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Two ruthenium(II) polypyridyl complexes were prepared with the {Ru(phen)2}2+ moiety and a third sterically non-hindering bidentate ligand, namely 2,2′-dipyridylamine (dpa) and N-benzyl-2,2′-dipyridylamine (Bndpa). Hence, complexes [Ru(phen)2(dpa)](PF6)2 (1) and [Ru(phen)2(Bndpa)](PF6)2 (2) were characterized and their photochemical behaviour in solution (acetonitrile and water) was subsequently investigated. Compounds 1 and 2, which do not exhibit notably distorted octahedral coordination environments, contrarily to the homoleptic “parent” compound [Ru(phen)3](PF6)2, experience two-step photoejection of the dpa and Bndpa ligand upon irradiation (1050–430 nm) for several hours. DNA-binding studies revealed that compounds 1 and 2 affect the biomolecule differently upon irradiation; while 2 solely modifies its electrophoretic mobility, complex 1 is also capable of cleaving it. In vitro cytotoxicity studies with two cancer-cell lines, namely A549 (lung adenocarcinoma) and A375 (melanoma), showed that both 1 and 2 are not toxic in the dark, while only 1 is significantly cytotoxic if irradiated, 2 remaining non-toxic under these conditions. Graphical abstract Light irradiation of the complex cation [Ru(phen)2(dpa)]2+ leads to the generation of transient Ru species that is present in the solution medium for several hours, and that is significantly cytotoxic, ultimately producing non-toxic free dpa and [Ru(phen)(OH2)2]2+.

QIAGEN Cancer Drug Targets Array file containing the raw data (Ct information) for the qPCR of all the conditions (both A549 and SW900 cell lines).

Supplementary Figures: Figure S1. Set of tambjamine analogues investigated; Figure S2. General reaction scheme; Figure S3. 1H NMR (DMSO-d6) of compound 3.HCl; Figure S4. 13C (DMSO-d6) of compound 3.HCl; Figure S5. HRMS (EI) of compound 3; Figure S6. 1H NMR (DMSO-d6) of compound 4.HCl; Figure S7. 13C (DMSO-d6) of compound 4.HCl; Figure S8. 13C dept (DMSO-d6) of compound 4.HCl; Figure S9. HRMS (EI) of compound 4.; Figure S10. 1H NMR (CDCl3) of compound 5.HCl; Figure S11. 13C NMR (CDCl3) of compound 5.HCl; Figure S12. 13C dept NMR (CDCl3) of compound 5.HCl; Figure S13. HRMS (EI) of compound 5.; Figure S14. 1H NMR (CDCl3) of compound 6.HCl; Figure S15. 13C NMR (CDCl3) of compound 6.HCl; Figure S16. HRMS (EI) of compound 6.; Figure S17. 1H NMR (CDCl3) of compound 7.HCl; Figure S18. 13C NMR (CDCl3) of compound 7.HCl; Figure S19. 13C dept NMR (CDCl3) of compound 7.HCl; Figure S20. HRMS (EI) of compound 7.; Figure S21. 1H NMR (CDCl3) of compound 8.HCl; Figure S22. 13C NMR (CDCl3) of compound 8.HCl; Figure S23. 13C dept NMR (CDCl3) of compound 8.HCl; Figure S24. HRMS (EI) of compound 8.; Figure S25. 1H NMR (CDCl3) of compound 9.HCl; Figure S26. 13C NMR (CDCl3) of compound 9.HCl; Figure S27. 13C dept NMR (CDCl3) of compound 9.HCl; Figure S28. HRMS (EI) of compound 9.; Figure S29. 1H NMR (CDCl3) of compound 10.HCl; Figure S30. 13C NMR (CDCl3) of compound 10.HCl; Figure S31. 13C dept NMR (CDCl3) of compound 10.HCl; Figure S32. HRMS (EI) of compound 10; Figure S33. 1H NMR (CDCl3) of compound 11.HOAc; Figure S34. 13C NMR (CDCl3) of compound 11.HOAc; Figure S35. 13C dept NMR (CDCl3) of compound 11.HOAc; Figure S36. HRMS (EI) of compound 11; Figure S37. Dose response curves in A549, DMS53, SW900 and H460 cells; Figure S38. Dose response curves in human lung tumor-derived primay cultures; Figure S39. Array validation using TaqMan gene expression assays; Figure S40. Cell cycle assay by flow cytometry; Figure S41. Western blot of apoptotic markers in the DMS53 cell line; Figure S42. Body weight recording; Supplementary Tables: Table S1. Applied biosystem probes list used for the TaqMan gene expression assays; Table S2. List of altered genes in the cancer drug targets QIAGEN RT2 Profiler Array for the SW900 cell line; Supplementary Materials and Methods: Synthesis and characterization; Cell viability; TaqMan assays; Gene expression; Cell cycle; Western blot; Mice weight.

Five metal-arene complexes of formula [MX2(η6-p-cymene)(diR(1-pyrenyl)phosphane)] (M = Os or Ru, X = Cl or I, R = isopropyl or phenyl) and symbolized as M_X2^R were synthesized and fully characterized,...

We have recently reported a series of piano-stool ruthenium(II) complexes of the general formula [RuCl2(η6-arene)(P(1-pyrenyl)R2R3)] showing excellent cytotoxic activities (particularly when R2 = R3 = methyl). In the present study, new members of this family of compounds have been prepared with the objective to investigate the effect of the steric hindrance of a bulky phosphane ligand, namely diisopropyl(1-pyrenyl)phosphane (L), on exchange reactions involving the coordinated halides (X = Cl, I). Two η6-arene rings were used, i.e. η6-methyl benzoate (mba) and η6-p-cymene (p-cym), and four complexes were synthesized, namely [RuCl2(mba)(L)] (1Cl2iPr), [RuI2(mba)(L)] (1I2iPr), [RuCl2(p-cym)(L)] (2Cl2iPr), and [RuI2(p-cym)(L)] (2I2iPr). Unexpectedly, all of the complexes exhibited poor cytotoxic activities after 24 h of incubation with cells, in contrast to the related compounds previously reported. However, it was observed that aged DMSO solutions of 2I2iPr (from 2 to 7 days) exhibited better activities in comparison to freshly prepared solutions and that the activity improved over “aging” time. Thorough studies were therefore performed to uncover the origin of this lag time in the cytotoxicity efficiency. The data achieved clearly demonstrated that compounds 2I2iPr and 2Cl2iPr were undergoing a series of transformation reactions in DMSO (with higher rates for the iodido complex 2I2iPr), ultimately generating cyclometalated species through a mechanism involving DMSO as a coordinated proton abstractor. The cyclometalated complexes detected in solution were subsequently prepared; hence, pure [RuCl(p-cym)(κ2C-diisopropyl(1-pyrenyl)phosphane)] (3CliPr), [RuI(p-cym)(κ2C-diisopropyl(1-pyrenyl)phosphane)] (3IiPr), and [Ru(p-cym)(κS-dmso)(κ2C-diisopropyl(1-pyrenyl)phosphane)]PF6 (3dmsoiPr) were synthesized and fully characterized. Remarkably, 3CliPr, 3IiPr, and 3dmsoiPr are all very efficient cytotoxic agents, exhibiting slightly better activities in comparison to the chlorido noncyclometalated complexes [RuCl2(η6-arene)(P(1-pyrenyl)R2R3)] described in an earlier report. For comparison purposes, the iodido compounds [RuI2(mba)(dimethyl(1-pyrenyl)phosphane)] (1I2Me) and [RuI2(p-cym)(dimethyl(1-pyrenyl)phosphane)] (2I2Me), bearing the less hindered dimethyl(1-pyrenyl)phosphane ligand, have also been prepared. The cytotoxic and chemical behaviors of 1I2Me and 1I2Me were comparable to those of their chlorido counterparts reported previously., Financial support from the Spanish Ministerio de Ciencia Innovación, y Universidades (Project Nos. CTQ2015-65040-P, RED2018-102471-T, PID2019-107006GB-C21, PGC2018-098630-B-I00, and CTQ2017-88446-R AEI/FEDER, UE) and from the Instituto de Salud Carlos III (ISCIIIFIS PI18/00441, FEDER) is acknowledged. A.G. thanks the Royal Society of Chemistry for financial support (RSC Research Fund grant RF19-7147). J.C. thanks the Spanish MICINN for a Ramoń y Cajal research contract (RYC2018-024692-I) and the Spanish Structures of Excellence María de Maeztu program (MDM-2017-0767). P.G. acknowledges the Institució Catalana de Recerca i Estudis Avançats (ICREA).

Overcoming resistance is one of the most challenging features in current anticancer therapy. Autophagy is a cellular process that confers resistance in some advanced tumors, since it enables cancer cells to adapt to stressful situations, such as anticancer treatments. Hence, the inhibition of this cytoprotective autophagy leads to tumor cells sensitization and death. In this regard, we designed a novel potent anionophore compound that specifically targets lysosomes, called LAI-1 (late-stage autophagy inhibitor-1), and evaluated its role in blocking autophagy and its potential anticancer effects in three lung cancer cell lines from different histological subtypes. Compared to other autophagy inhibitors, such as chloroquine and 3-Methyladenine, the LAI-1 treatment induced more potent anticancer effects in all tested cancer cells. LAI-1 was able to efficiently target and deacidify lysosomes, while acidifying cytoplasmic pH. Consequently, LAI-1 efficiently blocked autophagy, indicated by the increased LC3-II/I ratio and p62/SQSTM1 levels. Moreover, no colocalization was observed between autophagosomes, marked with LC3 or p62/SQSTM1, and lysosomes, stained with LAMP-1, after the LAI-1 treatment, indicating the blockage of autophagolysosome formation. Furthermore, LAI-1 induced cell death by activating apoptosis (enhancing the cleavage of caspase-3 and PARP) or necrosis, depending on the cancer cell line. Finally, LAI-1 sensitized cancer cells to the first-line chemotherapeutic agent cisplatin. Altogether, LAI-1 is a new late-stage autophagy inhibitor that causes lysosomal dysfunction and the blockage of autophagolysosome formation, as well as potently induces cancer cell death and sensitization to conventional treatments at lower concentrations than other known autophagy inhibitors, appearing as a potential new therapeutic approach to overcome cancer resistance., This research was supported by Consejería de Educación de la Junta de Castilla y León (grant BU067P20), MCIN/AEI/10.13039/501100011033 (grant PID2020-117610RB-I00) and Instituto de Salud Carlos III (grant PI18/00441), co-funded by the European Regional Development Fund. ERDF, a way to build Europe. P.F., D.A.-C. and I.C.-B. thank Consejería de Educación de la Junta de Castilla y León, European Social Fund and ERDF for their predoctoral (D.A.-C.) and postdoctoral (P.F. and I.C.-B.) contracts. C.B.-G. thanks the University of Barcelona for a predoctoral fellowship.

Survivin is a small protein that belongs to the inhibitor of apoptosis protein family. It is abundantly expressed in tumors compared with adult differentiated tissues, being associated with poor prognosis in many human neoplasms. This apoptotic inhibitor has a relevant role in both the promotion of cancer cell survival and in the inhibition of cell death. Consequently, aberrant survivin expression stimulates tumor progression and confers resistance to several therapeutic strategies in a variety of tumors. In fact, efficient survivin downregulation or inhibition results in spontaneous apoptosis or sensitization to chemotherapy and radiotherapy. Therefore, all these features make survivin an attractive therapeutic target to treat cancer. Currently, there are several survivin inhibitors under clinical evaluation, although more specific and efficient survivin inhibitors are being developed. Moreover, novel combination regimens targeting survivin together with other therapeutic approaches are currently being designed and assessed. In this review, recent progress in the therapeutic options targeting survivin for cancer treatment is analyzed. Direct survivin inhibitors and their current development status are explored. Besides, the major signaling pathways implicated in survivin regulation are described and different therapeutic approaches involving survivin indirect inhibition are evaluated. Finally, promising novel inhibitors under preclinical or clinical evaluation as well as challenges of developing survivin inhibitors as a new therapy for cancer treatment are discussed., Consejería de Educación, Junta de Castilla y León. Grant Number: BU092U16 Instituto de Salud Carlos III. Grant Number: FIS PI18/00441

A novel class of transmembrane anion carriers, the click-tambjamines, display remarkable anionophoric activities in model liposomes and living cells. The versatility of this building block for the generation of molecular diversity offers promise to develop future drugs based on this design., European Union’s Horizon 2020 research and innovation programme (TAT-CF project, grant agreement 667079), Instituto de Salud Carlos III (Grant PI18/00441) (co-funded by the European Regional Development Fund (ERDF), a way to build Europe) and “La Caixa” Foundation and Caja Burgos Foundation (CAIXAUBU004)

Mitochondrial metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. How genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Here, we show that homologous recombination‐defective (HRD) cancers rely on oxidative metabolism to supply NAD + and ATP for poly(ADP‐ribose) polymerase (PARP)‐dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components and a decline in the glycolytic Warburg phenotype. Hence, HRD cells are more sensitive to metformin and NAD + concentration changes. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. This feature is associated with a weak response to PARP inhibition in patient‐derived xenografts, emerging as a new mechanism to determine PARPi sensitivity. This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors., Homologous recombination‐defective (HRD) cancers need high levels of NAD + and ATP for alternative PARP‐dependent DNA repair. HRD cancer cells undergo a characteristic metabolic shift that include enhanced OXPHOS, opening new opportunities for treatment with OXPHOS inhibitors like metformin.

Lung cancer has become the leading killer cancer worldwide, due to late diagnosis and lack of efficient anticancer drugs. We have recently described novel natural-derived tambjamine analogues that are potent anion transporters capable of disrupting cellular ion balance, inducing acidification of the cytosol and hyperpolarization of cellular plasma membranes. Although these tambjamine analogues were able to compromise cell survival, their molecular mechanism of action remains largely unknown. Herein we characterize the molecular cell responses induced by highly active indole-based tambjamine analogues treatment in lung cancer cells. Expression changes produced after compounds treatment comprised genes related to apoptosis, cell cycle, growth factors and its receptors, protein kinases and topoisomerases, among others. Dysregulation of BCL2 and BIRC5/survivin genes suggested the apoptotic pathway as the induced molecular cell death mechanism. In fact, activation of several proapoptotic markers (caspase-9, caspase-3, and PARP) and reversion of the cytotoxic effect upon treatment with an apoptosis inhibitor (Z-VAD-FMK) were observed. Moreover, members of the Bcl-2 protein family suffered changes after tambjamine analogues treatment, with a concomitant protein decrease towards the prosurvival members. Besides this, it was observed cellular accumulation of ROS upon compound treatment and an activation of the stress-kinase p38 MAPK route that, when inhibited, reverted the cytotoxic effect of the tambjamine analogues. Finally, a significant therapeutic effect of these compounds was observed in subcutaneous and orthotopic lung cancer mice models. Taken together, these results shed light on the mechanism of action of novel cytotoxic anionophores and demonstrate the therapeutic effects against lung cancer., Spanish Government and EU funds through the Fondo de Investigaciones Sanitarias (FIS, project PI13/ 00089) and from La Marat o de TV3 Foundation (project 20132730) to R. P erez-Tom as. R. Ramos was supported by the Sociedad Espa~nola de Neumología y Cirugía Tor acica (SEPAR, Project 017/2013), R. Quesada by the Consejería de Educación de la Junta de Castilla y León (project BU340U13) and by the La Marat o de TV3 Foundation (project 20132732) and A. Villanueva by the FIS (project PI13/01339).

Autophagy is a tightly regulated catabolic process that facilitates nutrient recycling from damaged organelles and other cellular components through lysosomal degradation. Deregulation of this process has been associated with the development of several pathophysiological processes, such as cancer and neurodegenerative diseases. In cancer, autophagy has opposing roles, being either cytoprotective or cytotoxic. Thus, deciphering the role of autophagy in each tumor context is crucial. Moreover, autophagy has been shown to contribute to chemoresistance in some patients. In this regard, autophagy modulation has recently emerged as a promising therapeutic strategy for the treatment and chemosensitization of tumors, and has already demonstrated positive clinical results in patients. In this review, the dual role of autophagy during carcinogenesis is discussed and current therapeutic strategies aimed at targeting autophagy for the treatment of cancer, both under preclinical and clinical development, are presented. The use of autophagy modulators in combination therapies, in order to overcome drug resistance during cancer treatment, is also discussed as well as the potential challenges and limitations for the use of these novel therapeutic strategies in the clinic.

Lung cancer is the leading cause of cancer-related deaths worldwide, hence novel treatments for this malignancy are eagerly needed. Since natural-based compounds represent a rich source of novel chemical entities in drug discovery, we have focused our attention on tambjamines, natural compounds isolated from marine invertebrates that have shown diverse pharmacological activities. Based on these structures, we have recently identified the novel indole-based tambjamine analog 21 (T21) as a promising antitumor agent, which modulates the expression of apoptotic proteins such as survivin. This antiapoptotic protein plays an important role in carcinogenesis and chemoresistance. In this work, we have elucidated the molecular mechanism by which the anticancer compound T21 exerts survivin inhibition and have validated this protein as a therapeutic target in different lung cancer models. T21 was able to reduce survivin protein levels in vitro by repressing its gene expression through the blockade of Janus kinase/Signal Transducer and Activator of Transcription-3 (JAK/STAT3)/survivin signaling pathway. Interestingly, this occurred even when the pathway was overstimulated with its ligand interleukin 6 (IL-6), which is frequently overexpressed in lung cancer patients who show poor clinical outcomes. Altogether, these results show T21 as a potent anticancer compound that effectively decreases survivin levels through STAT3 inhibition in lung cancer, appearing as a promising therapeutic drug for cancer treatment.

Three Pt(II) complexes containing the natural ligands curcumin and caffeine, namely [Pt(curc)(PPh3)2]Cl (1), [PtCl(curc)(DMSO)] (2) (curc = deprotonated curcumin) and trans-[Pt(caffeine)Cl2(DMSO)] (3), were synthesized and fully characterized. The data obtained suggest that, for both 1 and 2, the anion of curcumin is coordinated to the platinum ion via the oxygen atoms of the β-diketonate moiety. Spectroscopic features reveal that in 2 and 3, a DMSO molecule is S-bonded to the metal centre. For 3, all data indicate a square-planar geometry formed by a 9-N bonded caffeine, two trans chloride anions and a DMSO. The three complexes undergo changes in solution upon incubation for 24 h; 1 and 2 release curcumin while 3 isomerizes from trans to cis configuration. The DNA-binding and cytotoxic properties of 1–3 were evaluated in vitro. Despite their structural similarity, curcuminate-containing 1 and 2 exhibit distinct DNA interactions. While 1 appears to intercalate between nucleobase pairs, inducing the oxidative degradation of the biomolecule, 2 behaves as a groove binder, by means of electrostatic forces. Caffeine-containing 3 exhibits a behaviour that is comparable to that of 2. Complexes 1 and 2 showed moderate to high cytotoxicity and selectivity against several cancer cell lines, while 3 is inactive. Compounds 1 and 2 can be further activated by visible-light irradiation.

In the present study, the potential anti-neoplastic properties of a series of ruthenium half-sandwich complexes of formula [Ru(η6-arene)Cl2(PR1R2(1-pyrenyl))] (η6-arene = p-cymene and R1 = R2 = methyl for 1; η6-arene = methylbenzoate and R1 = R2 = methyl for 2; η6-arene = p-cymene and R1 = R2 = phenyl for 3; η6-arene = methylbenzoate and R1 = R2 = phenyl for 4; η6-arene = p-cymene, R1 = methyl and R2 = phenyl for 5; η6-arene = methylbenzoate, R1 = methyl and R2 = phenyl for 6) have been investigated. The six structurally related organoruthenium(II) compounds have been prepared in good yields and fully characterized; the X-ray structures of three of them, i.e., 1, 2, and 4, were determined. Although the piano-stool compounds contain a large polycyclic aromatic moiety, viz. a 1-pyrenyl group, they do not appear to interact with DNA. However, all the piano-stool complexes show significant cytotoxic properties against five human cell lines, namely, lung adenocarcinoma (A549), melanoma (A375), colorectal adenocarcinoma (SW620), breast adenocarcinoma (MCF7), and nontumorigenic epithelial breast (MCF10A), with IC50 values in the micromolar range for most of them. In addition, the most active compound, i.e., 2, induces a remarkable decrease of cell viability, that is in the nanomolar range, against two human neuroblastoma cell lines, namely, SK-N-BE(2) and CHLA-90. Complexes 1-6 are all capable of inducing apoptosis, but with various degrees of magnitude. Whereas 1, 3, 5, and 6 have no effect on the cell cycle of A375 cells, 2 and 4 can arrest it at the G2/M phase; furthermore, 2 (which is the most efficient compound of the series) also stops the cycle at the S phase, behaving as the well-known anticancer agent cisplatin. Finally, 2 is able to inhibit/reduce the cell migration of neuroblastoma SK-N-BE(2) cells.

Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of sternness of CSCs leading to effective elimination of this cancer cell subpopulation., This work was supported by grants from the Spanish government and the EU (FIS PI13/00089, PIS PI12/02838, FIS PI12/00956 and RD12/0036/0025), a grant from La Marato de TV3 Foundation (20132730), a grant from SEPAR (17/2014), Consejeria de Educacion de la Junta de Castilla y Leon (Project BU340U13), Ministerio de Economia y competitividad/Instituto de Salud Carlos III (SAF2014-55700-P), and ICREA Academia-2010. E.H. thanks the Junta de Castilla y Leon (Consejeria de Educacion) and the Fondo Social Europeo for a PIRTU contract. We thank Benjamin Torrejon and Esther Castano from CCiT (Centres Cientifics i Tecnologics, Campus de Bellvitge, Universitat de Barcelona), Sandra Tejedor (Centro de Investigacion Principe Felipe), Luis Korrodi-Gregorio (UB), and Andrea Sancho (UBU) for their technical support.

Anion selective ionophores, anionophores, are small molecules capable of facilitating the transmembrane transport of anions. Inspired in the structure of natural product prodigiosin, four novel anionophores 1a-d, including a 1,2,3-triazole group, were prepared. These compounds proved highly efficient anion exchangers in model phospholipid liposomes. The changes in the hydrogen bond cleft modified the anion transport selectivity exhibited by these compounds compared to prodigiosin and suppressed the characteristic high toxicity of the natural product. Their activity as anionophores in living cells was studied and chloride efflux and iodine influx from living cells mediated by these derivatives was demonstrated. These compounds were shown to permeabilize cellular membranes to halides with efficiencies close to the natural anion channel CFTR at doses that do not compromise cellular viability. Remarkably, optimal transport efficiency was measured in the presence of pH gradients mimicking those found in the airway epithelia of Cystic Fibrosis patients. These results support the viability of developing small molecule anionophores as anion channel protein surrogates with potential applications in the treatment of conditions such as Cystic Fibrosis derived from the malfunction of natural anion transport mechanisms., European Union’s Horizon 2020 research and innovation programme under grant agreement No. 667079, La Marató de TV3 Foundation (20132730), Consejería de Educación de la Junta de Castilla y León (Projects BU340U13 and BU092U16)

Combining computational modeling, de novo compound synthesis, and in vitro and cellular assays, we have performed an inhibition study against the enhancer of zeste homolog 2 (EZH2) histone-lysine N-methyltransferase. This enzyme is an important catalytic component of the PRC2 complex whose alterations have been associated with different cancers. We introduce here several tambjamine-inspired derivatives with low micromolar in vitro activity that produce a significant decrease in histone 3 trimethylation levels in cancer cells. We demonstrate binding at the methyl transfer active site, showing, in addition, that the EZH2 isolated crystal structure is capable of being used in molecular screening studies. Altogether, this work provides a successful molecular model that will help in the identification of new specific EZH2 inhibitors and identify a novel class of tambjamine-derived EZH2 inhibitors with promising activities for their use in cancer treatment., Spanish Government (CTQ2016-79138-R; FIS PI13/00089), the Consejería de Educación de la Junta de Castilla y León (Projects BU340U13 and BU092U16), and from La Maratóde TV3 Foundation (20132730).

Prodigiosin is one of the most potent anion transporters in lipid bilayer membranes reported to date. Inspired by the structure of this natural product, we have recently designed and synthesised a new class of H+/Cl− cotransporters named ‘perenosins’. Here we report a new library of indole-based perenosins and their anion transport properties. The new transporters demonstrated superior transmembrane transport efficiency when compared to other indole-based transporters, due to favourable encapsulating effects from the substituents on the perenosin backbone. Anion transport assays were used to determine the mechanism of chloride transport revealing that the compounds function as ‘strict’ HCl cotransporters. Cell viability studies showed that some compounds specifically trigger late-onset cell death after 72 h with a unique correlation to the position of alkyl chains on the perenosins. Further investigations of cell death mechanism showed a mixture of cell cycle arrest and apoptosis was responsible for the observed decrease in cell viability.

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.

Melanoma is one of the most aggressive types of skin cancer and its incidence rate is still increasing. All existing treatments are minimally effective. Consequently, new therapeutic agents for melanoma treatment should be developed. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and anti-metastatic properties. The aim of this study was to evaluate the different signaling pathways involved in the cytotoxic effect of DM-1 on melanoma cells. The apoptotic process and cytoskeletal changes were evaluated by immunoblotting and immunofluorescence, respectively, in melanoma cells. After DM-1 treatment, SK-MEL-5 melanoma cells showed actin filament disorganization with spicule formation throughout the cytoskeleton and significant reduction of focal adhesion as well as they were present only at cell extremities, conferring a poor connection between the cell and the substrate. Besides this, there was significant filopodium retraction and loss of typical cytoskeleton scaffold. These modifications contributed to cell detachment followed by cell death. Furthermore, DM-1-induced apoptosis was triggered by multiple Bcl-2 proteins involved in both the extrinsic and the intrinsic apoptotic pathways. SK-MEL-5 cells showed a death mechanism mainly by Bcl-2/Bax ratio decrease, whereas A375 cells presented apoptosis induction by Mcl-1 and Bcl-xL downregulation. In SK-MEL-5 and A375 melanoma cells, there was a significant increase in the active form of caspase 9, and the inactive form of the effector caspase 3 was decreased in both cell lines. Expression of cleaved poly ADP ribose polymerase was increased after DM-1 treatment in these melanoma cell lines, demonstrating that the apoptotic process occurred. Altogether, these data elucidate the cellular and molecular mechanisms involved in the cytotoxicity induced by the antitumor agent DM-1 in melanoma cells.

Current pharmacological treatments for lung cancer show very poor clinical outcomes, therefore, the development of novel anticancer agents with innovative mechanisms of action is urgently needed. Cancer cells have a reversed pH gradient compared to normal cells, which favours cancer progression by promoting proliferation, metabolic adaptation and evasion of apoptosis. In this regard, the use of ionophores to modulate intracellular pH appears as a promising new therapeutic strategy. Indeed, there is a growing body of evidence supporting ionophores as novel antitumour drugs. Despite this, little is known about the implications of pH deregulation and homeostasis imbalance triggered by ionophores at the cellular level. In this work, we deeply analyse for the first time the anticancer effects of tambjamine analogues, a group of highly effective anion selective ionophores, at the cellular and molecular levels. First, their effects on cell viability were determined in several lung cancer cell lines and patient-derived cancer stem cells, demonstrating their potent cytotoxic effects. Then, we have characterized the induced lysosomal deacidification, as well as, the massive cytoplasmic vacuolization observed after treatment with these compounds, which is consistent with mitochondrial swelling. Finally, the activation of several proteins involved in stress response, autophagy and apoptosis was also detected, although they were not significantly responsible for the cell death induced. Altogether, these evidences suggest that tambjamine analogues provoke an imbalance in cellular ion homeostasis that triggers mitochondrial dysfunction and lysosomal deacidification leading to a potent cytotoxic effect through necrosis in lung cancer cell lines and cancer stem cells., Spanish government and the EU (FIS PI13/00089), Consejería de Educación de la Junta de Castilla y León (BU340U13 and BU092U16).

Synthetic transmembrane anion transporters (anionophores) have potential as tools for biomedical research and as therapeutic agents for diseases associated with anion-channel dysfunction. However, the possibility of H+ or OH- transport by anionophores has received little attention, and an anionophore selective for Cl- over H+/OH- is currently unavailable. Here, we show that depending on anionophore acidity, many anionophores facilitate electrogenic H+ or OH- transport, potentially leading to toxicity. Nevertheless, using several liposome-membrane-based assays, we identified two newly developed small molecules that promote electrogenic Cl- transport without effectively dissipating the transmembrane pH gradient, essentially mimicking the electrogenic cationophore valinomycin. The Cl- > H+/OH- selectivity of anionophores showed a consistent positive correlation with the degree of Cl- encapsulation and a negative correlation with the acidity of hydrogen-bond donors. Our study demonstrates that a valinomycin equivalent for Cl--selective transport is achievable., info:eu-repo/semantics/published

A series of fluorescent anion transporters consisting of a urea or thiourea group linked to a naphthalimide fluorophore have been synthesised and their anion transport properties studied. The compounds possess similar anion transport properties of (thio)urea-based anionophores that have previously been reported. Fluorescence studies in cells show all anionophores cross the plasma membrane and localise within the interior of cells. The most lipophilic, aromatic substituted transporters localise homogeneously throughout the cell and are toxic towards cancer cells with the highly fluorinated compound 6 being the most effective. The least lipophilic, alkyl substituted transporters localise in specific vesicles and are non-toxic to cells. This work provides new insight to the actions of anionophores in cells and may be useful in the design of novel antineoplastic agents.

The anticancer agent prodigiosin has been shown to act as an efficient immunosuppressant, eliciting cell cycle arrest at non-cytotoxic concentrations, and potent proapoptotic and antimetastatic effects at higher concentrations. Gene expression profiling of MCF-7 cells after treatment with a non-cytotoxic concentration of prodigiosin showed that expression of the p21WAF1/CIP1 gene, a negative cell cycle regulator was induced. In this study, we show that prodigiosin induces p21 expression leading to cell cycle blockade. Subsequently, we attempted to elucidate the molecular mechanisms involved in prodigiosin-mediated p21 gene expression. We demonstrate that prodigiosin induces p21 in a p53-independent manner as prodigiosin induced p21 in cells with both mutated and dominant negative p53. Conversely, the transforming growth factor-beta (TGF-beta) pathway has been found to be necessary for p21 induction. Prodigiosin-mediated p21 expression was blocked by SB431542, a TGF-beta receptor inhibitor. Nevertheless, this pathway alone is not enough to induce p21 expression. The TGF-beta family member (nonsteroidal anti-inflammatory drug)-activated gene 1/growth differentiation factor 15 (NAG-1) may activate this pathway, as it has previously been suggested to signal through the TGF-beta pathway and is overexpressed in response to prodigiosin treatment. We show that NAG-1 colocalizes with TGF-beta receptor type I, suggesting a possible interaction between them. Taken together, these results suggest the TGF-beta pathway is required for induction of p21 expression after prodigiosin treatment of MCF-7 cells.

Prodigiosin is a bacterial red pigment with cytotoxic properties and potential antitumor activity that has been tested against different cancerous cells. In this study we report the effect and mechanisms of action of prodigiosin against different human neuroblastoma cell lines: SH-SY5Y, LAN-1, IMR-32 (N-type) and SK-N-AS (S-type). We compare the anticancerous effect of prodigiosin with that of cisplatin at different concentrations during 24 h of exposure. Prodigiosin is more potent, with IC 50 values lower than 1.5 μM in N-type neuroblastoma cells and around 7 μM in the S-type neuroblastoma cell line. We describe prodigiosin as a proton sequestering agent that destroys the intracellular pH gradient, and propose that its main cytotoxic effect could be related to its action on mitochondria, where it exerts an uncoupling effect on the electronic chain transport of protons to mitochondrial ATP synthase. As a result of this action, ATP production is reduced but without decreasing in oxygen consumption. This mechanism of action differs from those induced by conventional chemotherapeutic drugs, suggesting a possible role for prodigiosin to enhance the effect of antitumor agents in the treatment of neuroblastoma.

Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) is a bacterial metabolite that has anticancer and antimetastatic properties. However, the molecular mechanisms responsible for these abilities are not fully understood. Gene expression profiling of the human breast cancer cell line MCF-7 treated with prodigiosin was analyzed by cDNA array technology. The majority of the significantly modified genes were related to apoptosis, cell cycle, cellular adhesion, or transcription regulation. The dramatic increase of the nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) made this gene an interesting candidate regarding the possible mechanism by which prodigiosin induces cytotoxicity in MCF-7 cells. Our results show that prodigiosin triggers accumulation of the DNA-damage response tumor-suppressor protein p53 but that NAG-1 induction was independent of p53 accumulation. Moreover, prodigiosin caused AKT dephosphorylation and glycogen synthase kinase-3β (GSK-3β) activation, which correlated with NAG-1 expression. Prodigiosin-induced apoptosis was recovered by inhibiting GSK-3β, which might be due, at least in part, to the blockade of the GSK-3β–dependent up-regulation of death receptors 4 and 5 expression. These findings suggest that prodigiosin-mediated GSK-3β activation is a key event in regulating the molecular pathways that trigger the apoptosis induced by this anticancer agent. [Mol Cancer Ther 2007;6(1):362–9]

Prodigiosin (PG) is a bacterial, red-pigmented antibiotic with immunosuppressive and apoptotic activities. To better understand its mechanisms of action, we tried to identify proteins associated with apoptosis induced by PG. For this purpose, the variation of protein expression on exposure to apoptotic concentrations of PG was examined, by high-resolution two-dimensional gel electrophoresis (2D-E), in the MCF-7 cancer cell line resistant to mitoxantrone (MCF-7-MR). Six PG apoptosis-associated protein spots were further characterized by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on a matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometer. The proteins identified were involved in various cellular functions, including cell defence, DNA repair and cellular organization. Our data provide novel information on cell response to PG, a new apoptotic drug with interesting anticancer activity.

In a search for new anticancer agents, we have identified serratamolide (AT514), a cyclodepsipeptide from Serratia marcescens 2170 that induces cell cycle arrest and apoptosis in various cancer cell lines. A cell viability assay showed that the concentrations that cause 50% inhibition (IC 50 ) in human cancer cell lines range from 5.6 to 11.5 μM depending on the cell line. Flow cytometry analysis revealed that AT514 caused cell cycle arrest in G 0 / G 1 or cell death, depending on the cell type and the length of time for which the cells were exposed to the drug. Subsequent studies revealed that AT514-induced cell death is caused by apoptosis, as indicated by caspases activation (8, 9, 2 and 3) and cleavage of poly (ADP-ribose) polymerase (PARP), release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, and the appearance of apoptotic bodies and DNA laddering. Alterations in protein levels of Bcl-2 family members might be involved in the mitochondrial disruption observed. AT514 induced p53 accumulation in wild-type p53 cells but cell death was observed in both deficient and wild-type p53 cells. Our results indicate that AT514 induces cell cycle arrest and apoptosis in breast cancer cells irrespectively of p53 status, suggesting that it might represent a potential new chemotherapeutic agent.

Prodigiosin (PG) is a secondary metabolite, isolated from a culture of Serratia marcescens, which has shown potent cytotoxicity against various human cancer cell lines as well as immunosuppressive activity. The purpose of this study was to evaluate the role of mitochondria in PG-induced apoptosis. Therefore, we evaluated the apoptotic action of PG in GLC4 small cell lung cancer cell line by Hoechst 33342 staining. In these cells, we examined mitochondrial apoptosis-inducing factor (AIF) and cytochrome c (cyt c) release to the cytosol in PG time-response studies. These findings suggest that PG induces apoptosis in both caspase-dependent and caspase-independent pathways.

The family of natural red pigments, called prodigiosins (PGs), characterised by a common pyrrolylpyrromethene skeleton, are produced by various bacteria. Some members have immunosuppressive properties and apoptotic effects in vitro and they have also displayed antitumour activity in vivo. Understanding the mechanism of action of PGs is essential for drug development and will require the identification and characterisation of their still unidentified cell target. Four possible mechanisms of action have been suggested for these molecules: (i) PGs as pH modulators; (ii) PGs as cell cycle inhibitors; (iii) PGs as DNA cleavage agents; (iv) PGs as mitogen-activated protein kinase regulators. Here, we review the pharmacological activity of PG and related compounds, including novel synthetic PG derivatives with lower toxicity and discuss the mechanisms of action and the molecular targets of those molecules. The results reported in this review suggest that PGs are a new class of anticancer drugs, which hold out considerable promise for the Pharmacological Industry.

Introduction: Lung cancer is the most frequent cause of cancer death worldwide, with an increasing incidence and a poor prognosis that depends on tumor stage at presentation. Characterization of molecular abnormalities that play a crucial role on lung cancer will allow the development of targeted therapies for this illness. Our aim is to test novel indol-based compounds chemically related to tambjamines and to characterize the alterations in signaling pathways caused by their treatment in order to identify their molecular targets. Materials and Methods: Cell lines representative of the four major groups of lung cancer types were selected to perform this study: A549 (adenocarcinoma), SW900 (squamous cell carcinoma), H460 (large cell carcinoma) and DMS53 (small cell lung carcinoma). Cell viability evaluation was performed by the standard MTT assay. In order to characterize the main transcriptional changes produced by the selected compounds, a QIAGEN array of genes related to human cancer drug targets was used. Transcriptional alteration of a set of genes was further corroborated by TaqMan gene expression assays in order to validate the array. Additionally, the protein expression levels of several apoptotic-related proteins were assessed by immunoblot in the A549 cell line exposed to IC25, IC50 and IC75 values of the selected compounds. Moreover, Z-VAD-FMK (a pan-caspase inhibitor) treatment was performed and the outcome analyzed using the MUSE cell count and viability assay kit in order to assess the reversion in viability upon compounds treatment. Lastly, the p38 specific inhibitor SB202190 was used to assess its potential reversion of survivin levels and apoptosis by Western blot. Results and Discussion: Cell viability was significantly reduced in a dose-dependent manner by tambjamine analogs in all the lung cancer cell lines studied. Our results, using gene expression arrays, showed the impact of our compounds in several targets from apoptosis (e.g. BCL2 and BIRC5/survivin) and cell cycle (e.g. AURKs, CDKs and PLKs), among others. The down-regulation of the anti-apoptotic proteins survivin and Bcl2, as well as the activation of several pro-apoptotic proteins (initiator caspases, effector caspases and final executor PARP protein) was analyzed by immunoblot. Furthermore, the inhibition of caspase activity significantly reversed the cytotoxic effect of the selected compounds. Finally, the p38 stress kinase was activated after compound treatment and experiments using a specific inhibitor showed to partially recover the down-regulation of survivin levels and the activation of apoptosis. All these results hint that after compounds treatment cell death process is triggered mainly by apoptosis and that survivin down-regulation might be the responsible for the induction of this process. Conclusion: Altogether, our selected compounds show more potent cytotoxic properties than the first-line treatment cisplatinum, altering the expression of several key signaling proteins in lung cancer cells, especially apoptotic related proteins. These compounds strongly down-regulate the survivin cellular pool thereby triggering apoptotic cell death via caspase activation. Acknowledgements: This work was supported by the grant from the Spanish government and the EU (FIS PI13/00089) and a grant from La Marató de TV3 Foundation (20132730). LKG holds a FCT (SFRH/BPD/91766/2012) fellowship. Citation Format: Pilar Manuel-Manresa, Luis Korrodi-Gregório, Ananda Marina Rodilla, Roberto Quesada, Vanessa Soto-Cerrato, Ricardo Pérez-Tomás. Novel indol-based tambjamine analogs down-regulate survivin in lung cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A04.

Macroautophagy, herein referred to as autophagy, is a biological process activated under diverse cellular stress conditions, such as starvation or oxidative stress. It involves the internalization of cytoplasmic portions, with or without organelles, within double-membrane vesicles called autophagosomes. These vesicles finally fuse with lysosomes, forming the autolysosomes, where the cargo is degraded due to the activity of the lysosomal enzymes. This catabolic process has primarily a cellular protective role but, depending on the context, it can also mediate an autophagic cell death. In fact, research related to the activation and impairment of autophagy is gaining importance as a novel anticancer therapeutic strategy in recent times. Therefore, identification of new potent and selective anticancer compounds is eagerly needed. In this view, we have synthesized novel and effective anion transporter molecules (anionophores), designed to disrupt the cellular ion homeostasis, therefore triggering cell death. The molecular mechanism implicated in their cytotoxic properties in lung cancer has been deeply analyzed in the present work. First, cytotoxic effects of our candidate compounds were evaluated by the MTT assay in four lung cancer cell lines representative of the main clinical histological subtypes as well as in cancer stem cells, a tumor cell subpopulation related to tumor initiation, progression and chemoresistance. A significant decrease in cell viability was observed in all of them, being compound 2 the most cytotoxic in all the cell lines and cancer stem cells tested. In order to characterize the cell death mechanisms involved in its cytotoxic activity, different molecular markers related to apoptosis and autophagy were analyzed. At low concentrations, we observed a very significant autophagosomal accumulation, detected through a high LC3II increase by Western blot and immunofluorescence, while some evidence of caspases activation is observed at higher concentrations. Moreover, p62 protein was also accumulated after treatment, suggesting impairment of the autophagic process and blockade of autophagosome processing. Interestingly, acidic lysosomal pH was totally disrupted by the compound, measured by acridine orange staining, which might be the cause of this autophagosomal accumulation. On the other hand, it was observed, by phase contrast and electron microscopy, that the compound also induced massive cytoplasmic vacuolization. We tested several markers to distinguish among different potential organelles that could have undergone this phenomenon. We used LAMP1, LC3II and EEA1 by immunofluorescence and we could exclude lysosomes, autophagosomes and early endosomes, respectively. Finally TOMM20, a mitochondrial marker, proved to be positive. That was subsequently corroborated by transfecting A549 cells with the subunit VIII of human cytochrome C oxidase mCherry-MITO7 plasmid. Therefore, mitochondrial swelling induced by the compound might be involved in the triggering of the autophagic process and provoking an energy failure which might participate in the cytotoxic effect observed. Altogether, these results show that these anion transporters have potent cytotoxic effects in lung cancer and cancer stem cells. They induce an imbalance in cellular ion homeostasis, which triggers mitochondrial swelling and lysosomal dysfunction leading to autophagosomal accumulation and finally cell death. Acknowledgements: This work was partially supported by grants from the Spanish government and the EU (FIS PI13/00089), La Marató TV3 Foundation (20132730) and SEPAR foundation (17/2014). LKG holds a FCT (SFRH/BPD/91766/2012) fellowship. Citation Format: Ananda Marina Rodilla, Luis Korrodi-Gregório, Pilar Manuel-Manresa, Roberto Quesada, Ricardo Pérez-Tomás, Vanessa Soto-Cerrato. Targeting autophagy through novel anionophores that induce lysosomal dysfunction. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A05.

Ten synthetic analogs of the marine alkaloids tambjamines, bearing aromatic enamine moieties, have been synthesized. These compounds proved to be highly efficient transmembrane anion transporters in model liposomes. Changes in the electronic nature of the substituents of the aromatic enamine or the alkoxy group of the central pyrrole group did not affect this anionophore activity. The in vitro activity of these compounds has also been studied. They trigger apoptosis in several cancer cell lines with IC50 values in the low micromolar range as well as modify the intracellular pH, inducing the basification of acidic organelles., Consejería de Educación de la Junta de Castilla y León (Project BU340U13) and Fundació la Maratón de TV3.

Additionally five N-methylated Sansalvamide A analogs are synthesized for comparison of biological and physical properties.

The main difficulty in the successful treatment of metastatic melanoma is that this type of cancer is known to be resistant to chemotherapy. Chemotherapy remains the treatment of choice, and dacarbazine (DTIC) is the best standard treatment. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and antimetastatic properties. The objective of this study was to evaluate the signaling pathways involved in melanoma cell death after treatment with DM-1 compared to the standard agent for melanoma treatment, DTIC. Cell death was evaluated by flow cytometry for annexin V and iodide propide, cleaved caspase 8, and TNF-R1 expression. Hoechst 33342 staining was evaluated by fluorescent microscopy; lipid peroxidation and cell viability (MTT) were evaluated by colorimetric assays. The antiproliferative effects of the drugs were evaluated by flow cytometry for cyclin D1 and Ki67 expression. Mice bearing B16F10 melanoma were treated with DTIC, DM-1, or both therapies. DM-1 induced significant apoptosis as indicated by the presence of cleaved caspase 8 and an increase in TNF-R1 expression in melanoma cells. Furthermore, DM-1 had antiproliferative effects in this the same cell line. DTIC caused cell death primarily by necrosis, and a smaller melanoma cell population underwent apoptosis. DTIC induced oxidative stress and several physiological changes in normal melanocytes, whereas DM-1 did not significantly affect the normal cells. DM-1 antitumor therapy in vivo showed tumor burden decrease with DM-1 monotherapy or in combination with DTIC, besides survival rate increase. Altogether, these data confirm DM-1 as a chemotherapeutic agent with effective tumor control properties and a lower incidence of side effects in normal cells compared to DTIC.

Highly potent but structurally simple transmembrane anion transporters are reported that function at receptor to lipid ratios as low as 1 : 1 000 000. The compounds, based on the simple ortho-phenylenediamine-based bisurea scaffold, have been studied for their ability to facilitate chloride/nitrate and chloride/bicarbonate antiport, and HCl symport processes using a combination of ion selective electrode and fluorescence techniques. In addition, the transmembrane transport of dicarboxylate anions (maleate and fumarate) by the compounds was examined. Molecular dynamics simulations showed that these compounds permeate the membrane more easily than other promising receptors corroborating the experimental efflux data. Moreover, cell based assays revealed that the majority of the compounds showed cytotoxicity in cancer cells, which may be linked to their ability to function as ion transporters.

Prodigiosin and obatoclax, members of the prodiginines family, are small molecules with anti-cancer properties that are currently under preclinical and clinical trials. The molecular target(s) of these agents, however, is an open question. Combining experimental and computational techniques we find that prodigiosin binds to the BH3 domain in some BCL-2 protein families, which play an important role in the apoptotic programmed cell death. In particular, our results indicate a large affinity of prodigiosin for MCL-1, an anti-apoptotic member of the BCL-2 family. In melanoma cells, we demonstrate that prodigiosin activates the mitochondrial apoptotic pathway by disrupting MCL-1/BAK complexes. Computer simulations with the PELE software allow the description of the induced fit process, obtaining a detailed atomic view of the molecular interactions. These results provide new data to understand the mechanism of action of these molecules, and assist in the development of more specific inhibitors of anti-apoptotic BCL-2 proteins., Spanish government and the European Union (FIS-PI10/00338) and from the ERC-2009-Adg 25027-PELE European project.

Background: TAS-102 (Lonsurf) consists of trifluridine (FTD) and tipiracil hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. The mechanism of FTD is considered to be based on its incorporation into DNA, however its intracellular metabolism is not completely defined. Here, we investigate the cellular uptake and phosphorylation of FTD, and substrate specificity of incorporation into DNA by DNA polymerase a. Material and Methods: Intra-cellular uptake of FTD was evaluated with nucleoside transporter specific inhibitors, NBMPR and dipyridamole. Substrate specificity for nucleoside kinase and nucleotide phosphorylase was evaluated with recombinant proteins. DNA elongation with FTD triphosphate (F3dTTP) by DNA polymerase a was analyzed. The levels of FTD incorporated into DNA were analyzed with [H]FTD or LC-MS/MS. Results: FTD was transported via ENT1 and ENT2 into cytoplasm, and was phosphorylated by thymidine kinase 1 as effectively as dThd. F3dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was degraded to monophosphate form. F3dTTP was inserted at the opposite site of adenine by DNA polymerase a. DNA extension was effectively carried out at the site of inserted FTD, and was evident on the single strand DNA template inserted FTD.The level of FTD incorporated into DNA was much higher than that of FdUrd in vitro, and FTD remained in DNA after the FTD removal from culturing medium. In vivo study also showed high FTD accumulation in tumor implanted in nude mice. Nucleosomal abnormalities such as swollen nuclei and decreased hetero-chromatin were observed in the cells treated with FTD. Conclusions: These results indicate that FTD is recognized and activated through the thymidine salvage pathway highly up-regulated in tumor, and incorporated into DNA during DNA replicating phase like the natural substrate dThd. FTD might exert antitumor activity by nucleosomal abnormalities caused by high level FTD incorporation into DNA. These unique properties might underlie the clinical benefit of prolonged survival of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU based chemotherapy.