salinomycin and Osteosarcoma

The growth factor receptor-bound protein 2 (Grb2)-Sos1 interaction, mediated by modular domains, plays an essential role in the oncogenic MAPK signaling pathway in osteosarcoma (OS). Recently, a dual-targeting peptide that targets the epidermal growth factor receptor and Grb2-Src homology 3 domain in OS cells was designed and synthesized.. We investigated the synergistic effects of the peptide and salinomycin (Sal), a chemotherapeutic drug with effective anti-OS properties in clinical therapy.. Flow cytometry was used to measure the targeting efficacy of the peptide. Migration and CCK-8 assays were used to explore whether Sal and the peptide could synergistically inhibit OS cell behavior. Western blotting was used to detect apoptosis.. Data were analyzed using the GraphPad Prism 5.01. Statistical analysis was performed using the Student's t-test for the direct comparisons and one-way analysis of variance for the comparisons among the multiple groups. Statistical significance was set at P < 0.05.. The peptide was shown to target OS cells. When applied together, Sal and the peptide synergistically inhibited OS cell migration, invasion, and proliferation through the inhibition of Grb2-Sos1. This synergistic treatment also promoted the apoptosis of OS cells and inhibited tumor volume in vivo.. These data provide valuable insights into the molecular mechanisms of OS and may be beneficial in clinical therapy.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Gene Expression Regulation, Neoplastic; GRB2 Adaptor Protein; Humans; Osteosarcoma; Peptides; Protein Kinase Inhibitors; Pyrans

A new straightforward gel permeation chromatography (GPC) method was developed to calculate the drug encapsulation efficiency and loading content of Poly(lactic acid) nanoparticles (PLA NPs) loaded with Salinomycin (Sal), exploiting the capability of this technique to separate a macromolecular/molecular mixture on the basis of the molecular weight of each component. The proposed GPC method allowed Sal detection until 1% of Sal content in PLA NPs, avoiding sample pre-treatments. The method was validated by wave voltammetry (SW) technique, using a slightly modified literature procedure, useful to detect Sal in the concentration range 0.4 ≤ C/μmol/L ≤ 12 (linear concentration range). PLA-based NPs were prepared by nanoprecipitation with either native and functionalized PLA. Specifically, folate-decorated PLA NPs (PLA-FA NPs) were obtained by CuAAC click functionalization of alkyne-grafted PLA with azide-folate. Sal-loaded NPs were characterized physicochemically and morphologically. They exhibited adequate physicochemical properties, good drug encapsulation efficiency (98 ± 0.5% and 99 ± 0.5%), and loading content (8.8 ± 0.1% and 8.9 ± 0.1% for PLA/Sal and PLA-FA/Sal NPs, respectively). The size of empty PLA NPs resulted smaller (90 ± 3.2 nm and 680 ± 15.3 nm, for PLA NPs and PLA-FA NPs respectively) than the correspondent drug-loaded NPs (110 ± 3.8 nm and 875 ± 20.5 nm, respectively). Their biological activity was assessed on osteosarcoma bulk cells MG63, healthy osteoblast cell line (hFOB1.19), and enriched osteosarcoma cancer stem cells (CSCs), showing cell-depending effect. Entrapped Sal maintained its cytotoxic effect on CSCs and MG63 cells, with a potency comparable to the free drug and no evident benefit was detected for folate-decorated PLA NPs respect to native PLA NPs. Graphical abstract.

Topics: Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Chromatography; Drug Carriers; Humans; Nanoparticles; Osteosarcoma; Polyesters; Pyrans

We previously developed salinomycin (sali)-entrapped nanoparticles labeled with CD133 aptamers which could efficiently eliminate CD133

Topics: AC133 Antigen; Animals; Anti-Bacterial Agents; Apoptosis; Aptamers, Nucleotide; Bone Neoplasms; Cell Proliferation; Drug Delivery Systems; ErbB Receptors; Female; Humans; Lipids; Mice; Mice, Nude; Nanoparticles; Neoplastic Stem Cells; Osteosarcoma; Polymers; Pyrans; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore. It was reported to anticancer activity on various cancer cell lines. In this study, salinomycin was examined on apoptosis and autophagy through generation of reactive oxygen species (ROS) in osteosarcoma U2OS cells. Apoptosis, autophagy, mitochondrial membrane potential (MMP) and ROS were analyzed using flow cytometry. Also, expressions of apoptosis- and autophagy-related proteins were determined by western blotting. As a result, salinomycin triggered apoptosis of U2OS cells, which was accompanied by change of MMP and cleavage of caspases-3 and poly (ADP-ribose) polymerase. And salinomycin increased the expression of autophagy-related protein and accumulation of acidic vesicular organelles (AVO). Salinomycin-induced ROS production promotes both apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-l-cysteine (NAC), a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of autophagy by 3-methyladenine (3 MA) enhanced the salinoymcin-induced apoptosis. Taken together, these results suggested that salinomycin-induced autophagy, as a survival mechanism, might be a potential strategy through ROS regulation in cancer therapy.

Topics: Antibiotics, Antineoplastic; Apoptosis; Autophagy; Bone Neoplasms; Cell Line, Tumor; Humans; Ionophores; Osteosarcoma; Pyrans; Reactive Oxygen Species

Malignant osteosarcoma (OS) is still a deadly disease for many affected patients. The search for the novel anti-OS agent is extremely urgent and important. Our previous study has proposed that salinomycin is a novel anti-OS agent. Here we characterized DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a primary salinomycin resistance factor in OS cells. DNA-PKcs inhibitors (NU7026, NU7441 and LY294002) or DNA-PKcs shRNA knockdown dramatically potentiated salinomycin-induced death and apoptosis of OS cells (U2OS and MG-63 lines). Further, forced-expression of microRNA-101 ("miR-101") downregulated DNA-PKcs and augmented salinomycin's cytotoxicity against OS cells. Reversely, over-expression of DNA-PKcs in OS cells inhibited salinomycin's lethality. For the mechanism study, we show that DNA-PKcs is required for salinomycin-induced pro-survival autophagy activation. DNA-PKcs inhibition (by NU7441), shRNA knockdown or miR-101 expression inhibited salinomycin-induced Beclin-1 expression and autophagy induction. Meanwhile, knockdown of Beclin-1 by shRNA significantly sensitized salinomycin-induced OS cell lethality. In vivo, salinomycin administration suppressed U2OS xenograft tumor growth in severe combined immuno-deficient (SCID) mice, and its anti-tumor activity was dramatically potentiated with co-administration of the DNA-PKcs inhibitor NU7026. Together, these results suggest that DNA-PKcs could be a primary resistance factor of salinomycin in OS cells. DNA-PKcs inhibition or silence may thus significantly increase salinomycin's sensitivity in OS cells.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; DNA-Activated Protein Kinase; Drug Resistance, Neoplasm; Drug Synergism; Humans; Mice; MicroRNAs; Morpholines; Neoplasm Transplantation; Nuclear Proteins; Osteosarcoma; Pyrans

Cancer stem cells (CSCs) possess the characteristics associated with normal stem cells and are responsible for cancer initiation, recurrence, and metastasis. CD133 is regarded as a CSCs marker of osteosarcoma, which is the most common primary bone malignancy in childhood and adolescence. Salinomycin, a polyether ionophore antibiotic, has been shown to kill various CSCs, including osteosarcoma CSCs. However, salinomycin displayed poor aqueous solubility that hinders its clinical application. The objective of this study was to develop salinomycin-loaded nanoparticles to eliminate CD133(+) osteosarcoma CSCs.. The salinomycin-loaded PEGylated poly(lactic-co-glycolic acid) nanoparticles (SAL-NP) conjugated with CD133 aptamers (Ap-SAL-NP) were developed by an emulsion/solvent evaporation method, and the targeting and cytotoxicity of Ap-SAL-NP to CD133(+) osteosarcoma CSCs were evaluated.. The nanoparticles are of desired particle size (~150 nm), drug encapsulation efficiency (~50%), and drug release profile. After 48 hours treatment of the Saos-2 CD133(+) osteosarcoma cells with drugs formulated in Ap-SAL-NP, SAL-NP, and salinomycin, the concentrations needed to kill 50% of the incubated cells were found to be 2.18, 10.72, and 5.07 μg/mL, respectively, suggesting that Ap-SAL-NP could be 4.92 or 2.33 fold more effective than SAL-NP or salinomycin, respectively. In contrast, Ap-SAL-NP was as effective as SAL-NP, and less effective than salinomycin in Saos-2 CD133(-) cells, suggesting that Ap-SAL-NP possess specific cytotoxicity toward Saos-2 CD133(+) cells. Ap-SAL-NP showed the best therapeutic effect in Saos-2 osteosarcoma xenograft mice, compared with SAL-NP or salinomycin. Significantly, Ap-SAL-NP could selectively kill CD133(+) osteosarcoma CSCs both in vitro and in vivo, as reflected by the tumorsphere formation and proportion of Saos-2 CD133(+) cells.. Our results suggest that CD133 is a potential target for drug delivery to osteosarcoma CSCs and that it is possible to significantly inhibit the osteosarcoma growth by killing CD133(+) osteosarcoma CSCs. We demonstrated that Ap-SAL-NP have the potential to target and kill CD133(+) osteosarcoma CSCs.

Topics: AC133 Antigen; Animals; Antigens, CD; Aptamers, Nucleotide; Cell Line, Tumor; Glycoproteins; Lactic Acid; Mice; Nanoparticles; Neoplastic Stem Cells; Osteosarcoma; Peptides; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Pyrans; Rats; Xenograft Model Antitumor Assays

Osteosarcoma is the most common primary bone tumor in children and adolescents and is typically associated with a poor prognosis. Tumor stem cells (TSCs) are presumed to drive tumor initiation and tumor relapse or metastasis. Hence, the poor prognosis of osteosarcoma likely results from a failure to target the osteosarcoma stem cells. Here, we have utilized three different methods to enrich TSCs in osteosarcoma and further evaluated whether salinomycin could selectively target TSCs in osteosarcoma. Our results indicated that sarcosphere selection, chemotherapy selection and stem cell marker OCT4 or SOX2 over-expression are all effective in the enrichment of TSCs from osteosarcoma cell lines. Further investigation found that salinomycin inhibited osteosarcoma by selectively targeting its stem cells both in vitro and in vivo without severe side effects, and the Wnt/β-catenin signaling pathway may be involved in this inhibition of salinomycin. Taken together, we have identified that salinomycin is an effective inhibitor of osteosarcoma stem cells, supporting the use of salinomycin for elimination of osteosarcoma stem cells and implying a need for further clinical evaluation.

Topics: Bone Neoplasms; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Humans; Immunohistochemistry; Neoplastic Stem Cells; Octamer Transcription Factor-3; Osteosarcoma; Pyrans; SOXB1 Transcription Factors; Wnt Signaling Pathway