Abstract
Metastasis is the main cause of cancer-related deaths. Anoikis is a type of apoptosis caused by cell detachment, and cancer cells become anoikis resistant such that they survive during circulation and can successfully metastasize. Therefore, sensitization of cancer cells to anoikis could prevent metastasis. Here, by screening for anoikis sensitizer using natural compounds, we found that pygenic acid A (PA), a natural compound from Prunella vulgaris, not only induced apoptosis but also sensitized the metastatic triple-negative breast cancer cell lines, MDA-MB-231 cells (human) and 4T1 cells (mouse), to anoikis. Apoptosis protein array and immunoblotting analysis revealed that PA downregulated the pro-survival proteins, including cIAP1, cIAP2, and survivin, leading to cell death of both attached and suspended cells. Interestingly, PA decreased the levels of proteins associated with anoikis resistance, including p21, cyclin D1, p-STAT3, and HO-1. Ectopic expression of active STAT3 attenuated PA-induced anoikis sensitivity. Although PA activated ER stress and autophagy, as determined by increases in the levels of characteristic markers, such as IRE1α, p-elF2α, LC3B I, and LC3B II, PA treatment resulted in p62 accumulation, which could be due to PA-induced defects in autophagy flux. PA also decreased metastatic characteristics, such as cell invasion, migration, wound closure, and 3D growth. Finally, lung metastasis of luciferase-labeled 4T1 cells decreased following PA treatment in a syngeneic mouse model when compared with the control. These data suggest that PA sensitizes metastatic breast cancer cells to anoikis via multiple pathways, such as inhibition of pro-survival pathways and activation of ER stress and autophagy, leading to the inhibition of metastasis. These findings suggest that sensitization to anoikis by PA could be used as a new therapeutic strategy to control the metastasis of breast cancer.
Highlights
Pygenic acid A (PA; 3-epicorosolic acid, corosolic acid) is a natural compound that is used in oriental medicine and is extracted from Prunella vulgaris, a perennial herb that is widely distributed throughout the world
After screening of thousand natural compounds for their ability to sensitize metastatic breast cancer cells to anoikis, we found PA candidates for inducing the apoptosis of cells under suspension conditions (Figure 1)
To test whether PA could sensitize cells to anoikis, cells in either attachment or suspension cultures were treated with PA, and cell growth was assessed by MTS assay
Summary
Pygenic acid A (PA; 3-epicorosolic acid, corosolic acid) is a natural compound that is used in oriental medicine and is extracted from Prunella vulgaris, a perennial herb that is widely distributed throughout the world. PA inhibited HER2(ERBB-2) signaling cascade, which is involved in cell cycle progression, in gastric cancer cells [3]. Tumor metastasis is a multistep process, involving the dissociation of cancer cells from the primary tumor site, intravasation allowing cells to enter blood stream, migration through the circulatory system, and extravasation, resulting in cells settling and growing in a secondary organ [5]. Normal cells are sensitive to anoikis and so undergo apoptosis when they become detached from the ECM. Cancer cells acquire anoikis-resistance when they become detached. Because of this anoikis resistance, cancer cells can survive in circulation and disseminate to secondary sites to form tumor metastases [7]. The sensitization of cancer cells to anoikis is critical for the control of metastasis
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