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.
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 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
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.
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
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
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 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
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
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
Monensin is a polyether ionophore antibiotic that is widely used in the control of coccidia in animals. Despite its significance in veterinary medicine, little is known about its mode of action and potential mechanisms of resistance in coccidian parasites. Here we show that monensin causes accumulation of the coccidian Toxoplasma gondii at an apparent late-S-phase cell cycle checkpoint. In addition, experiments utilizing a monensin-resistant T. gondii mutant show that this effect of monensin is dependent on the function of a mitochondrial homologue of the MutS DNA damage repair enzyme (TgMSH-1). Furthermore, the same TgMSH-1-dependent cell cycle disruption is observed with the antiparasitic ionophore salinomycin and the DNA alkylating agent methyl nitrosourea. Our results suggest a novel mechanism for the mode of action of monensin and salinomycin on coccidial parasites, in which the drug activates an MSH-1-dependent cell cycle checkpoint by an unknown mechanism, ultimately leading to the death of the parasite. This model would indicate that cell cycle disruption is an important mediator of drug susceptibility and resistance to ionophoric antibiotics in coccidian parasites.
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.
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.
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.
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.
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.
Cholangiocarcinoma is a strongly aggressive malignancy with a very poor prognosis. Effective therapeutic strategies are lacking because molecular mechanisms regulating cholangiocarcinoma cell growth are unknown. Furthermore, experimental in vivo animal models useful to study the pathophysiologic mechanisms of malignant cholangiocytes are lacking. Leptin, the hormone regulating caloric homeostasis, which is increased in obese patients, stimulates the growth of several cancers, such as hepatocellular carcinoma. The aim of this study was to define if leptin stimulates cholangiocarcinoma growth. We determined the expression of leptin receptors in normal and malignant human cholangiocytes. Effects on intrahepatic cholangiocarcinoma (HuH-28) cell proliferation, migration, and apoptosis of the in vitro exposure to leptin, together with the intracellular pathways, were then studied. Moreover, cholangiocarcinoma was experimentally induced in obese fa/fa Zucker rats, a genetically established animal species with faulty leptin receptors, and in their littermates by chronic feeding with thioacetamide, a potent carcinogen. After 24 weeks, the effect of leptin on cholangiocarcinoma development and growth was assessed. Normal and malignant human cholangiocytes express leptin receptors. Leptin increased the proliferation and the metastatic potential of cholangiocarcinoma cells in vitro through a signal transducers and activators of transcription 3–dependent activation of extracellular signal-regulated kinase 1/2. Leptin increased the growth and migration, and was antiapoptotic for cholangiocarcinoma cells. Moreover, the loss of leptin function reduced the development and the growth of cholangiocarcinoma. The experimental carcinogenesis model induced by thioacetamide administration is a valid and reproducible method to study cholangiocarcinoma pathobiology. Modulation of the leptin-mediated signal could be considered a valid tool for the prevention and treatment of cholangiocarcinoma.
Tetrandrine is a bisbenzylisoquinoline alkaloid found in Stephania tetrandra, a Chinese medicine commonly used as an anti-inflammatory. It has extensive pharmacological activity, including positive ion channel blockade and inhibition of multiple drug resistance proteins. These activities are very similar to that of salinomycin, a known drug targeting breast cancer initiation cells (TICs). Herein, we tested tetrandrine targeting of breast cancer TICs. SUM-149, an inflammatory breast cancer cell line and SUM-159, a non-inflammatory metaplastic breast cancer cell line were used in these studies. In proliferation assays using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), we found that the IC50 for inhibition of proliferation is 15.3 ± 4.1 μM for SUM-149 and 24.3 ± 2.1 μM for SUM-159 cells. Tetrandrine also inhibited mammosphere formation, a surrogate for breast cancer TICs growth in vitro with IC50 around 1 μM for SUM-149 and around 2 μM for SUM-159 cells. Tetrandrine has similar effects on the mammosphere formation from cells isolated from fresh patient sample. Moreover, tetrandrine decreases the aldehyde dehydrogenase (ALDH) positive population in SUM-159 by 45% ± 5.45% P = 0.005. In summary, tetrandrine demonstrates significant efficacy against in vitro surrogates for inflammatory and aggressive breast cancer TICs.
breast cancer; tetrandrine; cancer stem cells; tumor-initiating cells
The present study identified a novel salinomycin (Sal) sensitization mechanism in cancer. We tested whether Sal reduced proliferation in a high-density population by counting attached cell numbers after Sal treatment. Sal reduced proliferation in high-density cell populations. Longer exposure to Sal further reduced proliferation. Sal concentrations of 0.1 and 5 μM had similar sensitization effects, suggesting that Sal toxicity was minimal with longer exposure to a high-density cell population. The results suggest that Sal can be applied at a relatively low concentration for a longer time to overcome drug-resistant solid tumors. The 0.5 μM Sal treatment resulted in fewer attached cells than that of the 5 μM Sal treatment with a longer exposure. The lower Sal concentration mainly increased the number of easily detachable cells on the surface. In particular, 0.5 μM Sal increased cellular detachment of newly produced daughter cells. The easily-detachable cells were undergoing apoptosis. It seems that the 0.5 μM Sal treatment also increased cellular toxicity. These novel findings may contribute to the development of Sal-based therapy for patients with drug-resistant cancer or a high-density solid tumor.
salinomycin; apoptosis; high cell density; spaces among cells; cellular detachment; lower concentration
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.
Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor, and at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore, it is imperative that alternative treatment options are explored. We have previously shown that serotonin metabolism is dysregulated in cholangiocarcinoma leading to an increased secretion of serotonin, which has growth-promoting effects. Because serotonin and dopamine share the degradation machinery, we evaluated the secretion of dopamine from cholangiocarcinoma and its effects on cell proliferation. Using 4 cholangiocarcinoma cell lines and human biopsy samples, we demonstrated that there was an increase in mRNA and protein expression of the dopamine synthesis enzymes tyrosine hydroxylase and dopa decarboxylase in cholangiocarcinoma. There was increased dopamine secretion from cholangiocarcinoma cell lines compared to H69 and HIBEC cholangiocytes and increased dopamine immunoreactivity in human biopsy samples. Furthermore, administration of dopamine to all cholangiocarcinoma cell lines studied increased proliferation by up to 30% which could be blocked by the pretreatment of the D2 and D4 dopamine receptor antagonists, whereas blocking dopamine production by α-methyldopa administration suppressed growth by up to 25%. Administration of α-methyldopa to nude mice also suppressed cholangiocarcinoma tumor growth. The data presented here represent the first evidence that dopamine metabolism is dysregulated in cholangiocarcinoma and that modulation of dopamine synthesis may represent an alternative target for the development of therapeutic strategies.
Tyrosine hydroxylase; biliary cancer; Dopa decarboxylase; Monoamine oxidase A; biogenic amines
Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. Symptoms are usually evident after blockage of the bile duct by the tumor and, at this late stage, they are relatively resistant to chemotherapy and radiation therapy. Therefore it is imperative that alternative treatment options are explored. We present novel data indicating that the metabolism of serotonin is dysregulated in cholangiocarcinoma cell lines compared to normal cholangiocytes and in tissue and bile from cholangiocarcinoma patients. Specifically there was an increased expression of tryptophan hydroxylase 1 and a suppression of monoamine oxidase A expression (enzymes responsible for the synthesis and degradation of serotonin respectively) in cholangiocarcinoma. This resulted in an increased secretion of serotonin from cholangiocarcinoma and increased serotonin in the bile from cholangiocarcinoma patients. Increased local serotonin release may have implications on cholangiocarcinoma cell growth. Serotonin administration increased cholangiocarcinoma cell growth in vitro, whereas inhibition of serotonin synthesis decreases tumor cell growth both in vitro and in vivo. The data presented here represents the first evidence that serotonin metabolism is dysregulated in cholangiocarcinoma and that modulation of serotonin synthesis may represent an alternative target for the development of therapeutic strategies.
There are not many data available on antibiotics used solely in animals and almost exclusively for growth promotion. These products include bambermycin, avilamycin, efrotomycin, and the ionophore antibiotics (monensin, salinomycin, narasin, and lasalocid). Information is also scarce for bacitracin used only marginally in human and veterinary medicine and for streptogramin antibiotics. The mechanisms of action of and resistance mechanisms against these antibiotics are described. Special emphasis is given to the prevalence of resistance among gram-positive bacteria isolated from animals and humans. Since no susceptibility breakpoints are available for most of the antibiotics discussed, an alternative approach to the interpretation of MICs is presented. Also, some pharmacokinetic data and information on the influence of these products on the intestinal flora are presented.
Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.
Simvastatin is a cholesterol-lowering drug that is widely used to prevent and treat atherosclerotic cardiovascular disease. Simvastatin exhibits numerous pleiotropic effects including anti-cancer activity. However, the effect of simvastatin on cholangiocarcinoma has not been evaluated.
The aim of our study was to determine the effect of simvastatin on cholangiocarcinoma proliferation.
The effect of simvastatin was evaluated in five human cholangiocarcinoma cell lines (Mz-ChA-1, HuH-28, TFK-1, SG231, and HuCCT1) and normal cholangiocyte cell line (HiBEpiC).
We found that simvastatin stimulates a reduction in cell viability and apoptosis of cholangiocarcinoma cell lines, while in normal human cholangiocytes, HiBEpiC, simvastatin inhibits proliferation with no effect on apoptosis. Simvastatin-induced reduction of cell viability was partially blocked by pre-treatment with metabolites of the mevalonate pathway. In Mz-ChA-1 cells, pre-treatment with cholesterol alone stimulated an increase in the number of viable cells and fully restored cell viability following simvastatin treatment. Treatment with simvastatin triggered the loss of lipid raft localized Rac1 and reduction of Rac1 activity in Mz-ChA-1 cells. This effect was prevented by pre-treatment with cholesterol.
Collectively, our results demonstrate that simvastatin induces cholangiocarcinoma cancer cell death by disrupting Rac1/lipid raft colocalization and depression of Rac1 activity.
Cholangiocarcinoma; simvastatin; Rac1; apoptosis