Modulation of Tamoxifen Cytotoxicity by Caffeic Acid Phenethyl Ester in MCF-7 Breast Cancer Cells

Although Tamoxifen (TAM) is one of the most widely used drugs in managing breast cancer, many women still relapse after long-term therapy. Caffeic acid phenethyl ester (CAPE) is a polyphenolic compound present in many medicinal plants and in propolis. The present study examined the effect of CAPE on TAM cytotoxicity in MCF-7 cells. MCF-7 cells were treated with different concentrations of TAM and/or CAPE for 48 h. This novel combination exerted synergistic cytotoxic effects against MCF-7 cells via induction of apoptotic machinery with activation of caspases and DNA fragmentation, along with downregulation of Bcl-2 and Beclin 1 expression levels. However, the mammalian microtubule-associated protein light chain LC 3-II level was unchanged. Vascular endothelial growth factor level was also decreased, whereas levels of glutathione and nitric oxide were increased. In conclusion, CAPE augmented TAM cytotoxicity via multiple mechanisms, providing a novel therapeutic approach for breast cancer treatment that can overcome resistance and lower toxicity. This effect provides a rationale for further investigation of this combination.


Introduction
Breast cancer is the most prevalent cancer among the female population [1]. Despite the evolution in treating breast cancer, it is still the principal cause of cancer death in females [1,2].
Tamoxifen (TAM) is a widely used antiestrogenic drug for treating breast cancer patients [3]. Although there are many satisfying outcomes from the endocrine therapy with TAM, not all treated patients get the hopeful result. High concentration of TAM showed several hormonal dependent and independent effects. TAM can induce apoptosis of cancer cells via the involvement of a mitochondria-dependent pathway, the amendment of signaling proteins such as protein kinase C, and/or the upregulation of p53 [4]. Hence, a combination of lower concentrations of TAM with other drugs of synergistic antitumor effect might be of priority in the therapy of breast carcinomas.
Caffeic acid phenethyl ester (CAPE) is a polyphenolic compound existing in numerous medicinal plants and in propolis [5]. It is subjected to the action of blood esterase due to its aryl ester structure. The pharmacokinetic profiles of CAPE in rat tissues showed high values of volume of distribution and short elimination half-life after its systemic administration [6].
CAPE has a diversity of important biological activities including antibacterial, antiviral, and anticancer ones [7]. Moreover, at low doses, CAPE inhibits lipid peroxidation [8] and shows antioxidant activities [9].
Several reports have shed light on the impact of CAPE on cell cycle progression, cell proliferation, induction of cell cycle arrest, and apoptosis [10]. The in vitro and in vivo inhibitory effects of CAPE were predictable in plenty of cancer models, such as colon [11], lung cancers [12], and pancreatic carcinoma [13]. A remarkable finding is the ability of CAPE to exhibit differential toxicity against tumor cells without affecting normal cells. In this context, CAPE has no cytotoxic effect on normal nonmalignant cells as MCF-10A mammary cells [5,14].
The competency to increase response and reduce chemoresistance of cancer therapeutics via the use of the 2 Oxidative Medicine and Cellular Longevity combination therapy might thus be a significant advantage for cancer patients. Combination therapies promoting the effectiveness of TAM have been previously investigated in several studies, using compounds as vitamin E [15] and green tea [16].
Accordingly, this study examined the efficacy of TAM and CAPE with regard to multiple targets as apoptosis, autophagy, angiogenesis, and oxidative stress in breast cancer cells.

Materials and Methods
2.1. Drugs. TAM was a kind gift from El Amirya Pharmaceuticals Company (Alexandria, Egypt). It was dissolved in dimethyl sulfoxide (DMSO) at concentration 1 : 1 and stored at −20 ∘ C.
CAPE was purchased from Sigma-Aldrich Chemical Co. (ST. Louis, MO, USA). The compound was dissolved in DMSO at 100 mM concentration and stored at −20 ∘ C. Serial dilutions of both drugs were made in cell culture medium just prior to use, so that the final concentration of DMSO was about 0.1% (v/v).

Human Cancer Cell Line and Cell
Culture. Human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). MCF-7 cells were cultured in RPMI-1640 medium supplemented with heat-inactivated 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin, and 2 mM L-glutamine. Cells were incubated at 37 ∘ C in a humidified 5% CO 2 incubator.

Cytotoxicity Assay.
Cytotoxicity was determined using SRB method as described by Skehan et al. [17]. In brief, cells were seeded at a density of 3 × 10 3 cells/well in 96well microtiter plates. They were left to attach for 24 h before incubation with drugs. The old medium was discarded and was replaced with a fresh one containing drugs added alone or simultaneously with the specified concentrations.
In brief, the cells were treated with different concentrations of TAM (10, 20, 30, 40,  For each sample, three wells were used for every concentration and incubation was continued for 48 h. The same volume (200 L/well) of DMSO (1% v/v) was used as the vehicle control. At the end of incubation, cells were fixed with 20% trichloroacetic acid (TCA), stained with 0.4% sulforhodamine-B (SRB), and rinsed with 1% acetic acid. The bound protein stain was solubilized with Tris base (10 mM, pH 10.5) and the optical density (OD) of each well was measured spectrophotometrically at 570 nm using ELISA microplate reader (TECAN sunrise, Germany). The experiment was repeated 3 times and the mean values were estimated as fraction of cell survival as follows: OD (treated cells)/OD (control cells).
The IC 50 value (the required concentration to produce 50% inhibition of cell growth) of each drug was calculated using sigmoidal dose response curve-fitting models (Graph-Pad Prism software, version 5).
1 and 2 signify the respective concentrations of TAM and CAPE used in combination to produce a fixed level of inhibition, while 1 and 2 represent their concentrations that are alone able to produce the same magnitude of effect. If "CI" is less than 1, the effect of combination is synergistic, whereas if CI = 1 or >1, the effect is additive or antagonistic, respectively. The medium was then aspirated after 0, 2, 4, and 24 h intervals and centrifuged and the supernatant was stored at −20 ∘ C till HPLC assay.  [19]. Proteins were then separated using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protein was transferred onto a polyvinylidene difluoride (PVDF) membrane, which was blocked with 5% w/v nonfat dry milk and incubated overnight with the specific primary antibodies {mouse anti-human for caspase-9 monoclonal antibody (1 : 1000), rabbit LC 3 -II oligoclonal antibody (1 : 500), and rabbit anti-human -actin monoclonal antibody (1 : 1000)}. This step was followed by incubation with the appropriate diluted secondary antibody (1 : 2000 in PBS-T) for 1 h at room temperature. The blots were developed with Amersham ECL western blotting detection reagents and analysis system (GE Healthcare, UK) according to the manufacturer's protocol. Protein loading was corrected foractin and quantitation of band intensity was performed using Image J Software.

Determination of the Enzymatic Activity of Caspase-3 in Cell
Lysate. Caspase-3 activity was measured colorimetrically using Caspase-3 Colorimetric Assay kit (R&D, USA, Catalog number BF3100) according to the manufacturer's instructions based on the method of Fernandes-Alnemri et al. [20]. The cleavage of the peptide by the caspase released the chromophore pNA, which can be quantitated spectrophotometrically at a wavelength of 405 nm. Caspase-3 activity was expressed as optical density.   TCT GAC GGC AAC TTC AAC TG-3  Reverse 5 -TGG GTG TCC CAA AGT AGG AG-3 Beclin 1 2.11. DNA Fragmentation Analysis. DNA was extracted using QIAamp DNA Mini Kit according to the manufacturer's instructions. DNA was electrophoresed and visualized under ultraviolet light using 0.8% agarose gel stained with ethidium bromide.

Quantitative Real Time PCR Analysis.
Total RNA was extracted from cell culture by utilizing Thermo Scientific Gene JET RNA Purification Kit, following the manufacturer's protocol. cDNA was generated with M-MuLV reverse transcriptase using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. For real time PCR quantification, Thermo Scientific Maxima SYBR Green/ROX qPCR Master Mix kit was used. Briefly, in a 25 L reaction volume, 12.5 L of master mix, 2.5 L of primer assay, and 10 L of template cDNA (100 ng) were added to each well. Sequences of primers were described in Table 1. The PCR plate was subjected to 40 cycles of the following conditions: PCR activation at 95 ∘ C for 5 min, denaturation at 95 ∘ C for 5 sec, and annealing/extension at 60 ∘ C for 10 sec. The values of RT-PCR products were normalized with respect to GAPDH and then compared to controls. The relative expression was calculated from the 2 −ΔΔCT formula [21].

Determination of Glutathione (GSH).
Reduced glutathione was determined as described by Ellman [22]. In brief, cells were collected by trypsinization after treatment with TAM, CAPE, and their combination, as well as the control. Samples were then centrifuged at 1200 rpm for 5 min and the resultant cell pellet was suspended in 1 mL saline. Next, 500 L of cell suspension was mixed well with 25 L trichloroacetic acid (TCA) and the tubes were centrifuged at 3000 rpm for 10 min at 4 ∘ C. 100 L of the resultant supernatant was mixed thoroughly with 850 L of phosphate buffer followed by addition of 50 L Ellman's reagent. After 5 min, the absorbance was measured spectrophotometrically at 405 nm against a blank. Glutathione content was expressed as nmoles of GSH/mg protein.
2.14. Determination of Nitric Oxide (NO). Nitric oxide produced in cell culture media was estimated spectrophotometrically [23]. Briefly, cells were collected by trypsinization after treatment with TAM, CAPE, and their combination as well as the control. Next, 50 L of zinc sulfate solution was added to 250 L media and centrifuged at 17000 rpm. The resultant supernatant was treated with vanadium chloride (0.8% in 1 M HCl) and Griess reagent (prepared by mixing equal volumes of N-1-(naphthyl)ethylenediamine {0.1% in bidistilled water} and sulfanilamide {2% in 5% HCl}). Total NO content was expressed as g/mL.

Statistical
Analysis. Data were expressed as means ± SD. Differences among groups were tested using one-way analysis of variance (ANOVA) followed by a Tukey post hoc correction for multiple comparisons using SPSS (version 17.0). Significant differences were considered at value < 0.05. All figures were established using GraphPad Prism, version 5.

Ethical Aspects.
There is no need for ethical approval or informed consent since all experiments were carried out using MCF-7 breast cancer cell line.

Growth Inhibition of TAM, CAPE, and Their
Combination against MCF-7 Cell Line. Figure 1(a) shows the effect of different concentrations of TAM on the survival fraction of MCF-7 cells after 48 h exposure. As evident, there was a significant dose dependent decrease in the survival fraction compared to each respective control value. At 50 M, maximum cytotoxicity (76%) of TAM against MCF-7 was reached, whereas the IC 50 value was obtained at 20 M. When examining the cytotoxic effect of different combinations of TAM and CAPE (Figure 1(c)), we observed some cytotoxic actions upon decreasing the concentration of TAM and increasing that of CAPE. When combining the smallest dose of TAM (10 M) with different concentrations of CAPE, the IC 50 of CAPE decreased to a value ranging between 1 M and 10 M (Figure 1(d)). An isobologram analysis illustrated the synergistic effect of 10 M TAM and 4 M CAPE (CI = 0.9). Figure 2 showed that majority of the TAM was taken by the cells after

Effect of TAM, CAPE, and Their Combination on Caspase-3 Activity in MCF-7 Cell
Line. Treatment of MCF-7 cells with 10 M TAM produced 1.5-and 2.5-fold increases in caspase-3 enzymatic activity after 24 h and 48 h, respectively, as compared to the control values. Furthermore, 4 M CAPE exhibited approximately 2.5-fold increases at both treatment periods compared to the control groups. Upon treating the cells with both TAM and CAPE, the activity of caspase-3 was considerably enhanced almost 3-and 4-fold, respectively, as compared to the control values (Figures 4(a) and 4(b)). Figure 5, TAM, CAPE, and their combination enhanced apoptosis of MCF7 cancer cells by 59.54%, 45%, and 61%. It is worthy noting that the combination regimen exhibited the pronounced effect in this regard.   Figure 8(a), 10 M TAM when used alone resulted in significant increase in cellular GSH content to approximately 2-fold as compared to the control. On the other hand, treatment with 4 M CAPE resulted in insignificant increase in GSH content relative to the control values. However, the combination of TAM and CAPE significantly increased GSH level almost 3-fold when compared to the control cells.

Effect of TAM, CAPE, and Their Combination on NO Production in MCF7
Cells. TAM and CAPE significantly increased NO content as compared to vehicle-treated cells (Figure 8(b)). However, the combination significantly increased the NO values compared to either single treatment group.

Discussion
Since cancer is still a major threat to health worldwide, there is global demand for more affordable and effective therapeutic alternatives. Perhaps, combining anticancer drugs with natural nutrients will be promising in the therapy of cancer patients.
According to this background, we aimed to explore the effect of TAM and CAPE, as a novel combination regimen in treating human breast cancer, and also to determine their supposed mechanisms of action.
The data of the present study revealed a concentrationdependent cytotoxic effect of TAM on MCF-7. This result supports the previously reported data elucidating the effectiveness and usefulness of TAM as a chemotherapeutic agent in human MCF-7 cells [24]. Similarly, the cytotoxic effect in MCF-7 treated with CAPE was evidenced herein. This finding concurs with the report of Wu et al. [14] demonstrating a diversity of oncolytic effects of CAPE in preclinical models of human breast cancer. Based on that, it is plausible that the combination of TAM and CAPE produced a synergistic cytotoxic effect in MCF-7 cells as indicated by CI.
Regarding the cellular uptake of TAM, the present data showed that the majority of the TAM was taken up by the cells after 2 h treatment and that no significant change in its uptake was achieved upon cotreatment with CAPE as compared with TAM treated group in all the studied time intervals. This can be explained on the basis of the fact that the lipophilicity of TAM is the controlling factor responsible for its cellular uptake [25]. TAM was taken up by the cells through simple diffusion until establishing equilibrium after 2 h, as suggested by the present data. De Santana et al. have previously reported that TAM citrate reached maximum cellular uptake after 4 h when tested in dogs [26]. This finding somewhat concurs with the current one.
Our data also revealed that the incubation of MCF-7 cells with TAM, CAPE, and their combination provoked the activation of caspase-9 and increased significantly caspase-3 activity. These results pointed to the contribution of the intrinsic pathway of apoptosis where cytosolic cytochrome c binds with procaspase-9 and Apaf-1, forming the apoptosome that triggers the cleavage of procaspase-9. Active caspase-9 then switches on downstream executioner caspases, including caspase-3 [27].
The roles of mitogen-activated protein kinases (MAPK), mitochondrial permeability transition, and ceramide generation have been formerly implicated in TAM-induced apoptosis [28]. Likewise, activation of Fas-and Bax-mediated apoptosis and associated DNA fragmentation was detected in MCF-7 human breast cancer cells following treatment with CAPE [29]. Thus, it is obvious that CAPE could potentiate both the antiproliferative and apoptotic effects of TAM.
It is worthy noting that despite the significant increase in caspase-9 activity, seen in the combination regimen after 24 h, there was a decrease in its activity after 48 h when compared to either TAM or CAPE alone. This result might be attributed to the increased GSH level observed in this group. Actually, increased GSH level decreased the availability of free radicals, leading to attenuation of mitochondrial-dependent apoptosis.
TAM-and CAPE-triggered apoptosis was also supported by DNA fragmentation in MCF-7 cells following treatments. This effect might be attributed to the caspases activation mentioned before. Virtually, DNA fragmentation is a consequence of the activation of a specific DNase (CAD, caspase activated DNase), found complexed with ICAD (inhibitor of CAD) in proliferating cells. When cells undergo apoptosis, caspases, in particular caspase-3, dissociate the CAD-ICAD complex, freeing CAD that cleaves chromosomal DNA [30].
In the present investigation, both Bcl-2 and Beclin 1 expression levels were downregulated in all treated groups. This finding pointed to their interaction. Interestingly, Beclin 1 was found to contain a well-recognized -helical BH3 domain that allows it to dock into the hydrophobic groove of Bcl-2 [34]. Furthermore, decreased Beclin 1 expression level might be related to the plausible role of caspases in inactivating Beclin 1-induced autophagy. According to Wirawan et al. [34], caspases mediate cleavage of Beclin 1, abrogate its autophagic function, and generate a Beclin 1-C fragment that can enhance apoptosis by promoting the release of proapoptotic factors from the mitochondria. By this way, Beclin 1 switches from a proautophagic to a proapoptotic protein. In this context, Qadir et al. [35] have demonstrated that when autophagy is compromised in TAM treated MCF-7 cells, activation of the mitochondrial apoptotic pathway takes place and increased apoptosis is achieved, at least in part, through caspase-9.
Herein, no change in the protein level of LC3-II was detected in all treated groups. These results were in contrast with those of Hwang et al. [36] who found increased levels of LC3-II in MCF-7 cells exposed to TAM. Similarly, CAPE triggered activation of the autophagic response in C6 glioma cells by inducing an increase in LC3 [37].
The discrepancy between our result and other researchers may be due to time of detection of autophagosomes and interplay between autophagy and other players as oxidative stress, metabolites, and apoptosis.
The major problem was to quantify precisely autophagy levels in cancer cells. Indeed, LC3-II levels have been described as an accurate marker of the number of autophagosomes in cells. However, an accumulation of these intracellular vesicles can be also linked to an increase of autophagy induction or an inhibition of autophagosome degradation by the lysosomes. To our knowledge, no autophagy marker is currently available in order to discriminate between an increase or a decrease of overall autophagy flux in vivo [38].
The present study also wanted to clarify whether, or not, the tumor inhibitory effect of TAM + CAPE is mediated via angiogenesis inhibition in breast cancer. Previously, low serum VEGF levels were reported in breast carcinoma patients after TAM therapy [39]. However, the marked decrease in VEGF levels when combined with CAPE could be related to the additional angiostatic activity exerted by CAPE. According to El-Refaei and El-Naa [40], CAPE controls tumor growth by elevating the angiostatic factors and inhibiting the angiogenic ones. Moreover, CAPE was able to inhibit NF-B in human breast cancer MCF-7 cells [29]. Blockade of NF-B signaling has been suggested to inhibit angiogenesis and tumorigenicity in different types of cancer cells by suppressing the expression of VEGF [41].
Unexpectedly, the current study demonstrated that both TAM and CAPE alone increased GSH content, whereas their combination enhanced this effect compared to either treatment alone. These data suggested that increased cellular GSH content might have a role in TAM and CAPE-mediated effect. In line, Loo et al. [42] and Moreira et al. [43] have demonstrated that TAM exhibited an antioxidant effect and was able to induce the NO synthesis in vitro. Likewise, Singhal et al. [44] have revealed that low concentrations of polyphenolics can enhance the activity of -glutamyl cysteinyl synthetase and other GSH-linked detoxifying enzymes, preserving thus GSH level.
With respect to the effect of TAM and CAPE on NO production, we found an increase in NO level. Previously, TAM treatment has prevented the transformation of C3H10T1/2 murine fibroblast cell line and has augmented NO production via the induction of inducible nitric oxide synthase (iNOS) at concentrations blocking the cell transformation [42]. NO was found to exert a cytotoxic effect on tumor cells via inhibition of cellular proliferation [45]. Moreover, Ignarro et al. [46] have confirmed the inhibitory effect of arginine-NO pathway in vascular smooth muscle proliferation and have attributed their finding to the capacity of NO to inhibit ornithine decarboxylase. Furthermore, it has been reported that NO possesses a cytotoxic effect caused by its interaction with the Fe-containing rate-limiting enzyme of DNA synthesis, ribonucleotide reductase [47], and [Fe-S] cluster enzymes, as mitochondrial aconitase [48,49]. Moreover, Watts et al. [50] have reported that the cytotoxic and the antiproliferative effects of NO were attributed to its ability to induce iron and GSH efflux from tumor cells via the GSH transporter multidrug resistance-associated protein 1 (MRP1). In conclusion, this study demonstrates that CAPE enhanced TAM cytotoxicity via multitarget approach, including weakening of autophagy, strengthening of both apoptotic and angiostatic potentials, and finally augmentation of both cellular GSH and NO levels. In addition, the ability of CAPE to lower the effective dose of TAM provides a rationale for further experimental and clinical investigations of this combination.