Semi-synthetic salinomycin analogs exert cytotoxic activity against human colorectal cancer stem cells
Johannes Klose a, *, Sarah Kattner a, Bjo€rn Borgstro€m b, Claudia Volz a, Thomas Schmidt a, Martin Schneider a, Stina Oredsson c, Daniel Strand b, Alexis Ulrich a
Keywords: Salinomycin Salinomycin analogs Colorectal cancer Apoptosis
Cancer stem cells
A B S T R A C T
Salinomycin, a polyether antibiotic, is a well-known inhibitor of human cancer stem cells. Chemical modification of the allylic C20 hydroxyl of salinomycin has enabled access to synthetic analogs that display increased cytotoxic activity compared to the native structure. The aim of this study was to investigate the activity of a cohort of C20-O-acyl analogs of salinomycin on human colorectal cancer cell lines in vitro. Two human colorectal cancer cell lines (SW480 and SW620) were exposed to three C20-O- acylated analogs and salinomycin. The impact of salinomycin and its analogs on tumor cell number, migration, cell death, and cancer stem cell specifity was analyzed. Exposure of human colorectal cancer cells to the C20-O-acylated analogs of salinomycin resulted in reduced tumor cell number and impaired tumor cell migration at lower concentrations than salinomycin. When used at higher (micromolar) concentrations, these effects were accompanied by induction of apoptotic cell death. Salinomycin ana- logs further expose improved activity against cancer stem cells compared to salinomycin.
1. Introduction
Cancer cells are phenotypically heterogeneous and comprises subpopulations of cells that exhibit an increased tumorigenic po- tential, commonly referred to as cancer stem cells [1]. In 2009, the polyether ionophore antibiotic salinomycin was shown to exhibit selective inhibitory effects against such cells [2]. The efficacy has subsequently been confirmed in several types of cancer cells including prostate, gastric, and pancreatic cancer, hepatobiliary malignancies and glioblastoma [3e8]. The activity of salinomycin against colorectal cancer cells has been extensively studied in vitro and in vivo [9e12]. We have recently shown that salinomycin in- hibits colorectal cancer growth in mice more effectively than the common chemotherapeutic agent 5-Fluoruracil [10]. From a clinical perspective, there is concern that the utility of salinomycin may be limited by its toxic side effects. Accidental in- toxications in both humans and animals have been described [13,14]. A possible entry reducing the unwanted side effects of salinomycin is the development of structural analogs with an improved activity/toxicity profile.
We have recently synthesized a number of structurally modified salinomycin analogues [15e17]. Acylation of the C20 hydroxyl group, here exemplified by acetate, ethyl carbamate, and ethyl carbonate [18] which represent the most active derivative in each class (Supplementary Fig. S1), was shown to be beneficial in terms of increased basal toxicity compared to the natural product [15]. Significantly, such derivatives were also shown to exhibit a corre- sponding enhancement of activity also against putative cancer stem cells, and could be used at low nanomolar concentrations at which salinomycin was inactive [19]. Increased activity may be clinically advantageous, as lower drug concentrations could reduce toxic side effects. Here, we evaluate the activity of a small cohort of salinomycin analogs against human colorectal cancer cells. More specifically, the most active of each class of C20-acyl derivatives from prior studies .We show that the C20-O-acylated analogs of salinomycin exert considerably increased activity against two colorectal cancer cell lines compared to salinomycin in several in vitro-assays. These ef- fects are pronounced already at low micromolar concentrations where salinomycin exhibit only weak activity. Furthermore, the C20-O-acylated analogs exert increased activity against cancer stem cells compared to salinomycin. These findings indicate that semi-synthetic analogs of salinomycin can be used at lower con- centrations against colorectal cancer cells than salinomycin, which may carry implications for reducing the toxic side effects of salinomycin.
2. Materials and methods
2.1. Cell lines and culture
The two human CRC cell lines SW480 and SW620 were used to assess the effectiveness of salinomycin analogs in vitro. The cells were obtained from American Type Culture Collection (ATCC number: CCL-228 and CCL-227). SW480 cells were cultured in RPMI 1640 medium (Invitrogen); SW620 cells were culture in DMEM (Sigma Aldrich). All media were supplemented with 10% fetal calf serum, penicillin (50 U/ml) and streptomycin (50 mg/l). The cells were incubated at 37 ◦C and 5% CO2.
2.2. Chemicals and antibodies
Salinomycin sodium salt was purchased from Sigma Aldrich. The three C20-O-acylated analogs 20-ethyl carbonate, 20-N-ethyl carbamate and 20-acetate were synthesized as described previ- ously and used as their corresponding sodium salts [17,20]. All compounds were dissolved in dimethyl sulfoxide (DMSO) to obtain stock solutions (10 mM) and diluted with phosphate-buffered sa- line (PBS) to receive appropriate working solutions [4,19]. Stock
solutions were stored at —20 ◦C. The DMSO concentration in the
medium was 0.005% when using the compounds at a 0.05 mM
concentration.
2.3. Cell number assay
Human CRC cells (5 103) were cultured in 96-well flat bottom plates. Cells were exposed to increasing concentrations of salino- mycin or the analogs (0.05 mM, 0.1 mM, 1 mM and 10 mM) for 72 h. The effect of the evaluated compounds on cell number was assessed using the bromodeoxyuridine (BrdU) ELISA kit (Sigma Aldrich), which is based on BrdU incorporation into cellular DNA during proliferation, according to the manufacturer’s instructions. BrdU incorporation was determined by measuring the absorbance at 450 nm. Alternatively, cell proliferation was investigated using the WST-1 assay (Sigma Aldrich), which is based on the cleavage of the tetrazolium salt WST-1 into formazan by healthy cells. Soluble formazan formation was quantified by measuring the absorbance at 450 nm according to the manufacturers instructions. Addition- ally, cell numbers were analyzed applying the PrestoBlue Cell Viability Assay (ThermoFisher). This assay is a resazurin-based assay measuring reduced red fluorescent PrestoBlue dye by healthy cells at 538 nm as recommended by the manufacturer’s instructions.
2.4. Migration
Tumor cell migration was analyzed using transwell chambers (Corning Coster, Corning, NY, USA) provided with an 8 mm pore polycarbonate membrane. Human CRC cells (5 105 cells/well) were seeded in the upper compartment of the chamber and cultured using medium containing without fetal calf serum. The lower compartment was filled with culture medium containing 20% fetal calf serum acting as a chemo-attractant [5]. Cells were cultured in the absence or presence of each compound respectively (added into both inserts) for 72 h and for additional 72 h after removal of the compounds. Afterwards, the membrane was removed, fixed with ethanol, and stained with hematoxylin. The membranes were analyzed under a light microscope counting the number of migrated cells to the lower surface of the membrane in five randomly selected fields as described before [5]. The number of migrated cells were quantified using ImageJ software calculating the fraction of stained cells for every experimental condition in relation to untreated cells [21].
2.5. Cell death
Cell death after exposure to either compound was analyzed applying the AnnexinV apoptosis detection kit (BD Biosciences), Lactate dehydrogenase (LDH) Cytotoxicity Assay Kit (Thermo Fisher) and DeadEnd Fluorometric TUNEL system (Promega) following the manufacturer’s instructions as previously described [10,22].
2.6. ADELFOUR assay
The anti-cancer stem cell activity of the three analogs and sal- inomycin was analyzed applying the ADELFOUR kit (Stem Cell Technologies) according to the manufacturer’s protocol. 2 105 cells were either incubated with the ALDH1 inhibitor diethylaminobenzaldehyde (DEAB) and the ALDH1 substrate BODIPY-amino acetaldehyde or only BODIPY-amino acetaldehyde. DEAB-treated cells served as a control to set the ALDH1þ region for each sample using a Becton Dickinson flow cytometer as described
before [19].
2.7. RNA isolation and real-time PCR
Total RNA from tumor cells 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 (Life Technologies) for human leucine-rich-repeat-containing G-pro- tein-coupled-receptor 5 (Lgr5). Expression rates of the gene of in- terest were normalized to the expression of glyceraldehy-3- phosphat-dehydrogenase (GPDH). Primer sequences are listed in Supplementary Table S1. Salinomycin analogs reduce proliferation, inhibit migration and induce cell death in human colorectal cancer cells. SW480 cells were cultured in the absence or presence of increasing concentrations of salinomycin analogs (Acetate, Carbamate, and Carbonate) and salinomycin for 72 h. Tumor cell number was analyzed by (A) BrdU incorporation, (B) WST-1, and (C) PrestoBlue reduction, which are assumed to be proportional to the cancer cell number. Cancer cell migration was analyzed applying transwell- assays. After treatment the membranes were removed, fixed with ethanol and stained with hematoxylin. The membranes were analyzed under a light microscope counting the number of migrated cells to the lower surface of the membrane in five randomly selected fields and quantified using ImageJ software. Results are shown as representative images of stained membranes at a magnification of 100 (D) or as summary of at least 3 independent experiments as mean ± SEM (E). Detection of cell death was performed using LDH release assay (F); *p < 0.05, **p < 0.01 and ***p < 0.001 compared with salinomycin treatment. Scale bars ¼ 100 mM. 2.8. Statistical analysis Statistical analysis was performed using GraphPadPrism 6. Student's t-test or ANOVA analysis were applied as appropriate. Differences were regarded statistically significant with p < 0.05 compared to untreated cells, indicated as “control”. Results were expressed as mean ± standard error of the mean (SEM) of at least three independent experiments. 3. Results 3.1. Treatment with C20-O-acylated analogs results in increased reduction of cancer cell number compared to salinomycin At the outset, we investigated the anti-proliferative activity of acetate, carbamate, and carbonate in colon cancer cells using the BrdU assay. Each compound was investigated in two cancer cell lines, SW480 and SW620, using a range of concentrations (0.05, 0.1, 1, and 10 mM). The cells were treated for 72 h. SW480 cell prolif- eration was markedly reduced after exposure to the analogs at concentrations of 1 and 10 mM compared to salinomycin (Fig. 1A). Similar results were obtained when SW680 cells were used (Supplementary Fig. S2A). We also applied the WST-1 assay to investigate cancer cell proliferation, where WST-1 reduction is assumed proportional to the cell number. In SW480 cells, acetate, carbamate, and carbonate were more active than salinomycin applying concentrations of 0.1 mM for 72 h (Fig. 1B). In SW620 cells, the analogs exhibited a higher activity than salinomycin only when used at concentrations of 1 mM (Supplementary Figure S2 B). To further corroborate these findings, we employed a PrestoBlue assay to assess the effects of the salinomycin analogs on cancer cell proliferation. The number of SW480 cells was consistently decreased after exposure to acetate, carbamate, and carbonate even at low micromolar concentrations (0.05 mM). The anti-proliferative activity of the C20-O-acylated analogs was more pronounced compared to salinomycin (Fig. 1C). Exposure of SW620 cells with the C20-O-acylated analogs likewise resulted in a reduction of cancer cell number when con- centrations of 0.1 mM were used and increased activity compared to salinomycin (Supplementary Fig. S2C). 3.2. Treatment with C20-0-acylated analogs results in a reduced cancer cell migration compared to salinomycin In order to investigate the effects of the salinomycin analogs on colorectal cancer cell migration, a Boyden chamber migration assay was performed. The migration ability of SW480 cells was markedly impaired already after exposure to low concentrations of the C20- O-acylated analogs (0.05 mM, Fig. 2D E). This inhibition of tumor cell migration was more pronounced than the inhibitory effects of salinomycin on SW480 cell migration. The superior inhibitory ef- fects on tumor cell migration after exposure to the C20-O-acylated analogs compared to salinomycin were confirmed in SW620 cells (Supplementary Fig. S2D). The migration ability of the cancer cells was already impaired after exposure to drug concentrations of 0.05 mM. 3.3. C20-O-acylated analogs induce apoptotic cell death in human colorectal cancer cell lines To analyze induction of cell death of human colorectal cancer cells after exposure to the C20-O-acylated analogs, we initially examined LDH release in SW480 and SW620 cells. As shown in Fig. 1F, treatment of SW480 cells with the C20-O-acylated analogs resulted in increased LDH release. A higher activity of acetate and carbonate compared to salinomycin was observed when drug concentrations of 1 and 10 mM were used. Treatment with carba- mate did not result in extended LDH release compared to salino- mycin. In SW620 cells, high concentrations (1 and 10 mM) of acetate, carbamate, and carbonate likewise resulted in increased LDH release compared to salinomycin (Supplementary Fig. S2E). We further analyzed induction of apoptosis by measuring the amount of AnnexinV/PI positive cells. As demonstrated in Fig. 2A B, exposure of SW480 cells to the C20-O-acylated analogs resulted in induction of apoptosis (AnnexinV/PI positive cells). High doses (10 mM) of acetate and carbonate provoked an increased amount of apoptotic cells compared to salinomycin-treated cells. Exposure to carbamate was associated with equivalent amounts of apoptotic cells compared to exposure to salinomycin. Treatment of SW620 cells with the C20-O-acylated analogs was likewise accompanied by induction of apoptosis. Exposure to high concen- trations (1 and 10 mM) of acetate, carbamate, and carbonate resulted in an increased amount of apoptotic cells compared to salinomycin in SW620 cells (Supplementary Fig. S2F). Apoptotic cell death was also analyzed by TUNEL assay. As demonstrated in Fig. 2C, treatment with the C20-O-acylated ana- logs and salinomycin resulted in induction of apoptosis in SW480 cells after treatment for 72 h. According to the above- mentioned results, apoptosis induction was most prominent after exposure to acetate and carbonate. Comparable results were observed when SW620 cells were analyzed by TUNEL assay (data not shown). In all cell death assays, the cytotoxic effects of the C20- O-acylated analogs were observed at higher micromolar concen- trations (1 and 10 mM). 3.4. Colorectal cancer stem cell selectivity of C20-O-acylated analogs Aldehyde dehydrogenase 1 (ALDH1) has been recently identi- fied to label CRC stem cells [23]. Treatment of SW480 cells with increasing concentrations with the C20-O-acylated analogs and salinomycin resulted in a dose-dependent reduction of the ALDH1þ population already at low micromolar concentrations (Fig. 3A B). The cancer stem cell activity of the C20-O-acylated analogs was confirmed in SW620 cells (Supplementary Fig. S3A). We further investigated the anti-cancer stem cell effects of the C20-O-acylated analogs and salinomycin by analyzing the mRNA expression of Lgr5, which is involved in the colorectal cancer stem cell hierarchy and contribute to colorectal carcinogenesis [24]. Quantitative PCR after treatment with the C20-O-acylated analogs and salinomycin caused reduction of Lgr5 mRNA expression in SW480 cells (Fig. 3C), whereupon Lgr5 downregulation was more pronounced after exposure to salinomycin. The effects were also observed in SW620 cells (Supplementary Fig. S3B). 4. Discussion The aim of this study was to analyze the impact of semi- synthetic salinomycin analogs compared to salinomycin on hu- man colorectal cancer cell lines. The results confirm that the C20-O- acylated structural analogs of salinomycin exhibit an increased activity in human colorectal cancer cells relative to that of salino- mycin itself. Furthermore, the C20-O-acylated analogs also exert activity against colorectal cancer stem cells. The findings align with previous studies of these compounds on breast cancer cells [19]. The anti-cancer activity of salinomycin has been studied in detail over the past years [25]. Despite its cytotoxic effects against cancer cells and its cancer stem cell selectivity, the potential clinical usage of salinomycin remains questionable. Lack of knowledge of Induction of apoptosis of human colorectal cancer cells after treatment with salinomycin analogs. SW480 cells were cultured in the absence or presence of increasing concentrations of salinomycin analogs (Acetate, Carbamate, and Carbonate) and salinomycin for 72 h. Detection of apoptosis was performed using AnnexinV-FITC and PI staining and cells analyzed by flowcytometry. Results are displayed as representative dot blots (A) or as summary of at least 3 independent experiments as mean ± SEM (B). Alternatively, apoptosis was investigated by TUNEL assay (C). Results are shown as representative pictures of one out of three independent experiments. Scale bars ¼ 50 mM; *p < 0.05, **p < 0.01 and ***p < 0.001 compared with salinomycin treatment. the common underlying mode of action and its potential toxic side effects are major concerns before a pre-clinical study using the drug might be realistic. Chemical synthesis of structural analogs of sal- inomycin might contribute to solve this dilemma [15,17]. C20-O- acylated analogs of salinomycin also show cancer stem cell selec- tivity as identified in previous studies in human breast cancer stem cells [19,20]. To elucidate the activity of the C20-O-acylated analogs against colorectal cancer cells, we exposed two human colorectal cancer cell lines to different concentrations of the salinomycin analogs and compared its efficacy to salinomycin-treated cells. The following findings led us conclude that the analogs show increased activity against human colorectal cancer cells compared to salino- mycin. First, we demonstrated a dose-dependent reduction of cancer cell number after exposure to the salinomycin analogs indicating reduced cancer cell proliferation in three independent assays. In particular, we found that micromolar concentrations of the analogs result in increased anti-proliferative activity compared to salinomycin at the same concentrations. Second, cancer cell migration is inhibited upon treatment with micromolar concen- trations of the analogs. Again, salinomycin was less active when the 6 J. Klose et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7 Fig. 3. Anti-colorectal cancer stem cell activity of salinomycin analogs. SW480 cells were cultured in the absence or presence of increasing concentrations of salinomycin analogs (Acetate, Carbamate, and Carbonate) and salinomycin for 72 h. The activity against cancer stem cells was analyzed by measurement of the ALDH1þ population after treatment. Results are displayed as representative dot blots (A) or as summary of at least 3 independent experiments as mean ± SEM (B). Stem cell selectivity was further investigated by analysis of mRNA expression level of Lgr5 and shown as summary of at least 3 independent experiments as mean ± SEM (C); *p < 0.05 and **p < 0.01 compared with salinomycin treatment same drug concentrations were applied. Next, treatment with higher micromolar concentrations of the C20-O-acylated analogs is characterized by induction of apoptotic cell death as demonstrated by (i) LDH release assay, (ii) AnnexinV/PI staining and (iii) TUNEL assay. Alongside with the above-described observations, the pro- apoptotic effects mediated by the salinomycin analogs, are more pronounced than the effects of salinomycin. Finally, we demon- strate cancer stem cell selectivity of the salinomycin analogs as shown by reduction of ALDH1þ cells and reduced mRNA expression of the colorectal cancer stem cells specific gene Lgr5. As previously shown in breast cancer stem cells, the salinomycin analogs exert increased activity against colon cancer cells at low micromolar or even nanomolar concentrations [19]. This increased activity at low concentrations might have an important implication for further pre-clinical studies. Lower drug concentrations might be associated with less toxic side effects. This possibility is crucial, given that intoxication after salinomycin intake are described and the thera- peutic index is supposed to be narrow [14]. The results of our study result in several questions, which have to be answered in further studies. First, analysis of the activity of the analogs has to be performed in appropriate animal models. Second, the potential toxic side effects C20-O-acylated analogs should be investigated by treatment of benign cells and toxicity analysis in animal models. Not least, the underlying molecular mechanisms of cytotoxic activity of the C20-O-acylated analogs in human colorectal cancer have not been investigated so far. Based on our study, acetate and carbonate are the most prom- ising candidates for further investigations given that these salino- mycin analogs show the highest activity against colorectal cancer cells and can be easily synthesized to analyze colorectal cancer stem cell selectivity in vitro and in vivo [17]. In conclusion, by using several in vitro assays including prolif- eration, migration and apoptosis detection assays, the impact of three salinomycin analogs acylated at the C20 position on human colorectal cancer cells was analyzed. As a general observation, the cytotoxic activity of the analogs is higher than that of salinomycin when used at equivalent concentrations. At low micromolar con- centrations, the C20-O-acylated analogs inhibit cancer cell prolif- eration as well as cell migration. Treatment with concentrations in the high micromolar range results in induction of apoptotic cell death. At the same concentrations, salinomycin was less active. The C20-O-acylated analogs further exert activity against cancer stem cells. The increased activity of salinomycin analogs suggest that similar effects as that of salinomycin can be induced at lower concentrations which may be of value towards a potential pre- clinical usage of salinomycin or its analogs. Further studies to analyze the activity of the salinomycin analogs in animal models for colorectal cancer and toxic side effects of the drugs are under way, and will be reported in due course. Funding This study was supported by Heidelberger Stiftung Chirurgie to JK and the Swedish Cancer foundation. Author's contributions JK and AU designed research and analyzed the data; JK and SK performed the experiments; BB, CV, TS and MS helped analyzing the data; JK, SO, DS and AU wrote the paper. All authors read and approved the final manuscript. Acknowledgements The authors are thankful to Praveen Radhakrishnan for his assistance with mRNA expression analysis, to Marzena Knyssok- Sypniewski and Melanie Ho€fler for their technical support, and to Xiaoli Huang for her critical review of the manuscript. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2017.10.147. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2017.10.147. References [1] N.A. Lobo, Y. Shimono, D. Qian, M.F. Clarke, The biology of cancer stem cells, Annu. Rev. Cell Dev. Biol. 23 (2007) 675e699. [2] P.B. Gupta, T.T. Onder, G. Jiang, K. Tao, C. Kuperwasser, R.A. Weinberg, E.S. 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