In this study we demonstrate that resistance to apoptosis of CC cells can be overcome by treatment with Salinomycin. We show that two of three cell lines respond to Salinomycin-treatment with a significant degree of apoptosis independent of Caspase-3 activity. In addition, Salinomycin inhibits cell proliferation and cell migration. Of note, this accounts for all three tested cell lines.
Patient´s survival suffering from CC is poor and even CC calls for up to 15% of all primary liver malignancies, the molecular pathogenesis is unclear to the greatest possible extent
[2–4, 26]. Consequently, characterization of the molecular pathogenesis and development of innovative therapeutic strategies are imperatively required particularly since current approaches such as administration of Gemcitabine combined with Cisplatin are rather part of a palliative concept than a curative treatment strategy
. This is most likely due to apoptosis resistance of CC cells and subsequently weak efficacy of common chemotherapeutical regimes. The induction of apoptosis in human CC cells is barely observed
[25, 27] or only detectable after co-treatment of the cells with additional drugs or inhibiting RNAs
[28–30]. Accordingly, the understanding and the therapy of CC are characterized by nescience and ineffectiveness.
This is highlighted by the fact that even with Salinomycin which revealed capacity to provoke apoptosis in two of three tested human CC cell lines, EGI-1 remained to be unaffected in terms of being predispositioned to apoptosis. Exposure of Salinomycin to Mz-ChA-1 and TFK-1 cells, which were both originally isolated from an extrahepatic bile duct carcinoma
[21, 22], resulted in a high percentage of apoptotic tumor cells, while EGI-1 cells seem to be less susceptible for treatment with Salinomycin even after treatment with high concentrations. It has been reported that Salinomycin selectively affects malignant cells whereas non-malignant cells do not undergo apoptosis after treatment with Salinomycin
[20, 31, 32]. Given that EGI-1 cells are originally isolated from a poorly differentiated human bile duct adenocarcinoma
 and therewith undoubted are malignant, it remains unclear why these cells are nearly apoptosis-resistant to treatment with Salinomycin. These observations demand further investigations to elucidate potential escape mechanisms of tumor cells which might be important for a possible clinical application of Salinomycin in the future, indeed. Moreover, apoptosis-escape mechanisms of EGI-1 cells might explain in part the strong resistance of CC cells to chemotherapeutics in general.
However, the exact mechanisms by which Salinomycin induces apoptosis are still incomplete understood
. Salinomycin-induced apoptosis in human cancer cells is mediated by an uncommon pathway and independent of typical mechanism like activated caspases, death receptors like the CD95/DC95 ligand system or tumor suppressor protein p53
[15, 19]. Demonstrating that Salinomycin-induced apoptosis in human CC cells is independent of caspase-3 activation confirms that apoptosis is mediated through an uncommon pathway. Given that caspase-3 is activated both in the extrinsic and intrinsic pathway of apoptosis and plays a predominant role
[33, 34], it is astonishing that none of the common pathways seems to be involved. Although activated caspase-3 can be found in apoptotic CC cells after treatment with Lobaplatin in vitro another not yet discovered apoptotic pathway appears to be responsible for the effects of Salinomycin. Recently, it was reported that the Wingless type (Wnt)/β-catenin signaling pathway could be involved
. In chronic lymphocytic leukemia cells, Salinomycin inhibits the Wnt signaling cascade by blocking the phosphorylation of the Wnt co-receptor lipoprotein receptor related protein 6 (LRP6) causing impaired cell survival. These data are of great interest because in several tumor entities, LRP6 is over-expressed
. Even if not completely understood, Wnt signaling might also play an important role in the carcinogenesis of CC
 and recently, the effectiveness of several Wnt pathway inhibitors on human CC cells has been demonstrated
. Additionally, it was reported that Salinomycin induces apoptosis in prostate cancer cells via accumulation of reactive oxygen species and mitochondrial membrane depolarization
. Furthermore, Salinomycin inhibits prostate cancer growth via reduction of the expression of key oncogenes and induction of oxidative stress in cultured prostate cancer cells
. Taken together, several mechanisms are supposed to be responsible for the effects of Salinomycin to human cancer cells, which have to be investigated in greater detail in the near future.
Furthermore, we demonstrate that the proportion of non-apoptotic tumor cells following Salinomycin-treatment is sustainable affected, characterized by impaired tumor cell migration, reduced proliferation and cell cycle accumulation. These observations are noteworthy due to well-known counterproductive reactions of tumor cells that escaped apoptosis, including hyperproliferation. To further characterize the effects induced by Salinomycin particularly on the continuous apoptosis-resisting EGI-1 cells, we investigated the ability of human CC cells to migrate after drug exposure. Tumor cell migration and therewith the ability to form metastases is a hallmark of tumors. While Ketola et al. have described impaired migration of prostate cancer cells after treatment with Salinomycin in a wound-healing assay
; this is the first report that migration through a membrane is effectively inhibited. These observations disclose an additional anti-cancer effect of Salinomycin in which all three cell lines are included.
Furthermore, the assessment of CC cell proliferation with or without Salinomycin treatment revealed a significant reduction of cell division in the presence of the agent. Again, all three cell lines, even EGI-1 cells, have shown this effect. We further demonstrate that Salinomycin- treatment of human CC cells induced an enduring reduced proliferation even after the abolition of treatment. This long-lasting effect demonstrates that the proportion of human CC cells that have escaped apoptosis after Salinomycin-treatment are affected permanently. These observations might be of particular importance for the potential clinical use of Salinomycin in the future as a prolonged effect of Salinomycin in patients with CC could also be expected.
In addition, we were able to correlate the anti-proliferative effects of Salinomycin with the results of the cell cycle analyses. The impact of Salinomycin on human CC cells is reflected by cell cycle accumulation in the G2-phase. This finding is noticeable because others have demonstrated that treatment with Salinomycin in equal concentrations is associated with accumulation in the pre-G1-phase, indicating increased apoptosis
 Furthermore, in pre-treated human breast cancer with anti-mitotic drugs, Salinomycin abolishes G2-arrest and aneuploid cell formation
[17, 40]. In contrast, radiation-treated breast cancer cells accumulate in the G2-phase after treatment with Salinomycin
. Interestingly, Salinomycin-induced apoptosis in human leukemia cells is not accompanied by cell cycle arrest at all
. Thus, in respect to our data, it seems that the effects of Salinomycin on cell cycle are not consistent between human tumor cells of different origin. This again demonstrates the existing nebulosity concerning the biochemical mechanisms affected by Salinomycin.
Demonstrating the capability of Salinomycin to induce apoptosis and to interfere with tumor cell motility and proliferation in human CC cells, a potential and promising therapeutical approach for the treatment of CC might be discovered. Particularly, cancer entities with such calamitous prognosis like CC are tremendously dependent on innovative and sufficient therapy concepts. Thereby different human CC cell lines should be analyzed in regard to their susceptibility to Salinomycin-treatment. Furthermore, animal models have to be developed to investigate the impact of Salinomycin in vivo. To what extend Salinomycin will achieve to be a candidate for anti-cancer therapies in the future remains to be seen. Given that lethal intoxication in humans and animals are described
[42–44], potential clinical studies must be planned very thoughtful. Thus, finding the dosage of Salinomycin will be crucial for its application in prospective therapeutical regimes.