Cucurbitacin induces autophagy through mitochondrial ROS production which counteracts to limit caspase-dependent apoptosis

Abstract

NH2-terminal kinase; Baf-A 1 , bafilomycin A 1 ; 3-MA, 3-methyladenine; WM, wortmannin; NAC, N-acetylcysteine; BHA, butylated hydroxyanisole; Mito-TEMPO, (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride; DAPI, (4',6-diamidino-2-phenylindole; PARP, poly(ADP-ribose)polymerase; RET/PTC, rearranged in transformation/papillary thyroid carcinomas; LAMP-1, lysosomal-associated membrane protein 1; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; IM-54, 2-(1H-Indol-3-yl)-3-pentylamino-maleimide; t-BHP, tert-butyl hydroperoxide; siRNA, small interfering RNATargeted disruption of STAT3 function has proven to be a useful cancer therapeutic approach by inducing apoptotic cell death. Cucurbitacin is currently under development as a small molecule of STAT3 inhibitor to trigger cell death in many cancers. Here, we systematically studied the molecular mechanisms underlying cucurbitacin-induced cell death, in particular the involvement of autophagy. Treatment with cucurbitacin resulted in non-apoptotic cell death in a caspaseindependent manner. Notably, cucurbitacin enhanced excessive conversion of lipidated LC3 (LC3-II) and accumulation of autophagosomes in many cell types. Such autophagy and cell death induced by cucurbitacin were independent of its ability to inhibit STAT3 function, but mainly mediated by enhanced production of mitochondrial-derived reactive oxygen species (ROS), and subsequently activation of extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK). Interestingly, both the autophagy inhibitor wortmannin and knockdown of Atg5 or Beclin 1 failed to rescue the cells from cucurbitacin-induced cell death, as suppression of autophagy induced the mode of cell death to shift from autophagic cell death to caspase-dependent apoptosis. Thus the present study provides new insights into the molecular mechanisms underlying cucurbitacin-mediated cell death and supports cucurbitacin as a potential anti-cancer drug through modulating the balance between autophagic and apoptotic modes of cell death.

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

“…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. This is in agreement with Zhang et al (2012b), who found that cucurbitacin I enhances the production of mitochondrial-derived ROS. One likely possibility is that ROS generated by cucurbitacin I act directly on RhoA to cause its activation.…”

“…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.…”

“…Next, we investigated the underlying mechanisms leading to the modulation of small GTPases by cucurbitacin I. It has been recently reported that cucurbitacin I induces the formation of ROS, particularly from a mitochondrial compartment (Zhang et al, 2012b), and ROS have been implicated in the activation of Rho and Rho effectors (Jin et al, 2004). Therefore, we speculated that ROS could be involved in the activation of RhoA/ROCK and RhoA-mediated responses by cucurbitacin I.…”

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

“…Our results suggest that CuB-mediated HDACs inhibition altered the accessibility of various transcriptional factors to the promoters of the TSGs and TPG in H1299 cells. Furthermore, we also observed that CuB at 6 nmol/L concentration induced both class I and class II HDACs, which seems to be the cellular survival strategy to combat with the autophagic effects of CuB (32,33). The protein expressions of HATs, PCAF, and CBP were increased after CuB treatment, which might also help in inducing the expressions of the TSGs, as reported previously (34,35).…”

ROS activates JNK-mediated autophagy to counteract apoptosis in mouse mesenchymal stem cells in vitro

“…Reactive oxygen species (ROS) are toxic to transplanted stem cells, and the induction of apoptosis underlies the major mechanism of ROS-induced cytotoxicity [20][21][22][23] . Meanwhile, ROS can also activate autophagy in certain conditions [24][25][26] . The relationship between autophagy and apoptosis under oxidative conditions is complex [27] .…”