Salinomycin treatment reduces metastatic tumor burden by hampering cancer cell migration
© Kopp et al.; licensee BioMed Central Ltd. 2014
Received: 19 September 2013
Accepted: 24 January 2014
Published: 27 January 2014
Tumor spreading is the major threat for cancer patients. The recently published anti-cancer drug salinomycin raised hope for an improved treatment by targeting therapy-refractory cancer stem cells. However, an unambiguous role of salinomycin against cancer cell migration and metastasis formation remains elusive.
We report that salinomycin effectively inhibits cancer cell migration in a variety of cancer types as determined by Boyden chamber assays. Additionally, cells were treated with doxorubicin at a concentration causing a comparable low cytotoxicity, emphasizing the anti-migratory potential of salinomycin. Moreover, single-cell tracking by time-lapse microscopy demonstrated a remarkable effect of salinomycin on breast cancer cell motility. Ultimately, salinomycin treatment significantly reduced the metastatic tumor burden in a syngenic mouse tumor model.
Our findings clearly show that salinomycin can strongly inhibit cancer cell migration independent of the induction of cell death. We furthermore demonstrate for the first time that salinomycin treatment reduces metastasis formation in vivo, strengthening its role as promising anti-cancer therapeutic.
KeywordsSalinomycin Cancer Migration Cell motility Metastasis
Distant metastases are the major cause of death in patients suffering from cancer. In spite of this, there is a lack of effective treatments for patients with metastatic disease. The discovery and development of novel drugs which can potently inhibit cancer cell migration and hence prevent metastasis formation are therefore of great interest in order to prolong the survival of patients. Gupta et al. have recently found salinomycin to be a selective inhibitor of cancer stem cells (CSC) obtained from immortalized transformed HMLER cells by a stable E-cadherin knockdown. Salinomycin reduced the proportion of CSC more than 100-fold as compared to paclitaxel, a commonly used chemotherapeutic breast cancer drug . Subsequent studies in a variety of different cancer types including breast, blood, lung, pancreas and colon have revealed diverse mechanisms of salinomycin action against CSC resulting in an inhibition of proliferation or an induction of apoptosis and cell death . Very recently, some reports have indicated that salinomycin inhibits cancer cell migration in different cancer types [3–8]. However, when looking at these studies in more detail, some of them raise concerns regarding the salinomycin concentration used for the migration experiments. Moreover, the ultimate effect of salinomycin treatment on metastasis formation in vivo had yet to be elucidated.
In this study, we wanted to investigate whether salinomycin is able to inhibit migration in a variety of cancer types. In order to rule out that the inhibition of cell motility is due to unspecific cytotoxic effects, we focused on the use of salinomycin concentrations which only cause minor cytotoxicity. Finally, a syngenic mouse model for metastasis was utilized to prove the efficacy of salinomycin against tumor dissemination.
Salinomycin treatment effectively hampers migration in cancer cells
Time-lapse microscopy reveals an inhibition of the motility of MDA-MB-436 cells upon salinomycin treatment
Metastasis formation is reduced by salinomycin treatment in a syngenic intravenous mouse tumor model
In summary, salinomycin had considerable inhibitory effects on cell migration in several different cancer cell lines including MDA-MB-436 (breast), MDA-MB-231 (breast), 4T1-luc (breast), LLC (lung), COGA2 (colon) and COGA10 (colon) when applied at low dose. Selective targeting of CSC and induction of oxidative stress have been suggested to be responsible for this anti-migratory effect [4, 6]. Verdoodt et al. have recently demonstrated that salinomycin induces autophagy in colon and breast cancer cells , which might also hinder cells to migrate as autophagy has been shown to inhibit migration in hepatitis B virus-associated hepatocellular carcinoma and in HeLa cells [12, 13]. It is furthermore well established that salinomycin treatment leads indirectly to an influx of calcium ions into the cytoplasm most likely via the potassium/calcium antiporter [14, 15]. Gradients of calcium are crucial for polarized cell migration in mesenchymal cells. In the leading edge protrusion transient calcium flickers are induced by chemokines and regulate directional decision making . Similarly, it was speculated that detachment of the trailing edge is also calcium dependent . Recently, Witze et al.  demonstrated that Wnt5a induces ER mobilization to the trailing edge in migrating cells controlling calcium signaling via ER tubules, finally resulting in the activation of calpain proteases and substrate detachment. The collapse of the fine tuned calcium balance at both cellular edges by additional influx of calcium induced by salinomycin might lead to a breakdown of the calcium gradients hindering cells to execute directed movements.
In some of the previous studies in which they treated cells with salinomycin for migration experiments [4, 7, 8], relatively high salinomycin concentrations were used so that in our view the inhibitory effect on cell motility cannot be exclusively attributed to anti-migratory effects of salinomycin but rather to unspecific cytotoxicity. Here, we compared salinomycin- with doxorubicin-treated cells at concentrations resulting in approximately 85 – 90% viable cells. Since salinomycin treatment significantly reduced the migratory capacity of the breast and lung cancer cell lines to 50 – 55% in contrast to doxorubicin treatment, we conclude that salinomycin is able to inhibit cancer cell migration at low-toxic doses, independent of the induced cytotoxicity. In case of the migratory potential of the primary colon cancer cell lines, salinomycin treatment was not significantly superior to doxorubicin treatment due to higher standard deviations. However, in COGA2 cells treatment with non-toxic concentrations of salinomycin (cell viability of approximately 100%) reduced the number of migrated cells to 20 – 25%. The salinomycin concentration used for COGA10 cells (cell viability of approximately 75%) could at least reduce the number of migrated cells to 40 – 45%, albeit cytotoxic effects cannot be completely excluded in this setting. Exemplarily, we performed time-lapse microscopy of MDA-MB-436 cells to directly monitor the immediate effects of salinomycin on cell motility, i.e. the distance, the velocity and the direction of migrating cells, on a single-cell level. The videos taken from these experiments further underline the quantitative analyses of the cell motility. Hence, we showed, to our knowledge for the first time, that salinomycin inhibits migration of breast cancer cells and primary colon cancer cells independent of the induction of cell death. Consequently, we sought to explore the efficacy of salinomycin on tumor dissemination in vivo. Gupta et al. pre-treated 4T1 cells with salinomycin before they injected them intravenously into mice. After three weeks they obtained a smaller tumor burden upon salinomycin treatment in the lungs as determined by the lung tumor surface . In this study, we analyzed for the first time metastasis formation of intravenously injected firefly luciferase expressing 4T1-luc cells which were not pre-treated with salinomycin. The rationale behind this was to monitor the primary tumor formation in the lungs via bioluminescence imaging as well as to detect metastases in other organs via an ex vivo luciferase assay. Of note, metastasis formation in brain, spleen and kidneys from primary 4T1-luc tumors in the lungs was considerably reduced by salinomycin treatment, even though the growth of the primary lesion was not significantly hampered. Thus, our in vitro and in vivo results clearly demonstrate that salinomycin - initially a cancer stem cell-specific drug – inhibits the migration of various cancer cells and prevents tumor dissemination in mice. These findings raise hope for improved treatment options for cancer patients in the future.
aSupplement videos of the wound healing assay of MDA-MB-436 cells treated either with mock (control), doxorubicin or salinomycin are available on our homepage: http://www.cup.lmu.de/pb/aks/ewagner/projects.html.
Cancer stem cells.
The authors want to thank Prof. Angelika M Vollmar for providing the 4T1-luc cell line. We would also like to thank Laura Sellmer for conducting the migration assay of COGA cells and Miriam Hoehn for excellent technical support. This work was supported by the German Research Foundation SFB1032 project B4. Adam Hermawan was supported by a doctoral fellowship of the Islamic Development Bank, IDB (28/IND/P32).
- Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES: Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell. 2009, 138: 645-659. 10.1016/j.cell.2009.06.034View ArticlePubMedGoogle Scholar
- Naujokat C, Steinhart R: Salinomycin as a drug for targeting human cancer stem cells. J Biomed Biotechnol. 2012, 2012: 950658-PubMed CentralView ArticlePubMedGoogle Scholar
- Arafat K, Iratni R, Takahashi T, Parekh K, Al Dhaheri Y, Adrian TE, Attoub S: Inhibitory Effects of Salinomycin on Cell Survival, Colony Growth, Migration, and Invasion of Human Non-Small Cell Lung Cancer A549 and LNM35: Involvement of NAG-1. PLoS One. 2013, 8: e66931- 10.1371/journal.pone.0066931PubMed CentralView ArticlePubMedGoogle Scholar
- Dong TT, Zhou HM, Wang LL, Feng B, Lv B, Zheng MH: Salinomycin selectively targets ‘CD133’+‘ cell subpopulations and decreases malignant traits in colorectal cancer lines. Ann Surg Oncol. 2011, 18: 1797-1804. 10.1245/s10434-011-1561-2View ArticlePubMedGoogle Scholar
- He L, Wang F, Dai WQ, Wu D, Lin CL, Wu SM, Cheng P, Zhang Y, Shen M, Wang CF: Mechanism of action of salinomycin on growth and migration in pancreatic cancer cell lines. Pancreatology. 2013, 13: 72-78. 10.1016/j.pan.2012.11.314View ArticlePubMedGoogle Scholar
- Ketola K, Hilvo M, Hyotylainen T, Vuoristo A, Ruskeepaa AL, Oresic M, Kallioniemi O, Iljin K: Salinomycin inhibits prostate cancer growth and migration via induction of oxidative stress. Br J Cancer. 2012, 106: 99-106. 10.1038/bjc.2011.530PubMed CentralView ArticlePubMedGoogle Scholar
- Kusunoki S, Kato K, Tabu K, Inagaki T, Okabe H, Kaneda H, Suga S, Terao Y, Taga T, Takeda S: The inhibitory effect of salinomycin on the proliferation, migration and invasion of human endometrial cancer stem-like cells. Gynecol Oncol. 2013, 129: 598-605. 10.1016/j.ygyno.2013.03.005View ArticlePubMedGoogle Scholar
- Lieke T, Ramackers W, Bergmann S, Klempnauer J, Winkler M, Klose J: Impact of Salinomycin on human cholangiocarcinoma: induction of apoptosis and impairment of tumor cell proliferation in vitro. BMC Cancer. 2012, 12: 466- 10.1186/1471-2407-12-466PubMed CentralView ArticlePubMedGoogle Scholar
- Vecsey-Semjen B, Becker KF, Sinski A, Blennow E, Vietor I, Zatloukal K, Beug H, Wagner E, Huber LA: Novel colon cancer cell lines leading to better understanding of the diversity of respective primary cancers. Oncogene. 2002, 21: 4646-4662. 10.1038/sj.onc.1205577View ArticlePubMedGoogle Scholar
- Wiedmann RM, von Schwarzenberg K, Palamidessi A, Schreiner L, Kubisch R, Liebl J, Schempp C, Trauner D, Vereb G, Zahler S: The V-ATPase-inhibitor archazolid abrogates tumor metastasis via inhibition of endocytic activation of the Rho-GTPase Rac1. Cancer Res. 2012, 72: 5976-5987. 10.1158/0008-5472.CAN-12-1772View ArticlePubMedGoogle Scholar
- Verdoodt B, Vogt M, Schmitz I, Liffers ST, Tannapfel A, Mirmohammadsadegh A: Salinomycin induces autophagy in colon and breast cancer cells with concomitant generation of reactive oxygen species. PLoS One. 2012, 7: e44132- 10.1371/journal.pone.0044132PubMed CentralView ArticlePubMedGoogle Scholar
- Lan SH, Wu SY, Zuchini R, Lin XZ, Su IJ, Tsai TF, Lin YJ, Wu CT, Liu HS: Autophagy suppresses tumorigenesis of hepatitis B Virus-associated hepatocellular carcinoma through degradation of microRNA-224. Hepatology. 2013, doi:10.1002/hep.26659. [Epub ahead of print]Google Scholar
- Tuloup-Minguez V, Hamai A, Greffard A, Nicolas V, Codogno P, Botti J: Autophagy modulates cell migration and beta1 integrin membrane recycling. Cell Cycle. 2013, 12: 3317-3328.PubMed CentralView ArticlePubMedGoogle Scholar
- Boehmerle W, Endres M: Salinomycin induces calpain and cytochrome c-mediated neuronal cell death. Cell Death Dis. 2011, 2: e168- 10.1038/cddis.2011.46PubMed CentralView ArticlePubMedGoogle Scholar
- Wang F, He L, Dai WQ, Xu YP, Wu D, Lin CL, Wu SM, Cheng P, Zhang Y, Shen M: Salinomycin inhibits proliferation and induces apoptosis of human hepatocellular carcinoma cells in vitro and in vivo. PLoS One. 2012, 7: e50638- 10.1371/journal.pone.0050638PubMed CentralView ArticlePubMedGoogle Scholar
- Wei C, Wang X, Chen M, Ouyang K, Song LS, Cheng H: Calcium flickers steer cell migration. Nature. 2009, 457: 901-905. 10.1038/nature07577PubMed CentralView ArticlePubMedGoogle Scholar
- Lee J, Ishihara A, Oxford G, Johnson B, Jacobson K: Regulation of cell movement is mediated by stretch-activated calcium channels. Nature. 1999, 400: 382-386. 10.1038/22578View ArticlePubMedGoogle Scholar
- Witze ES, Connacher MK, Houel S, Schwartz MP, Morphew MK, Reid L, Sacks DB, Anseth KS, Ahn NG: Wnt5a directs polarized calcium gradients by recruiting cortical endoplasmic reticulum to the cell trailing edge. Dev Cell. 2013, 26: 645-657. 10.1016/j.devcel.2013.08.019View ArticlePubMedGoogle Scholar
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