Synthesis of New Chromene Derivatives Targeting Triple-Negative Breast Cancer Cells.

Breast cancer continues to be the leading cause of cancer-related deaths among women worldwide. The most aggressive type of breast cancer is triple-negative breast cancer (TNBC). Indeed, not only does TNBC not respond well to several chemotherapeutic agents, but it also frequently develops resistance to various anti-cancer drugs, including taxane mitotic inhibitors. This necessitates the search for newer, more efficacious drugs. In this study, we synthesized two novel chromene derivatives (C1 and C2) and tested their efficacy against a battery of luminal type A and TNBC cell lines. Our results show that C1 and C2 significantly and specifically inhibited TNBC cell viability but had no effect on the luminal A cell type. In addition, these novel compounds induced mitotic arrest, cell multinucleation leading to senescence, and apoptotic cell death through the activation of the extrinsic pathway. We also showed that the underlying mechanisms for these actions of C1 and C2 involved inhibition of microtubule polymerization and disruption of the F-actin cytoskeleton. Furthermore, both compounds significantly attenuated migration of TNBC cells and inhibited angiogenesis in vitro. Finally, we performed an in silico analysis, which revealed that these novel variants bind to the colchicine binding site in ß-tubulin. Taken together, our data highlight the potential chemotherapeutic properties of two novel chromene compounds against TNBC.

Rhus coriaria induces senescence and autophagic cell death in breast cancer cells through a mechanism involving p38 and ERK1/2 activation.

Here, we investigated the anticancer effect of Rhus coriaria on three breast cancer cell lines. We demonstrated that Rhus coriaria ethanolic extract (RCE) inhibits the proliferation of these cell lines in a time- and concentration-dependent manner. RCE induced senescence and cell cycle arrest at G1 phase. These changes were concomitant with upregulation of p21, downregulation of cyclin D1, p27, PCNA, c-myc, phospho-RB and expression of senescence-associated ß-galactosidase activity. No proliferative recovery was detected after RCE removal. Annexin V staining and PARP cleavage analysis revealed a minimal induction of apoptosis in MDA-MB-231 cells. Electron microscopy revealed the presence of autophagic vacuoles in RCE-treated cells. Interestingly, blocking autophagy by 3-methyladenine (3-MA) or chloroquine (CQ) reduced RCE-induced cell death and senescence. RCE was also found to activate p38 and ERK1/2 signaling pathways which coincided with induction of autophagy. Furthermore, we found that while both autophagy inhibitors abolished p38 phosphorylation, only CQ led to significant decrease in pERK1/2. Finally, RCE induced DNA damage and reduced mutant p53, two events that preceded autophagy. Our findings provide strong evidence that R. coriaria possesses strong anti-breast cancer activity through induction of senescence and autophagic cell death, making it a promising alternative or adjunct therapeutic candidate against breast cancer.

Carnosol induces ROS-mediated beclin1-independent autophagy and apoptosis in triple negative breast cancer.

BACKGROUND: In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer. RESULTS: We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS) and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol. CONCLUSION: In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.

Combined estrogen and ghrelin administration restores number of blood vessels and collagen type I/III ratio in the urethral and anal canal submucosa of old ovariectomized rats.

We compare the effects of estrogen and/or ghrelin on vascular counts and collagen I/III ratio of urethral and anal canal submucosa in old vs young-adult ovariectomized rats. Ovariectomized Fisher 344 rats (18 and 3 months old, n = 24 x 2) received 42 daily intraperitoneal 17-ss estradiol (10 microg/kg), ghrelin (2 microg/kg), both, or vehicle (n = 6 x 4 per group). Blood vessel counts and collagen I/III ratio were measured, respectively, by light microscopy and Western blot analysis with immunohistochemistry of ghrelin receptors. Estrogen significantly increased urethral and anal vascular counts and collagen I/III ratio in young-adult rats. In old rats, only combined estrogen/ghrelin administration significantly increased both variables. This was not observed with estrogen or ghrelin separately. Ghrelin receptors were immunostained in urethral and anal submucosa of all samples. Combined estrogen/ghrelin administration restored postovariectomy urethral and anal canal submucosal vessel number and collagen I/III ratio in old rats suggesting independent ageing effect.

The effect of ovariectomy on biomarkers of urogenital ageing in old versus young adult rats.

The aim of this study was to compare the effects of ageing and ovariectomy on biomarkers of urogenital ageing in old and young-adult rats. Fisher 344 rats (18- and 3-months-old, n = 6 x 2) underwent ovariectomy. Age-matched sham animals received no intervention (n = 6 x 2). One month later, biomarkers of urogenital ageing were evaluated (light microscopic count of urethral and anal canal submucosal blood vessels, Western blot analysis of urethral, and anal canal submucosal collagen I and III and cytoplasmic p27(kip1) expression in the striated urethral and anal sphincters and levator ani and gel electrophoresis of isomyosin I proportion in these muscles) and compared in all groups (n = 24). All biomarkers of urogenital ageing studied were significantly increased in old compared to young-adult sham rats. Ovariectomy significantly increased these changes further in old versus young-adult rats with either smaller or larger differential effect than ageing compared to young-adult sham animals. Ovariectomy significantly exacerbates normative urogenital ageing changes in rats.

Estrogen and ghrelin increase number of submucosal urethral and anal canal blood vessels in ovariectomized rats.

OBJECTIVES: Urinary and fecal control deteriorates after menopause, but it is not clear whether this is age or hormone related. This study investigates whether administration of estrogen and/or the anti-aging growth hormone-releasing peptide, ghrelin, improves the adverse effects of menopause/aging on urethral and anal canal submucosal blood vessel counts in middle-age rats. METHODS: Female Wistar rats (13 months old) underwent ovariectomy, followed 1 month later by intraperitoneal once-daily administration of 17-beta estradiol (10 microg/kg), ghrelin (2 microg/kg), both hormones, or vehicle (n = 6 in each of four groups) for 42 days. An age-matched sham group (n = 6) received no intervention. Submucosal blood vessels were counted by light microscopy in five randomly selected fields from five nonconsecutive sections (5 microm thick) per rat of formalin-fixed and paraffin-embedded tissue blocks of the urethra and anal canal stained with hematoxylin-eosin. The results are expressed as the mean vessel number per high power field (x400). RESULTS: Ovariectomy significantly reduced submucosal urethral and anal vascular counts below the sham values (7.41 +/- 0.98 versus 5.46 +/- 0.82, P = 0.003 and 7.16 +/- 1.11 versus 4.92 +/- 0.65, P = 0.0009, respectively). Estrogen restored the urethral counts (7.76 +/- 0.88, P = 0.5) and ghrelin or combined estrogen and ghrelin administration significantly increased the counts to greater than the sham counts (8.68 +/- 0.99, P = 0.04 and 9.72 +/- 1.21, P = 0.004, respectively). Estrogen, ghrelin, and combined estrogen and ghrelin administration also restored the anal counts to sham levels (7.26 +/- 0.97, P = 0.8; 6.56 +/- 0.78, P = 0.3; and 7.76 +/- 0.88, P = 0.3, respectively). CONCLUSIONS: Combined or individual replacement of estrogen and ghrelin produces a beneficial effect by reversing the ovariectomy-induced decrease in urethral and anal canal submucosal vessel numbers in middle-age rats.