably, the G-actin pool was rapidly depleted and actin aggregates were formed quickly after CuB treatment. A number of cytoskeleton-regulatory proteins were differentially regulated. Blockage of ROS production significantly reduced the G-actin depletion ability and the anti-tumor activity of CuB. These findings indicate that CuB induces rapid depletion of the G-actin pool through ROS-dependent actin aggregation in melanoma cells, which may at least partly account for its anti-tumor activity.

Cucurbitacin I Inhibits Rac1 Activation in Breast Cancer Cells by a Reactive Oxygen Species-Mediated Mechanism and Independently of Janus Tyrosine Kinase 2 and P-Rex1

“…Notably, activation of RhoA signaling and stress fiber formation by cucurbitacin I was dependent upon the generation of intracellular ROS. Recent studies in a number of cancer cellular models showed that cucurbitacins I and B induce the production of ROS (Yasuda et al, 2010;Zhang et al, 2011Zhang et al, , 2012b), as we observed in breast cancer cells. We found that the specific ROS mitochondrial antioxidant Mito-TEMPO prevented the effect of cucurbitacin I on Rac inhibition, arguing that mitochondrial-derived ROS have a prominent role in this effect.…”

Cucurbitacin B Induces Cell Cycle Arrest, Apoptosis and Autophagy Associated with G Actin Reduction and Persistent Activation of Cofilin in Jurkat Cells

“…Analysis of the cell cycle was performed as described previously [22]. In brief, the cells were fixed and stained with phosphate-buffered saline (PBS) containing 50 µg/ml propidium iodide and 30 µg/ml of RNase A. DNA content data were acquired using CELL Quest software on a flow cytometer (FACSCalibur; Becton Dickinson).…”

“…Moreover, several studies have reported that CuB activates the phosphorylation of Erk1/2 or has no significant effect on them [19,20,21], whereas others have indicated that CuB inhibits Erk1/2 phosphorylation [5,12]. Previous studies have also shown that CuB induces cell cycle arrest at the G 2 /M phase and formation of multinuclear cells in tumor cells [5,6,7,12,13,14,17], probably due to the disruption of the actin cytoskeleton [4,5,6,14,22]. Therefore, further investigations on the action mechanism of CuB are still needed.…”

Cucurbitacin B Alters the Expression of Tumor-Related Genes by Epigenetic Modifications in NSCLC and Inhibits NNK-Induced Lung Tumorigenesis

“…Recently, there has been growing interest in CuB-mediated anticancer activities, mainly mediated through induction of G 2 -M phase cell-cycle arrest, inhibition of the JAK/STAT pathway, and induction of apoptosis (13)(14)(15)(16)(17)(18)(19).…”