Inhibition of Human Enhancer of Zeste Homolog 2 with Tambjamine Analogs.

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.

Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem 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 stemness of CSCs leading to effective elimination of this cancer cell subpopulation.

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.

Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer.

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.

Novel Indole-based Tambjamine-Analogues Induce Apoptotic Lung Cancer Cell Death through p38 Mitogen-Activated Protein Kinase Activation.

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. Mol Cancer Ther; 16(7); 1224-35. ©2017 AACR.