Breast cancer is a substantial global health issue and is associated with the highest number of cancer related deaths in women [1].
A continuous need for new therapeutic approaches to breast cancer reflects its highly heterogeneous nature with many subtypes that can develop resistance to traditional treatment [18].
The aim of the present study was twofold. The first was to investigate the molecular chemotherapeutic effect of sorafenib as a multi- tyrosine kinase inhibitor in comparison to stigmasterol, a phytosterol with anticancer activity, on different signaling pathways in MCF-7 and MDA-MB-231 human breast cancer cells. The second was to assess the impact of a combination of sorafenib and stigmasterol on proliferation, angiogenesis, and apoptosis signaling involved in promoting breast cancer progression.
Cytotoxicity assays showed that sorafenib is a potent toxicant to cells in both lines, but stigmasterol cytotoxicity was weak; an IC50 was not estimated since even high concentrations did not cause 50% lethality. Previous studies reported similar results for sorafenib [19] and stigmasterol [20] cytotoxicity in breast cancer.
A combination of sorafenib and stigmasterol caused a decrease in the proliferation marker, Ki-67.This effect is ascribed to the inhibition of ERK signaling [9, 19, 21]. Inhibition of ERK prevents activation of downstream transcription factors and cell cycle regulators leading to diminishing cell proliferation [21].
A significant antiangiogenic effect was mediated by both compounds individually. Levels of p-ERK and its downstream transcription factor, nuclear factor kappa B (NF-kB) were reduced. Both agents also induced decreased expression of angiogenic mediator, VEGF-A, and its receptor, VEGFR-2.
A further decrease in NF-kB, VEGF-A and VEGFR-2 levels was observed in cells treated with a combination of agents. The effect of sorafenib on p-ERK is attributed to its inhibitory effect on RAF [4] and this effect was observed in a recent study using MCF-7 and MDA-MB 231 cells [19]. Stigmasterol inhibited the phosphorylation of ERK1/2 in a recent ovarian cancer study [9]. NF-kB [22, 23] and other transcription factors, such as FoxM1[21], were suppressed; as a result of ERK inhibition. Downregulation of NF-kB in this study is consistent with previous studies on sorafenib [24] and stigmasterol [25]. Inhibition of transcription factor, FoxM1, leads to downregulation of VEGF. This factor binds directly to Forkhead binding elements (FHRE) of the VEGF promoter that activates VEGF expression [21]. VEGF was also downregulated following the inhibition of NF-kB in MCF-7 and MDA-MB-231 cells [26, 27].
Both sorafenib and stigmasterol induced caspase-3 activity and decreased levels of the anti-apoptotic protein, Bcl-2. These responses were enhanced by combined treatment. Sorafenib exerted an efficient apoptotic effect in combination with other agents in a previous study carried on MCF-7 [28] and MDA-MB-231 cells [29]. Induction of apoptosis by stigmasterol was also reported in recent studies in ovarian [9], gastric [10, 11], hepatic [12] and gall bladder [13] cell lines. Apoptotic effects are ascribed to inhibition of both RAF-MEK-ERK and PI3K-AKT signaling by both agents [9, 10, 30]. These pathways are responsible for phosphorylation and inactivation of several factors, including pro-apoptotic Bad protein and caspase-9 [31, 32], leading to caspase-3 inhibition, and blocking of apoptotic signals. Apoptosis is also attributed to downregulation of NF-kB. This factor upregulates anti-apoptotic proteins, including Bcl-2, and transactivates inhibitors of apoptosis proteins (IAPs) [31]. In addition, reduction of anti-apoptotic Bcl-2 protein by the combination of sorafenib and stigmasterol promotes the release of cytochrome c and initiation of the intrinsic apoptotic cascade [31].
Thus, the combination of sorafenib and stigmasterol may prove useful for breast cancer treatment. Future studies are warranted to investigate the impact of this combination on several signaling pathways that mediate breast cancer pathogenesis.