Phosphoprotein Phosphatase 1 isoforms alpha and gamma respond differently to prodigiosin treatment and present alternative kinase targets in melanoma cells

Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological conditions, including cancer. Many studies have already addressed the role of protein kinases misregulation in cancer. However, much less is known about protein phosphatases influence. Phosphoprotein Phosphatase 1 (PPP1) is one of the major serine/threonine protein phosphatases who has three catalytic isoforms: PPP1CA, PPP1CB, and PPP1CC. Its function is achieved by binding to regulatory subunits, known as PPP1-interacting proteins (PIPs), which may prefer a catalytic isoform. Also, some inhibitors/enhancers may exhibit isoform specificity. Here we show that, prodigiosin (PG), a molecule with anticancer properties, promotes the formation of PPP1CA-AKT complex and not of PPP1CC-MAPK complex. Both, AKT and MAPK, are wellknown PIPs from two pathways that crosstalk and regulate melanoma cells survival. In addition, the analysis performed using surface plasmon resonance (SPR) technology indicates that PPP1 interacts with obatoclax (OBX), a drug that belongs to the same family of PG. Overall, these results suggest that PG might, at least in part, act through PPP1C/PIPs. Also, this study is pioneer in demonstrating PPP1 isoform-specific modulation by small molecules.publishe

Molecular Interactions of Prodiginines with the BH3 Domain of Anti-Apoptotic Bcl-2 Family Members

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

Monte Carlo Techniques for Drug Design: The Success Case of PELE

This chapter summarizes the most representative software packages that readily allow running Monte Carlo (MC) simulations in relevant scenarios for drug design. It explores in detail the Protein Energy Landscape Exploration (PELE) program, providing first the main characteristics of the technique, followed by an analysis of the different application studies in mapping protein‐ligand interactions. The ligand, formed by a rigid core and a set of rotatable side chains, is perturbed by translating and rotating it. PELE creates a list of perturbation poses, and then chooses the one with the lowest system energy. PELE was originally designed to map ligand migration pathways: its first applications aimed at finding exit pathways starting from ligand‐bound crystallographic structures. Additional applied studies have centered on modeling enzymatic mechanisms and engineering; the same techniques applied in mapping protein‐drug interactions can be used in the study of substrate recognition by enzymes.Along the development of PELE in the last years, we gratefully acknowledge financial support from the European Union (in particular from the ERC program) and from the Catalan and Spanish Governments. In addition we want to thank all present and past members from the EAPM lab. at BSC for their dedication and work.Peer ReviewedPostprint (author's final draft

Estudio de los mecanismos moleculares involucrados en las propiedades anticancerosas y antimetastásicas de las prodigininas. Identificación de mTOR como diana molecular en células de melanoma

[spa] El melanoma metastásico es un modelo de tumor especialmente agresivo y resistente a inmuno, radio y quimioterapia. La pobre prognosis de los pacientes y el preocupante incremento de casos en los últimos años hacen necesario el diseño y desarrollo de nuevos y mejorados agentes anticancerígenos, así como nuevas estrategias más eficaces. Las células de melanoma presentan alteraciones genéticas y moleculares que alteran el control de la proliferación y supervivencia celular. Estas alteraciones permiten superar las barreras de control del crecimiento y migración celular, aspectos asociados no sólo con la inmortalización y adquisición de las propiedades invasivas, sino que también les confieren resistencia frente a tratamientos quimioterapéuticos. Tanto la vía PI3K/AKT/mTOR, como la vía mitogénica mediada por MAPKs, son claves en la supervivencia y crecimiento celular. Ambas vías, a través de GSK-3 se encuentran implicadas en la sobreexpresión de proteínas anti-apoptóticas de la familia BCL-2. La sobreexpresión de esta familia de proteínas facilita que la célula escape a los mecanismos apoptóticos inducidos por agentes citotóxicos convencionales. Por esta razón, estrategias terapéuticas basadas en la inhibición de estas vías han mostrado su potencial en estudios clínicos. Este potencial radica en la activación simultánea de dos mecanismos de muerte celular: apoptosis y autofagia. De entre la familia de compuestos anticancerosos conocidos como prodigininas (PGs), en un principio, prodigiosina destacó por su marcado efecto pro-apoptótico en diferentes líneas tumorales. Actualmente, un derivado sintético de las PGs, obatoclax, que fue diseñado como molécula BH3 mimética, está siendo evaluado en ensayos clínicos en fase III. Sin embargo, los estudios realizados en melanoma muestran la modesta eficacia de obatoclax como agente individual, lo que hacía necesario estudios más exhaustivos sobre los mecanismos de acción de esta molécula. Un efecto citotóxico más marcado se ha obtenido mediante el tratamiento con prodigiosina. A pesar de que ambas moléculas han mostrado desencadenar similares mecanismos moleculares, las diferencias estructurales entre ambas moléculas serían críticas en su actividad. Los nuevos datos aportados contribuyen a un mejor entendimiento de los eventos moleculares que desencadenan la actividad anticancerosa y antimetastásica de las PGs. En este aspecto, la caracterización de PGs como inhibidores duales de mTORC1 y mTORC2 pone en evidencia la relevancia que estas moléculas podrían tener en un futuro en la terapia contra el cáncer. La interacción de PGs al centro activo de mTOR se confirmó mediante técnicas de resonancia de plasmón superficial (SPR), monitorizando las interacciones a tiempo real, usando biosensores ópticos. Los parámetros cinéticos y de afinidad mostraron que ambas moléculas forman complejos estables, específicos, y de alta afinidad a concentraciones nanomolares. Los ensayos de competición con ATP revelaron que no existe competición entre ATP y las PGs por su lugar de unión, sugiriendo que las PGs actúan como inhibidores ATP no miméticos. Un valor añadido de estas moléculas son sus probadas propiedades antimetastásicas. En este aspecto, demostramos que la inhibición de mTOR promueve la desestabilización del complejo ILK/rictor con la consecuente pérdida de señalización a través de la vía de las integrinas, crítica para el movimiento celular. Observamos que, todo ello conlleva a la desorganización del citoesqueleto, pérdida de los contactos focales y pérdida de la capacidad migratoria de las células. Finalmente, estrategias análogas fueron utilizadas para llevar a cabo estudios de relación estructura-actividad de las PGs. La estructura tripirrólica de prodigiosina (PG) contiene grupos amino y metoxi comunes en obatoclax (OBX) relacionados con su actividad citotóxica. El bloqueo de estos grupos mediante la adición del grupo aniónico F2B resultó en la pérdida de actividad de esta molécula. Estos resultados ponen de manifiesto la importancia de estos grupos en la actividad de PGs, y aportan nuevos datos para el diseño de nuevos derivados sintéticos más efectivos y menos tóxicos.[eng] MECHANISMS OF ACTION INVOLVED IN THE ANTICANCER AND ANTIMETASTATIC PROPERTIES OF PRODIGININES. Identification of mTOR as a molecular target in melanoma cells. Because of high chemotherapeutic resistance of metastatic melanoma are necessary new strategies with improved drugs, more effectives with lower toxicity. Anticancer activity of prodiginines has been described in vitro and in vivo in different cancer cell models, including melanoma. Prodiginines are tripyrrolic molecules from bacterial metabolism under pre-clinical and clinical evaluation. In this study we have focused our efforts on identified the molecular mechanisms of prodiginines involved in their cytotoxic effects. In melanoma cells, we have observed the activation of both cell death mechanisms: apoptosis and autophagy after prodigiosin treatment. Our results demonstrate that prodigiosin is a BH3-mimetic drug, as it was previously described for obatoclax (GX15-070). Obatoclax is a synthetic prodiginine derivative actually in clinical trials in phase III. Moreover, we have identified mTOR as a new molecular target of prodigiosin and obatoclax. PI3K/AKT/mTOR pathway is involved in cell survival, proliferation and migration. Different cross-talks and feedback loops regulate this pathway and, also, are involved in different compensatory mechanisms that confer chemoresistance to melanoma cells. Dual inhibition of mTORC1 and mTORC2 mediated by both prodiginines disrupted the negative feedback loop S6K/IRS-1. The interaction of both prodiginines in the active site of mTOR was monitorized by Surface Plasmon Resonance (SPR) assays. Nanomolar kinetic and affinity parameters indicated high affinity, stability and specificity of these interactions. Nevertheless, results showed that both prodiginines are not ATP-mimetic drugs. In silico models showed the detailed atomic view of the complexes indicating the critical residues involved in these interactions. Moreover, structure-activity relationship (SAR) studies corroborated the importance of pyrrole amino groups in the cytotoxic prodiginines activity mediated by mTOR inhibition. Finally, we observed that mTOR inhibition by prodiginines promotes actin filaments desorganization, decrease of focal adhesions and inhibition of cell migration. Our results suggest that prodiginines might inhibit integrin signalling through ILK/rictor complex. Alltogether, these results contribute to further understanding the molecular mechanisms of prodiginines and, also, their chemotherapeutical potential. Moreover, we provide new data about their structural properties that will allow the development of more-effective anticancer molecules

Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells

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

Molecular Interactions of Prodiginines with the BH3 Domain of Anti-Apoptotic Bcl-2 Family Members.

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

Antimetastatic Properties of Prodigiosin and the BH3-Mimetic Obatoclax (GX15-070) in Melanoma

Metastasis is the primary cause of death in cancer patients. Many current chemotherapeutic agents only show cytotoxic, but not antimetastatic properties. This leads to a reduction in tumor size, but allows cancer cells to disseminate, which ultimately causes patient death. Therefore, novel anticancer compounds with both effects need to be developed. In this work, we analyze the antimetastatic properties of prodigiosin and obatoclax (GX15-070), anticancer drugs of the Prodiginines (PGs) family. We studied PGs’ effects on cellular adhesion and morphology in the human primary and metastatic melanoma cell lines, SK-MEL-28 and SK-MEL-5, and in the murine melanoma cell line, B16F10A. Cell adhesion sharply decreased in the treated cells, and this was accompanied by a reduction in filopodia protrusions and a significant decrease in the number of focal-adhesion structures. Moreover, cell migration was assessed through the wound-healing assay and cell motility was severely inhibited after 24 h of treatment. To elucidate the molecular mechanisms involved, changes in metastasis-related genes were analyzed through a gene-expression array. Key genes related to cellular invasion, migration and chemoresistance were significantly down-regulated. Finally, an in vivo model of melanoma-induced lung metastasis was established and significant differences in lung tumors were observed in the obatoclax-treated mice. Altogether, these results describe, in depth, PGs’ cellular antimetastatic effects and identify in vivo antimetastatic properties of Obatoclax

Down-Regulation of miR-29b Is Essential for Pathogenesis of Uterine Leiomyoma

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Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells

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