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Mitochondrial Complex I Inhibitor Rotenone Induces Apoptosis through Enhancing Mitochondrial Reactive Oxygen Species Production*

  • Nianyu Li
    Affiliations
    Purdue University Cytometry Laboratories, Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907
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  • Kathy Ragheb
    Affiliations
    Purdue University Cytometry Laboratories, Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907
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  • Gretchen Lawler
    Affiliations
    Purdue University Cytometry Laboratories, Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907
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  • Jennie Sturgis
    Affiliations
    Purdue University Cytometry Laboratories, Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907
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  • Bartek Rajwa
    Affiliations
    Purdue University Cytometry Laboratories, Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907
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  • J. Andres Melendez
    Affiliations
    Center for Immunology & Microbial Disease, MC-151, Albany Medical College, Albany, New York 12208
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  • J. Paul Robinson
    Correspondence
    To whom correspondence should be addressed: Purdue University Cytometry Laboratories, Dept. of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907. Tel.: 765-494-0757; Fax: 765-494-0517;
    Affiliations
    Purdue University Cytometry Laboratories, Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:December 20, 2002DOI:https://doi.org/10.1074/jbc.M210432200
      Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.
      ROS
      reactive oxygen species
      PHPA
      p-hydroxyphenylacetate
      Mn-SOD
      magnesium superoxide dismutase
      HE
      hydroethidium
      EB
      ethidium bromide
      NAC
      N-acetylcysteine
      PI
      propidium iodide
      Tiron
      4,5-dihydroxy-1,3-benzene-disulfonic acid
      PBS
      phosphate-buffered saline
      HBSS
      Hank's balanced salt solution
      Me2SO
      dimethyl sulfoxide
      Z
      benzyloxycarbonyl
      The mitochondrial respiratory chain (complexes I-V) is the major site of ATP production in eukaryotes. Recently it was recognized that this organelle not only generates ATP, but also plays an important role in apoptosis (for reviews see Refs.
      • Green D.R.
      • Reed J.C.
      ,
      • Kroemer G.
      • Reed J.C.
      ,
      • Wang X.
      ).
      It is now clear that upon apoptotic stimulation mitochondria can release several proapoptotic regulators, including cytochromec (
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ), Smac/Diablo (
      • Du C.
      • Fang M.
      • Li Y.
      • Li L.
      • Wang X.
      ,
      • Verhagen A.M.
      • Ekert P.G.
      • Pakusch M.
      • Silke J.
      • Connolly L.M.
      • Reid G.E.
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      ), endonuclease G (
      • Li L.Y.
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      • Wang X.
      ), and apoptosis-inducing factor (
      • Susin S.A.
      • Lorenzo H.K.
      • Zamzami N.
      • Marzo I.
      • Snow B.E.
      • Brothers G.M.
      • Mangion J.
      • Jacotot E.
      • Costantini P.
      • Loeffler M.
      • Larochette N.
      • Goodlett D.R.
      • Aebersold R.
      • Siderovski D.P.
      • Penninger J.M.
      • Kroemer G.
      ), to the cytosol. These proapoptotic regulators will then activate cellular apoptotic programs downstream (for reviews see Refs.
      • Green D.R.
      • Reed J.C.
      ,
      • Kroemer G.
      • Reed J.C.
      ,
      • Wang X.
      ). The release of proapoptotic regulators is further regulated by the translocation of Bcl-2 family proteins (
      • Reed J.C.
      ,
      • Reed J.C.
      ). Although this evidence places mitochondria in the center of the apoptotic signaling pathway, the role of mitochondrial respiratory chain activity in apoptosis is still elusive.
      Inhibition of the mitochondrial respiratory chain by rotenone has been widely used to study the role of the mitochondrial respiratory chain in apoptosis (
      • Barrientos A.
      • Moraes C.T.
      ,
      • Isenberg J.S.
      • Klaunig J.E.
      ,
      • Chauvin C.
      • De Oliveira F.
      • Ronot X.
      • Mousseau M.
      • Leverve X.
      • Fontaine E.
      ). Some recent evidence showed that rotenone, a mitochondrial respiratory chain complex I inhibitor, could induce cell death in a variety of cells (
      • Barrientos A.
      • Moraes C.T.
      ,
      • Isenberg J.S.
      • Klaunig J.E.
      ,
      • Chauvin C.
      • De Oliveira F.
      • Ronot X.
      • Mousseau M.
      • Leverve X.
      • Fontaine E.
      ,
      • Higuchi M.
      • Proske R.J.
      • Yeh E.T.
      ,
      • Shimizu S.
      • Eguchi Y.
      • Kamiike W.
      • Waguri S.
      • Uchiyama Y.
      • Matsuda H.
      • Tsujimoto Y.
      ,
      • Sherer T.B.
      • Betarbet R.
      • Stout A.K.
      • Lund S.
      • Baptista M.
      • Panov A.V.
      • Cookson M.R.
      • Greenamyre J.T.
      ). The importance of mitochondrial respiratory chain complex I inhibitor-induced apoptosis was further recognized with the finding that tumor necrosis factor (TNF)-α could inhibit the mitochondrial respiratory chain at the mitochondrial complex I site (
      • Higuchi M.
      • Proske R.J.
      • Yeh E.T.
      ). However, contradictory reports showed that rotenone could inhibit apoptosis in other systems (
      • Vrablic A.S.
      • Albright C.D.
      • Craciunescu C.N.
      • Salganik R.I.
      • Zeisel S.H.
      ,
      • Torres-Roca J.F.
      • Tung J.W.
      • Greenwald D.R.
      • Brown J.M.
      • Herzenberg L.A.
      • Herzenberg L.A.
      • Katsikis P.D.
      ,
      • Deshpande S.S.
      • Angkeow P.
      • Huang J.
      • Ozaki M.
      • Irani K.
      ).
      It has been suggested that ROS1 play an important role in apoptosis, and several groups have shown that molecules that stimulate formation of ROS can result in apoptosis (
      • Heussler V.T.
      • Fernandez P.C.
      • Botteron C.
      • Dobbelaere D.A.
      ,
      • Kelso G.F.
      • Porteous C.M.
      • Coulter C.V.
      • Hughes G.
      • Porteous W.K.
      • Ledgerwood E.C.
      • Smith R.A.
      • Murphy M.P.
      ) and a process inhibited by antioxidants (
      • Koren R.
      • Hadari-Naor I.
      • Zuck E.
      • Rotem C.
      • Liberman U.A.
      • Ravid A.
      ,
      • Chrestensen C.A.
      • Starke D.W.
      • Mieyal J.J.
      ). Others reported production of ROS by a wide range of apoptotic stimuli, including TNF, ceramide, staurosporine, and UV radiation (
      • Gottlieb E.
      • Vander Heiden M.G.
      • Thompson C.B.
      ,
      • Garcia-Ruiz C.
      • Colell A.
      • Mari M.
      • Morales A.
      • Fernandez-Checa J.C.
      ,
      • Cai J.
      • Jones D.P.
      ,
      • Shaulian E.
      • Schreiber M.
      • Piu F.
      • Beeche M.
      • Wagner E.F.
      • Karin M.
      ). The mitochondrial respiratory chain is one of the most important sites of ROS production under physiological conditions (
      • Kroemer G.
      • Dallaporta B.
      • Resche-Rigon M.
      ,
      • Martinou J.C.
      ,
      • Tan S.
      • Sagara Y.
      • Liu Y.
      • Maher P.
      • Schubert D.
      ), and it has been long suspected that mitochondrial ROS play an important role in apoptosis. The mitochondrial-derived ROS are vital not only because mitochondrial respiratory chain components are present in almost all eukaryotic cells, but also because the ROS produced in mitochondria can readily influence mitochondrial function without having to cope with long diffusion times from the cytosol. Two sites in the respiratory chain, complex I and complex III, have been suggested to be the major ROS source (
      • Turrens J.F.
      • Boveris A.
      ,
      • Turrens J.F.
      • Alexandre A.
      • Lehninger A.L.
      ,
      • Turrens J.F.
      ). Based on stoichiometrical calculation, superoxide was suggested as the primary product, with hydrogen peroxide as the secondary product (
      • Cadenas E.
      • Boveris A.
      • Ragan C.I.
      • Stoppani A.O.
      ). Mitochondrial-derived ROS could be modified when the mitochondrial respiratory chain was interrupted under pathological conditions or by respiratory chain inhibitors (
      • Turrens J.F.
      • Boveris A.
      ,
      • Turrens J.F.
      ). Early reports showed that the complex I inhibitor rotenone and the complex b-c1 inhibitor antimycin could stimulate superoxide and hydrogen peroxide formation on submitochondrial particles (
      • Turrens J.F.
      • Boveris A.
      ,
      • Turrens J.F.
      • Freeman B.A.
      • Levitt J.G.
      • Crapo J.D.
      ). However, results of rotenone-induced mitochondrial ROS production measured at the cellular level appeared inconsistent with conflicting results reporting that rotenone could elevate cellular ROS production in some cases (
      • Barrientos A.
      • Moraes C.T.
      ,
      • Nakamura K.
      • Bindokas V.P.
      • Kowlessur D.
      • Elas M.
      • Milstien S.
      • Marks J.D.
      • Halpern H.J.
      • Kang U.J.
      ) while inhibiting cellular ROS production in others (
      • Vrablic A.S.
      • Albright C.D.
      • Craciunescu C.N.
      • Salganik R.I.
      • Zeisel S.H.
      ,
      • Schuchmann S.
      • Heinemann U.
      ,
      • Li Y.
      • Trush M.A.
      ).
      The present investigation studied the mechanism of rotenone-induced apoptosis. Our data show that rotenone can induce mitochondrial ROS production and that rotenone-induced mitochondrial ROS production is closely related to rotenone-induced apoptosis.

      DISCUSSION

      In this study we demonstrated that the ability of the mitochondrial respiratory chain complex I inhibitor rotenone to induce programmed cell death is closely related to its ability to induce mitochondrial ROS production. Previously, rotenone had been reported to enhance glutamate/malate-supported mitochondrial reactive oxygen species production on both bovine heart submitochondrial particles and rat heart intact mitochondria (
      • Turrens J.F.
      • Boveris A.
      ,
      • Hansford R.G.
      • Hogue B.A.
      • Mildaziene V.
      ). Our result confirmed that in isolated HL-60 cell mitochondria, rotenone induced mitochondrial ROS production through a similar mechanism. The induction of mitochondrial ROS production by rotenone had frequently been attributed to the ability of rotenone to block mitochondrial respiratory chain complex I, thereby increasing the formation of ubisemiquinone, the primary electron donor in mitochondrial superoxide generation. This also appeared to be the case for HL-60 mitochondria because the concentrations of rotenone that induced mitochondrial ROS corresponded to the concentrations of rotenone that inhibited cell respiration. In addition, rotenone at 0.5–1 μm resulted in a maximum decrease of cellular ATP level (∼63% of control). The respiratory uncoupler oligomycin, which shuts down electron input from both complex I and complex II, resulted in only 8% more inhibition on cellular ATP level (∼55% of control), indicating mitochondrial complex I substrates are the major substrates for the mitochondrial respiratory chain in HL-60 cells. For HL-60 cells, these results suggest that once rotenone inhibited mitochondrial respiratory chain complex I, the accumulation of pyruvate/malate in the mitochondria could create a condition that may well be similar to the isolated mitochondrial model.
      The whole-cell-level ROS measurement by flow cytometry using hydroethidine confirmed our findings on isolated mitochondria. Rotenone induced cellular ROS production dose-dependently. Rotenone could not induce cellular ROS production on ρ0 HL-60 cells, further confirming that rotenone-induced cellular ROS originated from mitochondria. At the cellular level, rotenone could enhance cellular ROS level in a similar pattern through a slightly different mechanism. Similar to the results from isolated mitochondria, increasing concentrations of rotenone greater than 0.5 μmproduced further but much slower increases in cellular ROS production. However, at lower concentration (100 nm), rotenone was able to increase cellular ROS level only slightly, whereas in the isolated mitochondria model, this concentration could induce a mitochondrial ROS production with close to 50% of maximum ROS production (for rotenone at 1 μm). The possible explanation is that at the cellular level, the cellular antioxidant system may have a greater impact on the decrease of ROS production.
      Rotenone has been reported to cause cell death in a variety of cell lines (
      • Barrientos A.
      • Moraes C.T.
      ,
      • Isenberg J.S.
      • Klaunig J.E.
      ,
      • Chauvin C.
      • De Oliveira F.
      • Ronot X.
      • Mousseau M.
      • Leverve X.
      • Fontaine E.
      ,
      • Higuchi M.
      • Proske R.J.
      • Yeh E.T.
      ,
      • Shimizu S.
      • Eguchi Y.
      • Kamiike W.
      • Waguri S.
      • Uchiyama Y.
      • Matsuda H.
      • Tsujimoto Y.
      ,
      • Sherer T.B.
      • Betarbet R.
      • Stout A.K.
      • Lund S.
      • Baptista M.
      • Panov A.V.
      • Cookson M.R.
      • Greenamyre J.T.
      ). However, whether this cytotoxicity leads to apoptosis or necrosis may depend upon cell type. Our results showed that in HL-60 cells rotenone induced cell death through an apoptotic mechanism. Cell cycle analysis showed a typical apoptotic subdiploid population after rotenone treatment. Chromatin condensation and DNA breakdown were also clearly observable by confocal microscopy. DNA laddering presented as a typical apoptotic DNA cleavage into 50 kbp and 200/1000 bp oligonucleosomal fragments. In addition, Western blots identified release of cytochrome c from the mitochondrial compartment to the cytosol after 0.5–1 μm rotenone treatment. Caspase 3 activation was also demonstrated. All these data indicated that in HL-60 cells rotenone-induced cell death occurs mainly via an apoptotic mechanism as opposed to necrosis. A possible mechanism might be that rotenone inhibits the mitochondrial respiratory chain at the complex I site, decreasing the cellular ATP level; however, the dependence of pyruvate/malate-supported ATP production varies in different cell types. It has already been well established that ATP can act as a switch between apoptosis and necrosis (
      • Stefanelli C.
      • Bonavita F.
      • Stanic I.
      • Farruggia G.
      • Falcieri E.
      • Robuffo I.
      • Pignatti C.
      • Muscari C.
      • Rossoni C.
      • Guarnieri C.
      • Caldarera C.M.
      ,
      • Eguchi Y.
      • Shimizu S.
      • Tsujimoto Y.
      ,
      • Leist M.
      • Single B.
      • Castoldi A.F.
      • Kuhnle S.
      • Nicotera P.
      ). A depleted cellular ATP level (usually to around 30% of control) has been shown to inhibit apoptosis (
      • Stefanelli C.
      • Bonavita F.
      • Stanic I.
      • Farruggia G.
      • Falcieri E.
      • Robuffo I.
      • Pignatti C.
      • Muscari C.
      • Rossoni C.
      • Guarnieri C.
      • Caldarera C.M.
      ,
      • Eguchi Y.
      • Shimizu S.
      • Tsujimoto Y.
      ,
      • Leist M.
      • Single B.
      • Castoldi A.F.
      • Kuhnle S.
      • Nicotera P.
      ). Therefore, in cell lines highly dependent on pyruvate/malate-supported ATP production, rotenone treatment might drastically decrease the cellular ATP level, which could switch cell death from apoptotic to necrotic. However, this is not the case in HL-60 cells, in which rotenone decreased cellular ATP to a moderate level (63%). Both our results and other reports show that even oligomycin decreases the cellular ATP level in HL-60 cells to around 50–60% of control after 24 h treatment (
      • Sweet S.
      • Singh G.
      ). In several other cell lines, such as Jurkat, HeLa, or thymocyte, the same concentrations of oligomycin can decrease the cellular ATP level to less than 20% of control level (
      • Stefanelli C.
      • Bonavita F.
      • Stanic I.
      • Farruggia G.
      • Falcieri E.
      • Robuffo I.
      • Pignatti C.
      • Muscari C.
      • Rossoni C.
      • Guarnieri C.
      • Caldarera C.M.
      ,
      • Eguchi Y.
      • Shimizu S.
      • Tsujimoto Y.
      ,
      • Leist M.
      • Single B.
      • Castoldi A.F.
      • Kuhnle S.
      • Nicotera P.
      ). While unlikely to be responsible for these results, the possible impact of high concentrations of rotenone on cellular glycolytic pathways has not been entirely excluded.
      Our results strongly suggest that in HL-60 cells, induction of mitochondrial ROS could well be the most significant mechanism of rotenone-induced apoptosis. The concentrations of rotenone that induced apoptosis were in the same range as the concentrations that induced mitochondrial ROS production. Rotenone-induced apoptotic cells in ρ0 HL-60 cells were much fewer than those in normal HL-60 cells. Several antioxidants (glutathione, vitamin C, andN-acetylcysteine) were shown to inhibit rotenone-induced cytochrome c release, caspase 3 activation, and DNA breakdown in HL-60 cells. Induction of mitochondrial ROS by rotenone also occurred very early (<1 h) compared with cytochrome crelease and caspase 3 activation, suggesting that ROS production is upstream of these apoptotic events.
      Results from Mn-SOD-overexpressing HT1080 cells confirmed our conclusion that rotenone induced apoptosis via an induction of mitochondrial ROS production. Mn-SOD is the superoxide dismutase that localizes in mitochondria. Mn-SOD is a primary component of the cellular defense system against oxidative toxicity since superoxide can react with hydrogen peroxide to generate singlet oxygen and hydroxyl radicals, which are more toxic than superoxide and hydrogen peroxide (
      • Freeman B.A.
      • Crapo J.D.
      ). In mitochondria, manganese superoxide dismutase is the major enzyme responsible for converting superoxide to hydrogen peroxide (
      • Boveris A.
      • Cadenas E.
      ,
      • Dionisi O.
      • Galeotti T.
      • Terranova T.
      • Azzi A.
      ,
      • Weisiger R.A.
      • Fridovich I.
      ). Overexpression of Mn-SOD inhibited rotenone-induced increase of cellular ROS, confirming that rotenone-induced cellular ROS production in HT1080 cells was from mitochondria. Consistent with our findings in HL-60 cells, overexpression of Mn-SOD also inhibited rotenone-induced cytochrome c release, caspase 3 activation, and DNA breakdown.
      The mechanism of rotenone-induced apoptosis is still elusive, and obscured by the occurrence of several concomitant events, including shutdown of the electron transfer through respiratory chain complex I, decreasing cellular ATP level, increasing mitochondrial ROS production, and decreasing mitochondrial membrane potential. Previously, the decrease of mitochondrial membrane potential and the opening of the mitochondrial permeability transition pore, but not ATP reduction, have been shown to be involved in rotenone-induced apoptosis (
      • Barrientos A.
      • Moraes C.T.
      ,
      • Isenberg J.S.
      • Klaunig J.E.
      ,
      • Chauvin C.
      • De Oliveira F.
      • Ronot X.
      • Mousseau M.
      • Leverve X.
      • Fontaine E.
      ). However, the role of rotenone-induced mitochondrial ROS production has not been fully investigated. Results from the current study identified mitochondrial ROS as playing a key role in rotenone-induced apoptosis.

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