3. RESULTS
3.2. Pentadecanoic acid effectively inhibits MCF-7 & MCF-7/SC cell proliferation
Various fatty acids may exert growth inhibitory effects depending on the cancer cell type. It has been reported that the saturated acid - oleic acid (C18:1) effectively inhibited the proliferation of breast [18], Tongue Squamous [19] and esophageal [20] cancer cells. Another unsaturated fatty acid, linoleic acid (C18:2) has been reported to exert cytotoxicity in colorectal cancer cells [21]. Therefore, screening of these fatty acids may provide the potential candidates for cancer therapy.
To investigate the effect of saturated fatty acids including pentadecanoic acid (C15:0), heptadecanoic acid (C17:0), and unsaturated fatty acid including oleic acid (C18:1), and linoleic acid (C18:2) in breast cancer cells, the MTT assay was conducted. As illustrated in Figure 2a, oleic acid and linoleic acid (unsaturated fatty acids) exhibited less cytotoxic in both MCF-7 and MCF-7/SC cells compared to pentadecanoic acid and heptadecanoic acid (saturated fatty acid). The effect of pentadecanoic acid in MCF-7 and MCF-7/SC cells was also compared in Figure 2b. As IC50 values of 41.94±4.06 µ M, heptadecanoic acid displayed more effective OCFA against MCF-7/SC cells compared to pentadecanoic acid (IC50 =
119±5.21 µ M). Heptadecanoic acid exerted the cytotoxic effects whereas pentadecanoic acid did not affect the proliferation of MCF-10A (non-tumorigenic epithelial cells) (Figure 2c), pentadecanoic acid may be an OCFA reliable for further experiments. Pentadecanoic acid treatment resulted in a reduction of MCF-7/SC cell viability and its cytotoxic were dose-dependent, with IC50 values of 155.5±9.55 µ M for 24 h post-incubation and 119±5.21 µ M for 48 h post-incubation (Figure 2d). Altogether, these results suggest that pentadecanoic acid is a suitable candidate for breast cancer treatment.
Figure 2. Pentadecanoic acid shows significant cytotoxic activity against 7 & MCF-7/SC cell proliferation. (a) The effect of unsaturated fatty acids: oleic acid (C18:1) and linoleic acid (C18:2) on breast cancer cells for 48 h. (b) The effect of saturated fatty acids:
pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) for 48 h. (c) The cytotoxic effect of saturated fatty acid on MCF-10A on breast cancer cells after 48 h. (e) The effect of
pentadecanoic acid treatment on MCF-7/SC cells for 24 h and 48 h. Results are shown as mean ± SD. (* p < 0.05).
3.3. Pentadecanoic acid supressed the migration and invasion capacity of MCF-7/SC cells
We evaluated whether pentadecanoic acid could inhibit the cell motility of MCF-7/SC cells by using wound healing, invasion assays, and Western blot. As presented in Figure 3a and 3b, pentadecanoic acid treatment at non-lethal concentration significantly supressed the migration and invasion capacity of MCF-7/SC were compared with the untreated group (Figure 3a, 3b). Previous studies have indicated that EMT has an important role in enhancing cancer cell motility. The anti-migration effects and anti-invasion effects of pentadecanoic acid were determined, we further examined the underlying mechanism of its action. Matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) are the members of MMPs - proteolytic enzymes of the extracellular matrix (ECM), closely related to EMT process of various types of cancer cells. Following this, the protein expressions of MMP2 and MMP9, as well as other EMT markers including Snail and Slug were detected to evaluate the effect of pentadecanoic acid on the motility capacity. Pentadecanoic acid remarkably decreased the expression of EMT-associated protein such as snail, slug, MMP2, and MMP9 on MCF-7/SC. All of these data indicated that pentadecanoic acid treatment could supress cell motility of MCF-7/SC cells.
Figure 3. Pentadecanoic acid suppressed EMT in MCF-7/SC cells. (a) The wound healing
assay was perfomed to evaluate the effect of pentadecanoic acid on cell migration. (b) Effect of pentadecanoic acid on cell invassion was evaluated by the Trans-well invassion assay. (c) Western blot analysis of EMT related-markers was conducted following 48 h of incubation with pentadecanoic acid. GAPDH was used as a loading control; Results are presented as mean ± SD. (* p < 0.05).
3.4. Pentadecanoic acid suppressed the stem cell-like properties of MCF-7/SC cells
As we found that pentadecanoic acid has more cytotoxic activity on MCF-7/SC cells than MCF-7 cells, we examined whether this OCFA could suppress the CSC population of MCF-7/SC cells. The characteristics of BCSCs were identified based on CD44+
/CD24-- expression, the enzymatic activity of ALDH and possess a capability to form mammospheres in non-adherent cultures. As illustrated in Figure 4a, the mammospheres formation capacity of MCF-7/SC cells was significantly diminished by pentadecanoic acid treatment. Furthermore, pentadecanoic acid treatment dramatically decreased the percentage of CD44+/CD24- cell population in a dose-dependent manner (Figure 4b).
Pentadecanoic acid treatment resulted in a reduction of ALDH activity was also observed in Figure 4c. To confirm this, we evaluated the effect of pentadecanoic acid on expression CSC markers by Western blot analysis. The results in Figure 4e and 4f, pentadecanoic acid caused decrease in expression levels of CSC markers such as MRP1, MDR1, CD44 and β-catenin, in a dose-dependent manner (Figure 4e) and in a time-dependent manner (Figure 4f). All of these data demonstrated that definite characteristics of CSCs are effectively suppressed by pentadecanoic acid treatment.
Figure 4. Pentadecanoic acid suppressed the stem cell-like properties of MCF-7/SC cells.
(a) Effects of pentadecanoic acid on mammosphere formation of MCF-7/SC (100×
magnification). (b,b’) FACs analysis showing reduction of CD24-/ CD44+ population on MCF-7/SC cells. (c) ALDEFLUOR assay kit of MCF-7/SC after exposure to pentadecanoic
acid for 48
h
. DEAB used as a negative control. (d) Western blot analysis for CSC markers after pentadecanoic acid (0, 50, 100, 150, 200 μM) treatment for 48 h. (e) Western blot analysis for CSC markers after pentadecanoic 100 μMtreatment for 6, 12, 24, 48 h. GAPDH was used as a loading control ; Results are shown as mean ± SD. (* p < 0.05).3.5. Pentadecanoic acid suppress cancer the JAK2/STAT3 signaling in MCF-7/SC cells.
Previous study reported that STAT3 can regulate several target oncogenes in breast carcinoma cells [22]. Previous studies reported that the activation of JAK2/STAT3 signaling play an essential role in breast CSCs development and progression included survival, apoptosis, metastasis, and chemoresistance [23]. Therefore, based on the effects of pentadecanoic acid on stem cell-like properties in MCF-7/SC, we examined whether treatment with pentadecanoic acid could inhibit JAK2/STAT3 signaling in MCF-7/SC. The
Western blot results indicated that pentadecanoic acid expose decreased the expression of JAK2, pJAK2, STAT3, pSTAT3 proteins in a dose-dependent manner (Figure 5a). The effect of pentadecanoic acid was supported by the diminished expression of these proteins in a time-dependent manner as illustrated in Figure 5b. Although pentadecanoic acid treatment could reduce both of total form and phosphorylated form of these protein, a dramatic reduction of the phosphorylated form compared to the total form were observed in Figure 5a and 5b. These data demonstrated that pentadecanoic acid attenuated the JAK2/STAT3 signaling pathway.
Previous studies reported that many upstream proteins that can activate JAK2/STAT3 signaling including Interleukin-6 (IL-6) [24-26], and the repression of IL-6/JAK2/STAT3 signaling activation leading to reduce migration, invasion as well as tumor aggressiveness of breast cancer[27]. To further understand the biological mechanism of pentadecanoic acid, we examined whether pentadecanoic acid treatment can prevent the function of IL-6 from activation JAK2/STAT3 signaling in MCF-7/SC cells. Pre-treat MCF-7/SC cells with Pentadecanoic acid at 150 M for 48 h. Before extracting protein from cells, stimulate MCF-7/SC with 20 ng/mL IL-6 for 15 mins. Our obtained result in Figure 5c showed an increase of pJAK2 and pSTAT3 protein levels caused by IL-6 stimulation in MCF-7/SC cells.
Nevertheless, pentadecanoic acid can dramatically inhibit the induction of phosphorylation of the JAK2/STAT3 level by IL-6. These data provided evidence for the novel role of pentadecanoic acid as an inhibitor of the IL-6/JAK2/STAT3 signaling pathway.
Figure 5. Pentadecanoic acid inhibited the JAK2/STAT3 signaling in MCF-7/SC cells. (a) Effects of different concentration pentadecanoic acid treatment for 48h on JAK2/STAT3 signaling in MCF-7/SC. (b) Effects of 100 µ M pentadecanoic acid treatment for 6, 12, 24, 48h on JAK2/STAT3 signaling in MCF-7/SC. (c) Effects of 150 µ M pentadecanoic acid treatment for 48h on preventing IL-6-induced JAK2/STAT3 signaling after 48h from treatment of Pentadecanoic acid. GAPDH was used as a loading control; Results are presented as mean
± SD. (* p < 0.05).
3.6. Pentadecanoic acid induce apoptosis in MCF-7/SC cells
Evasion of apoptosis is a basic feature of human cancer [28]. Screening natural compounds can kill cancer cells by inducing apoptosis is the promising strategy for cancer therapeutic [29-31]. Multiple studies have determined that the STAT3 signaling represses
apoptosis in cancer cells, includes breast cancer cells [32, 33]. Based on this, we evaluated the effect of pentadecanoic acid in promoting cell death by inducing apoptosis in MCF-7/SC.
We first analyzed MCF-7/SC cells after exposing with pentadecanoic acid at different concentration for 48 h, then AnnexinV/PI staining was performed to detect apoptosis population. The obtained results demonstrated the significance of late apoptosis by pentadecanoic acid and some early apoptosis at 48 h (Figure 6a). The connection between the induction of apoptosis and cell cycle arrest was reported in previous studies [34], we then investigated whether pentadecanoic could induce cell cycle arrest. As shown in Figure 6b, pentadecanoic acid treatment resulted in induction of sub-G1 accumulation compared with non-treated control cells. These data were further supported by Western blot results, in which increased expression of cleave form of apoptosis markers such as caspase-3,
caspase-7, caspase-8, caspase-9, PAPR. In addition, a decreased expression in total form of these proteins were also observed (Figure 6c).
Figure 6. Pentadecanoic acid promoted apoptosis in MCF-7/SC cells. (a) Results obtained after cell cycle analysis of MCF-7/SC cells treated with pentadecanoic acid at 0, 50, 100, 150
and 200 µ M for 48 h. (b) Results obtained after Annexin V/PI staining of MCF-7/SC cells treated with pentadecanoic acid at 0, 50, 100, 150 and 200 µ M for 48 h. (c) Protein levels of apoptosis markers were examined by western blot analysis following pentadecanoic acid (0, 50, 100, 150, 200 μM) expose for 48 h. GAPDH was used as a loading control; Results are shown as mean ± SD. (* p < 0.05).
3.7. Pentadecanoic acid enhances chemosensitivity of MCF-7/SC cells to Tamoxifen
To evaluate whether pentadecanoic acid enhances cytotoxic efficacy of tamoxifen (TAM)
in-vitro, MCF-7/SC cells were co-treated with pentadecanoic acid (50 and 100 μM) and TAM
(10 μM) for 48 h and cell viability was then examined by the MTT assay. A reduction in the
cell viability of MCF-7/SC was observed following treating with pentadecanoic acid and
TAM compared to the TAM treatment alone (Figure 7a). To explore whether pentadecanoic
acid and TAM had synergistic cytotoxic effects in MCF-7/SC cells, we calculated the
combination index (CI) values. Combined treatment containing 100 μM of pentadecanoic
acid and 10 μM of TAM after 48 h expose displayed highest synergistic inhibitory effects
with a CI value of 0.38 (Figure 7b). As illustrated in Figure 7c, Annexin V/PI staining showed
that combined treatment of pentadecanoic acid (50 and 100 μM) and TAM (10 μM) can
significantly promote apoptosis in MCF-7/SC cells compared to the control, TAM treatment
only or pentadecanoic acid treatment alone. Cell cycle analysis resulted in an increase in the
sub-G1 accumulation of MCF-7/SC cells after co-treatment with pentadecanoic acid and
TAM (Figure 7d). Highest sub-G1 population (50.05±3.78%) of MCF-7/SC cells was observed
at the combined treatment containing 100 μM of pentadecanoic acid and 10 μM of TAM
(Figure 7d). Furthermore, pentadecanoic acid and TAM combined treatment were further
supported by western blot analysis. As illustrated in Figure 7e, the combine treatment of
pentadecanoic acid and TAM resulted in an increased cleavage form of 7,
caspase-9 and PARP, whereas resulted in a reduction of the total form of these apoptosis markers.
Based on these observations, we conclude that pentadecanoic acid can enhance the in-vitro
cytotoxic efficacy of TAM and promote TAM induced apoptosis in MCF-7/SC cells.
Figure 7. Pentadecanoic acid enhances chemosensitivity of MCF-7/SC cells to Tamoxifen.
(a) Results obtained after MTT assay performance on MCF-7/SC cells co-treated with
pentadecanoic acid at 25, 50, 75, and 100 µ M and tamoxifen (TAM) at 10 µ M for 48 h. (b) Combination Index (CI) calculated for various concentrations of pentadecanoic acid and TAM treatment in MCF-7/SC cells. (c) Results obtained after cell cycle analysis of MCF-7/SC cells co-treated with pentadecanoic acid (50, 100 µ M) and TAM 10 µ M for 48 h. (e) Results obtained after Annexin V/PI staining of MCF-7/SC cells co-treated with pentadecanoic acid (50, 100 µ M) and TAM 10 µ M for 48 h. (e) Protein levels of apoptosis markers were examined by western blot analysis following pentadecanoic acid (50, 100 μM) plus TAM 10 μM exposure for 48 h. GAPDH was used as a loading control Results are shown as mean ± SD.
(* p < 0.05).
4. DISCUSSION
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
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