The CSCs play a key role in maintaining the tumor heterogeneity, driving cancer growth and drug resistance [35]. Therefore, the exploration of novel drug that can target cancer stem cells is a potential therapeutic to overcome therapy resistance. In recent years, fatty acids have become potential anti-cancer drug in cancer biology. However, still debate about particular types of fatty acids and their effects on cancer treatment. For instance, numerous types of fatty acids are not similar concerning their effects on breast cancer cell growth and death: the monounsaturated fatty acid oleate (C18:1) promotes the survival, whereas the anti-cancer function of saturated fatty acid palmitate (C16:0) was demonstrated by inducing apoptosis [36]. Saturated fatty acid, special is even chain fatty acid induce cell death have also reported in vitro and in vivo [15, 37-41]. Moreover, recent studies confirmed that effects of fatty acids against several types of cancer cells, e.g. lung cancer [16]. This compelling evidence indicated that OCFAs may exhibit anti-cancer effects on cancer cells.
Nevertheless, little information exists about the anti-tumor mechanism by OCFA influence breast cancer cell survival and metastasis, especially BCSCs. Our study showed that pentadecanoic acid, one of the most common compounds belong to OCFA, has a strong cytotoxic activity against human breast cancer stem cell line MCF-7/SC (Figure 2b,e). In this work, we examined the cytotoxic activity of pentadecanoic acid in non- breast cancer stem cell line (luminal-like) breast cancer cell line (MCF-7), breast cancer stem cell line (MCF-7/SC) and non-tumorigenic breast epithelial cell line (MCF-10A). Interestingly, pentadecanoic acid displayed selective cytotoxicity against BCSCs (MCF-7/SC) compared with luminal breast cancer MCF-7 (Figure 2d) whereas the highest concentration exerted non-cytotoxic effect on
non-tumorigenic breast epithelial cell line (MCF-10A) (Figure2c), indicating pentadecanoic may play as a novel anti-cancer reagent for BCSC.
In breast cancer patients, levels of cancer stem cells have positively correlated with the risk of poor prognosis, which may enhance the chemoresistance and metastasis in patients with highly malignant [42]. In this study, we demonstrated that the breast cancer stem cell line MCF-7/SC, which isolated from MCF-7 breast cancer cells showed more prominent stem cell properties than their parent cells by enhancing the CD44+/CD24- populations, accumulating lower ROS levels, increasing mammosphere formation as well as migration capacity (Figure 1a-e). Up-to-date evidence suggests that ALDH activity was accepted as a hallmark of cancer CSCs [4]. Moreover, MCF-7/SC cells displayed greater-level proteins including CD44, MDR1, and MRP1 than MCF-7 (Figure 1d). These proteins are CSC markers that frequently used [4]. Based on the selective cytotoxic effect on MCF-7/SC (Figure 2d), we hypothesis that this OCFA could eliminate the CSC population. To evaluate the effect of pentadeacanoic acid, we first examined the activity of pentadecanoic acid against BCSCs properties. Ours obtain results provided compelling evidence as to dramatically reduce the CD44+/CD24- population, the significant suppression of mammosphere evolution as well as ALDH activity (Figure 4a-c). As one of the most typical CSC markers and critical regulators of cancer stemness, CD44 is responsible for self-renewal, cell invasion and migration [43].
Therefore, CD44 is allowed as a marker for isolating or enriching CSCs by using separate or in combination with other cell surface markers [43]. As anticipated, upon pentadecanoic acid treatment, the CSC markers including CD44, β-catenin, MDR1, and MRP1 decrease significantly (Figure 4d-e). This is the first report that describes the effects of OCFA against BCSCs in vitro.
The EMT program is a cellular event that cells can move to another site. This process based on lose apical-basal polarity and cell-cell adhesion of epithelial cells [44]. EMT process is essential for tumor development by intensifying migratory, invasive properties, and becomes mesenchymal stem cells [45, 46]. The direct correlation between EMT progression and the development of CSCs have been demonstrated in the previous study, implying that the EMT program plays a deciding role in the generation and maintenance of CSCs or CSC-like cells [47, 48]. Up to now, many cellular and molecular related to EMT processes have been identified, and the most critical attention is the changes associated with a class of extracellular proteases, the MMPs [49]. Among the members, MMP2 and MMP9 are two well-known members of the MMP gene family [50, 51]. MMP2, also known as gelatinase A, promoted the malignant phenotype of cancer cells through causing the breakdown of the basement membrane and enhancing the local and distant invasion of tumor cells [52].
Besides MMP2, MMP9 (gelatinase B) driven angiogenesis by interrupting in the regulation of growth plate and recruitment of endothelial stem cells [53]. Previous studies also accepted both Snail and Slug are the zinc-finger regulatory transcription factors required in the EMT process of cancer cells, which is related to the aggressive clinical phenotype in breast cancer [54]. Our results found that the migration and invasion ability of MCF-7/SC significantly repressed upon pentadecanoic acid treatment (Figure 4a-b). Correlating with this, the expressions of MMP2, MMP9, Snail, and Slug also remarkably decreased compared with no treatment (Figure 4c), suggesting that pentadecanoic acid could suppress cell motility capacity through the suppression of EMT-related protein expression in MCF-7/SC cells.
A number of clinical evidence showed that the signal transducer and activator of transcription 3 (STAT3) are constitutively activated in almost cancer, covering more than 40%
of all breast cancer [55]. As an essential gene in generation and survival of the cancer cells,
STAT3 participates in cell proliferation, apoptosis, metastasis, and other cellular happenings including EMT in breast cancer [56-60]. Succinctly, when growth factors or cytokines bind to the related receptors on the cell surface, it leads to activation of receptor-associated tyrosine kinases [61]. The most notable is the Janus kinase, JAK family of kinases, this process leading to the recruitment and constitutive activation of STAT3 on the Tyr-705 residue [61]. The dimerization and nucleus translocation processes take place immediately after STAT3 was activated. Activated STAT3 acts as a regulator factor that can regulate the transcription of the target gene such as Bcl-2 families, c-Myc, Survivin, MMP2, MMP9 via binding to the interferon-gamma activated sequence (GAS) of promoters [59, 62-64]. A recent study indicated that U-STAT3 involved in the regulation of gene expression through bind to GAS sequences as a dimer or monomer [65, 66]. In the present investigation, upon treatment with pentadecanoic acid at both time and dose-dependent manner significantly inhibits JAK2/STAT3 signaling as shown in Figure 5a-b, provided the new biological function of pentadecanoic acid in BCSCs as a potent inhibitor of the JAK2/STAT3 pathway.
IL-6, a pro-inflammatory cytokine accepted as one of the most well-known upstream activators of the JAK2/STAT3 pathway [67]. As expected, our results showed in Figure 5c indicated that exposure to pentadecanoic acid resulted in a suppression IL-6-induces the JAK2/STAT3 signaling pathway. Taken together, these results provided compelling evidence for the first time that pentadecanoic acid can suppress cancer stem cell properties, decrease migration, and invasion capacity via inhibiting JAK2/STAT3 signaling.
As aforementioned, STAT3 regulates the transcription of a wide range of genes involved in apoptosis by binding to the specific promoter region both pro- and anti-apoptotic families [68, 69]. Hence, we hypothesis that the cell death mechanism behind processes was apoptosis. Play a critical role in regulating apoptotic processes, caspase, a
family of cysteine proteases, are accepted as the key mediators can cleave main cellular proteins [70]. There are two classes of caspases: the initiator caspases and the effector caspases [70]. The initiator caspases included caspase-3, csaspase-8, csaspase-9 and csaspase-10 whereas the effector caspases included csaspase-3, csaspase-6 and csaspase-7 [71]. Important, activation of caspase-3 is the central phenomenon that responsible for most of the cleavage events during the apoptosis process [72]. (ADP-ribose) polymerase-1 (PARP-1) is popular cellular substrates of caspases. One of the various events of apoptosis that have been accepted is the cleavage of PARP-1 by caspases [73]. Consistent with the reported finding of the critical role of caspases and PARP-1 during the apoptosis, our results showed a heightened expression of cleave caspase-3, -7, -8, -9 in the pathway upon pentadecanoic acid treatment (Figure 6a-c), which indicated the pentadecanoic acid treatment promote both the extrinsic and intrinsic apoptosis pathway in the BCSCs.
Resistance to TAM is the underlying cause of treatment failure that is frequently
observed in breast cancer patients receiving TAM [74].Therefore, developing new
therapeutic strategies to improve the clinical efficacy of Tamoxifen in breast cancer
treatments is necessary. Combined treatment of chemotherapeutics with natural drugs has
been reported to enhance the overall efficacy of chemotherapy treatments and minimize
adverse side effects [75]. Interesting, we observed that pentadecanoic acid can improve the
chemosensitivity of MCF-7/SC cells to TAM (Figure 7a and 7b). In addition, the results of
FACs analysis indicated an accumulation in the sub-G1 populations as well as an
accumulation of apoptosis population in MCF-7/SC cells following by combined treatment
containing 100 μM of pentadecanoic acid and 10 μM of TAM (Figure 7d and 7e). Moreover,
results of western blot experiments confirmed that combined treatment of TAM together
with pentadecanoic acid can increase the expression of apoptosis markers such as
cleave-caspase 7, cleave-cleave-caspase 9 and cleave-PARP in MCF-7/SC cells (Figure 7e), indicating the
potential use of petadecanoic acid/TAM combined therapy for breast cancer patients.