Background
Triple-negative breast cancer (TNBC) is a unique subset of human breast cancer that is characterized by negative estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status. TNBC accounts for approximately 15–20% of all breast cancer diagnosis [
1]. TNBC is far more aggressive and shows higher rates of relapse compared to other types of breast cancer [
2]. Tumor heterogeneity and lack of effective molecular targets contributes to the poor prognosis [
3,
4]. Chemotherapy is currently the mainstay of treatment for patients with TNBC. Although chemotherapy is effective in a subgroup of these cancers, it fails in the majority of patients [
5,
6]. Hence, there is an urgent clinical need to discover new molecular targets and develop drugs with minimal toxicity to treat patients with TNBC.
Osthole (7-methoxy-8-isopentenoxycoumarin) is a coumarin-derivative extract of
C. monnieri that has been shown to inhibit many pathological disorders. These include conditions such as allergies and inflammation [
7], diabetes [
8], as well as liver injury [
9]. In addition, osthole has been reported to beneficial inhibitory effects in multiple types of cancer, including hepatic carcinomas [
10], leukemia [
11], gastric cancer [
12], and lung cancer [
13]. Specifically in breast cancer cells, osthole inhibits the growth of breast cancer cells, at least in culture [
14,
15]. Taken together, the numerous studies conducted to date suggest that osthol possesses the potential to act in an inhibitory role in the progression of malignancies. However, the mechanisms of function and overall cellular effect of osthol toward particular cancers may not be the same. The mechanisms of action as well as its effect on TNBC cells are not currently known.
Here, we have investigated the effect of osthole in cultured TNBC cells as well as in a xenograft model of TNBC growth. We show that osthole inhibits the growth of TNBC cells and induces apoptosis. Using a high-throughput proteomis platform, we report for the first time, that osthole induces apoptosis in TNBC cells through the inactivation of signal transducer and activator of transcription-3 (STAT3) signaling pathway. In addition, osthole inhibited TNBC cell proliferation in mice implanted with TNBC cells. Our findings show that osthole is a therapeutic candidate in the treatment of patients with TNBC. We have also discovered a novel mechanism of the anti-cancer activities of osthole.
Methods
Reagents
Osthole (purity > 99%) and biotin were purchased from the Aladdin Chemicals (China) and was dissolved in DMSO. Biotinylated-osthole (purity > 97.8%) was designed and synthesized by Bocong Biotech (Guangzhou, China). Antibodies against cleaved-PARP (sc-56,196), Bax (sc-493), Bcl-2 (sc-492), Bcl-xl (sc-8392), MDM-2 (sc-965), CyclinB1 (sc-245), CDC2 (sc-54), Ki67 (sc-7846), GAPDH (sc-32,233), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, HRP-conjugated donkey anti-rabbit IgG, and PE-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against Phospho-STAT3 (Tyr705, Clone D3A7, 9145), STAT3 (12640S), and cleaved-caspase3 (9661S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ).
Cells culture
Human breast cancer cell lines (MDA-MB-231, BT-549, MDA-MB-468, and MCF-7 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 and MCF-7 cells were cultured in DMEM medium (Gibco, Eggenstein, Germany), BT-549 cells were cultured in RPMI-1640 medium (Gibco), and MDA-MB-468 were grown in L15 medium (Gibco). Media in all cases was supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 100 units/ mL penicillin, and 100 μg/mL streptomycin.
Cell viability assay
Human breast cancer cells were seeded in 96-well tissue culture plates at a density of 8000 per well, and allowed to attach overnight in complete growth media. Osthole were dissolved in DMSO and then diluted in medium to the desired final concentration (6.25, 12.5, 25, 50,100, 200, 400, and 800 μM). The following day, cells were treated with osthole at increasing concentrations for 24 h, 48 h, or 72 h, respectively. Cell viability was then measured through MTT assay.
Apoptosis and cell cycle analysis
Cells were plated in 60-mm dishes and allowed to attach overnight. Cells were then treated with osthole at 100, 150, or 200 μM. Following treatments, cells were fixed then labeled with FITC-conjugated Annexin V/PI (for apoptosis detection) or PI staining (for cell cycle detection). Analyses were performed using FACSCalibur flow cytometer. Data for apoptosis and cell cycle distribution was analyzed using FlowJo7.6 software.
To assess morphological changes associated with apoptosis, we stained cells with Hoechst 33258 (Beyotime Biotechnology, China). Cells were challenged with osthole at 100, 150, or 200 μM for 24 or 48 h. Cells were then fixed with 4% formaldehyde solution and stained with Hoechst 33258. Images were captured using a fluorescence microscope (Nikon, Japan). Five microscopic fields were randomly selected from each treatment group.
Western blot analysis
TNBC cells and tumor tissues were lysed and protein concentrations were measured by the Bradford assay (Bio-Rad, Hercules, CA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred to poly-vinylidene difluoride transfer membranes. Membranes were blocked for 1.5 h at room temperature using fresh 5% nonfat milk in TBST. Primary antibody incubations were carried out overnight at 4 °C. HRP-conjugated secondary antibodies were added for 1 h, and the bands were visualized by using ECL substrate (Bio-Rad). Densitometric measurements were performed using Image J (National Institute of Health, MD).
The cytoplasmic and nuclear extracts were prepared by using KeyGen biotech Nuclear and Cytoplasmic Extraction Reagent kit (Nanjing, China). Cells were washed with ice cold PBS, collected and mixed with Buffer A and Buffer B. The mixture was centrifuged for 10 min at 3000 xg at 4 °C. Supernatant was collected as cytosolic extract. Buffer C was added to the pellets for 1 h. Samples were then centrifuged at 14,000×g for 30 min to extract nuclear proteins. Protein concentrations by measured by the Bradford assay.
Proteome microarray assay and data analysis
Arrayit HuProt™ v2.0 19 K Human Proteome Microarrays (CDI Laboratories, Baltimore, MD) were blocked with 3% BSA for 1 h at room temperature. Biotinylated–osthole was diluted to 10 μM in blocking buffer and incubated on the proteome microarray for 1 h at room temperature. The arrays were washed with PBST and incubated with Cy3-Streptavidin at 1:1000 dilution (Sigma-Aldrich) for 1 h at room temperature. Finally, the microarray was spun dry and scanned with a GenePix 4200A microarray scanner (Molecular Devices, San Jose, CA). Data were analyzed by GenePix Pro 6.0 software. The signal to noise ratio (SNR) was defined as the ratio of median foreground value minus median background value. A cutoff was of SNR ≥ 1.1 was set.
Immunofluorescence staining
Cells were seeded in 35-mm cell culture dishes with glass bottom (NEST, Wuxi, China). After overnight culture in complete media, cells were serum-starved for 24 h. Cells were then treated with osthole for 32 h, followed by stimulation by 50 ng/mL IL-6 for 30 min. Cells were fixed in 4% paraformaldehyde/0.1% Triton-X100 solution. Primary anti-p-STAT3 antibody was added and samples incubated at 4 °C overnight. PE-conjugated goat anti-rabbit secondary antibody (1:200) was then added for 1 h. Cells were counterstained with DAPI. Images were captured using a fluorescence microscopy (Nikon, Japan).
Cell transfections
Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China). To express STAT3, MDA-MB-231 cells were seeded at 6 × 105 cells per dish into 60 mm plates. DMEM medium without antibiotics was added and cells were allowed to grow to 50–70% confluence. The medium was then changed to freshly prepared Opti-MEM medium. Four μg of STAT3 plasmid or control plasmid was dissolved in 6 μL Lipofectamine 2000 reagent and added onto cells. After 6 h, the medium was replaced with complete media containing 10% FBS. P-STAT3 and STAT3 expression in MDA-MB-231 cells was confirmed by Western blotting analysis after 48 h.
Breast cancer xenografts
All animal studies were in compliance with the Wenzhou Medical University’s Policy on the Care and Use of Laboratory Animals. Protocols for animal studies were approved by the Wenzhou Medical College Animal Policy and Welfare Committee. Five-week-old athymic BALB/c nu/nu female mice (18-22 g) were purchased from Vital River Laboratories (Beijing, China). Animals were housed at a constant room temperature with a 12 h:12 h light/dark cycle and fed a standard rodent diet and given water ad lib. The mice were divided into three experimental groups with six mice in each group. MDA-MB-231 cells were injected subcutaneously into the right flank at 5 × 106 cells in 100 μL of PBS per mouse. When tumors reached a volume of 50–150 mm3, mice were treated with osthole by intraperitoneal injections twice daily (100 or 200 mg/kg/d). Osthole was dissolved in 6% castor oil. Tumor volumes were determined by measuring length (l) and width (w) and calculating volume (V = 0.5 × l × w2) at the indicated time points. At the end of the study, mice were sacrificed and the tumors were removed. Heart, kidney, and liver tissues were also harvested to assess any toxicity associated with osthole treatment.
Tissue staining
All harvested tissues were fixed in 10% formaldehyde and embedded in formalin. Tissues were sectioned at 5-μm thickness. Heart, liver and kidney tissues were stained with hematoxylin and eosin (H&E). Tumor tissue sections were deparaffinized, rehydrated and incubated with primarily Ki-67, Bcl-2, Cleaved-caspase 3, MDM2 and CDC2 antibodies. HRP-conjugated secondary antibodies and diaminobenzidine (DAB) were used for detection.
Statistical analysis
Data shown as mean ± SEM with n ≥ 3 independent samples. Statistical analysis was performed with GraphPad Prism 5.0 software (San Diego, CA, USA). One-way ANOVA followed by Dunnett’s post hoc test was used for comparing more than two groups of data, and one-way ANOVA, non-parametric Kruskal–Wallis test, followed by Dunn’s post hoc test was used when comparing multiple independent groups. All other results were analyzed using t-test. Values of P < 0.05 were considered statistically significant. Post hoc tests were run only if F achieved P < 0.05 and there was no significant variance in homogeneity.
Discussion
In present study, we found that osthole inhibited the growth of TNBC cells by inducing cell cycle arrest apoptosis. Similarly, osthole treatment of mice bearing MDA-MB-231 TNBC cells showed reduced tumor growth and increased cell apoptosis. We also discovered that osthole mediates these beneficial inhibitory effects in TNBC cells through the suppression of STAT3. Specifically, we demonstrate that osthole binds to and inhibits the phosphorylation of STAT3, thus inhibiting its nuclear translocation. Collectively, these results suggest osthole has potential as a promising candidate for the treatment of TNBC.
A recent in vitro study on two invasive mammary carcinoma cell lines, MDA-MB-231 and 4 T1 cells, showed that osthole inhibited cell proliferation when used in combination with platycodin D [
16]. Platycodin D is a triterpene saponin and together with osthole reduced transforming growth factor-β receptor signaling in breast cancer cells. It should be noted that this suppression of signaling was seen in the combination treatment and it is not clear whether osthole along inhibits the pathway. Using a proteomic microarray containing 19,394 proteins, we identified 199 candidate targets to which osthole may bind. One of these candidate binding proteins was STAT3. STAT3 is a member of STAT transcription factors that mediate many aspects of immunity, proliferation, apoptosis and differentiation [
17,
18]. This wide range of cellular activities may explain the spectrum of beneficial effects seen with osthole in a variety of disease models. STAT3 is found constitutively phosphorylated in a number of human cancer cell lines and primary tumors [
19]. Evidence also suggests that constitutive activation of STAT3 is a point of convergence for malignant transformation at several levels [
20], including transformation, proliferation, invasion, and metastasis [
21‐
23]. STAT3 also has been reported to be constitutively active in TNBC [
21,
24]. Although there is no difference in the expression levels of STAT3 in ER+, HER2, and TNBC breast cancer subtypes, active and phosphorylated STAT3 has been shown to be restricted to TNBC [
25,
26]. Studies have shown that STAT3 signaling is critical for cell survival in TNBC [
26‐
28].
Tumor cells which lack STAT activation are more tolerant to small molecular inhibitors which block STAT3 signal pathway [
29‐
31]. Studies using normal mouse fibroblasts showed that blocking STAT3 signaling causes growth arrest but not apoptosis, suggesting that disruption of STAT3 pathway may not be grossly toxic [
32]. However, a large number of malignant transformations with associated constitutive STAT3 activation have been reported [
19,
33]. Studies have also shown that the suppression of constitutively active STAT3 leads to growth inhibition and apoptosis in tumor cell lines as well as in xenograft models [
24,
34,
35]. Similarly, we found that osthole decreased phosphorylated STAT3 levels in vitro and in vivo. Therefore, STAT3 protein modulation may be an important aspect of the anti-tumor activity of osthole. Moreover, treatment with osthole significantly reduced the levels of phosphorylated STAT3 induced by IL-6 suggesting that osthole selectively inhibits STAT3 phosphorylation. These findings offer the potential for preferential tumor cell killing and make STAT3 an attractive and promising target for therapeutic intervention in human cancer.