RESUMEN
A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.
Asunto(s)
Aldehídos/química , Glucemia/metabolismo , Insulina/química , Insulina/metabolismo , Acilación , Animales , Glucemia/efectos de los fármacos , Células CHO , Cricetulus , Humanos , Hidrazonas/química , Insulina/farmacología , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Tiazolidinas/químicaRESUMEN
Tumors are phenotypically heterogeneous and include subpopulations of cancer cells with stemlike properties. The natural product salinomycin, a K+-selective ionophore, was recently found to exert selectivity against such cancer stem cells. This selective effect is thought to be due to inhibition of the Wnt signaling pathway, but the mechanistic basis remains unclear. Here, we develop a functionally competent fluorescent conjugate of salinomycin to investigate the molecular mechanism of this compound. By subcellular imaging, we demonstrate a rapid cellular uptake of the conjugate and accumulation in the endoplasmic reticulum (ER). This localization is connected to induction of Ca2+ release from the ER into the cytosol. Depletion of Ca2+ from the ER induces the unfolded protein response as shown by global mRNA analysis and Western blot analysis of proteins in the pathway. In particular, salinomycin-induced ER Ca2+ depletion up-regulates C/EBP homologous protein (CHOP), which inhibits Wnt signaling by down-regulating ß-catenin. The increased cytosolic Ca2+ also activates protein kinase C, which has been shown to inhibit Wnt signaling. These results reveal that salinomycin acts in the ER membrane of breast cancer cells to cause enhanced Ca2+ release into the cytosol, presumably by mediating a counter-flux of K+ ions. The clarified mechanistic picture highlights the importance of ion fluxes in the ER as an entry to inducing phenotypic effects and should facilitate rational development of cancer treatments.
RESUMEN
The polyether ionophore salinomycin has recently captured much interest due to its potent activity against multi-drug resistant cancer cells and cancer stem cells. Previous studies have shown that either acylation of the C20 position or esterification/amidation of the C1 carboxylate moiety is beneficial in terms of biological properties. In this paper, we present the first analogs combining such modifications. Evaluation of the anti-proliferative activity against a series of cancer cell lines showed that acylation of the C20 hydroxyl group improves the activity of salinomycin C1 amides but not of the corresponding C1 esters. Importantly, the activity of several of the doubly modified analogs surpasses that of commonly used cytostatic drugs cisplatin and doxorubicin in the LoVo/DX multi-drug resistant cell line. All analogs were tested against primary acute lymphoblastic leukemia cells in standard cell viability assays; three were more potent than salinomycin. Further studies revealed that selected analogs induced characteristics of apoptotic cell death and increased expression of p53. Additionally, using an ex vivo model of breast tumor, tumor cell viability significantly decreased after treatment with salinomycin or its double-modified derivative (3a) in a time-dependent manner. The present findings indicate that double-modified salinomycin derivatives constitute promising lead compounds for targeting various types of cancer.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Piranos/química , Piranos/farmacología , Anciano , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Células Tumorales CultivadasRESUMEN
Salinomycin, a polyether antibiotic, is a well-known inhibitor of human cancer stem cells. Chemical modification of the allylic C20 hydroxyl of salinomycin has enabled access to synthetic analogs that display increased cytotoxic activity compared to the native structure. The aim of this study was to investigate the activity of a cohort of C20-O-acyl analogs of salinomycin on human colorectal cancer cell lines in vitro. Two human colorectal cancer cell lines (SW480 and SW620) were exposed to three C20-O-acylated analogs and salinomycin. The impact of salinomycin and its analogs on tumor cell number, migration, cell death, and cancer stem cell specifity was analyzed. Exposure of human colorectal cancer cells to the C20-O-acylated analogs of salinomycin resulted in reduced tumor cell number and impaired tumor cell migration at lower concentrations than salinomycin. When used at higher (micromolar) concentrations, these effects were accompanied by induction of apoptotic cell death. Salinomycin analogs further expose improved activity against cancer stem cells compared to salinomycin.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Piranos/farmacología , Acilación , Antibióticos Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Humanos , Células Madre Neoplásicas/patología , Piranos/químicaRESUMEN
The ionophore salinomycin has attracted attention for its exceptional ability to selectively reduce the proportion of cells with stem-like properties in cancer cell populations of varying origin. Targeting the tumorigenicity of such cells is of interest as they are implicated in recurrence, metastasis, and drug resistance. Structural derivatives of salinomycin are thus sought after, both as tools for probing the molecular mechanism(s) underlying the observed phenotype effects, and for improving selectivity and activity against cancer stem cells. Synthetic strategies for modification of each of the directly accessible functional groups of salinomycin are presented and the resulting library of analogues was investigated to establish structure-activity relationships, both with respect to cytotoxicity and phenotype selectivity in breast cancer cells. 20-O-Acylated derivatives stand out by exhibiting both improved selectivity and activity. Mechanistically, the importance of the ionophore properties of salinomycin is highlighted by a significant loss of activity by modifications directly interfering with either of the two primary ion coordinating motifs in salinomycin, the C11 ketone and the C1 carboxylate.
RESUMEN
The polyether ionophore salinomycin has recently gained attention due to its exceptional ability to selectively reduce the proportion of cancer stem cells within a number of cancer cell lines. Efficient single step strategies for the preparation of hydroxamic acid hybrids of this compound varying in N- and O-alkylation are presented. The parent hydroxamic acid, salinomycin-NHOH, forms both inclusion complexes and well-defined electroneutral complexes with potassium and sodium cations via 1,3-coordination by the hydroxamic acid moiety to the metal ion. A crystal structure of an cationic sodium complex with a noncoordinating anion corroborates this finding and, moreover, reveals a novel type of hydrogen bond network that stabilizes the head-to-tail conformation that encapsulates the cation analogously to the native structure. The hydroxamic acid derivatives display down to single digit micromolar activity against cancer cells but unlike salinomycin selective reduction of ALDH(+) cells, a phenotype associated with cancer stem cells was not observed. Mechanistic implications are discussed.
RESUMEN
BACKGROUND: Cancer stem cells (CSCs) have been invoked in resistance, recurrence and metastasis of cancer. Consequently, curative cancer treatments may be contingent on CSC selective approaches. Of particular interest in this respect is the ionophore salinomycin, a natural product shown to be 100-fold more active against CSCs than clinically used paclitaxel. We have previously reported that synthetic salinomycin derivatives display increased activity against breast cancer cell lines. Herein we specifically investigate the CSC selectivity of the most active member in each class of C20-O-acylated analogs as well as a C1-methyl ester analog incapable of charge-neutral metal ion transport. METHODS: JIMT-1 breast cancer cells were treated with three C20-O-acylated analogs, the C1-methyl ester of salinomycin, and salinomycin. The effects of treatment on the CSC-related CD44(+)/CD24(-) and the aldehyde dehydrogenase positive (ALDH(+)) populations were determined using flow cytometry. The survival ability of CSCs after treatment was investigated with a colony formation assay under serum free conditions. The effect of the compounds on cell migration was evaluated using wound-healing and Boyden chamber assays. The expression of vimentin, related to mesenchymal traits and expression of E-cadherin and ß-catenin, related to the epithelial traits, were investigated using immunofluorescence microscopy. RESULTS: Treatment with each of the three C20-acylated analogs efficiently decreased the putative CSC population as reflected by reduction of the CD44(+)/CD24(-) and ALDH(+) populations already at a 50 nM concentration. In addition, colony forming efficiency and cell migration were reduced, and the expression of the epithelial markers E-cadherin and ß-catenin at the cell surface were increased. In contrast, salinomycin used at the same concentration did not significantly influence the CSC population and the C1-methyl ester was inactive even at a 20 µM concentration. CONCLUSIONS: Synthetic structural analogs of salinomycin, previously shown to exhibit increased activity against cancer cells, also exhibited improved activity against CSCs across several assays even at nanomolar concentrations where salinomycin was found inactive. The methyl ester analog of salinomycin, incapable of charge-neutral metal ion transport, did not show activity in CSC assays, lending experimental support to ionophoric stress as the molecular initiating event for the CSC effects of salinomycin and related structures.
Asunto(s)
Neoplasias de la Mama/patología , Ionóforos/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Ionóforos/química , Células Madre Neoplásicas/metabolismo , Piranos/química , Vimentina/metabolismo , beta Catenina/metabolismoRESUMEN
Salinomycin, a naturally occurring polyether ionophore was recently found to selectively reduce the proportion of CD44(+)/CD24(-) cells, a phenotype associated with breast cancer stem cells. Subsequent studies from our group showed that chemical modification of the allylic C20 hydroxyl of salinomycin, located at the C-ring, can enhance the activity of derivatives against breast cancer cells over 5-fold compared to the native structure. Access to C-ring-modified salinomycin analogues is thus of interest from both a mechanistic and a synthetic perspective. Here, we report efficient strategies for gram scale synthesis of the natural product SY-1 (20-deoxy salinomycin), and a saturated analogue, 18,19-dihydro SY-1, for a comparative in vitro investigation of the biological profiles of these compounds with that of salinomycin. Across several assays, the deoxygenated structures required higher concentrations to elicit similar cellular responses to that of salinomycin. Similarly to salinomycin, SY-1 or 18,19-dihydro SY-1 treatment was found to reduce the proportion of CD44(+)/CD24(-) cells with essentially complete selectivity up to â¼IC25. Importantly, the proportion of CD44(+)/CD24(-) cells showed a pronounced U-shaped dose response curve for salinomycin and its derivatives, but not for paclitaxel. The concentration for maximum response in this assay followed differences in IC50 for salinomycin and its analogues, which emphasizes the importance of taking concentration dependence into account when comparing effects on the CD44(+)/CD24(-) phenotype. Small differences in the global conformation within the triad of compounds investigated together with differences in activity across assays emphasize the importance of substitution at C20 for the activity of salinomycin and its derivatives.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Ionóforos/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Piranos/farmacología , Antineoplásicos/química , Mama/efectos de los fármacos , Mama/patología , Neoplasias de la Mama/patología , Antígeno CD24/análisis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Receptores de Hialuranos/análisis , Ionóforos/química , Modelos Moleculares , Células Madre Neoplásicas/patología , Piranos/químicaRESUMEN
Salinomycin has found renewed interest as an agent for prevention of cancer recurrence through selectively targeting cancer stem cells. Strategies for generation of improved salinomycin analogs by individual modification of its hydroxyl groups are presented. An evaluation of the dose-response effects of the resulting library on breast cancer cell lines shows that acylation of the C20 hydroxyl can be used to improve IC50 values down to one fifth that of salinomycin.