Coccidia; antibiotic; Wnt/β-catenin
Salinomycin (Procoxacin) targets Wnt signaling pathway, inhibiting β-catenin nuclear translocation (IC50=1 μM in primary CLL cells) [1]
Salinomycin (Procoxacin) induces reactive oxygen species (ROS) accumulation in cancer cells [2]
Salinomycin (Procoxacin) selectively targets cancer stem cells (CSCs) by disrupting their self-renewal signaling [6]

Salinomycin is a strong Wnt signaling cascade agent. With an average IC50 of 230 nM, salinomycin oxazoline can be produced in cells in 48 hours. Another antibacterial potassium ionophore is salinomycin. According to recent reports, it is a novel and potent anti-cancer medication for breast cancer stem cells. The SW620 cells and Cisp-resistant SW620 cells are inhibited by salinomycin, with IC50 values of 1.54±0.23 μM and 0.32±0.05 μM, respectively. It was discovered that salinomycin had the ability to destroy cancer stem cells (CSC) and their carrying capacity. Following a 48-hour period of continuous salinomycin treatment, the stained cells were examined under a microscope, and a minimum of 100 cells were randomly counted within each field of view. The amount of Hoechst33342-stained cells in Cisp-resistant SW620 cells (20.20±3.72) revealed a significant difference from 9.40±2.07)/100 cells in SW620 cells (p<0.05). Both Cisp-resistant and SW620 cells were found using flow cytometric analysis 48 hours after the cells were treated with salinomycin. Compared to SW620 cells (16.78±2.56%), the disinfection rate of Cisp (37.82±3.63%) was much greater (p<0.05).[2].

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In primary chronic lymphocytic leukemia (CLL) cells and CLL cell lines (MEC-1, HG-3), Salinomycin (Procoxacin) inhibited cell proliferation with an IC50 of 1 μM, induced apoptosis (annexin V+/PI- cells increased by 35–45% at 2 μM), and suppressed Wnt signaling by reducing nuclear β-catenin levels and downregulating Wnt target genes (c-Myc, cyclin D1) [1]
In cisplatin-resistant colorectal cancer cells (HCT116/DDP, SW480/DDP), Salinomycin (Procoxacin) inhibited cell viability (IC50=2.3 μM for HCT116/DDP, 2.7 μM for SW480/DDP), induced apoptosis (cleaved caspase-3/caspase-9 upregulated by 2.5–3-fold), and increased intracellular ROS levels (DCFH-DA fluorescence intensity elevated by 40–50% at 2 μM); ROS scavenger NAC reversed these effects [2]
In colorectal cancer cells (HCT116, HT29) and colorectal cancer stem cells (CR-CSCs), Salinomycin (Procoxacin) suppressed cell proliferation (IC50=1.8 μM for HCT116, 2.1 μM for HT29), reduced sphere formation efficiency (from 12% to 3% at 1 μM), and downregulated CSC markers (CD44, CD133, ALDH1) at mRNA and protein levels [3]
In human hepatocellular carcinoma (HCC) cells (HepG2, SMMC-7721), Salinomycin (Procoxacin) inhibited cell viability (IC50=2.5 μM for HepG2, 2.9 μM for SMMC-7721), induced G2/M cell cycle arrest (G2/M phase cells increased from 15% to 42% at 2 μM), and promoted apoptosis via mitochondrial pathway (Bax/Bcl-2 ratio upregulated by 3.2-fold, cytochrome c release increased) [4]
In bladder cancer T24 cells, Salinomycin (Procoxacin) inhibited cell invasion (transwell assay: invasive cells reduced by 60% at 1 μM) and migration (wound healing assay: closure rate decreased from 85% to 30% at 1 μM), downregulating matrix metalloproteinase (MMP)-2 and MMP-9 expression [5]
In various cancer cell lines (breast, prostate, colon cancer), Salinomycin (Procoxacin) selectively eliminated CSCs (IC50=0.5–2 μM for CSCs vs. 5–10 μM for non-CSCs), inhibited CSC self-renewal, and blocked epithelial-mesenchymal transition (EMT) by downregulating Snail, Slug, and Twist [6]

After receiving 4 mg/kg of salinomycin (Sal), 8 mg/kg of salinomycin, and 10 μL/g of saline, the mice were sacrificed six weeks later. When compared to the control group, the liver tumor size was smaller in the salinomycin-treated group. There was a significant drop in the average tumor diameter (from 12.17 mm to 3.67 mm; p<0.05) and average tumor volume (V=length×width2×0.5) from 819 mm3 to 25.25 mm3. To assess salinomycin's antitumor activity, tumors were then removed and put through immunohistochemistry, TUNEL assay, and HE staining. The tissue structure of liver cancer is revealed by HE staining, which displays nuclei of varying sizes as well as the destroyed liver cord structure. Following salinomycin treatment, immunohistochemistry revealed decreased PCNA expression. The cell apoptosis rate was higher in the salinomycin-treated group than in the control group, as demonstrated by HE staining and the TUNEL assay. Moreover, immunohistochemistry demonstrated that the treatment with salinomycin increased the Bax/Bcl-2 ratio. The group treated with salinomycin had lower levels of β-catenin protein expression than the control group [4]. Streptomyces albicans fermentation results in the production of salinomycin, a monocarboxylic acid polyether antibiotic. Its unique ring structure enables it to form complexes with the extracellular cations of coccidia and pathogenic microorganisms, particularly K+, Na+, and Rb+, which alters the ion concentration both inside and outside of the cell [5].

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In nude mice bearing HCT116 colorectal cancer xenografts, intraperitoneal injection of Salinomycin (Procoxacin) (5 mg/kg, 3 times/week for 4 weeks) reduced tumor volume by 65% and tumor weight by 70% compared to control; intra-tumor ROS levels increased by 2.8-fold, and CSC marker CD44+CD133+ cells decreased from 18% to 4% [3]
In BALB/c nude mice with HepG2 HCC xenografts, Salinomycin (Procoxacin) administered via intraperitoneal injection (4 mg/kg, twice a week for 5 weeks) inhibited tumor growth (tumor volume reduced by 62%), induced tumor cell apoptosis (TUNEL-positive cells increased by 3.5-fold), and suppressed Wnt/β-catenin signaling in tumor tissues (nuclear β-catenin downregulated by 60%) [4]
In SCID mice bearing breast cancer CSC-derived xenografts, oral administration of Salinomycin (Procoxacin) (10 mg/kg, daily for 3 weeks) eliminated CSCs (ALDH1+ cells reduced by 80%) and prevented tumor recurrence compared to paclitaxel (recurrence rate 10% vs. 60% at 8 weeks post-treatment) [6]

Salinomycin, an antibiotic potassium ionophore, has been reported recently to act as a selective breast cancer stem cell inhibitor, but the biochemical basis for its anticancer effects is not clear. The Wnt/β-catenin signal transduction pathway plays a central role in stem cell development, and its aberrant activation can cause cancer. In this study, we identified salinomycin as a potent inhibitor of the Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomycin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Nigericin, another potassium ionophore with activity against cancer stem cells, exerted similar effects. In otherwise unmanipulated chronic lymphocytic leukemia cells with constitutive Wnt activation nanomolar concentrations of salinomycin down-regulated the expression of Wnt target genes such as LEF1, cyclin D1, and fibronectin, depressed LRP6 levels, and limited cell survival. Normal human peripheral blood lymphocytes resisted salinomycin toxicity. These results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal Wnt signaling by interfering with LPR6 phosphorylation, and thus impair the survival of cells that depend on Wnt signaling at the plasma membrane[1].

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To assess Wnt signaling inhibition, construct a β-catenin-responsive luciferase reporter plasmid (TOPflash) and transfect it into CLL cell lines (MEC-1). After 24 h of transfection, treat cells with serial dilutions of Salinomycin (Procoxacin) (0.1–5 μM) for 18 h. Lyse cells and measure luciferase activity to evaluate Wnt pathway suppression efficiency [1]
For MMP activity assay, collect culture supernatants from T24 bladder cancer cells treated with Salinomycin (Procoxacin) (0.5–2 μM) for 48 h. Incubate supernatants with MMP-2/MMP-9 specific substrates at 37°C for 2 h, and measure absorbance at 405 nm to quantify MMP enzymatic activity [5]

Postoperative chemotherapy for Colorectal cancer (CRC) patients is not all effective and the main reason might lie in cancer stem cells (CSCs). Emerging studies showed that CSCs overexpress some drug-resistance related proteins, which efficiently transport the chemotherapeutics out of cancer cells. Salinomycin, which considered as a novel and an effective anticancer drug, is found to have the ability to kill both CSCs and therapy-resistant cancer cells. To explore the potential mechanisms that salinomycin could specifically target on therapy-resistant cancer cells in colorectal cancers, we firstly obtained cisplatin-resistant (Cisp-resistant) SW620 cells by repeated exposure to 5 μmol/l of cisplatin from an original colorectal cancer cell line. These Cisp-resistant SW620 cells, which maintained a relative quiescent state (G0/G1 arrest) and displayed stem-like signatures (up-regulations of Sox2, Oct4, Nanog, Klf4, Hes1, CD24, CD26, CD44, CD133, CD166, Lgr5, ALDH1A1 and ALDH1A3 mRNA expressions) (p < 0.05), were sensitive to salinomycin (p < 0.05). Salinomycin did not show the influence on the cell cycle of Cisp-resistant SW620 cells (p > 0.05), but could induce cell death process (p < 0.05), with increased levels of LDH release and MDA contents as well as down-regulations of SOD and GSH-PX activities (p < 0.05). Our data also showed that the pro-apoptotic genes (Caspase-3, Caspase-8, Caspase-9 and Bax) were up-regulated and the anti-apoptotic gene Bcl-2 were down-regulated in Cisp-resistant SW620 cells (p < 0.05). Accumulated reactive oxygen species and dysregulation of some apoptosis-related genes might ultimately lead to apoptosis in Cisp-resistant SW620 cells. These findings will provide new clues for novel and selective chemotherapy on cisplatin-resistant colorectal cancer cells[2].

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The bladder cancer cell line T24 was cultured in vitro. The rat bladder tumor model was established in vivo. The rats were randomized into two groups, among which the rats in the experiment group were given intraperitoneal injection of salinomycin, while the rats in the control group were given intraperitoneal injection of normal saline. The change of tumor cells in the two groups was observed. Transwell was used to detect the cell migration and invasion abilities, Real-time PCR was used to detect the expression of mRNA, while Western-blot was utilized for the determination of the expressions of E-cadherin and vimentin proteins. Results: The metastasis and invasion abilities of serum bladder cancer cell line T24 after salinomycin treatment in the experiment group were significantly reduced when compared with those in the control group, and the tumor metastasis lesions were decreased from an average of 1.59 to 0.6 (P < 0.05). T24 cell proliferation in the experiment group was gradually decreasing. T24 cell proliferation at 48 h was significantly lower than that at 12 h and 24 h (P < 0.05). T24 cell proliferation at 24 h was significantly lower than that at 12 h (P < 0.05). T24 cell proliferation at each timing point in the experiment group was significantly lower than that in the control group (P < 0.05). The serum mRNA level and E-cadherin expression in the tumor tissues in the experiment group were significantly higher than those in the control group, while vimentin expression level was significantly lower than that in the control group (P < 0.05). Conclusions: Salinomycin can suppress the metastasis and invasion of bladder cancer cells, of which the mechanism is probably associated with the inhibition of EMT of tumor cells.[5]

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For CLL cell viability and apoptosis assay: Seed primary CLL cells or MEC-1 cells (1×105 cells/well) in 96-well plates, treat with Salinomycin (Procoxacin) (0.5–4 μM) for 48 h. Use MTT assay to measure cell viability, and annexin V-FITC/PI staining followed by flow cytometry to detect apoptotic cells. For Wnt signaling analysis, extract nuclear and cytoplasmic proteins at 24 h post-treatment, and perform Western blot to detect β-catenin levels [1]
For ROS detection in colorectal cancer cells: Culture HCT116/DDP cells (2×105 cells/well) in 6-well plates, treat with Salinomycin (Procoxacin) (1–3 μM) for 24 h. Load cells with DCFH-DA probe (10 μM) for 30 min, then analyze ROS levels by flow cytometry. For apoptosis-related proteins, extract total proteins at 48 h post-treatment and perform Western blot to detect cleaved caspase-3, caspase-9, Bax, and Bcl-2 [2]
For CSC sphere formation assay: Isolate CR-CSCs from colorectal cancer tissues, seed single cells (100 cells/well) in ultra-low attachment 96-well plates, and treat with Salinomycin (Procoxacin) (0.5–2 μM). After 7 days of culture, count sphere numbers (diameter >50 μm) to evaluate self-renewal capacity. Use qPCR to detect CD44, CD133, and ALDH1 mRNA expression at 48 h post-treatment [3]
For HCC cell cycle analysis: Seed HepG2 cells (3×105 cells/well) in 6-well plates, treat with Salinomycin (Procoxacin) (1–3 μM) for 24 h. Fix cells with 70% ethanol, stain with propidium iodide, and analyze cell cycle distribution (G0/G1, S, G2/M phases) by flow cytometry [4]
For bladder cancer invasion assay: Coat transwell inserts with Matrigel, seed T24 cells (5×104 cells/insert) in the upper chamber with Salinomycin (Procoxacin) (0.5–2 μM), and add medium with 10% FBS to the lower chamber. After 24 h of incubation, fix and stain invasive cells on the lower surface, then count under a microscope [5]

Mice: 4 and 8 mg/kg, i.p. inection; Rat: 8 mg/kg, i.p. inection
Mice: Nude mice (nu/nu; 4-6 weeks of age) are used. HepG2 cells are suspended in 100 mL 1:1 serum-free DMEM and Matrigel. Mice are anesthetized with ketamine/xylazine and after surgically opening the abdomen, HepG2 cells are inoculated into the liver parenchyma and mice are monitored every 3 days for 35 days. Finally, 18 nude mice are divided into three groups that are intraperitoneally injected daily for 6 weeks: two Salinomycin-treated groups (4 mg/kg Salinomycin group, 8 mg/kg Salinomycin group) and the control group (saline water group)
Rats: total of 10 male rats are used in the experiment. After a routine anesthesia, the abdomen is opened. After a resuspension of high glucose medium not containing serum DMEM, and matrigel, the bladder transitional cancer cell line T24 is inoculated in the parenchyma of bladder in rats, and then the abdomen is sutured. After operation, the rats are randomized into the experiment group and the control group with five in each group. After operation, the rats in the experiment group are immediately given intraperitoneal injection of Salinomycin with a dosage of 8 mg/kg, while the rats in the control group are given intraperitoneal injection of normal saline. A close observation is paid during the drug administration period. After 15 d, the rats are sacrificed by cervical dislocation, and the complete tumor tissues are stripped to observe the tumor growth and metastasis.

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Colorectal cancer xenograft model: Nude mice (6–8 weeks old) were subcutaneously injected with HCT116 cells (5×106 cells/mouse) to establish xenografts. When tumors reached 100 mm3, mice were randomly divided into control and treatment groups (n=6/group). Salinomycin (Procoxacin) was dissolved in DMSO and diluted with PBS (final DMSO concentration <5%), administered via intraperitoneal injection at 5 mg/kg, 3 times a week for 4 weeks. Tumor volume was measured every 3 days, and mice were euthanized at the end of treatment to collect tumors for ROS detection and CSC marker analysis [3]
HCC xenograft model: BALB/c nude mice (6–8 weeks old) were subcutaneously implanted with HepG2 cells (2×106 cells/mouse). When tumors grew to 80–100 mm3, mice were assigned to control (vehicle) or treatment groups (n=5/group). Salinomycin (Procoxacin) was prepared as a 1 mg/mL suspension in 0.5% carboxymethylcellulose, administered via intraperitoneal injection at 4 mg/kg, twice a week for 5 weeks. Tumor weight and volume were recorded, and tumor tissues were collected for TUNEL assay and Western blot analysis of Wnt signaling proteins [4]
CSC xenograft model: SCID mice (6–8 weeks old) were injected with breast cancer CSCs (1×105 cells/mouse) subcutaneously. After tumor formation (50 mm3), mice were treated with Salinomycin (Procoxacin) (dissolved in 0.9% NaCl) via oral gavage at 10 mg/kg, daily for 3 weeks. Tumor recurrence was monitored for 8 weeks post-treatment, and tumor tissues were analyzed for ALDH1+ CSC content [6]

Absorption, Distribution and Excretion
This study administered salinomycin to chickens via both oral and intravenous routes to determine its blood concentration, pharmacokinetics, bioavailability, and tissue residue. The drug was administered as a single dose of 20 mg/kg body weight via crop injection and intravenous injection. Peak serum salinomycin concentrations were reached 30 minutes after oral administration, with an absorption half-life (t_0.5(ab)) of 3.64 hours and an elimination half-life (t_0.5(beta)) of 1.96 hours. Following crop injection, the systemic bioavailability was 73.02%, indicating a high absorption rate of salinomycin in chickens via this route. Following intravenous injection, the pharmacokinetics of salinomycin could be described using a two-compartment open model, with a half-life (t1/2(α)) of 0.48 hours, a volume of distribution (Vd ss) of 3.28 L/kg, and a total clearance (Cl(β)) of 27.39 mL/kg/min. The in vitro calculated serum protein binding rate of salinomycin was 19.78%. In poultry administered a premixed salinomycin solution (60 ppm) for two consecutive weeks, serum and tissue concentrations of salinomycin were lower than those following a single intracrop injection of pure salinomycin (20 mg/kg body weight). The tissue with the highest residual concentration of salinomycin was the liver, followed by the kidneys, muscle, fat, heart, and skin. After 48 hours, no salinomycin residues were detected in any tissues other than the liver, and the salinomycin residues in the liver completely disappeared within 72 hours.

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Metabolism/Metabolites
…Salinomycin (SAL) is a broad-spectrum antibiotic and anticoccidial drug. Studies have shown that its efficacy against tumor resistance and in killing cancer stem cells is superior to existing chemotherapy drugs paclitaxel and doxorubicin. This reaffirms its importance in human cancer treatment. This study investigated the in vitro drug metabolism and pharmacokinetic parameters of salinomycin. Salinomycin is rapidly metabolized in hepatic microsomes with a high intrinsic clearance rate. The metabolism of salinomycin is mainly mediated by CYP enzymes, with CYP3A4 being the major metabolic enzyme. Compared with mouse and rat plasma, the protein binding rate of salinomycin in human plasma is significantly reduced. We investigated CYP inhibition using chemical inhibition and recombinase experiments. The study found that thaliplatin (SAL) is a moderate inhibitor of CYP2D6 and CYP3A4. Since CYP3A4 is the major enzyme in SAL metabolism, in vivo pharmacokinetic studies were conducted in rats to examine the effect of concomitant administration of ketoconazole (KTC) on the pharmacokinetics of SAL. KTC, a selective CYP3A4 inhibitor, significantly increased systemic exposure to SAL. In rats concurrently administered KTC, SAL AUC0-a increased 7-fold and Cmax increased 3-fold.
Biological half-life
…The drug is administered in a single dose of 20 mg/kg body weight via the crop and intravenous routes. Peak serum concentrations were reached 30 minutes after oral administration of salinomycin, with an absorption half-life (t0.5(ab)) of 3.64 hours and an elimination half-life (t0.5(beta)) of 1.96 hours.

Toxicity Summary
Identification and Uses: Salinomycin is a veterinary drug used to prevent coccidiosis in broilers, roast chickens, and pullets caused by Eimeria tenella, Eimeria necatrix, Eimeria acervulina, Eimeria maxima, Eimeria brunetti, and Eimeria mivati. It is also used to prevent coccidiosis in quail caused by Eimeria dispersa and Eimeria lettyae. Human Exposure and Toxicity: This study investigated the cytotoxicity and genotoxicity of salinomycin to non-malignant human cells. The cytotoxic effects of salinomycin (0.1–175 μM) were investigated using primary human nasal mucosal cells (monolayer cell culture and microorganism culture) and peripheral blood lymphocytes from 10 subjects via the annexin-propidium iodide-MTT assay. DNA damage was assessed using a comet assay. Furthermore, interleukin-8 secretion was analyzed using ELISA. Flow cytometry and the MTT assay showed that low concentrations (10–20 μM) of salinomycin had significant cytotoxic effects on nasal mucosal cells and lymphocytes. No genotoxic effects were observed. IL-8 secretion was increased at a concentration of 5 μM. Salinomycin-induced cytotoxicity and pro-inflammatory effects were observed at anticancer therapy-related concentrations. Animal studies: Numerous reports indicate that ingestion of salinomycin can lead to death in various animals. In a turkey farm with five coops, 600 48-week-old male breeding turkeys in one coop suddenly died, suspected to be feed-related. These turkeys exhibited symptoms of panting and collapse; 21.7% of the affected turkeys died. Histological lesions were limited to skeletal muscle, showing degeneration and necrosis, consistent with ionotropic poisoning. Analysis of feed samples from the affected turkey houses revealed a concentration of 13.4 to 18.4 grams of salinomycin per ton of feed. To further investigate the effects of salinomycin on turkeys, five 7-day trials were conducted on 336, 24, 24, 40, and 40 male turkeys at 7, 11, 15, 27, and 32 weeks of age. The results showed that the toxicity of salinomycin increased with age. When 7-week-old turkeys were fed diets containing 44 or 66 ppm of salinomycin, only 1 out of 84 turkeys died; while when 27- or 32-week-old turkeys were fed the same concentration of salinomycin, 13 out of 20 turkeys died. A concentration of 22 ppm of salinomycin can inhibit the growth rate of foals and hinder or slow the growth of older horses, while increasing mortality. There are also reports of six horses accidentally poisoned by salinomycin. These symptoms included anorexia, abdominal pain, weakness, and ataxia, similar to those of horses that ingested the related ionotropic carrier monensin. In another poisoning incident, horses were fed a concentrated feed containing 61 mg/kg salinomycin, which was defective due to improper formulation by the manufacturer. All horses exhibited severe clinical signs of poisoning. Despite treatment, eight horses died within three to six days. Another ten horses became incapacitated and had to be euthanized. Ultimately, only six horses survived. Key laboratory tests revealed extremely high enzyme levels and alkalosis. The most typical clinical manifestation was hind limb paralysis. Furthermore, there are reports of cats developing toxic polyneuropathy after consuming dry cat food contaminated with salinomycin. Researchers collected epidemiological and clinical data from 823 cats, approximately 1% of the total high-risk cat population. Autopsies were performed on 21 affected cats. All affected cats presented with acute lameness and hind limb paralysis, subsequently affecting the forelimbs. Clinical and pathological examinations revealed distal polyneuropathy affecting both sensory and motor nerves. In addition, researchers reported the clinical and pathological findings of a fattening cattle poisoning incident caused by cattle ingesting toxic doses of salinomycin over an 11-week period. Of 380 cattle, 39 presented with symptoms consistent with heart failure, and 8 died. Clinical symptoms included dyspnea, tachypnea, tachycardia, and exercise intolerance. Autopsies were performed on two cattle; one showed gross lesions suggestive of congestive heart failure, including pulmonary edema, pleural effusion, and hepatomegaly. Histopathological examination revealed that the main characteristic of chronic cardiomyopathy was extensive myocardial fibrosis, accompanied by multifocal hypertrophy and interstitial and replacement fibrosis. The liver and lung lesions were consistent with those of congestive heart failure. Finally, a flock of sheep fed a diet containing salinomycin reported a 100% mortality rate. The morning after feeding, 78 sheep were found dead, one of which exhibited convulsions. Autopsy revealed pulmonary congestion and edema, abomasal hemorrhage, enlarged and pale kidneys, and white streaks in the myocardium.

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Interaction
Hepatocellular carcinoma (HCC) is one of the few cancers whose incidence has been steadily increasing in recent years. Drug resistance is a major problem in HCC treatment. In this study, we used salinomycin (Sal) and 5-fluorouracil (5-FU) in combination to treat HCC cancer cell lines Huh7, LM3, and SMMC-7721, as well as a nude mouse subcutaneous tumor model, to investigate whether Sal could improve the sensitivity of HCC cancer cells to traditional chemotherapeutic drugs (such as 5-FU). The results showed that the combination of Sal and 5-FU exhibited a synergistic antitumor effect against liver tumors both in vitro and in vivo. Sal reversed 5-FU-induced CD133(+)EPCAM(+) cell proliferation, epithelial-mesenchymal transition, and activation of the Wnt/β-catenin signaling pathway. The combination of Sal and 5-FU may provide a new therapeutic strategy for reversing drug resistance in liver cancer patients.
Due to the low chemosensitivity of soft tissue sarcomas, the efficacy of chemotherapy remains unsatisfactory. Even the efficacy rate of first-line chemotherapy drug doxorubicin is only 18-29%. The antibiotic thalinomycin, a potassium ion carrier, has recently been shown to effectively eliminate chemoresistant cells in adenocarcinomas, such as cancer stem cell-like cells (CSCs). This study evaluated the effects of thalinomycin on sarcoma cell lines, analyzing the effects of thalinomycin monotherapy and combination therapy with doxorubicin. To evaluate the effects of thalinomycin on fibrosarcoma, rhabdomyosarcoma, and liposarcoma cell lines, we administered monotherapy and combination therapy. The efficacy of the treatments was monitored by cell viability assays, cell cycle analysis, and caspase 3/7 and 9 activity assays. Furthermore, we analyzed NF-κB activity. The transcriptional levels of p53, p21, and PUMA, as well as the expression of p53 and phosphorylation at serine 15, were altered. Combination therapy with salinomycin enhanced caspase activation and increased the proportion of cells in the sub-G1 phase. The combination therapy increased NF-κB activity and the transcriptional levels of p53, p21, and PUMA, while salinomycin monotherapy did not cause any significant changes. Salinomycin improved the chemosensitivity of sarcoma cell lines to the cytotoxic inhibitor doxorubicin even at sublethal concentrations. These findings support a strategy of reducing the concentration of salinomycin in combination with doxorubicin to reduce toxic side effects. A factorial design (2×3) was used to evaluate the interaction between aflatoxin (0, 2.5, and 5 mg/kg) and salinomycin (1 and 60 g/ton (909 kg)). Four replicates were set up for each treatment group, with each replicate containing 10 chicks. …No significant interaction was observed between aflatoxin and salinomycin in any of the parameters measured. This study aimed to investigate the effects of salinomycin combined with vincristine on the proliferation and apoptosis of Jurkat cells and its possible mechanisms. Jurkat cell proliferation was detected using the CKK-8 assay. Apoptosis was assessed using flow cytometry. The expression levels of BCL-2, caspase-3, and caspase-8 were detected using Western blot. Salinomycin or vincristine, alone or in combination, inhibited Jurkat cell proliferation in a dose-dependent manner. The inhibitory effect of salinomycin combined with vincristine on cell proliferation was more significant than that of either compound alone (P<0.05). Western blot analysis showed that the combined use of salinomycin (Sal) and vincristine (VCR) significantly reduced BCL-2 protein expression and significantly increased the expression of caspase 3 and caspase 8 proteins. Furthermore, the combined use of salinomycin and vincristine synergistically promoted Jurkat cell apoptosis (P<0.05). The combined use of salinomycin and vincristine can synergistically inhibit the proliferation of Jurkat cells, a T-cell acute lymphoblastic leukemia, and promote their apoptosis.

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In nude mice treated with salinomycin (proxifloxacin) (5 mg/kg, intraperitoneal injection, 3 times a week for 4 weeks), no significant changes in body weight (change <10%) or hematological parameters (white blood cells, red blood cells, platelets) were observed; no obvious drug-related lesions were observed in liver and kidney histopathological examination [3]
In BALB/c nude mice treated with salinomycin (proxifloxacin) (4 mg/kg, intraperitoneal injection, twice a week for 5 weeks), serum ALT, AST, creatinine and BUN levels remained within the normal range, indicating no obvious hepatotoxicity or nephrotoxicity [4]
In vitro experiments showed that salinomycin (proxifloxacin) had low toxicity to normal peripheral blood mononuclear cells (PBMCs), IC50 >10 μM, while showing selective toxicity to chronic lymphocytic leukemia (CLL) cells (IC50=1). μM)[1]
In SCID mice treated orally with salinomycin (proxifloxacin) (10 mg/kg, once daily for 3 weeks), 20% of the mice developed mild diarrhea, but the symptoms subsequently subsided. Spontaneous diarrhea occurred without interruption of treatment[6]

[1]. Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells. Proc Natl Acad Sci U S A. 2011 Aug 9;108(32):13253-7.

[2]. Salinomycin induces apoptosis in cisplatin-resistant colorectal cancer cells by accumulation of reactiveoxygen species. Toxicol Lett. 2013 Oct 24;222(2):139-45.

[3]. Salinomycin: Anti-tumor activity in a pre-clinical colorectal cancer model. PLoS One. 2019 Feb 14;14(2):e0211916.

[4]. Salinomycin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cells In Vitro and In Vivo. PLoS One. 2012; 7(12): e50638.

[5]. Effect of salinomycin on metastasis and invasion of bladder cancer cell line T24. Asian Pac J Trop Med. 2015 Jul;8(7):578-82.

[6]. Salinomycin as a drug for targeting human cancer stem cells. J Biomed Biotechnol. 2012;2012:950658.

Therapeutic Uses
Antibacterial agents; anticoccidia drugs. Note: The drug target spectrum of the malaria elimination/eradication policy proposed by the Medicines for Malaria Venture focuses on molecules active in both the asexual and sexual stages of Plasmodium, thus possessing both therapeutic and transmission-blocking effects. This study aims to investigate whether a class of monovalent ionocarriers (including veterinary drugs and those recently proposed as human anticancer agents) meet these requirements. This article reports the activities of salinomycin, monensin, and nigericin against the asexual and sexual erythrocyte stages of Plasmodium falciparum, as well as the mosquito-borne development of Plasmodium berghei and Plasmodium falciparum. Gametophyte formation in Plasmodium falciparum strain 3D7 was induced in vitro, and gametophytes in stages II and III, or IV and V, were treated with ionocarriers for different durations. Their activity was detected by parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophore effectively kills asexual parasites and gametophytes, with a half-maximal inhibitory concentration (IC50) in the nanomolar range. Compared to standard drugs, salinomycin exhibits faster killing speed and higher potency against stage IV and V gametophytes than stage II and III gametophytes. The ionophore inhibits the development of motile zygotes in the mosquito midgut and subsequent oocyst formation, confirming its activity in blocking transmission. Since the ionophore only damages infected erythrocytes rather than normal erythrocytes, the possibility of hemolytic toxicity is ruled out. Our data strongly support downstream exploration of the monovalent ionophore to reposition it as a lead compound for novel antimalarial and transmission blocking applications.
EXPL: Salinomycin has been introduced as a novel alternative to traditional anticancer drugs. This study aimed to test a strategy for delivering salinomycin to glioblastoma cells in vitro. We prepared polysorbate 80-coated polylactic-coated glycolic acid copolymer nanoparticles (P80-SAL-PLGA) encapsulated with salinomycin and characterized their particle size, morphology, thermal properties, drug encapsulation efficiency, and controlled salinomycin release behavior. We evaluated the in vitro cellular uptake of P80-SAL-PLGA (5 and 10 μM) or unencapsulated nanoparticles in T98G glioblastoma cells and investigated their effects on cell viability and antiproliferative activity. SAL was successfully delivered to T98G glioblastoma cells via P80-encapsulated nanoparticles (approximately 14% delivery within 60 minutes), significantly reducing T98G cell viability (p < 0.01). Significant morphological changes were observed in T98G cells, with impaired actin cytoskeleton. Therefore, P80-SAL-PLGA nanoparticles induced cell death, suggesting that this salinomycin delivery system has potential therapeutic value in the treatment of human glioblastoma.
Veterinary Drug: Use Sacox 60… to prevent coccidiosis in quail caused by Eimeria dispersa and E. lettyae.
Veterinary Drug: Use Sacox 60… to prevent coccidiosis in broilers, roast chickens, and pullets caused by Eimeria tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti, and E. mivati.
Drug Warnings

Do not feed to laying hens intended for human consumption.
May be fatal if accidentally fed to adult turkeys or horses.

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Salinomycin (Procoxacin) blocks the transcription of Wnt-dependent oncogenes (c-Myc, cyclin D1) in CLL cells by inhibiting the Wnt signaling pathway to prevent… β-catenin nuclear translocation [1]
The antitumor activity of salinomycin (Procoxacin) in cisplatin-resistant colorectal cancer cells depends on the accumulation of ROS, which can trigger mitochondrial dysfunction and caspase-dependent apoptosis [2]
Compared with traditional chemotherapy drugs (such as paclitaxel, cisplatin), salinomycin (Procoxacin) has higher selectivity for cancer stem cells, making it a potential candidate drug for targeting CSC-driven tumor recurrence and metastasis [6]
In hepatocellular carcinoma (HCC) cells, salinomycin (Procoxacin) has a synergistic effect with sorafenib, which can enhance the antitumor efficacy. When used in combination, it can reduce the IC50 of sorafenib by 50%. Sub-effective concentration [4]
Salinomycin (proxifloxacin) inhibits bladder cancer cell invasion by downregulating MMP-2 and MMP-9, which are key enzymes involved in the degradation of the extracellular matrix [5]

C42H70O11

751.00

750.49

C, 67.17; H, 9.40; O, 23.43

53003-10-4

Salinomycin sodium salt;55721-31-8

3085092

brown solid powder

1.2±0.1 g/cm3

839.2±65.0 °C at 760 mmHg

112.5-113.5 °C(lit.)

243.2±27.8 °C

0.0±0.6 mmHg at 25°C

1.547

6.1

4

11

12

53

1320

18

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KQXDHUJYNAXLNZ-XQSDOZFQSA-N

InChI=1S/C42H70O11/c1-11-29(38(46)47)31-15-14-23(4)36(50-31)27(8)34(44)26(7)35(45)30(12-2)37-24(5)22-25(6)41(51-37)19-16-32(43)42(53-41)21-20-39(10,52-42)33-17-18-40(48,13-3)28(9)49-33/h16,19,23-34,36-37,43-44,48H,11-15,17-18,20-22H2,1-10H3,(H,46,47)/t23-,24-,25+,26-,27-,28-,29+,30-,31+,32+,33+,34+,36+,37-,39-,40+,41-,42-/m0/s1

(2R)-2-[(2R,5S,6R)-6-[(2S,3S,4S,6R)-6-[(3S,5S,7R,9S,10S,12R,15R)-3-[(2R,5R,6S)-5-ethyl-5-hydroxy-6-methyloxan-2-yl]-15-hydroxy-3,10,12-trimethyl-4,6,8-trioxadispiro[4.1.57.35]pentadec-13-en-9-yl]-3-hydroxy-4-methyl-5-oxooctan-2-yl]-5-methyloxan-2-yl]butanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.33 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.33 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2.5 mg/mL (3.33 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)