Parthenolide generates reactive oxygen species and autophagy in MDA-MB231 cells. A soluble parthenolide analogue inhibits tumour growth and metastasis in a xenograft model of breast cancer

Abstract

Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and time-dependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1–3 h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused depletion of thiol groups and glutathione, activation of c-Jun N-terminal kinase (JNK) and downregulation of nuclear factor kB (NF-kB). During this first phase, parthenolide and DMAPT also stimulated autophagic process, as suggested by the enhanced expression of beclin-1, the conversion of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II and the increase in the number of cells positive to monodansylcadaverine. Finally, the drugs increased RIP-1 expression. This effect was accompanied by a decrement of pro-caspase 8, while its cleaved form was not detected and the expression of c-FLIPS markedly increased. Prolonging the treatment (5–20 h) ROS generation favoured dissipation of mitochondrial membrane potential and the appearance of necrotic events, as suggested by the increased number of cells positive to propidium iodide staining. The administration of DMAPT in nude mice bearing xenografts of MDA-MB231 cells resulted in a significant inhibition of tumour growth, an increment of animal survival and a marked reduction of the lung area invaded by metastasis. Immunohistochemistry data revealed that treatment with DMAPT reduced the levels of NF-kB, metalloproteinase-2 and -9 and vascular endothelial growth factor, while induced upregulation of phosphorylated JNK. Taken together, our data suggest a possible use of parthenolide for the treatment of TNBCs.

The oxygen radicals involved in the toxicity induced by parthenolide in MDA-MB-231 cells

“…In previous studies (13,15), we showed that parthenolide induces generation of ROS in MDA-MB-231 cells. The aim of the present study was to individuate the ROS produced under parthenolide treatment to specify the modalities of their production and the role exerted in the induction of cell death.…”

“…•-production in MDA-MB-231 cells as well (13,15), which could be responsible for the toxic effects caused by the drug. These effects consist in the depletion of thiol groups and glutathione, NF-κB inhibition, c-Jun N-terminal kinase (JNK) activation, dissipation of mitochondrial membrane potential (∆ψm) and necrotic effects (12,13).…”

“…These effects consist in the depletion of thiol groups and glutathione, NF-κB inhibition, c-Jun N-terminal kinase (JNK) activation, dissipation of mitochondrial membrane potential (∆ψm) and necrotic effects (12,13).…”

“…To determine membrane damage, the cells were stained with propidium iodide (PI), as previously reported (13). In particular, cells (8x10 3 /well) were incubated in culture medium and treated for various times with parthenolide, and PI (2.0 µg/ml medium) was then added and the incubation was protracted for an additional 15 min.…”

“…It has been shown that this drug exerts its effect by inducing generation of ROS (10) and inhibiting the nuclear factor κB (NF-κB) (11). We have previously shown that parthenolide rapidly stimulates ROS generation in human osteosarcoma MG-63 and melanoma SK-MEL-28 cells (12) as well as in breast cancer MDA-MB-231 cells (13). Furthermore, it has been reported that parthenolide induces the activity of NOX in prostate cancer PC3 cells (14).…”