Salinomycin raised hope to be effective in anti-cancer therapies due to its capability to overcome apoptosis-resistance in several types of cancer cells. Recently, its effectiveness against human hepatocellular carcinoma (HCC) cells both in vitro and in vivo was demonstrated. However, the mechanism of action remained unclear. Latest studies implicated interference with the degradation pathway of autophagy. This study aimed to determine the impact of Salinomycin on HCC-autophagy and whether primary human hepatocytes (PHH) likewise are affected. Following exposure of HCC cell lines HepG2 and Huh7 to varying concentrations of Salinomycin (0–10 µM), comprehensive analysis of autophagic activity using western-blotting and flow-cytometry was performed. Drug effects were analyzed in the settings of autophagy stimulation by starvation or PP242-treatment and correlated with cell viability, proliferation, apoptosis induction, mitochondrial mass accumulation and reactive oxygen species (ROS) formation. Impact on apoptosis induction and cell function of PHH was analyzed.
Constitutive and stimulated autophagic activities both were effectively suppressed in HCC by Salinomycin. This inhibition was associated with dysfunctional mitochondria accumulation, increased apoptosis and decreased proliferation and cell viability. Effects of Salinomycin were dose and time dependent and could readily be replicated by pharmacological and genetic inhibition of HCC-autophagy alone. Salinomycin exposure to PHH resulted in transient impairment of synthesis function and cell viability without apoptosis induction. In conclusion, our data suggest that Salinomycin suppresses late stages of HCC-autophagy, leading to impaired recycling and accumulation of dysfunctional mitochondria with increased ROS-production all of which are associated with induction of apoptosis.
Salinomycin is a polyether ionophore antibiotic that has recently been shown to induce cell death in human cancer cells displaying multiple mechanisms of drug resistance. The underlying mechanisms leading to cell death after salinomycin treatment have not been well characterized. We therefore investigated the role of salinomycin in caspase dependent and independent cell death in colon cancer (SW480, SW620, RKO) and breast cancer cell lines (MCF-7, T47D, MDA-MB-453).
We detected features of apoptosis in all cell lines tested, but the executor caspases 3 and 7 were only strongly activated in RKO and MDA-MB-453 cells. MCF-7 and SW620 cells instead presented features of autophagy such as cytoplasmic vacuolization and LC3 processing. Caspase proficient cell lines activated autophagy at lower salinomycin concentrations and before the onset of caspase activation. Salinomycin also led to the formation of reactive oxygen species (ROS) eliciting JNK activation and induction of the transcription factor JUN. Salinomycin mediated cell death could be partially inhibited by the free radical scavenger N-acetyl-cysteine, implicating ROS formation in the mechanism of salinomycin toxicity.
Our data indicate that, in addition to its previously reported induction of caspase dependent apoptosis, the initiation of autophagy is an important and early effect of salinomycin in tumor cells.
Salinomycin has been shown to control breast cancer stem cells, although the mechanisms underlying its anticancer effects are not clear. Deregulation of cell cycle regulators play critical roles in tumorigenesis, and they have been considered as anticancer targets. In this study, we investigated salinomycin effect on cell cycle progression using OVCAR-8 ovarian cancer cell line and multidrug-resistant NCI/ADR-RES and DXR cell lines that are derived from OVCAR-8. Parental OVCAR-8 cells are sensitive to several anticancer drugs, but NCI/ADR-RES and DXR cells are resistant to several anticancer drugs. However, salinomycin caused cell growth inhibition and apoptosis via cell cycle arrest at G1 in all three cell lines. Salinomycin inhibited signal transducer and activator of transcription 3 (Stat3) activity and thus decreased expression of Stat3-target genes, including cyclin D1, Skp2, and survivin. Salinomycin induced degradation of Skp2 and thus accumulated p27Kip1. Knockdown of Skp2 further increased salinomycin-induced G1 arrest, but knockdown of p27Kip1 attenuated salinomycin effect on G1 arrest. Cdh1, an E3 ligase for Skp2, was shifted to nuclear fractions upon salinomycin treatment. Cdh1 knockdown by siRNA reversed salinomycin-induced Skp2 downregulation and p27Kip1 upregulation, indicating that salinomycin activates the APCCdh1–Skp2–p27Kip1 pathway. Concomitantly, si-Cdh1 inhibited salinomycin-induced G1 arrest. Taken together, our data indicate that salinomycin induces cell cycle arrest and apoptosis via downregulation or inactivation of cell cycle-associated oncogenes, such as Stat3, cyclin D1, and Skp2, regardless of multidrug resistance.
salinomycin; Stat3; Skp2; multidrug resistance; cancer stem cells
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to exhibit therapeutic activity in cancer. However, many tumors remain resistant to treatment with TRAIL. Therefore, small molecules that potentiate the cytotoxic effects of TRAIL could be used for combinatorial therapy. Here we found that the ionophore antibiotic salinomycin acts in synergism with TRAIL, enhancing TRAIL-induced apoptosis in glioma cells. Treatment with low doses of salinomycin in combination with TRAIL augmented the activation of caspase-3 and increased TRAIL-R2 cell surface expression. TRAIL-R2 upmodulation was required for mediating the stimulatory effect of salinomycin on TRAIL-mediated apoptosis, since it was abrogated by siRNA-mediated TRAIL-R2 knockdown. Salinomycin in synergism with TRAIL exerts a marked anti-tumor effect in nude mice xenografted with human glioblastoma cells. Our results suggest that the combination of TRAIL and salinomycin may be a useful tool to overcome TRAIL resistance in glioma cells and may represent a potential drug for treatment of these tumors. Importantly, salinomycin+TRAIL were able to induce cell death of well-defined glioblastoma stem-like lines.
Cancer stem cells (CSC) are believed to play a crucial role in cancer recurrence due to their resistance to conventional chemotherapy and capacity for self-renewal. Recent studies have reported that salinomycin, a livestock antibiotic, selectively targets breast cancer stem cells 100-fold more effectively than paclitaxel. In our study we sought to determine the effects of salinomycin on head and neck squamous cell carcinoma (HNSCC) stem cells.
MTS and TUNEL assays were used to study cell proliferation and apoptosis as a function of salinomycin exposure in JLO-1, a putative HNSCC stem cell culture. MTS and trypan blue dye exclusion assays were performed to investigate potential drug interactions between salinomycin and cisplatin or paclitaxel. Stem cell-like phenotype was measured by mRNA expression of stem cell markers, sphere-forming capacity, and matrigel invasion assays. Immunoblotting was also used to determine expression of epithelial-mesenchymal transition (EMT) markers and Akt phosphorylation. Arrays by Illumina, Inc. were used to profile microRNA expression as a function of salinomycin dose.
In putative HNSCC stem cells, salinomycin was found to significantly inhibit cell viability, induce a 71.5% increase in levels of apoptosis, elevate the Bax/Bcl-2 ratio, and work synergistically with cisplatin and paclitaxel in inducing cell death. It was observed that salinomycin significantly inhibited sphere forming-capability and repressed the expression of CD44 and BMI-1 by 3.2-fold and 6.2-fold, respectively. Furthermore, salinomycin reduced invasion of HNSCC stem cells by 2.1 fold. Contrary to expectations, salinomycin induced the expression of EMT markers Snail, vimentin, and Zeb-1, decreased expression of E-cadherin, and also induced phosphorylation of Akt and its downstream targets GSK3-β and mTOR.
These results demonstrate that in HNSCC cancer stem cells, salinomycin can cause cell death and decrease stem cell properties despite activation of both EMT and Akt.
Salinomycin; Cancer stem cells; Head and neck squamous cell carcinoma; Akt; EMT; microRNA
The malignant osteoblastoma has poor prognosis, thus the search for novel and more efficient chemo-agents against this disease is urgent. Salinomycin induces broad anti-cancer effects both in
vivo and in
vitro, however, its role in osteoblastoma is still not clear.
Salinomycin induced both apoptosis and autophagy in cultured U2OS and MG-63 osteoblastoma cells. Inhibition of autophagy by 3-methyladenine (3-MA), or by RNA interference (RNAi) of light chain 3B (LC3B), enhanced salinomycin-induced cytotoxicity and apoptosis. Salinomycin induced a profound AMP-activated protein kinase (AMPK) activation, which was required for autophagy induction. AMPK inhibition by compound C, or by AMPKα RNAi prevented salinomycin-induced autophagy activation, while facilitating cancer cell death and apoptosis. On the other hand, the AMPK agonist AICAR promoted autophagy activation in U2OS cells. Salinomycin-induced AMPK activation was dependent on reactive oxygen species (ROS) production in osteoblastoma cells. Antioxidant n-acetyl cysteine (NAC) significantly inhibited salinomycin-induced AMPK activation and autophagy induction.
Salinomycin activates AMPK-dependent autophagy in osteoblastoma cells, which serves as a negative regulator against cell apoptosis. AMPK-autophagy inhibition might be a novel strategy to sensitize salinomycin’s effect in cancer cells.
Chemotherapy for soft tissue sarcomas remains unsatisfactory due to their low chemosensitivity. Even the first line chemotherapeutic agent doxorubicin only yields a response rate of 18-29%. The antibiotic salinomycin, a potassium ionophore, has recently been shown to be a potent compound to deplete chemoresistant cells like cancer stem like cells (CSC) in adenocarcinomas. Here, we evaluated the effect of salinomycin on sarcoma cell lines, whereby salinomycin mono- and combination treatment with doxorubicin regimens were analyzed.
To evaluate the effect of salinomycin on fibrosarcoma, rhabdomyosarcoma and liposarcoma cell lines, cells were drug exposed in single and combined treatments, respectively. The effects of the corresponding treatments were monitored by cell viability assays, cell cycle analysis, caspase 3/7 and 9 activity assays. Further we analyzed NF-κB activity; p53, p21 and PUMA transcription levels, together with p53 expression and serine 15 phosphorylation.
The combination of salinomycin with doxorubicin enhanced caspase activation and increased the sub-G1 fraction. The combined treatment yielded higher NF-κB activity, and p53, p21 and PUMA transcription, whereas the salinomycin monotreatment did not cause any significant changes.
Salinomycin increases the chemosensitivity of sarcoma cell lines - even at sub-lethal concentrations - to the cytostatic drug doxorubicin. These findings support a strategy to decrease the doxorubicin concentration in combination with salinomycin in order to reduce toxic side effects.
Apoptosis; Salinomycin; Doxorubicin; Malignant soft tissue tumors; Chemotherapy
Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.
salinomycin; endoplasmic reticulum stress; MTOR; autophagy; apoptosis
Medulloblastoma (MB) is the most common childhood brain tumor. Despite improved therapy and management, approximately 30% of patients die of the disease. To search for a more effective therapeutic strategy, the effects of salinomycin were tested on cell proliferation, cell death, and cell cycle progression in human MB cell lines. The results demonstrated that salinomycin inhibits cell proliferation, induces cell death , and disrupts cell cycle progression in MB cells. Salinomycin was also tested on the expression levels of key genes involved in proliferation and survival signaling and revealed that salinomycin down-regulates the expression of PDGFRβ, MYC, p21 and Bcl-2 as well as up-regulates the expression of cyclin A. In addition, the results reveal that salinomycin suppresses the expression of Hes1 and Hes5 in MB cells. Our data shed light on the potential of using salinomycin as a novel therapeutic agent for patients with MB.
Salinomycin; Medulloblastoma; PDGFRβ; MYC; Notch Signaling
Acquiring therapy resistance is one of the major obstacles in the treatment of patients with cancer. The discovery of the cancer stem cell (CSC)–specific drug salinomycin raised hope for improved treatment options by targeting therapy-refractory CSCs and mesenchymal cancer cells. However, the occurrence of an acquired salinomycin resistance in tumor cells remains elusive. To study the formation of salinomycin resistance, mesenchymal breast cancer cells were sequentially treated with salinomycin in an in vitro cell culture assay, and the resulting differences in gene expression and salinomycin susceptibility were analyzed. We demonstrated that long-term salinomycin treatment of mesenchymal cancer cells resulted in salinomycin-resistant cells with elevated levels of epithelial markers, such as E-cadherin and miR-200c, a decreased migratory capability, and a higher susceptibility to the classic chemotherapeutic drug doxorubicin. The formation of salinomycin resistance through the acquisition of epithelial traits was further validated by inducing mesenchymal-epithelial transition through an overexpression of miR-200c. The transition from a mesenchymal to a more epithelial-like phenotype of salinomycin-treated tumor cells was moreover confirmed in vivo, using syngeneic and, for the first time, transgenic mouse tumor models. These results suggest that the acquisition of salinomycin resistance through the clonal selection of epithelial-like cancer cells could become exploited for improved cancer therapies by antagonizing the tumor-progressive effects of epithelial-mesenchymal transition.
A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5–5 µM for 7 days) significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer.
The few currently available drugs for treatment of African trypanosomiasis are outdated and show problems with toxicity and resistance. Hence, there is an urgent need for the discovery and development of new anti-trypanosomal agents.
In this study, the ionophorous antibiotic salinomycin was investigated for its trypanocidal activity in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. The concentrations of salinomycin to reduce the growth rate by 50% and to kill the parasites were 0.31 μM and 1 μM, respectively. The trypanocidal action of the ionophore was shown to be the result of an influx of Na+ resulting in an increased intracellular Na+ concentration followed by cell swelling. This mode of action differs from the mechanism for the anti-cancer activity of salinomycin reported to be by induction of apoptosis.
Here we have shown that salinomycin is an effective agent against bloodstream forms of T. brucei and might be a potential candidate for treatment of African trypanosomiasis.
African trypanosomiasis; Trypanosoma brucei; Salinomycin; Drug screening
The polyether ionophoric antibiotics including monensin, salinomycin, and narasin, are widely used in veterinary medicine and as food additives and growth promoters in animal husbandry including poultry farming. Their effects on human health, however, are not fully understood. Recent studies showed that salinomycin is a cancer stem cell inhibitor. Since poultry consumption has risen sharply in the last three decades, we asked whether the consumption of meat tainted with growth promoting antibiotics might have effects on adipose cells. We showed in this report that the ionophoric antibiotics inhibit the differentiation of preadipocytes into adipocytes. The block of differentiation is not due to the induction of apoptosis nor the inhibition of cell proliferation. In addition, salinomycin also suppresses the transcriptional activity of the CCAAT/enhancer binding proteins and the peroxisome proliferator-activated receptor γ. These results suggest that the ionophoric antibiotics can be exploited as novel anti-obesity therapeutics and as pharmacological probes for the study of adipose biology. Further, the pharmacological effects of salinomycin could be a harbinger of its toxicity on the adipose tissue and other susceptible target cells in cancer therapy.
Adipogenesis; salinomycin; polyether antibiotic; ionophoric antibiotic; obesity
The natural polyether ionophorous antibiotics are used for the treatment of coccidiosis in poultry and ruminants. They are effective agents against infections caused by Gram-positive microorganisms. On the other hand, it was found that some of these compounds selectively bind lead(II) ions in in vivo experiments, despite so far no Pb(II)-containing compounds of defined composition have been isolated and characterized. To assess the potential of polyether ionophores as possible antidotes in the agriculture, a detailed study on their in vitro complexation with toxic metal ions is required. In the present paper we report for the first time the preparation and the structure elucidation of salinomycin complexes with ions of cadmium(II) and lead(II).
New metal(II) complexes of the polyether ionophorous antibiotic salinomycin with Cd(II) and Pb(II) ions were prepared and structurally characterized by IR, FAB-MS and NMR techniques. The spectroscopic information and elemental analysis data reveal that sodium salinomycin (SalNa) undergoes a reaction with heavy metal(II) ions to form [Cd(Sal)2(H2O)2] (1) and [Pb(Sal)(NO3)] (2), respectively. Abstraction of sodium ions from the cavity of the antibiotic is occurring during the complexation reaction. Salinomycin coordinates with cadmium(II) ions as a bidentate monoanionic ligand through the deprotonated carboxylic moiety and one of the hydroxyl groups to yield 1. Two salinomycin anions occupy the equatorial plane of the Cd(II) center, while two water molecules take the axial positions of the inner coordination sphere of the metal(II) cation. Complex 2 consists of monoanionic salinomycin acting in polydentate coordination mode in a molar ratio of 1: 1 to the metal ion with one nitrate ion for charge compensation.
The formation of the salinomycin heavy metal(II) complexes indicates a possible antidote activity of the ligand in case of chronic/acute intoxications likely to occur in the stock farming.
The aim of this study was to investigate the anti-proliferative effect of the salinomycin in cell proliferation and apoptosis in primary cultured human uterine leiomyoma cells.
Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Caspase-3 activity assay and DNA fragmentation assay were performed to determine the effect of apoptosis. The expression of apoptosis regulatory-related proteins was evaluated by western blot.
The cell viability and proliferation of uterine leiomyoma cells were significantly reduced by salinomycin treatment in a dose-dependent manner. DNA fragmentation assay results showed apoptotic cell death after salinomycin incubation. Salinomycin activated caspase-3, -8, and -9, causing apoptosis in uterine leiomyoma cells. Down-regulation of Bcl-2, XIAP, and FLIP with a concomitant increase in Bax, Fas, and DR5 were observed.
These results provided the first evidence that salinomycin induce both intrinsic and extrinsic apoptosis. Therefore, salinomycin may be a promising chemopreventive and therapeutic agent against human uterine leiomyoma.
Apoptosis; Extrinsic; Intrinsic; Leiomyoma; Salinomycin
Salinomycin, traditionally used as an anti-coccidial drug, has recently been shown to possess anti-cancer and anti-cancer stem cell (CSC) effects, as well as activities to overcome multi-drug resistance based on studies using human cancer cell lines, xenograft mice, and in case reports involving cancer patients in pilot clinical trials. Therefore, salinomycin may be considered as a promising novel anti-cancer agent despite its largely unknown mechanism of action.
This review summarizes the pharmacologic effects of salinomycin and presents possible mechanisms by which salinomycin exerts its anti-tumorigenic activities. Recent advances and potential complications that might limit the utilization of salinomycin as an anti-cancer and anti-CSC agent are also presented and discussed.
Salinomycin; cancer stem cell; toxicity; drug
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.
Salinomycin; Cancer; Migration; Cell motility; Metastasis
Cancer stem cells (CSCs) represent a subpopulation of tumor cells that possess self-renewal and tumor initiation capacity and the ability to give rise to the heterogenous lineages of malignant cells that comprise a tumor. CSCs possess multiple intrinsic mechanisms of resistance to chemotherapeutic drugs, novel tumor-targeted drugs, and radiation therapy, allowing them to survive standard cancer therapies and to initiate tumor recurrence and metastasis. Various molecular complexes and pathways that confer resistance and survival of CSCs, including expression of ATP-binding cassette (ABC) drug transporters, activation of the Wnt/β-catenin, Hedgehog, Notch and PI3K/Akt/mTOR signaling pathways, and acquisition of epithelial-mesenchymal transition (EMT), have been identified recently. Salinomycin, a polyether ionophore antibiotic isolated from Streptomyces albus, has been shown to kill CSCs in different types of human cancers, most likely by interfering with ABC drug transporters, the Wnt/β-catenin signaling pathway, and other CSC pathways. Promising results from preclinical trials in human xenograft mice and a few clinical pilote studies reveal that salinomycin is able to effectively eliminate CSCs and to induce partial clinical regression of heavily pretreated and therapy-resistant cancers. The ability of salinomycin to kill both CSCs and therapy-resistant cancer cells may define the compound as a novel and an effective anticancer drug.
Salinomycin is a polyether antibiotic with properties of an ionophore, which is commonly used as cocciodiostatic drug and has been shown to be highly effective in the elimination of cancer stem cells (CSCs) both in vitro and in vivo. One important caveat for the potential clinical application of salinomycin is its marked neural and muscular toxicity. In the present study we show that salinomycin in concentrations effective against CSCs exerts profound toxicity towards both dorsal root ganglia as well as Schwann cells. This toxic effect is mediated by elevated cytosolic Na+ concentrations, which in turn cause an increase of cytosolic Ca2+ by means of Na+/Ca2+ exchangers (NCXs) in the plasma membrane as well as the mitochondria. Elevated Ca2+ then leads to calpain activation, which triggers caspase-dependent apoptosis involving caspases 12, 9 and 3. In addition, cytochrome c released from depolarized mitochondria directly activates caspase 9. Combined inhibition of calpain and the mitochondrial NCXs resulted in significantly decreased cytotoxicity and was comparable to caspase 3 inhibition. These findings improve our understanding of mechanisms involved in the pathogenesis of peripheral neuropathy and are important to devise strategies for the prevention of neurotoxic side effects induced by salinomycin.
polyneuropathy; dorsal root ganglia; sodium calcium exchanger; cancer stem cells; salinomycin
We have shown that a sodium ionophore monensin inhibits prostate cancer cell growth. A structurally related compound to monensin, salinomycin, was recently identified as a putative cancer stem cell inhibitor.
The growth inhibitory potential of salinomycin was studied in a panel of prostate cells. To get insights into the mechanism of action, a variety of assays such as gene expression and steroid profiling were performed in salinomycin-exposed prostate cancer cells.
Salinomycin inhibited the growth of prostate cancer cells, but did not affect non-malignant prostate epithelial cells. Salinomycin impacted on prostate cancer stem cell functions as evidenced by reduced aldehyde dehydrogenase activity and the fraction of CD44+ cells. Moreover, salinomycin reduced the expression of MYC, AR and ERG, induced oxidative stress as well as inhibited nuclear factor-κB activity and cell migration. Furthermore, profiling steroid metabolites revealed increased levels of oxidative stress-inducing steroids 7-ketocholesterol and aldosterone and decreased levels of antioxidative steroids progesterone and pregnenolone in salinomycin-exposed prostate cancer cells.
Our results indicate that salinomycin inhibits prostate cancer cell growth and migration by reducing the expression of key prostate cancer oncogenes, inducing oxidative stress, decreasing the antioxidative capacity and cancer stem cell fraction.
salinomycin; prostate cancer; oxidative stress; cancer stem cells
The anti-tumor antibiotic salinomycin (Sal) was recently identified as a selective inhibitor of breast cancer stem cells; however, the effect of Sal on hepatocellular carcinoma (HCC) is not clear. This study aimed to determine the anti-tumor efficacy and mechanism of Sal on HCC. HCC cell lines (HepG2, SMMC-7721, and BEL-7402) were treated with Sal. Cell doubling time was determinated by drawing growth curve, cell viability was evaluated using the Cell Counting Kit 8. The fraction of CD133+ cell subpopulations was assessed by flow cytometry. We found that Sal inhibits proliferation and decreases PCNA levels as well as the proportion of HCC CD133+cell subpopulations in HCC cells. Cell cycle was analyzed using flow cytometry and showed that Sal caused cell cycle arrest of the various HCC cell lines in different phases. Cell apoptosis was evaluated using flow cytometry and Hoechst 33342 staining. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Several signaling pathways were selected for further mechanistic analyses using real time-PCR and Western blot assays. Compared to control, β-catenin expression is significantly down-regulated upon Sal addition. The Ca2+ concentration in HCC cells was examined by flow cytometry and higher Ca2+ concentrations were observed in Sal treatment groups. The anti-tumor effect of Sal was further verified in vivo using the hepatoma orthotopic tumor model and the data obtained showed that the size of liver tumors in Sal-treated groups decreased compared to controls. Immunohistochemistry and TUNEL staining also demonstrated that Sal inhibits proliferation and induces apoptosis in vivo. Finally, the role of Sal on in vivo Wnt/β-catenin signaling was evaluated by Western blot and immunohistochemistry. This study demonstrates Sal inhibits proliferation and induces apoptosis of HCC cells in vitro and in vivo and one potential mechanism is inhibition of Wnt/β-catenin signaling via increased intracellular Ca2+ levels.
Cancer stem cells (CSCs) are thought to be responsible for tumor initiation and recurrence after chemotherapy. Targeting CSCs and non-CSCs with specific compounds may be an effective approach to reduce lung cancer growth and metastasis. The aim of this study was to investigate the effect of salinomycin, a selective inhibitor of CSCs, with or without combination with paclitaxel, in a metastatic model. To evaluate the effect of these drugs in metastasis and tumor microenvironment we took advantage of the immunocompetent and highly metastatic LLC mouse model. Aldefluor assays were used to analyze the ALDH+/− populations in murine LLC and human H460 and H1299 lung cancer cells. Salinomycin reduced the proportion of ALDH+ CSCs in LLC cells, whereas paclitaxel increased such population. The same effect was observed for the H460 and H1299 cell lines. Salinomycin reduced the tumorsphere formation capacity of LLC by more than 7-fold, but paclitaxel showed no effect. In in vivo experiments, paclitaxel reduced primary tumor volume but increased the number of metastatic nodules (p<0.05), whereas salinomycin had no effect on primary tumors but reduced lung metastasis (p<0.05). Combination of both drugs did not improve the effect of single therapies. ALDH1A1, SOX2, CXCR4 and SDF-1 mRNA levels were higher in metastatic lesions than in primary tumors, and were significantly elevated in both locations by paclitaxel treatment. On the contrary, such levels were reduced (or in some cases did not change) when mice were administered with salinomycin. The number of F4/80+ and CD11b+ cells was also reduced upon administration of both drugs, but particularly in metastasis. These results show that salinomycin targets ALDH+ lung CSCs, which has important therapeutic effects in vivo by reducing metastatic lesions. In contrast, paclitaxel (although reducing primary tumor growth) promotes the selection of ALDH+ cells that likely modify the lung microenvironment to foster metastasis.
Salinomycin is used as an antibiotic in animal husbandry. Its implication in cancer therapy has recently been proposed. Present study evaluated the toxic effects of Salinomycin on male reproductive system of mice. Doses of 1, 3 or 5 mg/kg of Salinomycin were administered daily for 28 days. Half of the mice were sacrificed after 24 h of the last treatment and other half were sacrificed 28 days after withdrawal of treatment. Effects of SAL on body and reproductive organ weights were studied. Histoarchitecture of testis and epididymis was evaluated along with ultrastructural changes in Leydig cells. Serum and testicular testosterone and luteinizing hormones were estimated. Superoxide dismutase, reduced glutathione, lipid peroxidation, catalase and lactate dehydrogenase activities were measured. Spermatozoa count, morphology, motility and fertility were evaluated. Expression patterns of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage proteins (CYP11A1) were assessed by Western blotting. Salinomycin treatment was lethal to few mice and retarded body growth in others with decreased weight of testes and seminal vesicles in a dose dependent manner. Seminiferous tubules in testes were disrupted and the epithelium of epididymis showed frequent occurrence of vacuolization and necrosis. Leydig cells showed hypertrophied cytoplasm with shrunken nuclei, condensed mitochondria, proliferated endoplasmic reticulum and increased number of lipid droplets. Salinomycin decreased motility and spermatozoa count with increased number of abnormal spermatozoa leading to infertility. The testosterone and luteinizing hormone levels were decreased in testis but increased in serum at higher doses. Depletion of superoxide dismutase and reduced glutathione with increased lipid peroxidation in both testis and epididymis indicated generation of oxidative stress. Suppressed expression of StAR and CYP11A1 proteins indicates inhibition of steroidogenesis. Spermatogenesis was however observed in testis 28 days after Salinomycin withdrawal. The results indicate reversible dose-dependent adverse effects of Salinomycin on male reproductive system of mice.
Salinomycin is a polyether organic anion that is extensively used as a coccidiostatic antibiotic in poultry and commonly fed to ruminant animals to improve feed efficiency. However, salinomycin also causes severe toxicity when accidentally fed to animals in high doses. In addition, humans are highly sensitive to salinomycin and severe toxicity has been reported. Multidrug efflux transporters like P-glycoprotein (P-gp), BCRP, and MRP2 are highly expressed in the intestine and can restrict the oral uptake and tissue penetration of xenobiotics. The purpose of this study was to investigate whether the anionic drug salinomycin is a substrate for one or more of these efflux pumps. Salinomycin was actively transported by human MDR1 P-gp expressed in polarized MDCK-II monolayers but not by the known organic anion transporters human MRP2 and murine Bcrp1. Using P-gp-deficient mice, we found a marked increase in plasma salinomycin concentrations after oral administration and decreased plasma clearance after intravenous administration. Furthermore, absence of P-gp resulted in significantly increased brain penetration. P-gp-deficient mice also displayed clearly increased susceptibility to salinomycin toxicity. Thus far, P-gp was thought to affect mainly hydrophobic, positively charged or neutral drugs in vivo. Our data show that P-gp can also be a major determinant of the pharmacokinetic behavior and toxicity of an organic anionic drug. Variation in P-gp activity might thus directly affect the effective exposure to salinomycin and possibly to other anionic drugs and toxin substrates. Individuals with reduced or absent P-gp activity could therefore be more susceptible to salinomycin toxicity.
The cytokine TRAIL (tumor necrotic factor-related apoptosis-inducing ligand) selectively induces apoptosis in cancer cells, but cancer stem cells (CSCs) that contribute to cancer-recurrence are frequently TRAIL-resistant. Here we examined hitherto unknown effects of the dietary anti-carcinogenic compound phenethyl isothiocyanate (PEITC) on attenuation of proliferation and tumorigenicity and on up regulation of death receptors and apoptosis in human cervical CSC.
Cancer stem-like cells were enriched from human cervical HeLa cell line by sphere-culture method and were characterized by CSC-specific markers’ analyses (flow cytometry) and Hoechst staining. Cell proliferation assays, immunoblotting, and flow cytometry were used to assess anti-proliferative as well as pro-apoptotic effects of PEITC exposure in HeLa CSCs (hCSCs). Xenotransplantation study in a non-obese diabetic, severe combined immunodeficient (NOD/SCID) mouse model, histopathology, and ELISA techniques were further utilized to validate our results in vivo.
PEITC attenuated proliferation of CD44high/+/CD24low/–, stem-like, sphere-forming subpopulations of hCSCs in a concentration- and time-dependent manner that was comparable to the CSC antagonist salinomycin. PEITC exposure-associated up-regulation of cPARP (apoptosis-associated cleaved poly [ADP-ribose] polymerase) levels and induction of DR4 and DR5 (death receptor 4 and 5) of TRAIL signaling were observed. Xenotransplantation of hCSCs into mice resulted in greater tumorigenicity than HeLa cells, which was diminished along with serum hVEGF-A (human vascular endothelial growth factor A) levels in the PEITC-pretreated hCSC group. Lung metastasis was observed only in the hCSC-injected group that did not receive PEITC-pretreatment.
The anti-proliferative effects of PEITC in hCSCs may at least partially result from up regulation of DR4 and possibly DR5 of TRAIL-mediated apoptotic pathways. PEITC may offer a novel approach for improving therapeutic outcomes in cancer patients.
Apoptosis; TRAIL; Cancer stem cells; Death receptors; Phenethyl isothiocyanate