Breast cancer is a major health concern worldwide, and a leading cause of cancer-related deaths among women in the United States where over 245,000 new cases are diagnosed and 45,000 deaths occur each year (1). In the majority of diagnoses, this disease presents as a hormone-sensitive breast cancer that overexpressed estrogen receptor (ER), progesterone receptor (PR) and/or human epidermal growth factor receptor 2 (HER2/neu). These cancers typically depend on hormone-receptor signaling for tumor growth and progression. Consequently, conventional therapy against the majority of breast cancers targets these receptors (e.g., tamoxifen, a selective estrogen receptor modulator and herceptin, an antibody that inhibits HER) or attempts to inhibit enzymes involved in estrogen synthesis (e.g., aromatase inhibitors such as letrozole) (2–4). In contrast, the most deadly subtype of this disease is known clinically as triple-negative breast cancer (TNBC), and constitutes approximately 15% of invasive breast cancers. TNBCs lack expression of genes coding for ER, PR and HER2/neu, are not dependent on hormonal signaling for progression, and do not respond to conventional therapy (5).

TNBCs are frequently of the highly invasive and recurrent basal-like subtype (6). They are also associated with a variety of aberrations (e.g., higher incidence of TP53 mutations and the constitutive activation of pro-survival and inflammatory pathways such as MAPK and NF-κB), all of which may contribute to their growth, survival and resistance to chemotherapy (7–10). NF-κB signaling maintains an autocrine loop of pro-inflammatory cytokines such as IL-6 and TNF-α, promotes expression of pro-survival factors such as Bcl-xL, cIAP1 and cIAP2 and proteins involved in cell cycle progression (e.g., cyclin D1), as well as invasion and metastasis markers such as MMP-9 and vimentin (11–17). Constitutive activation of NF-κB in triple-negative breast cancer may contribute to its aggressiveness and ability to maintain hormone-independent growth. Inhibition of NF-κB signaling is thus a viable target for novel therapeutics against TNBC.

Plant-derived molecules have been a critical source of cancer therapeutic agents. Generally, there are four main classes of plant-derived anticancer drugs currently in clinical use: Vinca alkaloids, taxanes, epipodophyllotoxins and camptothecins; the former two have entered clinical use against breast cancer following successful trials (18,19). Studies have also identified other natural plant compounds as capable of exerting pleiotropic anticancer effects. Plumbagin, for example, was shown to inhibit the DNA-binding activity of NF-κB and induce apoptosis in triple-negative MDA-MB-231 breast cancer cells (20). Further, halofuginone inhibited the nuclear localization of NF-κB and AP1, which are critical transcriptional activators of matrix metalloproteinase-9 (MMP-9), thereby reducing migration and invasiveness of these cells (21). Curcumin, honokiol, resveratrol and pinocembrin, among others, have also been shown to possess potent anti-cancer effects (22–25). These positive outcomes highlight the importance of fully uncovering the untapped clinical potential of natural compounds.

Herein, we report on an extract from Lippia origanoides, a medicinal tropical plant native to South America for its potential anticancer effects on TNBC. Also known as 'oregano de monte' or mountain oregano, infusions of the plant have been used traditionally to alleviate gastrointestinal ailments and as topical analgesics (26). Towards a better understanding of L. origanoides actions and health benefits, the novel L. origanoides extract (LOE) used in this study was originally isolated by supercritical fluid extraction and characterized using HPLC-MS (27,28). The LOE was found to have numerous major components including pinocembrin, carvacrol, thymol and trans-β-caryophyllene. Studies using commercially available reagents of these compounds have shown they have pro-apoptotic and anti-proliferative effects on various cancer cell lines. For example, pinocembrin, a flavanone which comprises nearly 55% of the total LOE, has been shown to decrease viability and prevent epithelial-mesenchymal transition induced by TGF-β in Y-79 retinoblastoma cells (25). Further, trans-β-caryophyllene and α-humulene, two sesquiterpenes present in the extract, were shown to synergize and inhibit cell growth and proliferation in MCF-7 breast cancer cells (29). Strikingly, several components of LOE have shown inhibitory effects on NF-κB signaling, a key survival and proliferative pathway in TNBC (30–34). These reports support the idea that L. origanoides may be a source of novel components that can effectively target aggressive breast cancer.

Our results show that treatment with LOE leads to a G0/G1 phase halt and apoptosis in MDA-MB-231 triple-negative breast cancer cells without promoting necrotic cell death. Further, we reveal that cell cycle proteins and apoptotic markers, as well as key NF-κB regulatory molecules, are modulated by treatment with LOE, thereby shedding light on a mechanism of action behind the anticancer effects of LOE. These data provide an important first step towards defining the potential utility of LOE in the identification and development of novel therapeutic strategies for TNBC.

TNBC continues to be a monumental concern in women's health issues. As our understanding of the mechanisms that govern the initiation and progression of breast cancer grows, it has become apparent that conventional chemotherapeutics are limited by their inability to target subtypes that are inherently more aggressive while being drug resistant. One such subtype is TNBC, which lacks expression of ER, PR and HER2/neu, thereby rendering TNBC resistant to conventional endocrine-targeted therapy. In this study, we provide support that an extract from the tropical South American plant L. origanoides (LOE) may be a possible source for therapeutic agents against TNBCs. In brief, we utilized the MTT metabolic activity assay to show LOE induces a dose-dependent decrease in the viability of MDA-MB-231 mammary adenocarcinoma cells, a universal standard in vitro model of highly invasive and drug resistant TNBC. This effect was much greater compared to normal-like MCF10A mammary epithelial cells (Fig. 1). Our observation that LOE also significantly reduced cell viability in triple-negative Hras-transformed MCF10A invasive cells as well as the CRL2321 cell-line, recently shown to be the best in vitro phenotypic representative of grade 3 TNBC tumors (41), further confirms its anti-TNBC effects. This observation suggests the LOE induces molecular actions that target pathways specific to cancer cells. We also note that while CRL-2321 cells have a greater sensitivity to low doses of LOE than MDA-MB-231 cells, higher dosages of LOE do not induce decreases in CRL-2321 viability beyond 80%. Previous studies have shown that cancer cell line subpopulations and sub-lines can exhibit a high degree of transcriptome and phenotypic variability leading to observable differences in metastatic activity (42) and drug sensitivity (43). This suggests the possibility of heterogeneity within subpopulations of the CRL-2321 cell line allowing for compensatory survival pathways providing modest resistance to LOE treatment. The identification of novel mechanisms targeted by new small molecular inhibitors is of major interest in our continued studies of the actions of LOE.

Our study also quantified the molecular influences of the extract within cancer cells. Cyclin D1 is a protein which associates with cyclin-dependent kinase-4 and -6 (CDK-4 and -6) to form heterodimers that progress the cell cycle from G0/G1 to S phase (37). In addition, cellular inhibitor of apoptosis proteins (cIAPs) prevent apoptosis and permit cell cycle progression via binding and possibly inactivating executioner caspases, i.e., caspase-3 and -7 (38). cIAP2, is known to be constitutively expressed in MDA-MB-231 cells, and has been shown to protect against death-inducing ligands such as TNF-α by possibly enhancing NF-κB signaling through RIP1 activation as well as by binding and inhibiting caspase-3 (39). Herein, we observed that LOE induced G0/G1 phase halt in MDA-MB-231 cells (Fig. 2). Western blot analysis revealed the effects of LOE on cell cycle progression could be explained by a reduction in cyclin D1 and cIAP2 levels upon treatment of the cells with the extract (Fig. 4). These experiments begin to build an important framework for the actions of LOE on cell cycle regulation.

We next studied LOE's impact on MDA-MB-231 cell death. Upon triggering of the extrinsic pathway of apoptosis, procas-pase-8 is activated via dimerization-induced auto-cleavage and subsequently cleaves and activates the master 'executioner' caspase, caspase-3 (44,45). We revealed a potential mechanism behind the actions of the extract by showing LOE-induced apoptosis involved activation of caspase-8/-3 and cleavage of PARP (Figs. 5 and 6), hallmarks of extrinsic apoptotic pathway activation (36). Additionally, LOE induced apoptosis in these cells unaccompanied by necrotic cell death (Fig. 3). As cancer therapy is focused on preventing damage to non-targeted tissue areas, this result provides additional support for LOE as a source for potential therapeutics with desirable characteristics.

In previous work, Stashenko et al have characterized the major components of LOE (28). However, it remains to be defined which component, or combination of components, is responsible for the extract's apoptotic effects. Furthermore, it is imperative that major cellular pathways involved in LOE signal transduction be delineated. Based on previous studies involving components present in the LOE, one possible candidate pathway central to LOE-induced apoptosis is NF-κB signaling. Constitutive activation of this pathway has been previously linked to triple-negative breast cancer and blocking NF-κB signaling can spontaneously induce cell death via a caspase-8/-3-dependent mechanism or render cells susceptible to pro-apoptotic signals (40,46,47). In normal cells, ligand binding to TNFR1 is followed by rapid activation of the extrinsic pathway of apoptosis. However, in contrast to normal cells, ligand binding to TNFR1 in TNBC cells instead activates NF-κB signaling via membrane-recruitment of the scaffold protein RIP1, a Ser/Thr kinase (39). In brief, cIAP2 exerts E3 ubiquitin ligase activity to polyubiquitinate and activate RIP1, which goes on to phosphorylate the IKKα/β-NEMO complex. Active IKKs then phosphorylate IκB proteins which sequester p50/RelA (NF-κB) dimers to the cytoplasm, targeting the IκBs for polyubiquitination and subsequent degradation. This frees p50/RelA to translocate to the nucleus and act as a transcription factor for pro-survival genes such as cyclin D1 and cIAPs (48,49).

We hypothesized that components of LOE could interfere with NF-κB signaling, leading to a halt in the cell cycle and induction of apoptosis. Indeed, we showed LOE treatment resulted in a 40% decrease in protein levels of RIP1 in MDA-MB-231 cells (Fig. 7B). This may, at least in part, be explained by our previous observation that LOE treatment induced activation of caspase-8. Active caspase-8 has previously been shown to cleave RIP-1, thereby inhibiting pro-survival NF-κB signaling and inducing apoptosis (50). This result, coupled with our findings that LOE inhibits cyclin D1 and cIAP2, proteins activated by NF-κB signaling, while simultaneously stimulating caspase-8 dependent cell death, a characteristic of NF-κB inhibition, sheds critical light on the mechanistic foreground of LOE action. All together, these data highlight the potential of L. origanoides extract and its major constituents as cancer therapeutics.

In summary, we have shown that LOE promotes apoptosis in TNBC cells, while having a much reduced impact on cell death in normal mammary cells. We also found that LOE reduces cell viability, alters the cell cycle and primarily leads to tumor cell apoptosis and not necrosis. Further, we established some of the mechanisms of LOE actions, showing that it suppresses markers of cell cycle progression and cell survival while inducing the extrinsic pathway of apoptosis via caspase-8/-3 activation. Finally, we revealed that levels of RIP1, an upstream effector of pro-survival NF-κB signaling, are reduced upon LOE treatment. These data collectively support that LOE can be a valuable source for development of novel anticancer agents. Moving forward, beyond fully describing the cellular mechanisms of LOE action, a top priority will be identifying the major component or combination of components responsible for its bioactivity. Plant-derived compounds offer a vast source of small molecule inhibitors that could be used both in their native state as well as chemically modified to optimize their pharmacological activity. The bio-reactivity of the LOE-treatment in TNBC cells in vitro supports the idea that future treatments for individual cancer subtypes lie in naturally-derived chemicals.