Deregulation of lactate permeability using a small-molecule transporter (Lactrans-1) disturbs intracellular pH and triggers cancer cell death.

Due to the relevance of lactic acidosis in cancer, several therapeutic strategies have been developed targeting its production and/or regulation. In this matter, inhibition approaches of key proteins such as lactate dehydrogenase or monocarboxylate transporters have showed promising results, however, metabolic plasticity and tumor heterogeneity limits their efficacy. In this study, we explored the anticancer potential of a new strategy based on disturbing lactate permeability independently of monocarboxylate transporters activity using a small molecule ionophore named Lactrans-1. Derived from click-tambjamines, Lactrans-1 facilitates transmembrane lactate transportation in liposome models and reduces cancer cell viability. The results showed that Lactrans-1 triggered both apoptosis and necrosis depending on the cell line tested, displaying a synergistic effect in combination with first-line standard chemotherapeutic cisplatin. The ability of this compound to transport outward lactate anions was confirmed in A549 and HeLa cells, two cancer cell lines having distinct rates of lactate production. In addition, through cell viability reversion experiments it was possible to establish a correlation between the amount of lactate transported and the cytotoxic effect exhibited. The movement of lactate anions was accompanied with intracellular pH disturbances that included basification of lysosomes and acidification of the cytosol and mitochondria. We also observed mitochondrial swelling, increased ROS production and activation of oxidative stress signaling pathways p38-MAPK and JNK/SAPK. Our findings provide evidence that enhancement of lactate permeability is critical for cellular pH homeostasis and effective to trigger cancer cell death, suggesting that Lactrans-1 may be a promising anticancer therapy.

Small molecule anion carriers facilitate lactate transport in model liposomes and cells.

An excessive production of lactate by cancer cells fosters tumor growth and metastasis. Therefore, targeting lactate metabolism and transport offers a new therapeutic strategy against cancer, based on dependency of some cancer cells for lactate as energy fuel or as oncogenic signal. Herein we present a family of anionophores based on the structure of click-tambjamines that have proved to be extremely active lactate carriers across phospholipid membranes. Compound 1, the most potent lactate transmembrane carrier, was studied in HeLa cells. The use of a monocarboxylate transporters (MCTs) inhibitor proved that 1 is an active lactate transporter in living cells, confirming the results obtained in phospholipid vesicles. Moreover, an additive effect of compound 1 with cisplatin was observed in HeLa cells. Identification of active lactate anionophores working in living cells opens up ways to exploit this class of compounds as molecular tools and drugs addressing dysregulated lactate metabolism.

Cytotoxicity of osmium(II) and cycloosmated half-sandwich complexes from 1-pyrenyl-containing phosphane ligands.

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 MRX2 were synthesized and fully characterized, namely OsiPrCl2, OsiPrI2, OsPhCl2, OsPhI2 and RuPhI2. Furthermore, nine cyclometalated half-sandwich complexes of formula [MX-(η6-p-cymene)(k2C-diR(1-pyrenyl)phosphane)] (M = Os or Ru, X = Cl or I, R = isopropyl or phenyl) or [M(η6-p-cymene)(kS-dmso)(k2C-diR(1-pyrenyl)phosphane)]PF6 (M = Os or Ru, R = isopropyl or phenyl) and symbolized as c-MRX were prepared; hence, c-OsiPrCl, c-OsiPrI, c-OsiPrdmso, c-OsPhCl, c-OsPhI, c-OsPhdmso, c-RuPhCl, c-RuPhI and c-RuPhdmso were obtained and fully characterized. The crystal structures of ten out of the fourteen complexes were solved. All complexes exhibit notable cytotoxic properties against A549 (Lung Adenocarcinoma) human cells, with IC50 values ranging from 48 to 1.42 µM. In addition, complex c-OsiPrdmso shows remarkable toxic behaviours agains other cell lines, namely MCF7 (breast carcinoma), MCF10A (non-tumorigenic epithelial breast) and MDA-MB-435 (melanoma) human cells, as illustrated by IC50 values of 4.36, 4.71 and 2.32 µM, respectively. Finally, it has been found that OsiPrI2 affects the cell cycle of A549 cells, impeding their replication (i.e., the cell cycle is blocked), whereas OsPhI2 (namely with phenyl groups instead of isopropyl ones) does not induce this effect.

Steric hindrance, ligand ejection and associated photocytotoxic properties of ruthenium(II) polypyridyl complexes.

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. 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+.

A Novel Late-Stage Autophagy Inhibitor That Efficiently Targets Lysosomes Inducing Potent Cytotoxic and Sensitizing Effects in Lung Cancer.

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.

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.

Multi-Smart and Scalable Bioligands-Free Nanomedical Platform for Intratumorally Targeted Tambjamine Delivery, a Difficult to Administrate Highly Cytotoxic Drug.

Cancer is one of the leading causes of mortality worldwide due, in part, to limited success of some current therapeutic approaches. The clinical potential of many promising drugs is restricted by their systemic toxicity and lack of selectivity towards cancer cells, leading to insufficient drug concentration at the tumor site. To overcome these hurdles, we developed a novel drug delivery system based on polyurea/polyurethane nanocapsules (NCs) showing pH-synchronized amphoteric properties that facilitate their accumulation and selectivity into acidic tissues, such as tumor microenvironment. We have demonstrated that the anticancer drug used in this study, a hydrophobic anionophore named T21, increases its cytotoxic activity in acidic conditions when nanoencapsulated, which correlates with a more efficient cellular internalization. A biodistribution assay performed in mice has shown that the NCs are able to reach the tumor and the observed systemic toxicity of the free drug is significantly reduced in vivo when nanoencapsulated. Additionally, T21 antitumor activity is preserved, accompanied by tumor mass reduction compared to control mice. Altogether, this work shows these NCs as a potential drug delivery system able to reach the tumor microenvironment, reducing the undesired systemic toxic effects. Moreover, these nanosystems are prepared under scalable methodologies and straightforward process, and provide tumor selectivity through a smart mechanism independent of targeting ligands.

Piano-Stool Ruthenium(II) Complexes with Delayed Cytotoxic Activity: Origin of the Lag Time.

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.

Tumors defective in homologous recombination rely on oxidative metabolism: relevance to treatments with PARP inhibitors.

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.

Click-tambjamines as efficient and tunable bioactive anion transporters.

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.

Targeting Autophagy for Cancer Treatment and Tumor Chemosensitization.

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.

The Natural-Based Antitumor Compound T21 Decreases Survivin Levels through Potent STAT3 Inhibition in Lung Cancer Models.

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.

DNA-binding and in vitro cytotoxic activity of platinum(II) complexes of curcumin and caffeine.

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 ß-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.

Therapeutic strategies involving survivin inhibition in cancer.

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.

Highly Cytotoxic Ruthenium(II)-Arene Complexes from Bulky 1-Pyrenylphosphane Ligands.

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.

Small molecule anionophores promote transmembrane anion permeation matching CFTR activity.

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.

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.

Indole-based perenosins as highly potent HCl transporters and potential anti-cancer agents.

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.

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.

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.