Lonidamine

Lonidamine Extends Lifespan of Adult Caenorhabditis elegans by Increasing the Formation of Mitochondrial Reactive Oxygen Species

Authors S. Schmeisser1,2, K. Zarse1, M. Ristow1,3

Key words
●▶ glucose
●▶ nutrition
●▶ antioxidants

Abstract

Compounds that delay aging in model organ- isms may be of significant interest to antiaging medicine, since these substances potentially provide pharmaceutical approaches to promote healthy lifespan in humans. The aim of the study was to test whether pharmaceutical concentra- tions of the glycolytic inhibitor lonidamine are capable of extending lifespan in a nematodal model organism for aging processes, the round- worm Caenorhabditis elegans. Several hundreds of adult C. elegans roundworms were maintained on agar plates and fed E. coli strain OP50 bacte- ria. Lonidamine was applied to test whether it may promote longevity by quantifying survival in the presence and absence of the compound. In addition, several biochemical and metabolic assays were performed with nematodes exposed to lonidamine. Lonidamine significantly extends both median and maximum lifespan of C. elegans when applied at a concentration of 5 micromolar by 8 % each. Moreover, the com- pound increases paraquat stress resistance, and promotes mitochondrial respiration, culminat- ing in increased formation of reactive oxygen species (ROS). Extension of lifespan requires activation of pmk-1, an orthologue of p38 MAP kinase, and is abolished by co-application of an antioxidant, indicating that increased ROS for- mation is required for the extension of lifespan by lonidamine. Consistent with the concept of mitohormesis, lonidamine is capable of promot- ing longevity in a pmk-1 sensitive manner by increasing formation of ROS.

Introduction

Promotion of longevity and in particular exten- sion of healthy lifespan (also named ‘healthspan’) is of eminent interest to most humans. Specific mutations have been shown to extend the lifespan of model organisms dramatically [1–8], while more readily available interventions, including calorie restriction, extend life expect- ancy of model organisms, however, less strik- ingly [9]. Accordingly, considerable effort has been invested to identify naturally occurring and/or pharmaceutical compounds that promote lon- gevity in model organisms. A number of such compounds have been identified in recent years, including rapamycin [10–12], resveratrol [12–17],
2-deoxy-D-glucose [18], and phytochemicals [19]. We have tested here whether lonidamine (LND) at pharmacological doses may be effective in extending lifespan of a metazoal model organ- ism, C. elegans. Lonidamine (LND) is a derivative of indazole-3-carboxylic acid first developed as antispermatogenic drug and later employed as potentially antineoplastic drug for cancer treat- ment [20]. Numerous existing evidences suggest that LND belongs to the group of mitochondria targeting agents [21–24]. The drug has been tested in phase II and III trials of metastatic breast cancer and ovarian cancer [25–28]. Thereby, it is assumed that the antineoplastic effects are medi- ated by the ability of LND to cause a disruption of the cellular energy metabolism and induction of apoptosis [21, 29]. Accordingly, LND is able to reduce aerobic glycolysis in neoplastic cells by affecting the mitochondrially bound hexokinase (which is highly present in malignant cells) [30]. Moreover, it inhibits mitochondrial respiration both in cancer and normal cells by interfering with the NAD-FAD dehydrogenase complex [20, 30–32]. A study which was done on isolated mitochondria indicates that LND, similar to arsenite, targets the mPTP [21, 33]. The mPTP represents a polyprotein complex that is located at the contact sites between the inner and outer mitochondrial membrane [34]. Opening of this pore leads to a disruption of the mitochondrial transmembrane potential, release of pro-apoptogenic factors such as cytochrome C and other hallmarks of the intrinsic apoptotic pathway [23]. Interestingly, the mitochondrially bound hexokinase is associ- ated to the mPTP, whereas glucose metabolism inhibits perme- ability transition [35]. Thus, suppressing mitochondrially bound hexokinase by LND and a subsequent activation of the transition pore, could link the energy disrupting properties to direct mito- chondrial effects and induction of apoptosis [23, 36, 37]. There- fore, LND represents a drug, which acts as a metabolic inhibitor with mitochondria as the main site of action. Applying this com- pound, we observed a significant extension of C. elegans lifespan following induction of mitochondrial hormesis.

Materials and Methods

Compounds

Lonidamine, N-acetyl-L-cysteine (NAC), and paraquat were obtained from Sigma-Aldrich (Munich, Germany).

HepG2-based determination of ATP

HepG2 cells were maintained in RPMI containing 10 % (v/v) FBS (Biochrom AG, Berlin, Germany) and 1 % antibiotic solution (100 U/ml penicillin, and 10 μg/ml streptomycin (P/S), obtained from Biochrom AG, Berlin, Germany) in a humidified atmos- phere of 5 % CO2 at 37 °C.
For experiment, 35 000 cells were seeded into each well of a 96-well plate. After 24 h, medium was changed to RPMI 0.5 % FBS and 1 % P/S.

After another 6 h, medium was changed again, whereas treatment compounds were included (1:1000).Determination of ATP and protein content has been described previously [38]. Briefly, after the 6 h of incubation period, reac- tions were stopped by placing the plates on ice. ATP content was subsequently examined using a commercial available luciferin/ luciferase-based bioluminescence assay (CellTiter-Glo, Promega, Madison, USA) according to the manufacturer’s instructions.

The cells were lysed using the applied lysis buffer. An aliquot was removed for protein determination (as normalization to the luminescence signal). Protein content was quantified with bicin- choninic acid assay kit (BCA Protein Assay, Thermo Fischer Sci- entific, Rockford, USA). The remaining lysate was supplemented with a bioluminescent buffer containing luciferin/luciferase. Luminescence was measured using a luminometer (FLUOstar Optima, BMG, Offenburg, Germany). To calculate the absolute ATP amount, an ATP standard curve was determined in parallel.

Compound treatment

Treatment of C. elegans was carried out on NGM agar plates con- taining the respective compound. Thereby, agar plates were pre- pared from the same batch of NGM agar, whereas treatment plates were supplemented with the compound and control plates with the corresponding solvent as described [39]. After plates were poured and dried for about 30 min, they were sealed and stored at 4 °C.While C. elegans maintenance was done on NGM agar spotted with alive OP50 E. coli, compound treatment was always per- formed in presence of heat inactivated bacteria (OP50 HIT) to avoid interference by the xenobiotic-metabolizing activity of E. coli [40]. To obtain heat-inactivated bacteria, an overnight culture of E. coli was treated for 30 min at 65 °C. The bacteria sus- pension was then concentrated 20-fold by centrifugation (30 min 3 200 g) within S-buffer containing 10 mM MgSO4 and 5 μg/ml cholesterol as described [14]. Furthermore, treatment NGM plates were prepared with 100 μg/ml ampicillin. It should be noted that this treatment causes an inactivation of metabolic activity rather than complete lethality. NAC was applied the entire life time of the nematodes starting from egg stadium.

Lifespan assays

All lifespan assays were performed at 20 °C according to stand- ard protocol and as previously described [18]. Briefly, a C. elegans population was treated with hypochlorite/NaOH solution to synchronize the population at day zero of the lifespan. Eggs were transferred to freshly spotted plates to allow hatching and devel- opment. Soon after L4, around 150 nematodes were manually transferred to fresh incubation plates containing the respective compound or the solvent as control and spotted with a lawn of heat inactivated OP50 E. coli.
For the first 10–14 days, worms were transferred every day and afterwards 3–4 times a week. Nematodes that show no reaction to gentle stimulation were scored as dead. Those animals that crawled off the plates or display a premature, non-natural death due to internal hatching or bursting were censored (i. e., excluded from statistics on day of premature death).

Stress resistance

Resistance to lethal oxidative stress derived from paraquat was determined with minor modifications as previously described [18]. Nematodes were treated with the respective compound for 6 days starting at L4 stage on NGM agar containing the com- pound and spotted with heat-inactivated bacteria. To avoid mix- ing of the population, worms were washed and transferred to fresh plates every day, whereas adult worms were separated from larvae and eggs by removing the supernatant several times after gravity-based separation. Following this pretreatment period, worms were transferred manually to fresh NGM plates containing 10 mM paraquat spotted with a lawn of heat-inacti- vated OP50 following by daily determining the survival rate until all nematodes were dead. As described for lifespan analysis, worms were count as censored in case of internal hatching, crawling off, and bursting.

Oxygen consumption

Respiration was quantified using a DW1/AD Clark-type elec- trode (Hansatech, King’s Lynn, England) as previously described [18]. After individual incubation period, worms were harvested, washed, and transferred into the DW1 chamber. Oxygen con- sumption was monitored for at least 10 min. Afterwards, worms were carefully removed from the chamber and collected for a subsequent protein determination. The worms were sonicated 3 times and centrifuged for 10 min at 12 000 × g. Supernatant was used for protein determination using BCA.

Mitochondrial formation of reactive oxygen species

To quantify mitochondrial ROS production, we used a commer- cially available dye, which enter the mitochondria in a reduced state (MitoTracker Red CM-H2X ROS; obtained from Invitrogen). The endogenous oxidation due to mitochondrial ROS production leads to an increase in fluorescence which could be measured (excitation 570 nm; emission 620 nm).

In order to elucidate mtROS production in C. elegans, worms were treated with LND according to the description above. Following that incubation period, nematodes were shifted to plates spotted with a mixture of dead OP50 (30 min at 65 °C in a water bath) and dye (final conc. 0.5 μM). Worms were incubated with the bacteria/ dye mixture for 2 h with a subsequent 1 h recovery period on fresh plates (w/o dye) to eliminate artificial results due to large amounts of intestinal dye. Worms were washed at least 3 times.

Lonidamine promotes nematodal oxygen consumption Since the data depicted in ●▶ Fig. 1a insinuate that LND may pro- mote mitochondrial metabolism, we quantified nematodal oxy- gen consumption after exposure to LND for varying time periods, transferred to new plates. Finally, nematodes were distributed into wells of a FLUOTRACTM 96-Well plate (Greiner Bio-One, Fric- kenhausen, Germany) and fluorescence was determined using a Fluorometer (FLUOstar Optima, BMG, Offenburg, Germany; Ex: 570 nm, Em: 610 nm; well-scanning mode). To normalize the flu- orescence signal after measurement, worms were recovered from the wells and protein was analyzed as given above.

Western blotting

Protein analysis was done as described [18]. Worms were treated with Lonidamine for 120 h, harvested, washed and flash frozen in liquid nitrogen. The remaining frozen worm pellet was ground in a nitrogen-chilled mortar together with protease and phos- phatase inhibitors, CompleteTM protease inhibitor cocktail (Roche), 2 mM sodium fluoride, 2 mM sodium orthovanadate, 1 mM PMSF, and 2 mM EDTA (all from Sigma-Aldrich). The homoge- nate was sonicated 3 times and centrifuged for 10 min at 12 000 × g. The supernatant was used both for protein quantifica- tion and subsequent western blot analysis. Antibodies against the phosphorylated p38 and α-tubulin were obtained from Cell Signaling (Boston, MA, USA).

Results

HepG2-based identification of ionidamine as a potentially lifespan-extending compound In an effort to identify compounds that potentially extend lifespan in C. elegans, we commonly applied candidate com- pounds to HepG2 cells. Hereby we aimed to identify compound that activate mitochondrial metabolism, as quantified by an increase in cellular ATP content. Applying LND to this assay at a concentration of 50 micromolar, an increase in ATP content normalized to protein content of 25 % was observed (●▶ Fig. 1a).

Lonidamine increases stress resistance

In order to elucidate whether the pretreatment of nematodes with LND potentially increase ROS defense mechanism with a subsequently rise in resistance against lethal oxidative stress, LND treated worms were exposed to a high dose of paraquat.

Paraquat is a continuous ROS inducer know to exert lethal effects on C. elegans, whereas compounds that increase survival during exposure to paraquat are likely to extend lifespan as well [18]. Nematodes were exposed to LND at a concentration of 5 micro- molar for 6 days, and then exposed to paraquat at a lethal con- centration of 10 millimolar. As depicted in ●▶ Fig. 1b, C. elegans survive significantly longer when pretreated with LND. This indicates that LND increases resistance against paraquat-medi- ated ROS production to culminate in reduced lethality.

Lonidamine increases life expectancy

Applying LND at a concentration of 5 micromolar to C. elegans extends life span by 1.7 days (8 %). Maximum lifespan was extended by 2.5 days (8 %) (see ●▶ Table 1 for details). Hence, LND is capable of extending C. elegans lifespan significantly (p < 0.001) (●▶ Fig. 1c). Lonidamine increases mitochondrial ROS formation in C. elegans Increased respiration, as shown in ●▶ Fig. 2a, may be indicative of increased mitochondrial ROS (mtROS) formation. To test this possibility, we have used redox-sensitive fluorescent dye which accumulates in the mitochondrial compartment to obtain a rela- tive quantification of mtROS in nematodes in the presence and absence of LND. Consistent with the constitutive increase in res- piration (●▶ Fig. 2a), we observed increased formation of mtROS in the presence of LND for 5 days (●▶ Fig. 2b), indicating that increased respiration following LND exposure is indeed promot- ing mtROS formation in C. elegans. Increased ROS formation is required for lifespan extension The fact that LND increases both lifespan (●▶ Fig. 1c) as well as mtROS formation (●▶ Fig. 2b) raises the possibility that mtROS are instrumental or even required for promoting lifespan in C. elegans. To test this possibility, we have repeated the lifespan assays in the presence of an antioxidant, NAC, which is known to significantly reduce the formation of mtROS in C. elegans, as pre- viously shown [18]. Applying LND in the presence of the antioxi- dant fully prevents the lifespan-extending effects of LND in C. elegans (●▶ Fig. 2c) indicating that increased mtROS formation is indeed required for lifespan extension by LND. p38 mitogen-activated protein kinase/pmk-1 signaling acts as a ROS sensor There is ample evidence that ROS activate a number of down- stream signaling molecules, including p38 MAP kinase. We have first extracted proteins from nematodes that have been exposed to LND for 5 days and compared abundance of phosphorylated p38 MAP kinase/pmk-1 in comparison to untreated worms. We observed an increased abundance of phosphorylated pmk-1 (●▶ Fig. 3a), while basal expression of pmk-1 could not be quan- tified due to the lack of an appropriate antibody. Nevertheless this finding may indicate that activation of pmk-1 by mtROS is required for lifespan extension in C. elegans. To test this hypoth- esis, the lifespan assays were repeated in the presence and absence of LND in nematodes that lack functional pmk-1, named KU25. Consistent with the hypothesis that pmk-1 acts as a ROS sensor, LND fails to extend lifespan in nematodes lacking pmk-1. Discussion To potentially support the ongoing search for compounds that may promote human health especially at higher age, we show here that LND promotes longevity in a nematodal model organ- isms, the roundworm C. elegans. Most importantly, the lifespan- extending effects of LND depend on increased formation of mtROS to activate pmk-1, supporting and mechanistically extending the concept of mitochondrial hormesis, also known as mitohormesis. Fig. 3 Lonidamine mediates lifespan extension through activation of p38 mitogen-activated protein kinase. Panel a depicts results of a Western blot analysis using a primary antibody against the phosphorylated pmk-1/ p38 after 120 h of exposure to lonidamine as well as a primary antibody against α-tubulin (loading control); note that no antibody against basal pmk-1 is available. Panel b shows lifespan assays using nematodes lack- ing pmk-1 in the presence (grey triangles) and absence (black circles) of lonidamine. LND is a compound that has previously been shown to act as metabolic inhibitor [22, 32, 41]. Accordingly, it was reported that LND is capable to inhibit the mitochondrially bound hexokinase II [30]. Moreover, LND interfere also with the mitochondrial transition pore (mPTP) to cause a decrease in mitochondrial membrane potential as well as reduced ATP production [21, 33]. Thus, LND belongs to the group of mitochondrial targeting agents which are able to compromises mitochondrial function [21–24]. Hence, we could observe that short term LND exposure reduces oxygen consumption by 37 %. This early event, however, gets compensated and culminates finally in an increase respira- tion that is associated with an increased mtROS production. Recent evidences suggested that mitochondrial derived ROS are not only involved into cell damaging mechanisms, which is com- monly accepted for high oxidative stress, but also serves as important signal molecules that are involved into cellular signal transduction and regulation of a variety of processes. Among these processes, the induction of endogenous defense mecha- nisms as a secondary response to a stressful condition is assumed to contribute to longevity [42–57]. Thus, a lifetime low dose oxidative stress with a subsequent sec- ondary induction of defense mechanisms could delay the aging process as well as the mortality in case of very high ROS expo- sure, that is, due to paraquat treatment. This clearly indicates, in regard to the formation of potential harmful ROS, that a low dose exposure could be beneficial by increasing C. elegans lifespan and stress resistance, whereas high amounts of ROS-production exerts detrimental properties and premature death which is demonstrated by the short overall lifespan within the paraquat stress assay. Since the current study has been performed in the model organ- isms C. elegans, it is unclear whether our results can be extrapo- lated to mammals or even humans. Hence, further studies will have to show whether LND has any effect on mammalian human health span and/or longevity. However, other compounds that have been identified by using a similar, metazoan-based approach have been shown to be effective in rodents [10–16]. Taken together, these findings indicate that LND extends C. ele- gans lifespan in a mitohormetic manner, moreover suggesting that this compound may be worth evaluating in mammals and potentially humans in regards to prevention of aging and age- associated diseases. Acknowledgements C. elegans strains used in this work were provided by the Caenorhabditis Genetics Center (Univ. of Minnesota), which is funded by the NIH National Center for Research Resources (NCRR). The authors thank Beate Laube, Annett Müller, and Wal- traud Scheiding for excellent technical assistance. 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