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Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer
Abstract
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 favors 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 analyze for the first time the anticancer effects of tambjamine analogues, a group of highly effective anion selective ionophores, at the cellular and molecular level. 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.
... To date, many small-molecule anion transporters have been reported based on ureas [4,32], thioureas [13,33], sulfonamides [34,35], squaramides [5,16,36], pyrroles [14,37], indoles [15,38], imidazoles/benzimidazoles [18,39,40] and triaminopyrimidines [41] (Fig. 1). These small-molecule compounds do not only have potent anion transport activity, but also show strong tumor inhibition [3,42]. ...
... As the concentration of chloride anions in lysosomes (~80 mM) is higher than that in cytoplasm (5e40 mM), these compounds may make chloride anions flow out of the lysosomes, and at the same time, protons leak from the lysosomes or bicarbonate anions enter the lysosomes. As the lysosomal pH increases, the activity of lysosomal enzymes decreases, which interferes with the normal physiological processes of subsequent lysosomal functions, such as autophagy [14,16]. In 2018, we reported the first example of a lysosome-targeting fluorescent ionophore 24 (Fig. 8A) [63]. ...
... In addition, western blot assays showed that compound 5 can up-regulate the levels of LC3-II and p62 proteins, indicating that compound 5 interferes with autophagy. In the same year, Rodilla et al. found that tambjamine 20 (Fig. 7) can not only disturb the anion balance and cause mitochondrial swelling, but also cause lysosomal dysfunction and lead to the inhibition of autophagy [14]. In the acridine orange experiments, they observed that in the presence of compound 20, the red fluorescence of A549 cells disappeared, indicating that it can increase the pH of intracellular acidic organelles. ...
The balance of normal anion concentrations in cells provides basis for maintaining cellular morphology and function. Disrupting the homeostasis of cellular anions and lysosomal pH, in particular with high selectivity for cancer cells over normal cells may serve as a promising approach for the treatment of cancers. Small-molecule organic compounds with transmembrane anion transport activity, namely synthetic anion transporters are able to destroy the homeostasis of cellular anions, in particular chloride anions to trigger cell death and thus may be developed as a new class of anti-tumor drugs. This paper reviews the latest advance in the investigation into the in vitro anion transport, promising anti-tumor activity and probable mechanism of biological action of synthetic anion transporters. The strategies for optimizing the biological activity of synthetic anion transporters and improving the selectivity for cancer cells over normal cells are also discussed.
... During the past decades, considerable interest has been attracted in identifying small-molecular anion transporters that are capable of efficiently mediating the transport of anions across lipid membranes [1,2]. By modulating the intracellular pH [3,4], or disrupting the cellular ionic homeostasis in cancer or bacteria cells [5,6], effective anion transporters are able to induce cell death and thereby may serve as a new therapy for cancers and bacterial infections [1,7]. For example, Gale et al. have shown that pyridine diamide-strapped calix [4]pyrroles are able to induce coupled chloride/sodium transport both in liposomal models and cells, and promote cell death by increasing the intracellular concentrations of chloride and sodium ions [8]. ...
... The in vitro anionophoric activity of m-Bimbe, (CF 3 ) 2 -Bimbe, MeO(CF 3 ) 2 -Bimbe and NO 2 (CF 3 ) 2 -Bimbe in HeLa cell lines, was further studied using vital staining with acridine orange (AO), a cell-permeable dye [3,4]. When protonated and accumulated in acidic compartments such as lysosomes, AO exhibits a characteristic orange fluorescence emission, whereas it emits green fluorescence when the acidic compartments are basified. ...
... Notably, the basifying ability in the order of NO 2 (CF 3 ) 2 -Bimbe > (CF 3 ) 2 -Bimbe > MeO(CF 3 ) 2 -Bimbe > m-Bimbe, parallels their ionophoric activity in liposomal models ( Table 1). The proton/chloride symport observed in the liposomal models, is thought to be responsible for the increase of the internal pH although other mechanisms (e.g., Cl À /HCO 3 À ) may also be involved [3,4]. ...
In this paper we show that a series of 1,3-bis(benzimidazol-2-yl)benzene (m-Bimbe) derivatives exhibit excellent performance as transmembrane anion transporters with anticancer activity. The transport efficiency of m-Bimbe and its derivatives has been firstly optimized by adding a strong electron-withdrawing nitro group at the 5-position of the central phenyl subunits to enhance the CH···anion interactions. Evidences for the interactions were obtained from ESI MS, spectrophotometric and 1H NMR titrations. These compounds exhibit potent anionophoric activity in both liposomal models and live cells. In particular, the 5-nitrated derivatives having nitro or trifluoromethyl groups at the benzimidazoloyl subunits exhibit 2370- and 1721-fold enhanced anionophoric activity with the EC50 values as low as 36 and 50 nM, respectively. These compounds can disturb the cellular homeostasis of chloride anions, modify the intracellular pH and induce the basification of acidic organelles. Most of this series of m-Bimbe derivatives exhibit potent cytotoxicity toward the tested human solid tumor cell lines, and the 5-nitrated derivative bearing trifluoromethyl groups at the benzimidazoloyl subunits is the most active with the IC50 value in the low micromolar range. Mechanistic studies suggest that the transport of chloride anions across the cellular membranes plays a critical role in the cytotoxic effect and these compounds induce cell death probably via an apoptotic process.
... On the other hand, ion-homeostasis disturbing agents, such as small molecules with ionophoric activity have shown to effectively induce intracellular acidification and trigger cancer cell death [20]. The effect of these compounds relies mostly on the facilitated transport of chloride, calcium and potassium, and their disturbance usually promotes oxidative environments that trigger the activation of cellular stress signaling pathways and programmed or unprogrammed forms of cell death [21][22][23][24]. Thereby, it is feasible to propose that other relevant anions in the specific context of cancer, such as lactate, could be interesting and more specific targets. ...
... Our results showed that Lactrans-1 is also able to trigger apoptotic or necrotic cell death depending on the cell line assessed, an effect previously reported with other click-tambjamine analogues [22,29]. A predominant apoptotic cell death evaluated through the activation of the executioner caspase 3 and cleavage of its substrate PARP was observed in HeLa cells after treatment at different time points (beyond 8 h). ...
... Intracellular ion stability is crucial for maintaining cell osmotic pressure and proper physiological functions, whereas imbalances in intracellular ion homeostasis will lead to cell death. Studies have shown that drastic changes in intracellular ion concentrations, such as Cl − , Ca 2+ , and K + , can lead to cell death through apoptosis, autophagy, necroptosis, ferroptosis, oxidative stress, and endoplasmic reticulum (ER) stress [7][8][9][10][11]. In this context, various synthetic structures have been proposed to mimic the physiological transport function in living systems [12][13][14][15][16][17][18][19][20]. ...
... Studies have shown that disruption of intracellular ion concentration homeostasis can activate multiple pathways such as apoptosis, autophagy, pyroptosis, ferroptosis, oxidative stress, and ER stress, ultimately leading to cell death [7][8][9][10][11]. Based on the above theory, we investigated whether PTU, TFPTU, and BTFPTU could disrupt the ion homeostasis of osteosarcoma cells and kill them. ...
Background
Osteosarcoma represents a serious clinical challenge due to its widespread genomic alterations, tendency for drug resistance and distant metastasis. New treatment methods are urgently needed to address those treatment difficulties in osteosarcoma to improve patient prognoses. In recent years, small-molecule based anion transporter have emerged as innovative and promising therapeutic compound with various biomedical applications. However, due to a lack of efficient delivery methods, using ion transporters as therapeutic drugs in vivo remains a major challenge.
Result
Herein, we developed self-assembled supramolecular drugs based on small-molecule anion transporters, which exhibited potent therapeutic effect towards osteosarcoma both in vitro and in vivo. The anion transporters can disrupt intracellular ion homeostasis, inhibit proliferation, migration, epithelial-mesenchymal transition process, and lead to osteosarcoma cell death. RNA sequencing, western blot and flow cytometry indicated reprogramming of HOS cells and induced cell death through multiple pathways. These pathways included activation of endoplasmic reticulum stress, autophagy, apoptosis and cell cycle arrest, which avoided the development of drug resistance in osteosarcoma cells. Functionalized with osteosarcoma targeting peptide, the assembled supramolecular drug showed excellent targeted anticancer therapy against subcutaneous xenograft tumor and lung metastasis models. Besides good tumor targeting capability and anti-drug resistance, the efficacy of the assembly was also attributed to its ability to regulate the tumor immune microenvironment in vivo.
Conclusions
In summary, we have demonstrated for the first time that small-molecule anion transporters are capable of killing osteosarcoma cells through multiple pathways. The assemblies, OTP-BP-L, show excellent targeting and therapeutic effect towards osteosarcoma tumors. Furthermore, the supramolecular drug shows a strong ability to regulate the tumor immune microenvironment in vivo. This work not only demonstrated the biomedical value of small-molecule anion transporters in vivo, but also provided an innovative approach for the treatment of osteosarcoma.
... Supporting these results, LAI-1 anionophoric activity was observed across the lysosomal membrane in lung cancer cells, since acridine orange staining demonstrated an increase in lysosomal pH after 1 h upon treatment with increasing concentrations of LAI-1. An increase in lysosomal pH was also previously found after cell treatment with different anionophoric molecules, such as tambjamine analogs or obatoclax; however, these molecules also target other organelles [30][31][32]. The maintenance of an acidic environment inside lysosomes is crucial for their proper function, as the hydrolases that degrade damaged proteins or organelles are only functional at an acidic pH [33]. ...
... However, the results observing the nucleus staining with Hoechst and propidium iodide also suggested that this apoptosis occurred together with a necrotic cell death, being predominant in DMS53 cells. Similar results have been reported for ionophoric compounds, such as tambjamine analogs, prodiginines, obatoclax and salinomycin, inducing both apoptosis and also necrosis [29,31,[40][41][42]. In our case, at basal levels, these cancer cells would be inducing cytoprotective autophagy, and after being blocked by our drug, it would trigger cell death. ...
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.
... Literature protocols were used to carry out the vesicle preparations [43], 1 H NMR titration [17,37,38,[44][45][46][47][48][49][50], pH discharge [51,52], calcein leakage [53], chloride efflux [54,55] and U-tube assay [56] experiments of each of compounds 1-4. Measurement of cytotoxicity & the mechanism of biological actions Literature protocols were used to carry out the AO staining [22,[57][58][59][60][61], MQAE assay [22,29], SBFI AM assay [29], MTT-based cytotoxicity assay [22], Hoechst staining [22,60,61] and JC-1 staining [22,62]. ...
... For this purpose, we used AO to stain human cervical cancer HeLa cells and observed the changes in the fluorescence of AO. Because AO exhibits characteristic pH-sensitive fluorescence that changes from orange to green when pH increases, it has been widely used as a fluorescent reporter for the pH change within organelles [57][58][59]. As shown in Supplementary Figure 52, vital staining of the HeLa cells with AO led to the presence of granular orange fluorescence in the cytoplasm of organelles. ...
Aim: Anion/cation symport across cellular membranes may lead to cell apoptosis and be developed as a strategy for new anticancer drug discovery. Methodology: Four aza-crown ether-squaramide conjugates were synthesized and characterized. Their anion recognition, anion/cation symport, cytotoxicity and probable mechanism of action were investigated in details. Conclusion: These conjugates are able to form ion-pairing complexes with chloride anions and facilitate the transmembrane transport of anions via an anion/cation symport process. They can disrupt the cellular homeostasis of chloride anions and sodium cations and induce the basification of acidic organelles in live cells. These conjugates exhibit moderate cytotoxicity toward the tested cancer cells and trigger cell apoptosis by mediating the influx of chloride anions and sodium cations into live cells.
... 5 Several classes of synthetic anionophores have been utilized as vectors for the disruption of anion homeostasis in cellulo. [6][7][8] The dysregulation of chloride influx with perenosins can induce apoptosis and cell-cycle arrest, 9 and some squaramide transporters have been shown to disrupt autophagy through altering the lysosomal pH. 10 There has been less research focus on the applicability of anionophores that might function as antimicrobial agents. 11 This is somewhat surprising given the documented antimicrobial activity of many cationophore natural products, such as monensin, salinomycin, and lasalocid. ...
... Soto-Cerrato, Pérez Tomàs et al. analyzed in detail the cellular and molecular mechanisms of action of two marine alkaloids (i.e., tambjamines), bearing aromatic enamine moieties T5 and T6 (Table 3) [130]. Both promoted transmembrane Cl − and HCO 3 − transport in liposomes. ...
Ion channels and transporters typically consist of biomolecules that play key roles in a large variety of physiological and pathological processes. Traditional therapies include many ion-channel blockers, and some activators, although the exact biochemical pathways and mechanisms that regulate ion homeostasis are yet to be fully elucidated. An emerging area of research with great innovative potential in biomedicine pertains the design and development of synthetic ion channels and transporters, which may provide unexplored therapeutic opportunities. However, most studies in this challenging and multidisciplinary area are still at a fundamental level. In this review, we discuss the progress that has been made over the last five years on ion channels and transporters, touching upon biomolecules and synthetic supramolecules that are relevant to biological use. We conclude with the identification of therapeutic opportunities for future exploration.
... Authors later analysed the cellular and molecular mechanisms of killing the lung cancer cells and cancer stem cells by these tambjamine anionophores (23 b-23 d, 24 a-24 d, and 25 b). [33] The transmembrane transport caused imbalance in cell homeostasis activated mitochondrial dysfunction and lysosomal deacidification. These events consequently generated potential cytotoxic effects through necrosis in cancer cell lines. ...
Several life‐threatening diseases, also known as ‘Channelopathies’ are linked to irregularities in ion transport proteins. Significant research efforts have fostered the development of artificial transport systems that facilitates to restore the functions of impaired natural transport proteins. Indeed, a few of these artificial ionophores demonstrate the rare combination of transmembrane ion transport and important biological activity, offering early promises of suitability in ‘channel replacement therapy’. In this review, structural facets and functions of both cationophores and anionophores are discussed. Ionophores that are toxic to various bacteria and yeast, could be exploited as antimicrobial agent. Nevertheless, few non‐toxic ionophores offer the likelihood of treating a wide range of genetic diseases caused by the gene mutations. In addition, their ability to disrupt cellular homeostasis and to alter lysosomal pH endow ionophores as promising candidates for cancer treatment. Overall, critically outlining the advances in artificial ionophores in terms of in vitro ion transport, possible modes of action and biological activities enables us to propose possible future roadmaps in this research area.
... 18 Quesada et al have shown that tambjamine analogues provoke an imbalance in the homeostasis of cellular anions and the disruption triggers lysosomal deacidification leading to a potent cytotoxic effect through necrosis in lung cancer cell lines and cancer stem cells. 19 We have also demonstrated that anion transport-active iridium complexes are able to alkalize lysosomes and deactivate lysosomal Cathepsin B enzyme, thus leading to an inhibition in the autophagic flux. 20 Inspired by these findings, we reasoned that an anion transporter equipped with a lysosome-targeting group might serve as a useful research tool to investigate the function of anion transporters within cells. ...
Two lysosome-targeting fluorescent anion transporters derived from coumarins, trifluoromethylated arylsquaramides and morpholines were synthesized, and their specificity and efficiency to target and alkalize lysosomes were investigated. They are able to target lysosomes specifically. Compared with the previous analogue without trifluoromethyl substituents, these two conjugates, in particular the one having a 3,5-bis(trifluoromethyl) substituent, exhibit significantly higher ability to facilitate the transport of chloride anions, alkalize lysosomes and reduce the activity of lysosomal Cathepsin B enzyme. The present finding suggests that improving the anionophoric activity of lysosome-targeting fluorescent anion transporters is favorable to the efficiency to alkalize lysosomes and deactivate lysosomal Cathepsin B enzyme.
... Soto-Cerrato and Tomá s recently studied the effect of synthetic tambjamine analogs 85 and 86 ( Figure 50C) in several lung cancer cell lines and cancer stem cells (CSCs). 94 They demonstrated that compound 85a causes a significant decrease in cell viability, either in CSCs derived from cell lines (A549 and SW900) or in CSCs derived from tumor-patient samples (FIS302 and FIS303). After treatment with compounds 85 and 86, lysosomal deacidification, massive cytoplasmic vacuolization, and mitochondrial swelling were observed, and finally, several proteins involved in stress response, autophagy, and apoptosis were activated. ...
This review covers advances in anion complexation in the years 2017 and 2018. The review focuses on the applications of anion receptor chemistry, including sensing, self-assembly, extraction, transport, catalysis, and fundamental advances in the area.
... Ionophores can also disrupt lysosomal pH, impairing the autophagy process. Tambjamine analogues are anion selective ionophores derived from the naturally occurring tambjamines and induce mitochondrial swelling and autophagy blockade with cytotoxic effects in lung cancer cells and cancer stem cells (CSCs) [149]. Monensin, nigericin, and lasalocid are cation ionophores, but only monensin presents selectivity for lysosomes [150]. ...
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.
... 12 Another relevant cellular event occurring because of the facilitated transport mediated by anionophores is lysosomal dysfunction. 13 Lysosomes function as the cellular digestive system by degrading and recycling intracellular material. Lysosomes contain enzymes, which are active at acidic pH, and actively pump protons from the cytosol, resulting in a characteristic acidic pH as well as high internal chloride concentration. ...
In this issue of Chem, Pellois, Gabbaï, and colleagues introduce pnictogenium cations as useful transmembrane anion transporters, and Gale, Sessler, Injae, and co-workers provide insights into the cellular mechanism elicited by some of these compounds, leading to the induction of apoptosis in cancer cells.
... 9,10 Such processes may be used to induce apoptosis, thus opening the door to applications in cancer therapy. [11][12][13][14][15] While the use of organic receptors dominates the domain of anion transport, a series of recent reports indicate that Lewis acidic main-group compounds may constitute promising platforms, as in the case of the pnictogen-, chalcogen-, and halogenbond-donor derivatives B, 16 C, 16 D, 17 and E, 18 which transport chloride ions through artificial lipid bilayers ( Figure 1). These precedents, as well as the knowledge we have derived from our work on main-group Lewis acids as anion sensors, [19][20][21][22] ...
Our work on the complexation of fluoride anions using group 15 Lewis acids has led us to investigate the use of these main group compounds as anion transporters. In this paper, we report on the anion transport properties of tetraarylstibonium and tetraarylbismuthonium cations of the general formula [Ph3PnAr]+ with Pn = Sb or Bi and with Ar = phenyl, naphthyl, anthryl, or pyrenyl. Using EYPC-based large unilamellar vesicles, we show that these main group cations transport hydroxide, fluoride and chloride anions across phospholipid bilayers. A comparison of the properties of [Ph3SbAnt]+ and [Ph3BiAnt]+ (Ant = 9-anthryl) illustrates the favorable role played by the Lewis acidity of the central pnictogen element with respect to the anion transport. Finally, we show that [Ph3SbAnt]+ accelerates the fluoride-induced hemolysis of human red blood cells, an effect that we assign to the transporter-facilitated influx of toxic fluoride anions.
... Cell autophagy induced by prodigiosin was mediated through AMPKα and phosphoinositide 3-kinase (PI3K) class III/Akt signaling pathway to inhibit mTOR and Beclin-1 to induce p62/ LC3 (Cheng et al., 2017). Tambjamine analogues showed cytotoxic activities through necrosis in lung cancer cell lines due to the imbalance in cellular ion homeostasis that led to mitochondrial dysfunction and lysosomal deacidification (Rodilla et al., 2017). Homoharringtonine, a clinically approved drug for leukemia, sensitized cancer cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced necroptosis through the pathway involving RIPK1/RIPK3/MLKL (Philipp et al., 2015). ...
Alkaloids are important chemical compounds that serve as a rich source for drug discovery. Numerous alkaloids screened from medicinal plants and herbs showed antiproliferative and anticancer effects on wide category of cancers both in vitro and in vivo. Vinblastine, vinorelbine, vincristine, and vindesine have already been successfully developed as anticancer drugs. The available and up-to-date information on the ethnopharmacological uses in traditional medicine, phytochemistry, pharmacology and clinical utility of alkaloids were collected using various resources (PubMed, ScienceDirect, Google Scholar and Springerlink). In this article, we provide a comprehensive and critical overview on naturally-occurring alkaloids with anticancer activities and highlight the molecular mechanisms of action of these secondary metabolites. Furthermore, this review also presents a summary of synthetic derivatives and pharmacological profiles useful to researchers for the therapeutic development of alkaloids. Based on the literature survey compiled in this review, alkaloids represent an important group of anticancer drugs of plant origin with enormous potential for future development of drugs for cancer therapy and management.
... To date, considerable effort has been devoted to developing artificial ion transporters that have anticancer activity. 23,24,[80][81][82][83] However, most of this effort has been empirical in nature. ...
Recently, we showed that synthetic anion transporters DSC4P-1 and SA-3 had activity related to cancer cell death. They were found to increase intracellular chloride and sodium ion concentrations. They were also found to induce apoptosis (DSC4P-1) and both induce apoptosis and inhibit autophagy (SA-3). However, determinants underlying these phenomenological findings were not elucidated. The absence of mechanistic understanding has limited the development of yet-improved systems. Here, we show that three synthetic anion transporters, DSC4P-1, SA-3, and 8FC4P, induce osmotic stress in cells by increasing intracellular ion concentrations. This triggers the generation of reactive oxygen species via a sequential process and promotes caspase-dependent apoptosis. In addition, two of the transporters, SA-3 and 8FC4P, induce autophagy by increasing the cytosolic calcium ion concentration promoted by osmotic stress. However, they eventually inhibit the autophagy process as a result of their ability to disrupt lysosome function through a transporter-mediated decrease in a lysosomal chloride ion concentration and an increase in the lysosomal pH.
... Indeed, at least theoretically, such ionophores might be used as substitutes to compensate for the defective function of CFTR 44,50 . As shown in this paper, consistent with precedent reports 19,51 , chloride ionophores that can dissipate pH gradients, such as the compounds studied here that have been shown to be capable of transporting both chloride and protons across lipid bilayers, block autophagic flux in a peculiar manner, arguing against their therapeutic utility. Indeed, it appears to be advantageous to restore autophagic turnover in cystic fibrosis 12,15,17 , implying that autophagy inhibition would ultimately Fig. 5 Contribution of TFEB and ATG5 in LC3 relocation induced by the SQs. ...
Cystic fibrosis is a disease caused by defective function of a chloride channel coupled to a blockade of autophagic flux. It has been proposed to use synthetic chloride transporters as pharmacological agents to compensate insufficient chloride fluxes. Here, we report that such chloride anionophores block autophagic flux in spite of the fact that they activate the pro-autophagic transcription factor EB (TFEB) coupled to the inhibition of the autophagy-suppressive mTORC1 kinase activity. Two synthetic chloride transporters (SQ1 and SQ2) caused a partially TFEB-dependent relocation of the autophagic marker LC3 to the Golgi apparatus. Inhibition of TFEB activation using a calcium chelator or calcineurin inhibitors reduced the formation of LC3 puncta in cells, yet did not affect the cytotoxic action of SQ1 and SQ2 that could be observed after prolonged incubation. In conclusion, the squaramide-based synthetic chloride transporters studied in this work (which can also dissipate pH gradients) are probably not appropriate for the treatment of cystic fibrosis yet might be used for other indications such as cancer.
... To use these substances in cells, with the expectation to employ them as therapeutic drugs, is a big challenge, since this molecules are derivatives of cytotoxic natural products. Indeed, substances like obatoclax have been proposed as antitumor chemotherapeutic (Hernando et al., 2014b;Ko et al., 2014;Rodilla et al., 2017). Indeed, an effort to reduce the prodigiosine related molecules has been successful preparing the triazole derivatives of prodigiosine Hernando et al., 2018). ...
Background and Purpose
Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease that originates from the defective function of the CF transmembrane conductance regulator (CFTR) protein, a cAMP‐dependent anion channel involved in fluid transport across epithelium. Because small synthetic transmembrane anion transporters (anionophores) can replace the biological anion transport mechanisms, independent of genetic mutations in the CFTR, such anionophores are candidates as new potential treatments for CF.
Experimental Approach
In order to assess their effects on cell physiology, we have analysed the transport properties of five anionophore compounds, three prodigiosines and two tambjamines. Chloride efflux was measured in large uni‐lamellar vesicles and in HEK293 cells with chloride‐sensitive electrodes. Iodide influx was evaluated in FRT cells transfected with iodide‐sensitive YFP. Transport of bicarbonate was assessed by changes of pH after a NH4⁺ pre‐pulse using the BCECF fluorescent probe. Assays were also carried out in FRT cells permanently transfected with wild type and mutant human CFTR.
Key Results
All studied compounds are capable of transporting halides and bicarbonate across the cell membrane, with a higher transport capacity at acidic pH. Interestingly, the presence of these anionophores did not interfere with the activation of CFTR and did not modify the action of lumacaftor (a CFTR corrector) or ivacaftor (a CFTR potentiator).
Conclusion and Implications
These anionophores, at low concentrations, transported chloride and bicarbonate across cell membranes, without affecting CFTR function. They therefore provide promising starting points for the development of novel treatments for CF.
... The cytotoxic activity of these compounds is related to their ionophoric activity 13,14 , and a related derivative, obatoclax, has shown potential as anticancer agent in the clinic 15 . Indeed, we and others have studied the cytotoxicity of anionophores and their potential applications as antitumor chemotherapeutics delineating the relationships between their ionophoric activity and the triggered cellular mechanism associated to their action [16][17][18][19][20][21] . ...
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.
... To investigate this further, Annexin-V was used as a marker with flow cytometry analysis to evaluate apoptosis in MDA-MB-231 cells after treatments with 2 (best transporter) and 6 (most potent) at their respective IC 50 values. It is known that synthetic transporters can disrupt ion homeostasis in cells and induce cell death via an apoptotic pathway 3,22,32,63,64 . ...
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.
The incidence and mortality of cancer are increasing, making it a leading cause of death worldwide. Conventional treatments such as surgery, radiotherapy, and chemotherapy face significant limitations due to therapeutic resistance. Autophagy, a cellular self-degradation mechanism, plays a crucial role in cancer development, drug resistance, and treatment. This review investigates the potential of autophagy inhibition as a therapeutic strategy for cancer. A systematic search was conducted on Embase, PubMed, and Google Scholar databases from 1967 to 2024 to identify studies on autophagy inhibitors and their mechanisms in cancer therapy. The review includes original articles utilizing in vitro and in vivo experimental methods, literature reviews, and clinical trials. Key terms used were “Autophagy”, “Inhibitors”, “Molecular mechanism”, “Cancer therapy”, and “Clinical trials”. Autophagy inhibitors such as chloroquine (CQ) and hydroxychloroquine (HCQ) have shown promise in preclinical studies by inhibiting lysosomal acidification and preventing autophagosome degradation. Other inhibitors like wortmannin and SAR405 target specific components of the autophagy pathway. Combining these inhibitors with chemotherapy has demonstrated enhanced efficacy, making cancer cells more susceptible to cytotoxic agents. Clinical trials involving CQ and HCQ have shown encouraging results, although further investigation is needed to optimize their use in cancer therapy. Autophagy exhibits a dual role in cancer, functioning as both a survival mechanism and a cell death pathway. Targeting autophagy presents a viable strategy for cancer therapy, particularly when integrated with existing treatments. However, the complexity of autophagy regulation and the potential side effects necessitate further research to develop precise and context-specific therapeutic approaches.
Six novel click-tambjamines (1-6) bearing an alkyl chain of varying length linked to the imine moiety have been formulated in nanostructured lipid carriers (NLCs) to evaluate their transmembrane anion transport activity both when free (i.e., not encapsulated) and nanoformulated. Nanostructured lipid carriers (NLCs) are an example of drug delivery systems (DDSs) that stand out because of their versatility. In this work we show that NLCs can be used to efficiently formulate highly lipophilic anionophores and experiments conducted in model liposomes reveal that these formulations are adequate to deliver anionophores without compromising their transport activity. This result paves the way to facilitate the study of highly lipophilic anionophores and their potential use as future drugs.
The majority of cellular physiological processes depend on natural ion channels, which are pore-forming membrane-embedded proteins that let ions flow across the cell membranes selectively. This selective movement of ions across the membranes balances the osmolality within and outside the cell. However, mutations in the genes that encode essential membrane transport proteins or structural reorganisation of these proteins can cause life-threatening diseases like cystic fibrosis. Artificial ion transport systems have opened up a way to replace dysfunctional natural ion channels to cure such diseases through channel replacement therapy. Moreover, recent research has also demonstrated the ability of these systems to kill cancer cells, reigniting interest in the field among scientists. Our contributions to the recent progress in the design and development of artificial chloride ion transporters and their effect on biological systems have been discussed in this review. This review would provide current vistas and future directions toward the development of novel ion transporters with improved biocompatibility and desired anti-cancer properties. Additionally, it strongly emphasises stimuli-responsive ion transport systems, which are crucial for obtaining target-specificity and may speed up the application of these systems in clinical therapeutics.
Global warming and eutrophication accelerated Microcystis blooms, causing water quality deterioration. Cyanocides produced by cyanobactericidal bacteria have attracted substantial attention for controlling Microcystis blooms owing to their environmental friendliness. However, their application is still in the early stages, highlighting the need for the discovery of novel cyanocides. Herein, we isolated and purified a novel macrocyclic tambjamine produced by Paucibacter aquatile DH15, named paucibactin A. The cyanocidal activity of paucibactin A (1 μg/mL) against Microcystis (10⁶ cells/mL) was 81.6% after 24 h of exposure. Paucibactin A killed Microcystis by destroying its structural integrity, photosynthetic and antioxidant systems, and macromolecules. The expression of the key genes involved in these fundamental processes in Microcystis was downregulated after paucibactin A treatment. In a microcosm study, paucibactin A (1 μg/mL) exhibited selective cyanobactericidal activity against Microcystis and Dolichospermum but not against other cyanobacteria. However, 87.4% of paucibactin A was degraded by photolysis in 24 h. Cyanobactericidal processes promoted the growth of eukaryotic microalgae, possibly by altering the competitive relationship and inducing changes in the bacterial community and interaction network. Collectively, our findings suggest that paucibactin A could be an effective cyanobactericidal compound for controlling Microcystis and Dolichospermum blooms.
Numerous naturally available phytochemicals have potential anti-cancer activities due to their vast structural diversity. Alkaloids have been extensively used in cancer treatment, especially lung cancers, among the plant-based compounds. However, their utilization is limited by their poor solubility, low bioavailability, and inadequacies such as lack of specificity to cancer cells and indiscriminate distribution in the tissues. Incorporating the alkaloids into nanoformulations can overcome the said limitations paving the way for effective delivery of the alkaloids to the site of action in sufficient concentrations, which is crucial in tumor targeting. Our review attempts to assess whether alkaloid nanoformulation can be an effective tool in lung cancer therapy. The mechanism of action of each alkaloid having potential is explored in great detail in the review. In general, Alkaloids suppress oncogenesis by modulating several signaling pathways involved in multiplication, cell cycle, and metastasis, making them significant component of many clinical anti-cancerous agents. The review also explores the future prospects of alkaloid nanoformulation in lung cancer. So, in conclusion, alkaloid based nanoformulation will emerge as a potential gamechanger in treating lung cancer in the near future.
Lysosomal pH is an important modulator for many cellular processes. An agent that is capable of regulating lysosomal pH may find a wide range of potential applications in the field of biomedicine. In this study, we describe the synthesis of a family of morpholinyl-bearing arylsquaramides as small-molecule lysosomal pH modulators. These compounds are able to efficiently facilitate the transmembrane transport of chloride anions as mobile carriers across vesicular and cellular phospholipid membranes. They are capable of specifically alkalizing liposomes, disrupting the homeostasis of lysosomal pH and inactivivating lysosomal Cathepsin B enzyme. Anion transport is considered as the probable mechanism of action for the high efficiency of these compounds to modulate lysosomal pH. The present findings present a novel means to efficiently regulate lysosomal pH, which is in contrast to the methods shown by conventional lysosomal pH modulators that generally function by either acting as a weak base/acid, or releasing a basic/acidic component in lysosomal environments to change lysosomal pH.
Roseophilin is an alkaloid structurally related to prodiginines . The intriguing pharmacological properties of these derivatives have prompted us to prepare synthetic compounds 1–3 inspired by their structure and to explore their transmembrane anion transport activity. The methoxyfuran heterocycle impacts the anionophoric activity of the compounds as a result of the reduced hydrogen-bonding ability and electrostatic repulsions between the oxygen in the furan ring and the anions. The position of the furan was also found to be crucial for determining their anion transport activity. Overall, replacement of the characteristic methoxypyrrole moiety of prodiginines and tambjamines by the methoxyfuran found in roseophilin is detrimental to their ability as anion carriers, suggesting that the biological activity of roseophilin is likely not related to their potential activity as anion carriers. Compound 2, bearing a furan ring attached to a dipyrromethene moiety, was found to be the most active anion carrier.
Membrane transport proteins fulfill important regulatory functions in biology with a common trait being their ability to respond to stimuli in the environment. Various small-molecule receptors, capable of mediating transmembrane transport, have been successfully developed. However, to confer stimuli-responsiveness on them poses a fundamental challenge. Here we demonstrate photocontrol of transmembrane transport and electric potential using bis(thio)ureas derived from stiff-stilbene. UV–vis and ¹H NMR spectroscopy are used to monitor E–Z photoisomerization of these bis(thio)ureas and ¹H NMR titrations reveal stronger binding of chloride to the (Z)-form than to the (E)-form. Additional insight into the binding properties is provided by single crystal X-ray crystallographic analysis and DFT geometry optimization. Importantly, the (Z)-isomers are much more active in transmembrane transport than the respective (E)-isomers as shown through various assays. As a result, both membrane transport and depolarization can be modulated upon irradiation, opening up new prospects toward light-based therapeutics as well as physiological and optopharmacological tools for studying anion transport-associated diseases and to stimulate neuronal activity, respectively.
Various artificial transmembrane transporters, designed to function through mobile carrier or channel mechanisms, have been developed in the past decade. With the aid of structural manipulation and by employing either discrete chemical entities or self-assembled nanostructures, progress has been made in achieving the selective recognition and transmembrane transport of key ions. The ability to perturb intracellular pH or disrupt intracellular ion homeostasis makes transmembrane ion transporters of interest as potential therapeutics that might see use as cancer treatments or as antibacterial agents. In this review, recent progress in the area of artificial transmembrane ion transporter research is summarized with an emphasis on applications involving anticancer research and antibiotic applications. The examples chosen for highlights are meant to be illustrative of key themes involving synthetic ion transport rather than comprehensive. Nevertheless, it is anticipated that this review will provide a useful entry point for the general reader and set the stage for further progress in the area.
Pathological mechanisms underlying many aspects of lung disease are incompletely understood. Attempts to identify such mechanisms have revealed an important, cell-specific role for autophagy in lung health and several lung pathologies. This chapter discusses the role of autophagy in lung development and maturation and several lung diseases such as lung cancer, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary arterial hypertension, and interstitial lung diseases; lung infection and inflammation are also discussed. We also examine the dual role of autophagy and its contribution to cell fate decisions under these disease conditions. Finally, we discuss the current autophagy-based therapies for each of these lung diseases.
Oligomers with distinct self‐folding and self‐assembly behaviour are constructed by using m‐pyridine–urea repeating units, an important feature of which is that the urea carbonyl tends to point outward and the NH unit tends to direct inward. Among these oligomers, the trimer forms nanotubes, which can efficiently transport Cl⁻ ions.
Abstract
The m‐pyridine urea (mPU) oligomer was constructed by using the intramolecular hydrogen bond formed by the pyridine nitrogen atom and the NH of urea and the intermolecular hydrogen bond of the terminal carbonyl group and the NH of urea. Due to the synergistic effect of hydrogen bonds, mPU oligomer folds and exhibits strong self‐assembly behaviour. Affected by folding, mPU oligomer generates a twisted plane, and one of its important features is that the carbonyl group of the urea group orientates outwards from the twisted plane, while the NHs tend to direct inward. This feature is beneficial to NH attraction for electron‐rich species. Among them, the trimer self‐assembles into helical nanotubes, and can efficiently transport chloride ions. This study provides a novel and efficient strategy for constructing self‐assembled biomimetic materials for electron‐rich species transmission.
The m‐pyridine urea (mPU) oligomer was constructed by using the intramolecular hydrogen bond formed by the pyridine nitrogen atom and the NH of urea and the intermolecular hydrogen bond of the terminal carbonyl group and the NH of urea. Due to the synergistic effect of hydrogen bonds, mPU oligomer folds and exhibits strong self‐assembly behaviour. Affected by folding, mPU oligomer generates a twisted plane, and one of its important features is that the carbonyl group of the urea group orientates outwards from the twisted plane, while the NHs tend to direct inward. This feature is beneficial to NH attraction for electron‐rich species. Among them, the trimer self‐assembles into helical nanotubes, and can efficiently transport chloride ions. This study provides a novel and efficient strategy for constructing self‐assembled biomimetic materials for electron‐rich species transmission.
The use of synthetic ion transporters for alteration of the concentration of ions across cell membranes has drawn attention from scientists over the last two decades. This ion transport property has been sensibly used to reduce the viability of cancer cells mainly due to the disruption of their ion homeostasis, leading to the perturbation of their abnormal pH gradient. The use of the proanionophore strategy has been recently adopted to increase cellular deliverability and reduce unwanted cytotoxicity towards normal cells. Meanwhile, various anionophores exhibiting non-toxic behavior in epithelial cells have shown a great propensity to be used as a putative treatment for channelopathies like cystic fibrosis. Anionophores with Cl⁻ ion transport mediated antibacterial activities have also been successfully used against clinically relevant bacterial strains, many of which are tolerant towards commercial antibiotics. Recent developments in the chloride ion transport mediated biological activities of anionophores have been mainly discussed in this article. It also highlights other aspects, such as design criteria, targeted delivery options, and others, which can be useful for the further development of selective anionophores for specific biological applications.
The tambjamines are a small group of bipyrrolic alkaloids that, collectively, display a significant range of biological activities including antitumor, antimicrobial and immunosuppressive properties. The key objective of the present study was to undertake preclinical assessments of tambjamine J (T‐J) so as to determine its in vivo antitumor effects. To that end, sarcoma 180 cells were transplanted in mice and the impacts of the title compound then evaluated using a range of protocols including hematological, biochemical, histopathological, genotoxic and clastogenic assays. As a result it was established that this alkaloid has a significant therapeutic window and effectively reduces tumor growth (by 40 % and 79 % at doses of 10 and 20 mg/kg/day, respectively). In this regard it displays similar antitumor activity to the anticancer agent cyclophosphamide and alters animal weight in an analogous manner.
The development of discrete molecular carriers for anions and supramolecular anion channels are reviewed followed by an overview of the use of these systems in biological systems as putative treatments for diseases such as cystic fibrosis and cancer.
Bis‐imidazole‐based anion transporters have been complexed to AuIII to switch off their anion transport properties. In the presence of reducing agents such as GSH the gold is sequestered from the transporter and transport is switched on. This provides a method of targeting anion transporters to tissue with higher concentrations of GSH including tumors.
Abstract
Anion transporters have shown potential application as anti‐cancer agents that function by disrupting homeostasis and triggering cell death. In this research article we report switchable anion transport by gold complexes of anion transporters that are “switched on” in situ in the presence of the reducing agent GSH by decomplexation of gold. GSH is found in higher concentrations in tumors than in healthy tissue and hence this approach offers a strategy to target these systems to tumors.
Anion transporters have shown potential application as anti‐cancer agents that function by disrupting homeostasis and triggering cell death. In this research article we report switchable anion transport by gold complexes of anion transporters that are “switched on” in situ in the presence of the reducing agent GSH by decomplexation of gold. GSH is found in higher concentrations in tumors than in healthy tissue and hence this approach offers a strategy to target these systems to tumors.
A simple di(thioamido)carbazole 1 serves as a potent multispecific transporter for various biologically relevant oxyanions, such as drugs, metabolites and model organic phosphate. The transport kinetics of a wide range...
The anion‐binding and transport properties of an extensive library of thiophene‐based molecules are reported. Seventeen bis‐urea positional isomers, with different binding conformations and lipophilicities, have been synthesized by appending α‐ or β‐thiophene or α‐, β‐, or γ‐benzo[b]thiophene moieties to an ortho‐phenylenediamine central core, yielding six subsets of positional isomers. Through ¹H NMR, X‐ray crystallography, molecular modelling, and anion efflux studies, it is demonstrated that the most active transporters adopt a pre‐organized binding conformation capable of promoting the recognition of chloride, using urea and C−H binding groups in a cooperative fashion. Additional large unilamellar vesicle‐based assays, carried out under electroneutral and electrogenic conditions, together with N‐methyl‐d‐glucamine chloride assays, have indicated that anion efflux occurs mainly through an H⁺/Cl⁻ symport mechanism. On the other hand, the most efficient anion transporter displays cytotoxicity against tumor cell lines, while having no effects on a cystic fibrosis cell line.
Spannungsabhängiger Ionentransport: Eine Gruppe von synthetischen Anionentransportern mit einem Tetraharnstoff‐Makrocyclus erleichtert den HCl‐Transport, der durch Anlegen eines Membranpotentials abgeschaltet werden kann. Diese Eigenschaft steht im Zusammenhang mit der starken Lipidphosphat‐Kopfgruppenbindung und dem Fehlen von Chlorid‐Uniport‐Aktivität.
Abstract
Synthetic anion transporters that facilitate transmembrane H⁺/Cl⁻ symport (cotransport) have anti‐cancer potential due to their ability to neutralize pH gradients and inhibit autophagy in cells. However, compared to the natural product prodigiosin, synthetic anion transporters have low‐to‐modest H⁺/Cl⁻ symport activity and their mechanism of action remains less well understood. We report a chloride‐selective tetraurea macrocycle that has a record‐high H⁺/Cl⁻ symport activity similar to that of prodigiosin and most importantly demonstrates unprecedented voltage‐switchable transport properties that are linked to the lack of uniport activity. By studying the anion binding affinity and transport mechanisms of four other anion transporters, we show that the lack of uniport and voltage‐dependent H⁺/Cl⁻ symport originate from strong binding to phospholipid headgroups that hampers the diffusion of the free transporters through the membrane, leading to an unusual H⁺/Cl⁻ symport mechanism that involves only charged species. Our work provides important mechanistic insights into different classes of anion transporters and a new approach to achieve voltage‐switchability in artificial membrane transport systems.
Synthetic anion transporters that facilitate transmembrane H⁺/Cl⁻ symport (cotransport) have anti‐cancer potential due to their ability to neutralize pH gradients and inhibit autophagy in cells. However, compared to the natural product prodigiosin, synthetic anion transporters have low‐to‐modest H⁺/Cl⁻ symport activity and their mechanism of action remains less well understood. We report a chloride‐selective tetraurea macrocycle that has a record‐high H⁺/Cl⁻ symport activity similar to that of prodigiosin and most importantly demonstrates unprecedented voltage‐switchable transport properties that are linked to the lack of uniport activity. By studying the anion binding affinity and transport mechanisms of four other anion transporters, we show that the lack of uniport and voltage‐dependent H⁺/Cl⁻ symport originate from strong binding to phospholipid headgroups that hampers the diffusion of the free transporters through the membrane, leading to an unusual H⁺/Cl⁻ symport mechanism that involves only charged species. Our work provides important mechanistic insights into different classes of anion transporters and a new approach to achieve voltage‐switchability in artificial membrane transport systems.
The Prodiginine family consists of primarily red-pigmented tripyrrole secondary metabolites that were first characterized in the Gram-negative bacterial species Serratia marcescens and demonstrates a wide array of biological activities and applications. Derivatives of prodiginine have since been characterized in the marine γ-proteobacterium, Pseudoalteromonas. Although biosynthetic gene clusters involved in prodiginine synthesis display homology among genera, there is an evident structural difference in the resulting metabolites. This review will summarize prodiginine biosynthesis, bioactivity, and gene regulation in Pseudoalteromonas in comparison to the previously characterized species of Serratia, discuss the ecological contributions of Pseudoalteromonas in the marine microbiome and their eukaryotic hosts, and consider the importance of modern functional genomics and classic DNA manipulation to understand the overall prodiginine biosynthesis pathway.
Anion transporters have attracted substantial interest due to their ability to induce cell apoptosis by disrupting cellular anion homeostasis. In this paper we describe the synthesis, anion recognition, transmembrane anion transport and cell apoptosis-inducing activity of a series of fluorinated 1,3-bis(benzimidazol-2-yl)benzene derivatives. These compounds were synthesized from the condensation of 1,3-benzenedialdehyde or 5-fluoro-1,3-benzenedialdehyde with the corresponding 1,2-benzenediamines and fully characterized. They are able to form stable complexes with chloride anions, and exhibit potent liposomal and in vitro anionophoric activity. Their anion transport efficiency may be ameliorated by the total number of fluorine atoms, and the enhanced anionophoric activity was a likely consequence of the increased lipophilicity induced by fluorination. Most of these fluorinated bisbenzimidazoles exhibit potent cytotoxicity toward the selected cancer cells. Mechanistic investigations suggest that these compounds are able to trigger cell apoptosis probably by disrupting the homeostasis of chloride anions.
In this paper we present the first example of a lysosome-targeting fluorescent ionophore. Specifically, we synthesized a squaramide derivative bearing a coumarin fluorophore and a morpholinyl group, and found that it was able to target and efficiently deacidify lysosomes. In contrast, an analogue without a morpholinyl group exhibits much lower ability to localize in lysosomes and is much less active in regulating the lysosomal pH.
The marine bacterium Pseudoalteromonas tunicata produces the bipyrrole antibiotic tambjamine YP1. This natural product is built from common amino acid and fatty acid building blocks in a biosynthetic pathway that is encoded in the tam operon which contains 19 genes. The exact role that each of these Tam proteins plays in tambjamine biosynthesis is not known. Here, we provide evidence that TamA initiates the synthesis and controls the chain length of the essential tambjamine fatty amine tail. Sequence analysis suggests the unusual TamA is comprised of an N-terminal adenylation (ANL) domain fused to a C-terminal acyl carrier protein (ACP). Mass spectrometry analysis of recombinant TamA revealed the surprising presence of bound C11 and C12 acyl-adenylate intermediates. Acylation of the ACP domain was observed upon attachment of the phosphopantetheine (4’-PP) arm to the ACP. We also show that TamA can transfer fatty acids ranging in chain length from C6-C13 to an isolated ACP domain. Thus TamA bridges the gap between primary and secondary metabolism by linking fatty acid and pyrrole biosynthetic pathways.
The unraveling of the cellular effects of anion transporters is key to their potential development as apoptosis-inducing or autophagy-disrupting therapeutics. Here, we conducted a systematic study of the cellular responses to the anion transporter prodigiosin by using a pH on/off responsive near-infrared (NIR)-fluorescent probe in HeLa and LAMP1-GFP-transfected HeLa cell lines. The sequence of localized and global cellular acidity changes and the resulting outcomes induced by the anion transporter were visualized with high temporal and spatial resolution. The results show that prodigiosin causes the pH within the lysosomal lumen to rise, after which a non-organelle-specific increase in acidity of the cytosol takes place, which prompts cells to undergo apoptosis. This was confirmed by the quantification of NIR-emissive lysosomes, the intra-cellular fluorescence intensity, and fluorescence volume over time. This NIR probe overcame the limitations of acridine orange, which to date have severely restricted researchers in this field.
Anion transporters play a vital role in cellular processes and their dysregulation leads to a range of diseases such as cystic fibrosis, Bartter's syndrome and epilepsy. Synthetic chloride transporters are known to induce apoptosis in cancer cell lines. Herein, we report macrocycles that are easily synthesized and externally functionalized by pendant membrane-permeable groups. Among a variety of chains appended onto the macrocycle scaffold, cholesterol is found to be the best with an EC50 value of 0.44 µM. The macrocycle is highly anion-selective and transports ions via an OH¯/X¯ antiport mechanism. The macrocycle is an interesting scaffold for ion-transport as it is able to discriminate between various anions and shows a preference for SCN¯ and Cl¯. Such anion-selective transporters are highly attractive model systems to study ion-transport mechanisms and could potentially be of high therapeutic value.
Poorly differentiated and anaplastic thyroid carcinomas are very aggressive, almost invariably lethal neoplasms for which no effective treatment exists. These tumors are intrinsically resistant to cell death, even when their driver oncogenic signaling pathways are inhibited.
We have undertaken a detailed analysis, in mouse and human thyroid cancer cells, of the mechanism through which Obatoclax, a pan-inhibitor of the anti-apoptotic proteins of the BCL2 family, effectively reduces tumor growth in vitro and in vivo.
We demonstrate that Obatoclax does not induce apoptosis, but rather necrosis of thyroid cancer cells, and that non-transformed thyroid cells are significantly less affected by this compound. Surprisingly, we show that Obatoclax rapidly localizes to the lysosomes and induces loss of acidification, block of lysosomal fusion with autophagic vacuoles, and subsequent lysosomal permeabilization. Notably, prior lysosome neutralization using different V-ATPase inhibitors partially protects cancer cells from the toxic effects of Obatoclax. Although inhibition of autophagy does not affect Obatoclax-induced cell death, selective down-regulation of ATG7, but not of ATG5, partially impairs Obatoclax effects, suggesting the existence of autophagy-independent functions for ATG7. Strikingly, Obatoclax killing activity depends only on its accumulation in the lysosomes, and not on its interaction with BCL2 family members.
Finally, we show that also other lysosome-targeting compounds, Mefloquine and LLOMe, readily induce necrosis in thyroid cancer cells, and that Mefloquine significantly impairs tumor growth in vivo, highlighting a clear vulnerability of these aggressive, apoptosis-resistant tumors that can be therapeutically exploited.
Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal alkalinization contributes to the cytotoxic activity of obatoclax.
The specific autophagic elimination of mitochondria (mitophagy) plays the role of quality control for this organelle. Deregulation
of mitophagy leads to increased number of damaged mitochondria and triggers the cell death. The deterioration of mitophagy
has been hypothesized to underlie the pathogenesis of several neurodegenerative diseases, most notably Parkinsons disease.
Although some of the biochemical and molecular mechanisms of mitochondrial quality control described in details physiological
or pathological triggers of mitophagy are still not fully characterised. Here we show that induction of mitophagy by mitochondrial
uncoupler FCCP is independent of the effect of mitochondrial membrane potential but dependent on acidification of the cytosol
by FCCP. Ionophore nigericin also reduces cytosolic pH and induces PINK1/PARKIN independent mitophagy. The increase of intracellular
pH with monensin suppresses effects of FCCP and nigericin on mitochondrial degradation. Thus, a change in intracellular pH
is a regulator of mitochondrial quality control.
The molecular mechanism of Salinomycin's toxicity is not fully understood. Various studies reported that Ca(2+), cytochorme c, and caspase activation play a role in Salinomycin-induced cytotoxicity. Furthermore, Salinomycin may target Wnt/β-catenin signaling pathway to promote differentiation and thus elimination of cancer stem cells. In this study, we show a massive autophagic response to Salinomycin (substantially stronger than to commonly used autophagic inducer rapamycin) in prostrate-, breast cancer cells, and to lesser degree in human normal dermal fibroblasts. Interestingly, autophagy induced by Salinomycin is a cell protective mechanism in all tested cancer cell lines. Furthermore, Salinomycin induces mitophagy, mitoptosis and increased mitochondrial membrane potential (∆Ψ) in a subpopulation of cells. Salinomycin strongly, and in time-dependent manner decreases cellular ATP level. Contrastingly, human normal dermal fibroblasts treated with Salinomycin show some initial decrease in mitochondrial mass, however they are largely resistant to Salinomycin-triggered ATP-depletion. Our data provide new insight into the molecular mechanism of preferential toxicity of Salinomycin towards cancer cells, and suggests possible clinical application of Salinomycin in combination with autophagy inhibitors (like i.e. clinically-used Chloroquine). Furthermore, we discuss preferential Salinomycin's toxicity in the context of Warburg effect.
The cancer stem cell (CSC) concept has important therapeutic implications, but its investigation has been hampered both by a lack of consistency in the terms used for these cells and by how they are defined. Evidence of their heterogeneous origins, frequencies and their genomic, as well as their phenotypic and functional, properties has added to the confusion and has fuelled new ideas and controversies. Participants in The Year 2011 Working Conference on CSCs met to review these issues and to propose a conceptual and practical framework for CSC terminology. More precise reporting of the parameters that are used to identify CSCs and to attribute responses to them is also recommended as key to accelerating an understanding of their biology and developing more effective methods for their eradication in patients.
Significance:
Lysosomes are organelles in which cellular degradation occurs in a controlled manner, separated from other cellular components. As several pathways terminate in the lysosome, lysosomal dysfunction has a profound impact on cell homeostasis, resulting in manifold pathological situations, including infectious diseases, neurodegeneration, and aging.
Recent advances:
Lysosomal biology demonstrates that in addition to regulating the final steps of catabolic processes, lysosomes are essential up-stream modulators of autophagy and other essential lysosomal pathways.
Future directions and critical issues:
Lysosomal membrane permeabilization offers therapeutic potential in the treatment of cancer, though the molecular regulators of this process remain obscure. This review focuses on recent discoveries in lysosomal function and dysfunction, primarily in in vivo situations.
Ion channels contribute to virtually all basic cellular processes, including such crucial ones for maintaining tissue homeostasis as proliferation, differentiation, and apoptosis. The involvement of ion channels in regulation of programmed cell death, or apoptosis, has been known for at least three decades based on observation that classical blockers of ion channels can influence cell death rates, prolonging or shortening cell survival. Identification of the central role of these channels in regulation of cell cycle and apoptosis as well as the recent discovery that the expression of ion channels is not limited solely to the plasma membrane, but may also include membranes of internal compartments, has led researchers to appreciate the pivotal role of ion channels plays in development of cancer. This review focuses on the aspects of programmed cell death influenced by various ion channels and how dysfunctions and misregulations of these channels may affect the development and progression of different cancers.
The tambjamine alkaloids and related synthetic analogs are potent transmembrane anion tranporters promoting bicarbonate/chloride exchange in model phospholipid liposomes and discharging pH gradients in living cells.
Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores are 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.
Ion channels serve many functions apart from electrical signal transduction:chemical signalling (Ca 2þ as a second messenger), transepithelial transport, regulation of cytoplasmic or vesicular ion concentration and pH, and regulation of cell volume. Therefore, ion channel dysfunction can cause diseases in many tissues. The list of human diseases known to be associated with defects in ion channels has grown considerably during the past years. This review gives a short overview of known channelopathies, and focuses in particular on recent findings and on channelopathies that have significantly advanced our physiological insight. Ion channels provide pores for the passive diffusion of ions across biological membranes. They are often highly selective for a particular ionic species, leading to a classification into sodium (Na þ ), potassium (K þ ), calcium (Ca 2þ ), chloride (Cl � ) and unspecific cation channels. The direction of net ion transport, which is associated with an electric current, depends on the electrochemical gradient for the relevant ionic species. These gradients are established by an interplay of active pumps, co-transporters and ion channels. Ion channels can close and open in a process called gating. This allows many types of regulation. Thus, there are ligand-gated channels (e.g. postsynaptic GABA- or glutamate-receptor channels), voltagegated, swelling- or stretch-activated, and heat- or cold-activated channels. In addition, channels may be regulated by calcium, pH, phosphorylation and lipids. Many, but not all, channels are oligomers of identical or homologous pore-forming a subunits. They are often present in a complex with b and sometimes g subunits, which may be essential for their function or may modulate their properties. Channels reside not only in the external (plasma) membrane, but also in membranes of intracellular organelles such as the endoplasmic reticulum, endosomes, lysosomes and mitochondria. Gap junction channels connect the cytoplasm of adjacent cells and are formed by closely opposed hemichannels in their respective plasma membranes. While the role of ion channels in generating the electric currents (the basis of neuronal signalling) is probably known best, channels have many other functions. For instance, ion channels are crucial for the transepithelial transport of salt and water, for the regulation of cellular volume and pH, for the acidification of intracellular organelles, and (in particular in the case of Ca 2þ channels), for
Researchers discuss applications of supramolecular anion recognitions. The researchers state that fluoride anions are an important target in supramolecular chemistry due to the importance of fluoride in biological and medical processes. Many other fluoride sensors based on deprotonation of hydrogen-bond donors have been reported from extensive studies conducted by other researchers. The researchers also state that silicon is another Lewis-acidic center with high fluorophilicity that deserves special attention when developing fluoride sensors.
Nearly 60 years ago, lysosomes were first described in the laboratory of Christian de Duve, a discovery that significantly contributed to him being awarded a share of the 1974 Nobel Prize in Physiology or Medicine for elucidating "the structural and functional organization of the cell". Initially thought of as a simple waste degradation facility of the cells, these organelles recently emerged as signalling centres with connections to major cellular processes. This minireview will give an overview of the many roles of lysosomal proteins in two of these processes, cell death and autophagy. We will discuss both resident lysosomal proteins as well those that temporarily associate with lysosomes to influence autophagy and cell death pathways. Particular focus will be given to studies in mammalian cells and in vivo systems. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
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.
In this Account, we discuss the development of new lipid bilayer aniontransporters based on the structure of anionophoric natural products (the prodigiosins) and purely synthetic supramolecular systems. We have studied the interaction of these compounds with human cancer cell lines, and, in general, the most active anion transporter compounds possess the greatest anti-cancer properties.
Initially, we describe the anion transport properties of synthetic mol-ecules that are based on the structure of the family of natural products known as the prodiginines. Obatoclax, for example, is a prodiginine derivative with an indole ring that is currently in clinical trials for use as an anti-cancer drug. The anion transport properties of the compounds were correlated with their toxicity toward small cell human lung cancer GLC4 cells. We studied related compounds with enamine moieties, tambjamines, that serve as active transporters. These molecules and others in this series could depolarize acidic compartments within GLC4 cells and trigger apoptosis. In a study of the variation of lipophilicity of a series of these compounds, we observed that, as log P increases, the anion transport efficiency reaches a peak and then decreases.
In addition, we discuss the anion transport properties of series of synthetic supramolecular anion receptor species. We synthesized trisureas and thioureas based on the tren backbone, and found that the thiourea compounds effectively transport anions. Fluorination of the pendant phenyl groups in this series of compounds greatly enhances the transport properties. Similar to our earlier results, the most active anion transporters reduced the viability of human cancer cell lines by depolarizing acidic compartments in GLC4 cells and triggering apoptosis.
In an attempt to produce simpler transporters that obey Lipinski’s Rule of Five, we synthesized simpler systems containing a single urea or thiourea group. Once again the thiourea systems, and in particular a thiourea with a pendant indole group, transported anions efficiently. A series of related compounds containing a pendant trifluoromethyl group showed enhanced transport and significant anticancer properties.
Researchers still need to determine of the exact mechanism of how these compounds depolarize acidic organelles within cancer cells. However, this work shows that these transporters based upon both natural products and purely synthetic supramolecular systems transport anions, depolarize acidic compartments within cancer cells and trigger apoptosis.
The development of small-molecule lipid-bilayer anion transporters for potential future use in channel replacement therapy for the treatment of diseases caused by dysregulation of anion transport (such as cystic fibrosis), and in treating cancer by perturbing chemical gradients within cells, thus triggering apoptosis, is an area of intense current interest. This Minireview looks at recent developments in the design of small-molecule transmembrane anion transporters and focuses on the progress so far in employing these compounds in biological systems.
Autophagy, a vital catabolic process that degrades cytoplasmic components within the lysosome, is an essential cytoprotective response to pathologic stresses that occur during diseases such as cancer, ischemia, and infection. In addition to its role as a stress-response pathway, autophagy plays an essential quality-control function in the cell by promoting basal turnover of long-lived proteins and organelles, as well as by selectively degrading damaged cellular components. This homeostatic function protects against a wide variety of diseases, including neurodegeneration, myopathy, liver disease, and diabetes. This review discusses our current understanding of these two principal functions of autophagy and describes in detail how alterations in autophagy promote human disease. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease Volume 8 is January 24, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Mitochondria are primarily responsible for providing the contracting cardiac myocyte with a continuous supply of ATP. However, mitochondria can rapidly change into death-promoting organelles. In response to changes in the intracellular environment, mitochondria become producers of excessive reactive oxygen species and release prodeath proteins, resulting in disrupted ATP synthesis and activation of cell death pathways. Interestingly, cells have developed a defense mechanism against aberrant mitochondria that can cause harm to the cell. This mechanism involves selective sequestration and subsequent degradation of the dysfunctional mitochondrion before it causes activation of cell death. Induction of mitochondrial autophagy, or mitophagy, results in selective clearance of damaged mitochondria in cells. In response to stress such as ischemia/reperfusion, prosurvival and prodeath pathways are concomitantly activated in cardiac myocytes. Thus, there is a delicate balance between life and death in the myocytes during stress, and the final outcome depends on the complex cross-talk between these pathways. Mitophagy functions as an early cardioprotective response, favoring adaptation to stress by removing damaged mitochondria. In contrast, increased oxidative stress and apoptotic proteases can inactivate mitophagy, allowing for the execution of cell death. Herein, we discuss the importance of mitochondria and mitophagy in cardiovascular health and disease and provide a review of our current understanding of how these processes are regulated.
AMP-activated protein kinase (AMPK) is an important sensor of cellular energy status, and is involved in cell growth and autophagy through mammalian target of rapamycin complex 1 (mTORC1). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, leads to AMPK activation and Parkin-dependent mitophagy, respectively. However, the detailed biochemical mechanism of how CCCP induces autophagy or mitophagy has not been investigated yet. Here, we showed that CCCP inhibits mTORC1 independently of AMPK, although CCCP induces AMPK activation. Using wild type (WT) and AMPKα1/α2 double knockout (DKO) MEFs, we observed that CCCP promotes endogenous LC3 lipidation and formation of RFP-LC3 puncta, indicating autophagosome or autolysosome, in an AMPK-independent manner. Finally, we also revealed that the percentage of CCCP-dependent colocalization between mitochondria and RFP-LC3 puncta is similar both in WT and AMPKα1/α2 DKO MEFs. Based on these data, we concluded that AMPK is not essential in regulation of CCCP-induced autopahgy including mitophagy.
Although cancer is a diverse set of diseases, cancer cells share a number of adaptive hallmarks. Dysregulated pH is emerging as a hallmark of cancer because cancers show a 'reversed' pH gradient with a constitutively increased intracellular pH that is higher than the extracellular pH. This gradient enables cancer progression by promoting proliferation, the evasion of apoptosis, metabolic adaptation, migration and invasion. Several new advances, including an increased understanding of pH sensors, have provided insight into the molecular basis for pH-dependent cell behaviours that are relevant to cancer cell biology. We highlight the central role of pH sensors in cancer cell adaptations and suggest how dysregulated pH could be exploited to develop cancer-specific therapeutics.
Progress through the cell mitotic cycle requires precise timing of the intrinsic molecular steps and tight coordination with the environmental signals that maintain a cell into the proper physiological context. Because of their great functional flexibility, ion channels coordinate the upstream and downstream signals that converge on the cell cycle machinery. Both voltage- and ligand-gated channels have been implicated in the control of different cell cycle checkpoints in normal as well as neoplastic cells. Ion channels mediate the calcium signals that punctuate the mitotic process, the cell volume oscillations typical of cycling cells, and the exocytosis of autocrine or angiogenetic factors. Other functions of ion channels in proliferation are still matter of debate. These may or may not depend on ion transport, as the channel proteins can form macromolecular complexes with growth factor and cell adhesion receptors. Direct conformational coupling with the cytoplasmic regulatory proteins is also possible. Derangement or relaxed control of the above processes can promote neoplasia. Specific types of ion channels have turned out to participate in the different stages of the tumor progression, in which cell heterogeneity is increased by the selection of malignant cell clones expressing the ion channel types that better support unrestrained growth. However, a comprehensive mechanistic picture of the functional relations between ion channels and cell proliferation is yet not available, partly because of the considerable experimental challenges offered by studying these processes in living mammalian cells. No doubt, such studies will constitute one of the most fruitful research fields for the next generation of cell physiologists.
The chemical investigation of Azorean nudibranch mollusk Tambja ceutae led us to isolate a new member of the tambjamine family, tambjamine K (1). The bryozoan Bugula dentata, prey of the nudibranch, was also analyzed and found to contain compound 1 in very small amounts together with known blue pigment 2 and tambjamines A (3) and B (4). The structure of tambjamine 1 was elucidated by the interpretation of the spectroscopic data as well as by the comparison with related compounds. Compounds 1 and 2 possess antiproliferative activity, in particular, tambjamine K (1) displayed high cytotoxicity against both tumor and non-tumor mammalian cells.
Autophagy is a highly conserved bulk protein degradation pathway responsible for the turnover of long-lived proteins, disposal of damaged organelles, and clearance of aggregate-prone proteins. Thus, inactivation of autophagy results in cytoplasmic protein inclusions, which are composed of misfolded proteins and excess accumulation of deformed organelles, leading to liver injury, diabetes, myopathy, and neurodegeneration. Although autophagy has been considered non-selective, growing lines of evidence indicate the selectivity of autophagy in sorting vacuolar enzymes and in the removal of aggregate-prone proteins, unwanted organelles and microbes. Such selectivity by autophagy enables diverse cellular regulations, similar to the ubiquitin-proteasome pathway. In this review, we introduce the selective turnover of the ubiquitin- and LC3-binding protein 'p62' through autophagy and discuss its physiological significance.
Screens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here an approach to screening for agents with epithelial CSC-specific toxicity. We implemented this method in a chemical screen and discovered compounds showing selective toxicity for breast CSCs. One compound, salinomycin, reduces the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates the ability to identify agents with specific toxicity for epithelial CSCs.
The p62 protein, also called sequestosome 1 (SQSTM1), is a ubiquitin-binding scaffold protein that colocalizes with ubiquitinated protein aggregates in many neurodegenerative diseases and proteinopathies of the liver. The protein is able to polymerize via an N-terminal PB1 domain and can interact with ubiquitinated proteins via the C-terminal UBA domain. Also, p62/SQSTM1 binds directly to LC3 and GABARAP family proteins via a specific sequence motif. The protein is itself degraded by autophagy and may serve to link ubiquitinated proteins to the autophagic machinery to enable their degradation in the lysosome. Since p62 accumulates when autophagy is inhibited, and decreased levels can be observed when autophagy is induced, p62 may be used as a marker to study autophagic flux. Here, we present several protocols for monitoring autophagy-mediated degradation of p62 using Western blots, pulse-chase measurement of p62 half-life, immunofluorescence and immuno-electron microscopy, as well as live cell imaging with a pH-sensitive mCherry-GFP double tag. We also present data on species-specificity and map the epitopes recognized by several commercially available anti-p62 antibodies.
The tetrazolium salt (MTT) method involving conversion of MTT to coloured formazan by cells serving as indirect measurements of cell growth/cell kill has been reported by several groups, although technical problems have been encountered. The present investigation was undertaken in order to delineate what laboratory variables have direct influence on the sensitivity and reproducibility of the method. The pH of the extraction buffer was of the utmost importance, since it was demonstrated that a pH greater than 5 would give rise to false signals. Furthermore, modifying the composition of the extraction buffer, all formazan dye grains were solubilised, totally. A direct comparison with published methods demonstrated that only the modified method would yield 100% higher signals without increasing the background. In contrast to previous reports, it was shown that phenol red does not interfere with the measurements and no washing steps are required since all ingredients can be added subsequently. Serum proteins at concentrations up to 25% have no influence on the result. All samples can be measured in an ELISA scanner at 570 nm with little intra-assay variation.
Ion channels serve many functions apart from electrical signal transduction: chemical signalling (Ca(2+) as a second messenger), transepithelial transport, regulation of cytoplasmic or vesicular ion concentration and pH, and regulation of cell volume. Therefore, ion channel dysfunction can cause diseases in many tissues. The list of human diseases known to be associated with defects in ion channels has grown considerably during the past years. This review gives a short overview of known channelopathies, and focuses in particular on recent findings and on channelopathies that have significantly advanced our physiological insight.
Opening of high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT). The MPT is a causative event, leading to necrosis and apoptosis in hepatocytes after oxidative stress, Ca(2+) toxicity, and ischemia/reperfusion. CsA blocks opening of permeability transition pores and protects cell death after these stresses. In contrast to necrotic cell death which is a consequence of ATP depletion, ATP is required for the development of apoptosis. Reperfusion and the return of normal pH after ischemia initiate the MPT, but the balance between ATP depletion after the MPT and ATP generation by glycolysis determines whether the fate of cells will be apoptotic or necrotic death. Thus, the MPT is a common pathway leading to both necrotic and apoptotic cell death after ischemia/reperfusion.
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.
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.
Cells are continuously exposed to a variety of environmental stresses and have to decide 'to be or not to be' depending on the types and strength of stress. Among the many signaling pathways that respond to stress, mitogen-activated protein kinase (MAPK) family members are crucial for the maintenance of cells. Three subfamilies of MAPKs have been identified: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38-MAPKs. It has been originally shown that ERKs are important for cell survival, whereas JNKs and p38-MAPKs were deemed stress responsive and thus involved in apoptosis. However, the regulation of apoptosis by MAPKs is more complex than initially thought and often controversial. In this review, we discuss MAPKs in apoptosis regulation with attention to mouse genetic models and critically point out the multiple roles of MAPKs.
Prodigiosin (PG) is a red pigment produced by Serratia marcescens with pro-apoptotic activity in haematopoietic and gastrointestinal cancer cell lines, but no marked toxicity in non-malignant cells. Breast cancer is the most frequent malignancy among women in the European Union and better therapies are needed, especially for metastatic tumors. Moreover, multidrug resistance is a common phenomenon that appears during chemotherapy, necessitating more aggressive treatment as prognosis worsens. In this work, we extend our experiments on PG-induced apoptosis to breast cancer cells. PG was potently cytotoxic in both estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines. Cytochrome c release, activation of caspases-9, -8 and -7 and cleavage of poly (ADP-ribose) polymerase protein typified the apoptotic event and caspase inhibition revealed that PG acts via the mitochondrial pathway. In a multidrug-resistant subline of MCF-7 cells that over-expresses the breast cancer resistance protein, the cytotoxic activity of PG was slightly reduced. However, flow-cytometry analysis of PG accumulation and efflux in MCF-7 sublines showed that PG is not a substrate for this resistance protein. These results suggest that PG is an interesting and potent new pro-apoptotic agent for the treatment of breast cancer even when multidrug resistance transporter molecules are present.
Chloromethyl-X-rosamine (CMXRos) and MitoTracker Green (MTG) have proved to be useful dyes with which to measure mitochondrial function. CMXRos is a lipophilic cationic fluorescent dye that is concentrated inside mitochondria by their negative mitochondrial membrane potential (MMP). MTG fluorescence has been used as a measure of mitochondrial mass independent of MMP. The fluorescence ratio of the two dyes is a relative measure of the MMP independent of mitochondrial mass. Because MTG was recently reported to be sensitive to MMP, we have reevaluated the effects of loss of MMP on MTG and CMXRos fluorescence, using both flow cytometry and laser scanning confocal microscopy (LSCM).
Using flow cytometry, the relative fluorescence of CMXRos, R123, and MTG was determined in human lymphoblastoid cell lines (LCLs) with or without carbonyl cyanide p-trifluoromethoxylphenyl-hydrazone (FCCP), used to collapse the MMP. LSCM analysis was also used to evaluate the effect of FCCP on MTG and CMXRos fluorescence of mouse cells and viable lenses in culture. The cytotoxicity of the dyes was determined using flow analysis of endogenous NADH fluorescence. The sensitivity of MTG fluorescence to H(2)O(2) was also evaluated using flow cytometry.
CMXRos fluorescence was dependent on MMP, whereas MTG fluorescence was not affected by MMP, using either flow or LSCM. Specific staining of mitochondria was seen with both dyes in all cell types tested, without evidence of cytotoxicity, as determined by NADH levels. H(2)O(2) damage slightly increased MTG staining of cells.
Our results indicate that CMXRos is a nontoxic sensitive indicator of relative changes in MMP, whereas MTG is relatively insensitive to MMP and oxidative stress, using both flow and LSCM analyses, provided optimal staining conditions are used. In addition, these dyes can be useful for the study of mitochondrial morphology and function in whole tissues, using LSCM.
(Chemical Equation Presented) Pro and anti: Recognition of chloride ion and through-membrane H+/Cl- ion transport ("symport") could account for the biological effects (anticancer activity) of prodigiosin, as inferred from model studies involving pyrrole-based anion receptors designed to mimic the key features of this naturally occurring pigment (see picture).
Lysosomal hydrolases participate in the digestion of endocytosed and autophagocytosed material inside the lysosomal/autolysosomal compartment in acute cell death when released into the cytosol and in cancer progression following their release into the extracellular space. Lysosomal alterations are common in cancer cells. The increased expression and altered trafficking of lysosomal enzymes participates in tissue invasion, angiogenesis and sensitization to the lysosomal death pathway. But lysosomal heat-shock protein 70 locally prevents lysosomal-membrane permeabilization. Similarly, alterations in the autophagic compartment are linked to carcinogenesis and resistance to chemotherapy. Targeting these pathways might constitute a novel approach to cancer therapy.
Lung cancer was relatively uncommon at the turn of the 20th century, and has increased in prevalence at alarming rates, particularly because of the augmented trend in smoking, so that it is now the most common cause of cancer death in the world. As almost a quarter of these cancers are of small cell in origin, it seems only appropriate that small cell lung cancer receives ample attention, rather than seemingly to have been overlooked over the last 10-15 years. Despite its generally late presentation and high risk of dissemination, it is exceptionally sensitive to chemo-radiotherapy. This review looks at the diverse options of treatment that have been used over the last few years and tries to highlight the best available. As more than 50% of patients diagnosed with lung cancer are over 70 years of age and various studies have shown that older people respond just as well as their younger counterparts, with similar results in response rates, toxicity and outcomes, it is imperative that the older generation are not disregarded in terms of age being a contraindication to therapy.
Lung cancer is a worldwide problem. At the time of diagnosis, 50% of patients have advanced incurable disease. Different chemotherapy combinations--with or without targeted therapies--yield similar results despite the continuous efforts of clinicians. However, molecular biological studies have already shed a great deal of light on the existence of multiple genetic aberrations that can be useful for customizing treatment. mRNA transcripts involved in DNA repair pathways, such as ERCC1 and BRCA1, confer selective resistance to cisplatin or taxanes, whereas thioredoxin confers a broad spectrum of chemoresistance. Polymorphisms in DNA repair genes and methylation of checkpoint genes in circulating serum DNA could become important predictive markers of survival in certain cisplatin-based regimens. Epidermal growth factor receptor tyrosine kinase mutations are the crux of targeted therapies, whereas epithelial-mesenchymal transitions and HER3 mRNA levels are promising ancillary markers for treatment with epidermal growth factor receptor tyrosine kinase inhibitors.
Mitochondria are the essential site of aerobic energy production in eukaryotic cells. Reactive oxygen species (ROS) are an inevitable by-product of mitochondrial metabolism and can cause mitochondrial DNA mutations and dysfunction. Mitochondrial damage can also be the consequence of disease processes. Therefore, maintaining a healthy population of mitochondria is essential to the well-being of cells. Autophagic delivery to lysosomes is the major degradative pathway in mitochondrial turnover, and we use the term mitophagy to refer to mitochondrial degradation by autophagy. Although long assumed to be a random process, increasing evidence indicates that mitophagy is a selective process. This review provides an overview of the process of mitophagy, the possible role of the mitochondrial permeability transition in mitophagy and the importance of mitophagy in turnover of dysfunctional mitochondria.
Bacterial prodiginines are a family of red-pigmented, tripyrrolic compounds that display numerous biological activities, including antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive and anticancer properties. Recently, significant progress has been made in understanding the biosynthesis and regulation of bacterial prodiginines. An understanding of the biosynthesis of prodiginines will allow engineering of bacterial strains capable of synthesizing novel prodiginines through rational design and mutasynthesis experiments. Bacterial prodiginines and synthetic derivatives are effective proapoptotic agents with multiple cellular targets, and they are active against numerous cancer cell lines, including multidrug-resistant cells, with little or no toxicity towards normal cell lines. A synthetic derivative, GX15-070 (Obatoclax), developed through structure-activity relationship studies of the pyrrolic ring A of GX15, is in multiple Phase I and II clinical trials in both single and dual-agent studies to treat different types of cancer. Therefore, prodiginines have real therapeutic potential in the clinic.
Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development, and homeostasis. Autophagy principally serves an adaptive role to protect organisms against diverse pathologies, including infections, cancer, neurodegeneration, aging, and heart disease. However, in certain experimental disease settings, the self-cannibalistic or, paradoxically, even the prosurvival functions of autophagy may be deleterious. This Review summarizes recent advances in understanding the physiological functions of autophagy and its possible roles in the causation and prevention of human diseases.
Advances in fluorescent protein development over the past 10 years have led to fine-tuning of the Aequorea victoria jellyfish color palette in the emission color range from blue to yellow, while a significant amount of progress has been achieved with reef coral species in the generation of monomeric fluorescent proteins emitting in the orange to far-red spectral regions. It is not inconceivable that near-infrared fluorescent proteins loom on the horizon. Expansion of the fluorescent protein family to include optical highlighters and FRET biosensors further arms this ubiquitous class of fluorophores with biological probes capable of photoactivation, photoconversion, and detection of molecular interactions beyond the resolution limits of optical microscopy. The success of these endeavors certainly suggests that almost any biological parameter can be investigated using the appropriate fluorescent protein-based application.
Marine organisms have been shown to be potential sources of bioactive compounds with pharmaceutical applications. Previous chemical investigation of the nudibranch Tambja eliora led to the isolation of the alkaloid tambjamine D. Tambjamines have been isolated from marine sources and belong to the family of 4-methoxypyrrolic-derived natural products, which display promising immunosuppressive and cytotoxic properties. Their ability to intercalate DNA and their pro-oxidant activity may be related to some of the biological effects of the 4-methoxypyrrolic alkaloids. The aim of the present investigation was to determine the cytotoxic, pro-oxidant and genotoxic properties of tambjamine D in V79 Chinese hamster lung fibroblast cells. Tambjamine D displayed a potent cytotoxic effect in V79 cells (IC50 1.2 microg/mL) evaluated by the MTT assay. Based on the MTT result, V79 cells were treated with different concentrations of tambjamine D (0.6, 1.2, 2.4 and 4.8 microg/mL). After 24h, tambjamine D reduced the number of viable cells in a concentration-dependent way at all concentrations tested, assessed by the trypan blue dye exclusion test. The hemolytic assay showed that the cytotoxic activity of tambjamine D was not related to membrane disruption (EC50>100 microg/mL). Tambjamine D increased the number of apoptotic cells in a concentration-dependent manner at all concentrations tested according to acridine orange/ethidium bromide staining, showing that the alkaloid cytotoxic effect was related to the induction of apoptosis. MTT reduction was stimulated by tambjamine D, which may indicate the generation of reactive oxygen species. Accordingly, treatment of cells with tambjamine D increased nitrite/nitrate at all concentrations and TBARS production starting at the concentration corresponding to the IC50. Tambjamine D, also, induced DNA strand breaks and increased the micronucleus cell frequency as evaluated by comet and micronucleus tests, respectively, at all concentrations evaluated, showing a genotoxic risk induced by tambjamine D.
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