Background
The pro-apoptotic effects of the polyether antibiotic Salinomycin (Sal) in cancer stem cells were first described by Gupta and co-workers [
1] and confirmed in succeeding studies in cancer cells of solid and non-solid malignancies (reviewed in [
2,
3]). The precise mode of action of Sal is still not completely understood and it is plausible that it differs among the diverse types of cancer cells.
Colorectal cancer (CRC) is the third leading cause of death in the western world [
4]. Given that patients’ prognosis in advanced stage of disease is limited and colorectal liver metastases are the most frequent cancer-related death, innovative therapeutic approaches are of utmost importance. The impact of Sal on CRC cells has been already demonstrated [
5‐
7]. In vitro, Sal reduces the CD133
+ subpopulation of human CRC cells and inhibits epithelial-mesenchymal transition (EMT) [
5]. The effect of Sal on CRC has been further explained by induction of autophagy and accumulation of reactive oxygen species [
6,
8]. However, there are no data available analyzing the impact of Sal on CRC in vivo.
Hence, the aim of this study was to establish a mouse model to investigate the effectiveness of Sal against CRC growth in vivo. Furthermore, we analyzed the impact of Sal on Wnt signaling in human CD133
+and CD133
- CRC cells. Aberrant Wnt signaling is regarded as crucial for the oncogenesis of CRC [
9,
10] and inhibitory effects of Sal on Wnt signaling in other types of cancer but not CRC have been demonstrated before [
11].
Methods
Cell lines and culture
The murine CRC cell line MC38 [
12,
13] was provided by H. Abken (University of Cologne, Germany). CT 26 cells were purchased from the American Type Culture Collection (sub-clone ATTC® CRL2638™) [
13]. The human CRC cell line SW620 [
14,
15] was obtained from (ATCC); HT29 [
15] cells were purchased from the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures. Cells were cultured in DMEM (Sigma Aldrich) and RPMI 1640 medium (Invitrogen), respectively, supplemented with 10% fetal calf serum, penicillin (50 U/ml) and streptomycin (50 mg/l) at 37°C and 5%CO
2.
Chemicals and antibodies
Sal and 5-FU were purchased from Sigma Aldrich. Sal was dissolved in dimethyl sulfoxide (DMSO) for in vitro analysis [
16] or in corn oil for in vivo applications [
17]. 5-FU was dissolved in phosphate buffered saline (PBS). Stock solutions were stored at -20°C. The CD133 antibody for flow cytometry and cell sorting was purchased from Miltenyi (clone AC133). Antibodies for cleaved (c-) PARP, LRP6 (C47E12), phosphorylated (P-) LRP6 (Ser1490), β-Actin, and β-Tubulin (TU-20) for protein analysis were obtained from Cell Signaling Technology.
Flow cytometric analysis and cell sorting for CD133+/- cells
Analysis of CD133 positivity was performed according to the manufacturers instructions and as described before [
18]. In brief, cells were washed with PBS and stained with a Phycoerythyrin (PE)-conjugated CD133 antibody. Signal enhancement was performed by a two-step FASER procedure (Fluorescence Amplification by Sequential Employment of Reagents). Appropriate isotype antibodies served as control. Cell sorting was performed on a FACS Aria II (Beckton Dickinson). Representative setups before cell sort are depicted in Additional file
1: Figure S1 A + B. The purity of CD133
+/CD133
- cells was analyzed before the experiments were performed (see Additional file
1: Figure S1 C + D). CD133
+/CD133
- cells were maintained in culture for one passage after sorting.
RNA isolation and real-time PCR
Total RNA from tumor cells and tumor tissues was isolated by an RNA extraction kit (Qiagen). cDNA synthesis and real-time (RT)-PCR were performed using the first strand cDNA synthesis kit (Fermentas) and SYBR Green Master Mix kit (Roche) applying specific primers for human or murine Cyclin D1, Fibronectin, lymphoid enhancer-binding factor 1 (LEF-1) and leucine-rich-repeat-containing G-protein-coupled-receptor 5 (Lgr5). Expression rates of the genes of interest were normalized to the expression of glyceraldehy-3-phosphat-dehydrogenase (GPDH). Primer sequences are listed in Additional file
2: Table S1.
Western blotting
After drug treatment for 48 h nuclear protein was isolated (Life Technologies). Protein content was determined applying the BCA Protein Assay Kit (Life Technologies). Equal amounts of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes (Milipore). The membranes were incubated overnight with primary antibodies against human LRP6, P-LRP6, and β-catenin. After washing, membranes were incubated and developed with a horseradish peroxidase-conjugated secondary antibody (Life Technologies). β-Tubulin served as internal control. Densitometry quantitative analysis was performed applying Image J software (NIH).
Proliferation
5 × 103 murine or human CRC cells were cultured in 96-well flat bottom plates. Cells were exposed to increasing concentrations of Sal (1, 2, 5 and 10 μM), to 1 μM 5-FU alone, or to combined Sal and 5-FU for different time periods: either for 24 or 48 h under treatment, or additionally cultured in medium alone for another 24 or 48 h. Cell proliferation was measured using the WST-1 assay, which is based on the cleavage of the tetrazolium salt WST-1 into formazan by healthy cells. After the end of treatment, WST-1 reagent (Roche) was added, followed by further incubation for 4 h. Formazan formation was quantified by measuring the absorbance at 450 nm according to the manufaturer’s instructions.
Migration
Murine CRC cell migration was further analyzed using an in vitro scratch assay as described before [
19]. In brief, 0.5 × 10
6 MC38 and CT26 cells were cultured in 6-well plates until confluence. A scratch was created in the middle of the monolayer and cells were treated with either Sal or 5-FU alone, or combined Sal and 5-FU. Cell migration was assessed by phase-contrast microscopy (Zeiss, Jena, Germany) and images were captured at the beginning of treatment and after 24 and 48 h. Open wound area was calculated using TScratch software (Swiss Federal Institute of Technology Zurich) as described before [
20].
Tumor cell migration was further investigated using transwell-chambers (Cell Biolabs) equipped with an 8 μm pore polycarbonate membrane according to Boyden [
21]. 1 × 10
5 cells were seeded in the upper compartment of the membrane in culture medium without fetal calf serum. The lower compartment of the chamber was filled with culture medium containing 20% fetal calf serum. Cells were cultured in the absence or presence of either Sal or 5-FU alone or combined Sal and 5-FU for 48 h and analyzed immediately or further incubated with fresh culture medium for another 48 h. Afterwards the cells in the upper compartment of the membrane were removed using a cotton swab. Membranes were stained with crystal violet solution, migrated cells on the lower side of the membrane were isolated from the membrane and quantified by measurement of the absorbance at 540 nm according to the manufaturer’s instructions.
Invasion
Tumor cell invasiveness was analyzed by seeding 1 × 10
5 cells in Matrigel-coated membranes of transwell-chambers (Cell Biolabs) according to the manufaturer’s instructions and as described before [
22]. Tumor cell invasion assay was further performed as described for tumor cell migration assay (see above).
Cell death
Cells were analyzed for apoptosis, late apoptosis or necrosis induction following exposure to either Sal or 5-FU alone or combined Sal and 5-FU for 24 h applying the AnnexinV apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions as previously described [
16,
23]. AnnexinV positive cells were regarded as apoptotic cells; AnnexinV/PI positive cells were regarded as late apoptotic and PI positive cells were regarded as necrotic cells. Cell death was further evaluated by quantification of DNA fragmentation in cultured MC38 and CT26 cells using the HT Titer TACS Assay Kit (Trevigen) according to the manufacturer’s instructions and as described before [
24]. Tumor cell death was analyzed applying the Lactate dehydrogenase (LDH) Cytotoxicity Assay Kit (Thermo Fisher) following the manufacturer’s protocol and as described before [
25].
Cell cycle analysis
Cell cycle analysis was performed after exposure to either Sal, 5-FU or Sal and 5-FU as indicated above for 24 h applying the CellTest Plus Reagent Kit (BD Biosciences) according to the manufaturer’s instructions as described before [
23]. Analysis was performed using the Mod Fit LT Software (Verity House Software).
Animal models and treatment
Animal experiments were carried out in 6–10 week-old BALB/c-mice purchased from Charles River Laboratories. Animals were housed under standard conditions with free access to food and water under constant environmental conditions with a 12-h day-night-cycle. Isoflurane was used for inhalation anesthesia. For a subcutaneous tumor model, 1 × 106 CT26 cells were injected in 50 μl Matrigel (BD Biosciences) into the flank. After 7 days the animals were randomized into 5 treatment groups. The animals were treated daily either with corn oil (control group), 8 mg/kg 5-FU, 4 mg/kg Sal or a combination of 4 mg/kg Sal and 8 mg/kg 5-FU intraperitoneally. Tumor volume was assessed daily during chemotherapy for a total of 14 days.
For the orthotopic CRC model, an abdominal midline incision was performed, the cecum exposed, and 0.5 × 106 CT26 cells (in 50 μl Matrigel) were injected into the cecal wall. Afterwards, the cecum was rinsed with distilled water to kill leaking tumor cells and repositioned into the abdomen. Alternatively, to assess the efficacy of Sal treatment in colorectal liver metastases, after laparotomy the portal vein was exposed and 0.5 × 106 CT26 were injected into the vein. The abdomen was closed using PDS 6-0 running suture. The skin was closed using a 7 mm skin stapler. Mice were checked routinely every day. Explorative laparotomy was performed after 5, 7 and 21 days to evaluate tumor growth during further treatment. Adequate tumour growth was observed after 7 days, and the animals were randomly divided into 5 groups. After end of treatment, mice were sacrificed by cervical dislocation, tumors were harvested, tumor volume assessed and tumor tissue was either snap frozen in liquid nitrogen or embedded in paraffin for further analysis. Based on H&E staining, the metastatic area within the livers was determined morphometrically applying Image J software. 20 pictures from each H&E stained slide (10 slides per animal) were randomly taken and the metastatic lesions marked. Pixels within the marked areas were related to the overall pixel count by Image J software and means ± SD were calculated and expressed as percentage of metastatic area in correlation to the whole liver.
Terminal desoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay
TUNEL assay for apoptosis detection was performed on tissue slides after removal of paraffin applying the DeadEnd Fluorometric TUNEL system (Promega) according to the manufaturer’s instructions.
Immunohistochemistry
Paraffin fixed tissue samples were cut into sections of 5 μm and routine hematoxylin and eosin (H&E) staining was performed to evaluate histomorphological features.
Statistical analysis
Statistical analysis was performed using GraphPadPrism 6. Student’s t-test or ANOVA analysis were applied as appropriate. For all in vivo experiments, we gained for an effect (Cohen d) < 2. We supposed an effect d (d = Δ/σ with Δ = relevant difference; σ = standard deviation) of 1.5. Hence, we needed 9 animals per group to confirm this effect α = 0.05 with a power of 80%. We calculated a fault rate of n = 3 animals per group. Student’s t-test was used for analysis.
Differences were regarded statistically significant with p < 0.05 compared to untreated cells which are indicated as “control” below. Results were expressed as mean ± SD of at least three independent experiments.
Discussion
In this study we demonstrate that Sal exerts a growth-inhibiting effect on murine CRC in vivo. Furthermore, we provide evidence that the pro-apoptotic effect of Sal on human CD133+ CRC cells is associated with impaired Wnt signaling and reduced expression of Wnt target genes.
The potential of Sal to treat CRC has been demonstrated in vitro before [
5,
6,
8]. However, the evidence for the effectiveness of Sal in CRC in vivo is still missing. We therefore established a murine model to investigate the impact of Sal on CRC in vivo. First, we demonstrated that Sal exerts its pro-apoptotic effect in two murine CRC cell lines in a dose-dependent manner. This is accompanied by inhibition of tumor cell migration and invasion. Interestingly, in contrast to other observations before, an additive synergistic effect of Sal and 5-FU in vitro was not observed [
26‐
28].
The obtained data providing the effectiveness of Sal in murine CRC in vitro encouraged us to establish an in vivo-model for CRC. First, we induced subcutaneous CRC growth in BALB/c-mice. Treatment with Sal resulted in inhibition of tumor growth compared to control or 5-FU-treatment. Orthotopic tumor growth in the cecum of the mice was likewise abolished after treatment with Sal. Additionally, Sal inhibited colorectal spread in the liver of mice. Treatment with Sal alone or in combination with 5-FU was superior combined to 5-FU alone. Given that CRC liver metastasis are the leading cause for CRC-related death [
4] this observation promises an important clinical impact. The effectiveness of Sal in vivo has been described before predominantly in subcutaneous tumor models [
1,
7,
17,
26,
29‐
31]. Our data demonstrate the effectiveness of Sal in clinical relevant CRC models for the first time in a convincing extent.
Next, we gained to dissect a molecular mechanism of Sal. Based on the observation that Sal reduces the CD133
+ and therewith stem-like cell signature subpopulations of human CRC cells [
5], we investigated if the mode of action of Sal in CD133
+ and CD133
- cells varies. We focused on Wnt signaling in human CD133
+ and CD133
- CRC cell lines given that aberrations in Wnt signaling cascade and its impact on CRC development are well known [
9,
10,
32‐
34]. Additionally, the inhibitory effect of Sal on Wnt signaling has been described in other tumor entities before, including leukemia, breast, pancreatic, prostate, and lung cancer cells [
11,
26,
35‐
38]. Hence, the hypothesis that Salinomycin acts as an inhibitor of Wnt signaling is not new. On the other hand, there is no data available indicating that Sal has an influence on Wnt signaling in CRC. The following findings led us to the conclusion that interference with the Wnt/β-catenin pathway might be responsible for the pro-apoptotic effect of Sal in human CRC cells.
First, we exposed SW620 and HT29 and the CD133+/- subpopulations of each cell line separately to Sal and confirmed the toxic effect on all CRC cell subpopulations. Interestingly, the effect of Sal on the CD133+ and CD133- subpopulations did not differ regarding impairment of proliferation and induction of apoptosis.
Next, we observed blocking of LRP6-phosphorylation by Sal and reduced LRP6 levels in both CD133
+ and CD133
- cells. The phosphorylation of the LRP6 co-receptor is crucial for the activation of the Wnt/β-catenin pathway [
11,
36] and in aberrant Wnt signaling-associated carcinogenesis [
39] of CRC. Reduced phosphorylation of LRP6 after exposure to Sal was first described in chronic lymphocytic leukemia cells [
36]. Our results further confirm the data obtained by Lu and co-workers in another study on Sal and Wnt signaling. They observed suppression of LRP6-expression in prostate and breast cancer cells after Sal-treatment [
37]. Additionally, they also described reduced expression of the Wnt target genes Cyclin D1 and Survivin [
37]. We further analyzed the expression profiles of selected Wnt target genes. Fibronectin and Lgr5 mRNA expression were both down-regulated upon treatment with Sal in SW620 and HT29 cells. The importance of Fibronectin and its spliced variant extra domain A (EDA) has been characterized as essential for the phenotype and the tumorigenic properties of CD133
+/CD44
+ CRC cells [
40]. Furthermore, a correlation between Fibronectin EDA-level and stage of disease and chemoresistance of CRC patients was reported [
40]. Given that silencing of EDA resulted in downregulation of Wnt/β-catenin signaling and the inhibitory effects of Sal on Fibronectin expression in our study, Sal might be regarded as an inhibitor of the regulatory Fibronectin/Wnt/β-catenin signaling loop in human CRC cells [
41].
Lgr5 is a Wnt target gene that acts as a receptor for the Wnt agonist R spondin [
42,
43]. The implications of Lgr5 expression and its tumorigenic activity in human CRC were described before indicating a crucial role in stem-like cells of CRC [
44,
45]. Silencing of Lgr5 resulted in reduced proliferation, migration and colony formation and decreased tumorigenic activity in vivo [
44]. Zhou et al. reported that Sal is able to overcome Cisplatin-resistance in human CRC cells displaying stem-like signatures, including increased Lgr5 expression [
8]. To the best of our knowledge, our study is the first to show that treatment with Sal directly inhibits Lgr5 expression in human CRC cells. Having in mind the impaired tumorigenicity after silencing of Lgr5, the toxic effect of Sal in CRC cells are plausible.
Acknowledgements
The authors are thankful to Praveen Radhakrishnan for his assistance with protein and mRNA expression analysis and to Marzena Knyssok-Sypniewski for her technical support.