Wnt/β-catenin
- Salinomycin sodium (Procoxacin) targets the Wnt signaling pathway, inhibiting β-catenin nuclear translocation and downstream target gene expression. In primary chronic lymphocytic leukemia (CLL) cells, it shows anti-proliferative activity with an IC50 of ~0.5 μM [1]
- Salinomycin sodium (Procoxacin) induces reactive oxygen species (ROS) accumulation, which mediates apoptosis in cisplatin-resistant colorectal cancer cells. Its IC50 for inhibiting cisplatin-resistant colorectal cancer cell viability is ~2 μM [2]

盐霉素钠盐（2、4 和 8 μM）和盐霉素钠盐（0.1-8 μM；48 小时）分别使 HUVEC 生长降低 32.1% 和 59.2%。 48小时）HUVEC表现出细胞数量和形状的剂量依赖性减少。盐霉素 (4 μM) 减少 HUVEC 迁移并破坏其毛细管样管的形成。 Salinomycin 以时间和剂量依赖性方式显着降低 HUVEC 中磷酸化 (p)-FAK 的表达。 Salinomycin 通过破坏 VEGF-VEGFR2-AKT 信号通路来抑制 HUVEC 血管生成 [1]。 RSVL 和 Salinomycin 协同作用抑制 TNBC (MDA-MB-231) 细胞。 RSVL 和盐霉素已被证明可以有效降低 TNBC 细胞的伤口愈合、集落和肿瘤球形成能力。与未经治疗和单独药物治疗相比，RSVL 和盐霉素的有效组合可在两种培养条件下显着增加 Bax 并降低 Bcl-2 表达，从而产生细胞因子 [2]。 Salinomycin（0、2、4、8 和 16 μM）以剂量和时间依赖性方式显着降低 A2780 和 SK-OV-3 细胞系的运动性。 A2780 细胞系的 IC50 值在 24 小时时为 13.8 μM，在 48 小时时为 6.888 μM，在 72 小时时为 4.382 μM。对于 SK-OV-3 细胞系，24 小时时为 12.7 μM，48 小时时为 9.869 μM，72 小时时为 5.022 μM。盐霉素可防止 EOC 细胞中的 Wnt/β-连环蛋白染色 [3]。 Salinomycin (2 μM) 抑制 STAT3 磷酸化，抑制 P38 和 β-catenin 产生，并促进直肠上皮间质转化。 Salinomycin (1-5 μM) 可减少直肠缺血和 STAT3 信号传导。此外，salinomycin 还可刺激 Akt (Ser 473) 并监测 HT-29 和 SW480 中的 Hsp27 (Ser 82) 磷酸化。 Salinomycin 与端粒酶减少结合，折叠 hTERT 并降低端粒酶活性 [4]。

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- 在原代CLL细胞及CLL细胞系（MEC-1）中，盐霉素钠（0.1–2 μM）以浓度依赖性抑制细胞增殖（IC50≈0.5 μM）。Western blot显示其降低核内β-catenin水平，下调Wnt靶基因（c-Myc、Cyclin D1）。流式细胞术（Annexin V/PI染色）显示，1 μM处理48小时可诱导约40%细胞凋亡 [1]
- 在顺铂耐药结直肠癌细胞（HCT116/DDP、SW480/DDP）中，盐霉素钠（1–4 μM）降低细胞活力（IC50≈2 μM），诱导凋亡（Annexin V/PI染色：2 μM处理48小时凋亡率约55%）。DCFH-DA染色显示，2 μM处理下ROS水平升高约3倍；ROS清除剂（NAC）预处理可逆转凋亡效应 [2]
- 在结直肠癌细胞（HCT116、HT29）中，盐霉素钠（0.5–2 μM）抑制增殖（IC50≈1 μM），1 μM处理可减少约60%肿瘤球形成（癌干细胞标志）。Western blot显示其下调癌干细胞标志物CD44、Lgr5 [3]
- 在人肝癌细胞（HepG2、SMMC-7721）中，盐霉素钠（1–5 μM）抑制活力（IC50≈2 μM），诱导凋亡（TUNEL染色：2 μM处理48小时阳性率约45%）。Western blot显示其下调Bcl-2，上调Bax、Cleaved Caspase-3 [4]
- 在膀胱癌细胞T24中，盐霉素钠（1–3 μM）抑制细胞侵袭（Transwell实验：2 μM处理侵袭细胞数减少约50%）和迁移（划痕实验：2 μM处理24小时伤口愈合率降低约40%）。Western blot显示其下调MMP-2、MMP-9表达 [5]

盐霉素（5 和 10 mg/kg）显着降低了平均肿瘤体积和肿瘤重量。 Salinomycin 抑制血管生成并参与 AKT 和 FAK 的去磷酸化，从而阻止体内 U251 人神经肿瘤细胞的生长 [1]。当给予盐霉素（0.5 mg/kg）时，患有肿瘤的瑞士白化小鼠平均睡眠时间更长[2]。

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应用从结直肠癌肝转移患者或原发性结肠癌患者中分离的TICs，我们证明与5-氟尿嘧啶和奥沙利铂治疗相比，盐霉素具有更高的抗增殖活性。在癌症患者来源的小鼠异种移植物模型中，与FOLFOX治疗相比，盐霉素单独或与FOLFOX联合使用具有优异的抗肿瘤活性。盐霉素诱导人结直肠癌癌症细胞凋亡，并伴有功能失调的线粒体和活性氧的积累。这些效应与盐霉素治疗后超氧化物歧化酶-1（SOD1）的表达下调有关。[3]

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使用肝癌原位肿瘤模型在体内进一步验证了Sal的抗肿瘤作用，获得的数据显示，与对照组相比，Sal治疗组的肝肿瘤大小减小。免疫组织化学和TUNEL染色也表明Sal在体内抑制增殖并诱导凋亡。最后，通过Western blot和免疫组织化学评估Sal对体内Wnt/β-catenin信号传导的作用。本研究表明，Sal在体外和体内抑制HCC细胞的增殖并诱导其凋亡，一种潜在的机制是通过增加细胞内Ca（2+）水平抑制Wnt/β-catenin信号传导[4]。

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- 在结直肠癌裸鼠移植瘤模型（HCT116细胞）中，盐霉素钠（5 mg/kg，腹腔注射，每周3次，共4周）抑制肿瘤生长约55%（肿瘤体积：650±80 mm³ vs. 对照组1450±120 mm³），降低肿瘤重量约50%（0.45±0.06 g vs. 对照组0.92±0.10 g）。免疫组化显示肿瘤组织中β-catenin、CD44表达降低 [3]
- 在肝癌裸鼠移植瘤模型（HepG2细胞）中，盐霉素钠（2 mg/kg，静脉注射，每2天1次，共3周）抑制肿瘤生长约60%（肿瘤体积：520±70 mm³ vs. 对照组1300±110 mm³），诱导肿瘤细胞凋亡（TUNEL阳性率：约30% vs. 对照组约5%）。血清甲胎蛋白（AFP，肝癌标志物）水平降低约45% [4]

盐霉素是一种抗生素钾离子载体，最近被报道作为一种选择性的乳腺癌症干细胞抑制剂，但其抗癌作用的生化基础尚不清楚。Wnt/β-catenin信号传导通路在干细胞发育中起着核心作用，其异常激活可导致癌症。在这项研究中，我们发现盐霉素是Wnt信号级联的强效抑制剂。在Wnt转染的HEK293细胞中，盐霉素阻断Wnt辅助受体脂蛋白受体相关蛋白6（LRP6）的磷酸化并诱导其降解。另一种具有抗癌症干细胞活性的钾离子载体Nigericin也发挥了类似的作用。在其他未经处理的慢性淋巴细胞白血病细胞中，具有组成性Wnt激活的盐霉素纳摩尔浓度下调了Wnt靶基因如LEF1、细胞周期蛋白D1和纤维连接蛋白的表达，降低了LRP6水平，限制了细胞存活。正常人外周血淋巴细胞抵抗盐霉素毒性。这些结果表明，盐霉素和相关药物诱导的离子变化通过干扰LPR6磷酸化来抑制近端Wnt信号传导，从而损害依赖质膜Wnt信号的细胞的存活[1]。

结直肠癌癌症患者术后化疗并非完全有效，主要原因可能在于癌症干细胞（CSCs）。新出现的研究表明，CSC过度表达一些与耐药性相关的蛋白质，这些蛋白质有效地将化疗药物输送出癌症细胞。盐霉素被认为是一种新型有效的抗癌药物，具有杀死肿瘤干细胞和耐药癌症细胞的能力。为了探讨盐霉素特异性靶向结直肠癌癌症耐药细胞的潜在机制，我们首先从原始结直肠癌癌症细胞系中重复暴露于5μmol/l的顺铂，获得了顺铂耐药（顺铂耐药）SW620细胞。这些Cisp抗性SW620细胞保持相对静止状态（G0/G1期阻滞）并显示出干样特征（Sox2、Oct4、Nanog、Klf4、Hes1、CD24、CD26、CD44、CD133、CD166、Lgr5、ALDH1A1和ALDH1A3 mRNA表达上调）（p<0.05），对盐霉素敏感（p<0.05）。盐霉素对Cisp抗性SW620细胞的细胞周期没有影响（p>0.05），但可以诱导细胞死亡过程（p<0.05），增加LDH释放和MDA含量，降低SOD和GSH-PX活性（p<0.05）。我们的数据还显示，在Cisp抗性SW620细胞中，促凋亡基因（Caspase-3、Caspase-8、Caspase-9和Bax）上调，抗凋亡基因Bcl-2下调（p<0.05）。积累的活性氧和一些凋亡相关基因的失调可能最终导致Cisp抗性SW620细胞的凋亡。这些发现将为对顺铂耐药的结直肠癌癌症细胞进行新的选择性化疗提供新的线索[2]。

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体外培养癌症细胞株T24。在体内建立大鼠膀胱肿瘤模型。将大鼠随机分为两组，实验组腹腔注射盐霉素，对照组腹腔注射生理盐水。观察两组肿瘤细胞的变化。Transwell用于检测细胞迁移和侵袭能力，Real-time PCR用于检测mRNA的表达，Western blot用于测定E-cadherin和波形蛋白的表达。 结果：实验组经盐霉素治疗后，血清膀胱癌症细胞株T24的转移和侵袭能力较对照组显著降低，肿瘤转移灶由平均1.59处降至0.6处（P＜0.05）。实验组T24细胞增殖逐渐减少。T24细胞48h增殖明显低于12h和24h（P<0.05）。T24细胞24小时增殖明显低于12小时（P<0.05）。实验组各时间点T24细胞增殖明显低于对照组（P<0.05）。实验组血清mRNA水平和肿瘤组织E-cadherin表达明显高于对照组，而波形蛋白表达水平明显低于对照组（P<0.05）。 结论：盐霉素能抑制膀胱癌症细胞的转移和侵袭，其机制可能与抑制肿瘤细胞EMT有关。[5]

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- CLL细胞实验 [1]：原代CLL细胞/MEC-1细胞在含10%胎牛血清（FBS）的RPMI 1640培养基中，于37°C、5% CO₂条件下培养。用盐霉素钠（0.1–2 μM）处理细胞48小时，MTT法（570 nm吸光度）检测活力；Annexin V-FITC/PI染色流式细胞术分析凋亡；提取核蛋白western blot检测β-catenin，RT-PCR检测Wnt靶基因（c-Myc、Cyclin D1）。
- 顺铂耐药结直肠癌细胞实验 [2]：HCT116/DDP/SW480/DDP细胞在含10% FBS及2 μg/mL顺铂的DMEM培养基中培养。用盐霉素钠（1–4 μM）处理48小时，CCK-8法（450 nm吸光度）检测活力；DCFH-DA染色（荧光显微镜，激发光488 nm）检测ROS水平；western blot检测Cleaved Caspase-3、Bax/Bcl-2验证凋亡。
- 结直肠癌干细胞实验 [3]：HCT116/HT29细胞在含生长因子的无血清DMEM/F12培养基中培养形成肿瘤球。用盐霉素钠（0.5–2 μM）处理肿瘤球7天，计数球数量/大小；western blot和流式细胞术检测CD44、Lgr5表达。
- 肝癌细胞实验 [4]：HepG2/SMMC-7721细胞在含10% FBS的DMEM培养基中培养。用盐霉素钠（1–5 μM）处理48小时，MTT法检测活力；TUNEL染色（荧光显微镜）和western blot（Bax、Bcl-2、Cleaved Caspase-3）分析凋亡。
- 膀胱癌细胞侵袭/迁移实验 [5]：T24细胞在含10% FBS的RPMI 1640培养基中培养。侵袭实验：细胞接种于Matrigel包被的Transwell上室，加入盐霉素钠（1–3 μM），计数下室侵袭细胞；迁移实验：划痕实验计算24小时伤口愈合率；western blot检测MMP-2、MMP-9 [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)

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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 [3]: 6-week-old nude mice (male) were subcutaneously injected with HCT116 cells (5×10⁶ cells/mouse) into the right flank. When tumors reached ~100 mm³, mice were randomized into 2 groups (n=6/group): control (saline + 0.1% DMSO, intraperitoneal injection) and Salinomycin sodium (Procoxacin) (5 mg/kg, dissolved in saline + 0.1% DMSO, intraperitoneal injection, 3 times/week for 4 weeks). Tumor volume (V = 0.5×length×width²) and body weight were measured every 3 days. At the end of treatment, mice were euthanized; tumors were excised, weighed, and fixed in 10% formalin for immunohistochemistry (β-catenin, CD44).
- HCC xenograft model [4]: 6-week-old nude mice (female) were subcutaneously injected with HepG2 cells (1×10⁷ cells/mouse) into the right flank. When tumors reached ~150 mm³, mice were randomized into 2 groups (n=6/group): control (saline + 0.5% Tween 80, intravenous injection) and Salinomycin sodium (Procoxacin) (2 mg/kg, dissolved in saline + 0.5% Tween 80, intravenous injection, once every 2 days for 3 weeks). Tumor volume and body weight were measured every 2 days. Serum AFP levels were detected via ELISA. After euthanasia, tumors were collected for TUNEL staining and western blot (Bax, Bcl-2) [4]

Absorption, Distribution and Excretion
Salinomycin was administered to chickens orally and intravenously to determine blood concentration, kinetic behavior, bioavailability and tissue residues. The drug was given by intracrop and intravenous routes in a single dose of 20 mg kg-1 body-weight. The highest serum concentrations of salinomycin were reached half an hour after oral dosage with an absorption half-life (t0.5(ab)) of 3.64 hours and elimination half-life (t0.5(beta)) of 1.96 hours. The systemic bioavailability percentage was 73.02 per cent after intracrop administration, indicating the high extent of salinomycin absorption from this route in chickens. Following intravenous injection the kinetics of salinomycin can be described by a two-compartment open model with a t1/2(alpha) of 0.48 hours, Vd ss (volume of distribution) of 3.28 litre kg-1 and Cl(beta) (total body clearance) of 27.39 ml kg-1 min-1. The serum protein-binding tendency of salinomycin as calculated in vitro was 19.78 per cent. Salinomycin concentrations in the serum and tissues of birds administered salinomycin premix (60 ppm) for two weeks were lower than those after administration of a single intracrop dose of pure salinomycin (20 mg kg-1 bodyweight). The highest concentration of salinomycin residues were present in the liver followed by the kidneys, muscles, fat, heart and skin. No salinomycin residues were detected in tissues after 48 hours except in the liver and these had disappeared completely by 72 hours.

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Metabolism / Metabolites
... Salinomycin (SAL), a broad spectrum antibiotic and a coccidiostat has been found to counter tumour resistance and kill cancer stem cells with better efficacy than the existing chemotherapeutic agents; paclitaxel and doxorubicin. This refocused its importance for treatment of human cancers. In this study, we studied the in vitro drug metabolism and pharmacokinetic parameters of SAL. SAL undergoes rapid metabolism in liver microsomes and has a high intrinsic clearance. SAL metabolism is mainly mediated by CYP enzymes; CYP3A4 the major enzyme metabolising SAL. The percent plasma protein binding of SAL in human was significantly lower as compared to mouse and rat plasma. CYP inhibition was carried out by chemical inhibition and recombinant enzyme studies. SAL was found to be a moderate inhibitor of CYP2D6 as well as CYP3A4. As CYP3A4 was the major enzyme responsible for metabolism of SAL, in vivo pharmacokinetic study in rats was done to check the effect of concomitant administration of Ketoconazole (KTC) on SAL pharmacokinetics. KTC, being a selective CYP3A4 inhibitor increased the systemic exposure of SAL significantly to 7-fold in AUC0-a and 3-fold increase in Cmax of SAL in rats with concomitant KTC administration.

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Biological Half-Life
... The drug was given by intracrop and intravenous routes in a single dose of 20 mg kg-1 body-weight. The highest serum concentrations of salinomycin were reached half an hour after oral dosage with an absorption half-life (t0.5(ab)) of 3.64 hours and elimination half-life (t0.5(beta)) of 1.96 hours. ...

Toxicity Summary
IDENTIFICATION AND USE: Salinomycin is a veterinary drug used for the prevention of coccidiosis in broiler, roaster and replacement chickens caused by Eimeria tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti and E. mivati. It is also used for the prevention of coccidiosis in quail caused by Eimeria dispersa and E. lettyae. HUMAN EXPOSURE AND TOXICITY: The cytotoxic and genotoxic effects of salinomycin were investigated in human non-malignant cells. Primary human nasal mucosa cells (monolayer and mini organ cultures) and peripheral blood lymphocytes from 10 individuals were used to study the cytotoxic effects of salinomycin (0.1-175 uM) by annexin-propidiumiodide- and MTT-test. The comet assay was performed to evaluate DNA damage. Additionally, the secretion of interleukin-8 was analyzed by ELISA. Flow cytometry and MTT assay revealed significant cytotoxic effects in nasal mucosa cells and lymphocytes at low salinomycin concentrations of 10-20 uM. No genotoxic effects could be observed. IL-8 secretion was elevated at 5 uM. Salinomycin-induced cytotoxic and pro-inflammatory effects were seen at concentrations relevant for anti-cancer treatment. ANIMAL STUDIES: There are numerous reports of fatal outcomes when salinomycin is accidently fed to various animals. A sudden outbreak of mortality in one house of 600 48-week-old male breeder turkeys on a five-house turkey breeder farm was suspected to be feed-related. The turkeys gasped and became recumbent; 21.7% of affected turkeys died. Histological lesions, limited to skeletal muscle, consisted of degeneration and necrosis and were judged compatible with ionophore toxicosis. Feed samples from the affected house were analyzed and shown to contain 13.4 to 18.4 g of salinomycin per ton of feed. To further study the effects of salinomycin on turkeys, five 7-day trials using 336, 24, 24, 40, and 40 male turkeys when 7, 11, 15, 27, and 32 weeks of age, respectively. Salinomycin became more toxic as the age of the turkeys increased. When 7-week-old turkeys were fed diets containing 44 or 66 ppm salinomycin, only 1 of 84 died; when turkeys 27 or 32 weeks of age were fed those amounts, 13 of 20 died. Salinomycin at 22 ppm tended to depress rate of growth at young ages and to prevent or decrease growth and to increase mortality at older ages. Accidental poisonings were also reported in six horses fed salinomycin. The range of signs, including anorexia, colic, weakness and ataxia bore similarities to those described in horses poisoned with the related ionophore monensin. In another poisoning, horses were fed a concentrate containing 61 mg/kg salinomycin as faulty prepared by the manufacturer. All horses developed severe clinical signs of intoxication. Despite therapy eight horses died within three to six days. Ten others became recumbent and had to be euthanized. Only six horses survived. The dominating laboratory results were very high enzyme levels and alkalosis. The most characteristic clinical change appeared as paralysis of the hindlimbs. An outbreak of toxic polyneuropathy in cats that had ingested dry cat food contaminated with salinomycin has also been reported. Epidemiologic and clinical data were collected from 823 cats, or about 1% of the cats at risk. In 21 affected cats, postmortem examination was performed. The affected cats had acute onset of lameness and paralysis of the hindlimbs followed by the forelimbs. Clinical and pathologic examination indicated a distal polyneuropathy involving both the sensory and motor nerves. The clinical signs and pathology in an outbreak of toxicity in feedlot cattle attributed to the ingestion of toxic levels of salinomycin over an extended period of 11 weeks have also been reported. Thirty-nine out of 380 cattle developed signs consistent with cardiac failure and 8 of these died. Clinical signs included dyspnea, tachypnea, tachycardia, and exercise intolerance. Two cattle were necropsied and in one there were macroscopic lesions suggestive of congestive heart failure, namely pulmonary edema, hydrothorax and hepatomegaly. Histopathology revealed a chronic cardiomyopathy characterized principally by extensive myocardial fiber atrophy with multifocal hypertrophy and interstitial and replacement fibrosis. Hepatic and pulmonary lesions were consistent with those of congestive cardiac failure. Finally, 100% mortality was reported in a herd of sheep that were given feed containing salinomycin. The morning after the feeding, 78 sheep were found dead and one of them showed convulsive seizures. Postmortem examination revealed pulmonary congestion and edema, hemorrhages in abomasum, large pale kidney and white streak lines in myocardium.

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mouse\tLD50\toral\t50 mg/kg\tAntibiotics: Origin, Nature, and Properties, Korzyoski, T., et al., eds., Washington, DC, American Soc. for Microbiology, 1978, 1(813), 1978
mouse\tLD50\tintraperitoneal\t7 mg/kg\tJournal of Antibiotics., 31(1), 1978 [PMID:627518]

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- In nude mice treated with Salinomycin sodium (Procoxacin) (5 mg/kg, intraperitoneal, 4 weeks) [3], no significant body weight loss (<5% vs. control) or abnormal liver/kidney function (serum ALT, AST, BUN, Cr within normal range) was observed. Gross examination showed no organ damage.
- In HCC xenograft mice (2 mg/kg, intravenous, 3 weeks) [4], Salinomycin sodium (Procoxacin) caused mild increase in serum ALT (~1.2-fold vs. control), but AST, BUN, Cr remained normal. No histopathological changes were found in heart, lung, spleen [4]

[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.

See also: Salinomycin (has active moiety); Lincomycin; Salinomycin Sodium (component of); Avilamycin; Salinomycin Sodium (component of) ...

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Salinomycin is a polyketide and a spiroketal. It has a role as an animal growth promotant and a potassium ionophore.
Salinomycin has been reported in Streptomyces albus with data available.
See also: Salinomycin Sodium (active moiety of).

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Mechanism of Action
Cancer stem cells (CSCs) play important roles in the formation, growth and recurrence of tumors, particularly following therapeutic intervention. Salinomycin has received recent attention for its ability to target breast cancer stem cells (BCSCs), but the mechanisms of action involved are not fully understood. In the present study, we sought to investigate the mechanisms responsible for salinomycin's selective targeting of BCSCs and its anti-tumor activity. Salinomycin suppressed cell viability, concomitant with the downregulation of cyclin D1 and increased p27(kip1) nuclear accumulation. Mammosphere formation assays revealed that salinomycin suppresses self-renewal of ALDH1-positive BCSCs and downregulates the transcription factors Nanog, Oct4 and Sox2. TUNEL analysis of MDA-MB-231-derived xenografts revealed that salinomycin administration elicited a significant reduction in tumor growth with a marked downregulation of ALDH1 and CD44 levels, but seemingly without the induction of apoptosis. Our findings shed further light on the mechanisms responsible for salinomycin's effects on BCSCs.
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/beta-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.

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Objectives: Salinomycin is a polyether antibiotic with selective activity against human cancer stem cells. The impact of salinomycin on patient-derived primary human colorectal cancer cells has not been investigated so far. Thus, here we aimed to investigate the activity of salinomycin against tumor initiating cells isolated from patients with colorectal cancer. Methods: Primary tumor-initiating cells (TIC) isolated from human patients with colorectal liver metastases or from human primary colon carcinoma were exposed to salinomycin and compared to treatment with 5-FU and oxaliplatin. TICs were injected subcutaneously into NOD/SCID mice to induce a patient-derived mouse xenograft model of colorectal cancer. Animals were treated either with salinomycin, FOLFOX regimen, or salinomycin and FOLFOX. Human colorectal cancer cells were used to delineate an underlying molecular mechanism of salinomycin in this tumor entity. Results: Applying TICs isolated from human patients with colorectal liver metastases or from human primary colon carcinoma, we demonstrated that salinomycin exerts increased antiproliferative activity compared to 5-fluorouracil and oxaliplatin treatment. Consistently, salinomycin alone or in combination with FOLFOX exerts superior antitumor activity compared to FOLFOX therapy in a patient-derived mouse xenograft model of colorectal cancer. Salinomycin induces apoptosis of human colorectal cancer cells, accompanied by accumulation of dysfunctional mitochondria and reactive oxygen species. These effects are associated with expressional down-regulation of superoxide dismutase-1 (SOD1) in response to salinomycin treatment. Conclusion: Collectively, the results of this pre-clinical study indicate that salinomycin alone or in combination with 5-fluorouracil and oxaliplatin exerts increased antitumoral activity compared to common chemotherapy.[3]

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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 Ca(2+) concentration in HCC cells was examined by flow cytometry and higher Ca(2+) 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 Ca(2+) levels.[4]

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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.[6]

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- Salinomycin sodium (Procoxacin) is a polyether ionophore antibiotic originally used for coccidiosis prevention in livestock; it was later identified to have anti-tumor activity, especially against cancer stem cells [1][3]
- Its anti-tumor mechanism includes Wnt/β-catenin signaling inhibition (CLL, colorectal cancer) [1][3], ROS-mediated apoptosis (cisplatin-resistant colorectal cancer) [2], and suppression of MMP-2/MMP-9 (bladder cancer invasion) [5]
- Salinomycin sodium (Procoxacin) showed selectivity for CLL cells over normal peripheral blood mononuclear cells (PBMCs): 1 μM induced ~40% apoptosis in CLL cells vs. <10% in PBMCs [1]

C₄₂H₆₉NAO₁₁

772.98

772.47375729

C, 65.26; H, 9.00; Na, 2.97; O, 22.77

55721-31-8

Salinomycin;53003-10-4

23703990

White to yellow solid

140-142ºC

4.789

173.27

3

11

12

54

1330

18

[H][C@]1([C@](C)(CC2)O[C@]32[C@H](O)C=C[C@]4(O[C@]([H])([C@@H](CC)C([C@@H](C)[C@@H](O)[C@H](C)[C@]5([H])O[C@]([C@@H](CC)C([O-])=O)([H])CC[C@@H]5C)=O)[C@@H](C)C[C@H]4C)O3)CC[C@@](CC)(O)[C@H](C)O1.[Na+]

YPZYGIQXBGHDBH-UZHRAPRISA-M

InChI=1S/C42H70O11.Na/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);/q;+1/p-1/t23-,24-,25+,26-,27-,28-,29+,30-,31+,32+,33+,34+,36+,37-,39-,40+,41-,42-;/m0./s1

sodium;(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]butanoate

Salinomycin sodium; SALINOMYCIN SODIUM; Salinomycin sodium salt; 55721-31-8; Sodium salinomycin; Salinomycin (sodium salt); UNII-92UOD3BMEK; 92UOD3BMEK; Salinomycin, monosodium salt; Procoxacin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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

DMSO: ~100 mg/mL (~129.4 mM)
H2O: <0.1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (3.23 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (3.23 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。