Effect of carboxylic ionophores on lysosomal protein degradation in rat hepatocytes

doi:10.1016/0014-4827(83)90377-4

Experimental Cell Research

Volume 149, Issue 1, November 1983, Pages 27-35

https://doi.org/10.1016/0014-4827(83)90377-4 Get rights and content

Abstract

Three different carboxylic ionophores (monensin, nigericin and lasalocid) were each found capable of causing a relatively complete block of the lysosomal (i.e., methylamine-sensitive) protein degradation in isolated rat hepatocytes. Monensin was found to be the most specific in action, as it had no effect on non-lysosomal degradation and did not bring about any substantial inhibition of protein synthesis. Morphometric examination of electron micrographs revealed that monensin causes an accumulation of early forms of autophagic vacuoles and blocks the swelling of lysosomes seen in the presence of methylamine. The results indicate that monensin inhibits lysosomal protein degradation by affecting lysosomal pH.

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Cited by (45)

Polyethers isolated from the marine actinobacterium Streptomyces cacaoi inhibit autophagy and induce apoptosis in cancer cells
2019, Chemico-Biological Interactions
Citation Excerpt :
Polyether ionophores have recently gathered attention in autophagy inhibition. It was reported that Mon and Nig treatment caused accumulation of autophagic vacuoles inside the cell [11] by inhibiting the terminal fusion stage of autophagy pathway along with the accumulation of autophagy flux markers LC3-II and p62 [12,13]. In addition, two polyether antibiotics, viz. salinomycin and maduramicin, were reported to inhibit the autophagic flux in several cancer cell lines [14,15].
Polyether compounds, a large group of biologically active metabolites produced by Streptomyces species have been reported to show a variety of bioactivity such as antibacterial, antifungal, antiparasitic, antiviral, and tumour cell cytotoxicity. Since some of these compounds target cancer stem cells and multi-drug resistant cancer cells, this family of compounds have become of high interest. In this study, three polyether-type metabolites (1–3), one of which was a new natural product (3), were isolated from the marine derived Streptomyces cacaoi via antimicrobial activity-guided fractionation studies. As several polyether compounds with structural similarity such as monensin have been linked with autophagy and cell death, we first assessed the cytotoxicity of these three compounds. Compounds 2 and 3, but not 1, were found to be cytotoxic in several cell lines with a higher potency towards cancer cells. Furthermore, 2 and 3 caused accumulation of both autophagy flux markers LC3-II and p62 along with cleavage of caspase-3, caspase-9 and poly (ADP-ribose) polymerase 1 (PARP-1). Interestingly, prolonged treatment of the compounds caused a dramatic downregulation of the proteins related to autophagasome formation in a dose dependent manner. Our findings provide insights on the molecular mechanisms of the polyether-type polyketides, and signify their potency as chemotherapeutic agents through inhibiting autophagy and inducing apoptosis.
Cytotoxicity of anticancer candidate salinomycin and identification of its metabolites in rat cell cultures
2018, Toxicology in Vitro
Citation Excerpt :
SAL impairs the flow of ions (K+/Na+) through cellular membranes, which is responsible for both pharmacological and toxicological action. It leads to Ca2+ overload within cells followed by decreased cellular energy and inhibits lysosomal protein degradation by affecting pH inside the organelle (Grinde, 1983; Boehmerle and Endres, 2011; Sommer et al., 2016; Zou et al., 2017). The determined median effective concentrations of the ionophore (EC50 < 0.39 μg/ml) alone or in co-action with tiamulin or prednisolone confirm strong inhibition of lysosomal activity.
Salinomycin (SAL) is a polyether antibiotic, which is commonly used as a coccidiostat and has recently shown to exhibit anticancer activity. The toxic action of the drug may be connected with the extent and routes of its biotransformation. The cytotoxic potential of SAL and its combination with tiamulin and prednisolone was investigated using three cell models from rat: primary hepatocytes, hepatoma cells (FaO) and myoblasts (L6). The four biochemical endpoints were assessed: mitochondrial and lysosomal activity, total cell protein content and membrane integrity. The metabolites of SAL in the medium from cell cultures were determined using LC-MS/MS. The cytotoxicity of SAL was time-, concentration- and cells dependent. The most sensitive endpoint was the inhibition of lysosomal activity. Tiamulin increased SAL cytotoxicity, whereas the opposite results were observed for prednisolone. Primary hepatocytes were the most efficient in SAL biotransformation both in terms of its intensity and number of produced metabolites. The range of the cytotoxicity and mode of salinomycin interaction with tiamulin and prednisolone cannot be explained by the biotransformation alone.
Intracellular pH modulates autophagy and mitophagy
2016, Journal of Biological Chemistry
Citation Excerpt :
Induction of mitophagy with the alternative compound nigericin, which induces significant acidification of cytosol but does not induce mitochondrial depolarization, suggesting that a decrease in pHcyt can be a stimulus for relocation of mitochondria to lysosomes. The role of carboxylic ionophores (including nigericin and monensin) in lysosomal protein degradation was shown previously, and interestingly monensin inhibited autophagy (29). Thus, monensin and nigericin produced opposite effects on pH and on autophagy and mitophagy that again, suggests the effect of intracellular pH on mitochondrial and protein degradation in lysosome acidification is to inhibit this process.
The specific autophagic elimination of mitochondria (mitophagy) plays the role of quality control for this organelle. Deregulation of mitophagy leads to an increased number of damaged mitochondria and triggers cell death. The deterioration of mitophagy has been hypothesized to underlie the pathogenesis of several neurodegenerative diseases, most notably Parkinson disease. Although some of the biochemical and molecular mechanisms of mitochondrial quality control are described in detail, physiological or pathological triggers of mitophagy are still not fully characterized. Here we show that the induction of mitophagy by the mitochondrial uncoupler FCCP is independent of the effect of mitochondrial membrane potential but dependent on acidification of the cytosol by FCCP. The ionophore nigericin also reduces cytosolic pH and induces PINK1/PARKIN-dependent and -independent mitophagy. The increase of intracellular pH with monensin suppresses the effects of FCCP and nigericin on mitochondrial degradation. Thus, a change in intracellular pH is a regulator of mitochondrial quality control.
Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak
2012, Journal of Biological Chemistry
Citation Excerpt :
The structure and properties of nigericin are similar to those of monensin, although monensin has a preference for Na+ instead of K+ (25, 38). Like nigericin, monensin treatment disrupts lysosomal function (27, 39). Immunoblotting with anti-LC3 antibody and quantitative analysis showed that the time-dependent increase in LC3-II following monensin treatment was not exacerbated by the presence of chloroquine, indicating that monensin also impaired autophagic flux in MN9D cells (supplemental Fig. S3, A and B).
Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K⁺/H⁺ ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.
Background: Exact mechanisms underlying regulatory roles for the Bcl-2 family remain to be delineated.
Results: Overexpressed Bak blocked ionophore-induced impairment of autophagic flux in neuronal cells.
Conclusion: Bak plays critical roles in maintaining autophagic flux and vacuole homeostasis.
Significance: Our findings contribute to better understanding of the Bcl-2 family's potential as a novel therapeutic strategy in autophagy-associated neurodegeneration.
Disruption of lysosome function promotes tumor growth and metastasis in Drosophila
2010, Journal of Biological Chemistry
Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the Ras^V12 cells. Knocking down either of the two other components of the Class C VPS complex, carnation (car) and vps16A, also renders Ras^V12 cells capable for uncontrolled growth and metastatic behavior. Finally, chemical disruption of the lysosomal function by feeding animals with antimalarial drugs, chloroquine or monensin, leads to malignant tumor growth of the Ras^V12 cells. Taken together, our data provide evidence for a causative role of lysosome dysfunction in tumor growth and invasion and indicate that members of the Class C VPS complex behave as tumor suppressors.
Carboxylic ionophores in malaria chemotherapy: The effects of monensin and nigericin on Plasmodium falciparum in vitro and Plasmodium vinckei petteri in vivo
1996, Life Sciences
Chloroquine is widely used in malaria chemotherapy. Due to its weak base properties, this drug accumulates in the parasite food vacuole where it acts initially by raising the pH of this organelle. thereby reducing the digestion of hemoglobin by the parasite and preventing its growth. Nevertheless, alkalinization of the food vacuole and inhibition of lysosomal protein degradation could also be achieved by means of carboxylic ionophores such as monensin and nigericin. These drugs intercalate into intracellular organelle membranes and exchange protons for K⁺ or Na⁺. In the present study, we show that monensin and nigericin exhibit $in vitro$ intrinsic antimalarial activities at nanomolar and picomolar range, respectively, on $P.falciparum$ and thereby appear 25 fold and 30,000 fold more potent than chloroquine. The very low IC50 values exhibited by these two ionophores prompted us to test their antimalarial activities $in vivo$ on $Plasmodium vinckei$ $petteri$ . We found that the ED₅₀ and ED₉₀ values were respectively 1.1 $mg kg$ and 3.5 $mg kg$ for monensin; 1.8 $mg kg$ and 4.6 $mg kg$ for nigericin. In addition, when treated with monensin at 10 $mg kg$ , 100% of the infected mice were cured. Interestingly, nigericin can be combined with monensin and we show that this combination is synergic. Thus, this finding would allow the use of lower doses of these ionophores and prevent occurrence of drug resistance. Carboxylic ionophores can be viewed as a new strategy in malaria chemotherapy.

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^☆: The work was supported by a grant from A/S Norsk Varekrigsforsikrings Fond.

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Effect of carboxylic ionophores on lysosomal protein degradation in rat hepatocytes☆

Abstract

Biochem pharmacol

Cell

Biochim biophys acta

Biochim biophys acta

Exp cell res

Methods cell biol

Exp cell res

Ann rev biochem

J cell biol

J exp med

J cell biol