Research Communication Rotenone Induces Apoptosis in Human Lung Cancer Cells by Regulating Autophagic Flux

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1 Research Communication Rotenone Induces Apoptosis in Human Lung Cancer Cells by Regulating Autophagic Flux Wensi Hu 1 Hui Tian 1 * Weiming Yue 1 Lin Li 1 Shuhai Li 1 Cun Gao 1 Libo Si 1 Lei Qi 1 Ming Lu 1 Bin Hao 2 Shuyu Shan 2 1 Department of Thoracic Surgery, Qilu Hospital, Shandong University, Jinan, China 2 Department of Thoracic Surgery, Central Hospital of ZiBo, ZiBo, China Abstract Reactive oxygen species (ROS) are at the center of many physiological and pathological processes. ROS generated due to oxidative stress can potentiate both cancer initiation and progression. Rotenone, which is an inhibitor of the mitochondrial electron transport chain complex I, results in the activation of NOX2 and release of ROS, and has been shown to display anticancer activity through the induction of apoptosis in various cancer cells. The mechanistic link between rotenonedependent activation of NOX2 and induction of apoptosis is still elusive. In this study, we used the human lung cancer A549 cells to study the molecular mechanism(s) involved between rotenone-dependent activation of NOX2 and impairment of autophagic machinery. We report that acute exposure to rotenone induced mild NOX2-dependnet oxidative stress, which impaired the autophagic flux, resulting in cytosolic accumulation of LC3 and p62/stsqm1. We further show that this induction occurs through the PI3K/Akt/mTORC1 signaling pathway. We furthermore show that chronic exposure to rotenone lead to excessive NOX2-dependent ROS generation, increases autophagy, and decreases p62 level via increasedautophagic flux. Taken together, this study is the first mechanistic elucidation of how rotenone can be used to potently target cancer cells without overhauling the entire cellular machinery. VC 2016 IUBMB Life, 68(5): , 2016 Keywords: oxidative stress; human lung cancer; A549 cells Introduction Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, nitric oxide, and highly reactive hydroxyl radicals, play VC 2016 International Union of Biochemistry and Molecular Biology Volume 68, Number 5, May 2016, Pages *Address correspondence to: Hui Tian, Department of Thoracic Surgery, Qilu Hospital, Shandong University, No.107, Wenhua West Road, Jinan , China. Tel: Fax: thqlyy@126.com Grant sponsor: Natural Science Foundation of Shandong Province; Grant number: ZR2013HM089. Received 24 December 2015; Accepted 17 February 2016 DOI /iub.1493 Published online 26 March 2016 in Wiley Online Library (wileyonlinelibrary.com) pivotal role in regulating signaling molecules in cells but, in excess, induces oxidative stress, which leads to the cellular injury (1). Oxidative stress has been reported to be involved in the pathogenesis of a wide spectrum of human diseases, inclusive of but not limited to neurodegenerative diseases, inflammatory diseases, diabetes, aging, cardiovascular disorders, and cancer (1). ROS generated due to oxidative stress can potentiate both cancer initiation and progression by promoting genome instability, cellular proliferation, inhibition of anoikis and apoptosis, promoting invasion, metastasis, and neoangiogenesis (1,2). However, sustained ROS generation can be detrimental to the parent cell and this strategy has been used for chemotherapeutic targeting of the ROS generation machinery. Failure in ROS-scavenging activity of antioxidants pinpoints an urgent need to identify the specific source of ROS generation and characterize the molecular mechanism how oxidative stress leads to the pathogenesis of neurodegenerative diseases. 388 IUBMB Life

2 FIG 1 Activation of Nox2-mediated ROS generation post-rotenone treatment. (A) A549 cells were treated or untreated with 0.5 mm rotenone for 6 h. Some cells were preincubated with 5 mm gp91 ds for 1 h before the 6 h of rotenone treatment. For control experiments, some cells were treated with 5 mm gp91 ds tat for 7 h and hydrogen peroxide (H 2 O 2 ) for 15 min. ROS generation was measured by monitoring DCF-DA fluorescence intensity. (B) Bar diagram indicates quantitative analysis of ROS generation. Results are represented as means 6 standard deviation (SD). *P and #P <0.01 versus all groups. $P < 0.05 among groups as indicated. Up-regulation of NADPH oxidase (PHOX), a major superoxideproducing enzyme complex, has recently been associated with cancer pathogenesis (1,2). Upon stimulation, the cytosolic subunits of PHOX (p47phox, p67phox, p40phox, and the small Rho GTPase, Rac1, or Rac2) translocate to the membrane and binds with membrane-bound subunits p22phox and NOX2 (gp91phox) to assemble the active NADPH oxidase enzyme complex(nox2 complex) that catalyzes the reduction of oxygen to superoxide (3). Recent studies have elucidated that NOX2 is up-regulated in different human cancers (4 6). However, the molecular mechanism of Nox2-dependent inflammatory responses in carcinogenesis is still unknown. Rotenone, which is an inhibitor of the mitochondrial electron transport chain complex I, results in the activation of NOX2 (7) and the generation of ROS (3). Rotenone has been shown to display anticancer activity through the induction of apoptosis in various cancer cells, including the human lung cancer A549 cells (8). In A549 cells, rotenone was shown to induce apoptosis through ROS generation and Bcl-XL/p53- mediated amplification mechanisms (8). However, the mechanistic link between rotenone-dependent activation of NOX2 and cancer pathogenesis is still not clear, which was the primary objective of the current study. Materials and Methods Cell Lines and Treatment Conditions A549 cells were obtained from the ATCC (Manassas, VA) and cultured in DMEM (Life Technologies, Carlsbad, CA) containing 10% FBS. Cells were treated with rotenone (Sigma Aldrich, Beijing, China) at indicated doses and time frames. Where indicated, cells were pretreated with 5 mm of gp91 ds tat (Anaspec, Fremont, CA), an inhibitor of NADPH oxidase assembly (9), or treated with hydrogen peroxide (H 2 O 2 ) (Sigma Aldrich) or Bafilomycin A, which prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes, at the indicated doses and time frame. Cell Lysis and Western Blot Cells were lysed in buffer containing 25 mm Tris-HCl ph 7.4, 150 mm NaCl, 1 mm EDTA, 1% NP-40, and 5% glycerol containing complete, Mini protease inhibitor cocktail (Roche Diagnostics, Beijing, China). Ten micrograms of whole cell lysate was resolved on a NuPAGE 4-20% gel (Life Technologies), transferred to an Immobilon PVDF membrane (Millipore, Billerica, MA), and probed with indicated antibodies (LC3 Novus Biologicals; p62//sqstm1, total Akt, and P-Akt (ser473)). All HU et al. 389

3 IUBMB LIFE FIG 2 blots were subsequently stripped, and reprobed for GAPDH to confirm equal loading. The blots were imaged using ECL Plus western blotting substrate (Pierce, Rockford, IL) and HyBlot CL autoradiography film (Denville Scientific, Metuchen, NJ). Detection of ROS Production ROS production was detected using the Total ROS/Superoxide detection kit (Enzo LifeSciences, Boppard, Shanghai, China). Immunofluorescence Immunofluorescence detection of LC3 was done as described previously (10). Statistical Analysis Data are presented as mean 6 standard deviation (SD) except otherwise stated. When two groups were compared, the Student s t-test was used unless otherwise indicated and a P value <0.05 was considered significant. Results Nox2-dependent chronic activation of oxidative stress by rotenone treatment causes an increase in LC3 expression level. Immunofluorescence analysis of LC3 expression in A549 cells treated or untreated with 500 nm rotenone for 6 h. Some cells were preincubated with 5 mm gp91 ds tat for 1 h before the 6 h of rotenone treatment. Blue color DAPI staining represents nucleus. Scale bar, 40 mm. Mild Activation of NOX2 Impairs Autophagic Flux in a PI3K-Akt-mTORC1-Dependent Pathway To understand the role of acute activation of Nox2-dependnet oxidative stress on autophagy, we treated A549 cells with 500 nm rotenone for 6 h. We found that rotenone treated cells showed an increase in ROS generation compared with untreated cells as evidenced by an increase in DCF-DA fluorescence, which was significantly suppressed in the cells preincubated with the NOX2-specific inhibitor gp91 ds tat (Figs. 1A and 1B). ROS generation post-h 2 O 2 treatment was used as a positive control (Figs. 1A and 1B). Next, we tested the effect of NOX2-dependent acute activation of oxidative stress on the autophagy marker, LC3 s expression. Immunofluorescence analysis showed an increase in LC3 expression levels compared with untreated cells (Fig. 2), which were partially inhibited upon inhibition of Nox2 activity with gp91 ds tat (Fig. 2). NOX2-dependnet increase in LC3 level could possibly be either by increase in expression level due to activation of autophagic stimuli or by impairment in autophagic flux, resulting in accumulation of these proteins. We did not find any significant effect of rotenone on transcript level of lysosomalassociated membrane protein 1 (LAMP1) or its transcriptional regulator, transcription factor EB (TFEB) (data not shown) (11). We thus tested autophagic flux by monitoring p62/ SQSTM1 level and using bafilomycin-a as a negative regulator of autophagic flux (12). Increase in p62 level upon rotenone treatment and suppression of this increment by preincubation with gp91 ds indicated impairment of autophagic flux by Nox2-activation (Fig. 3A). Failure to increase in LC3II, and p62 level by bafilomycin-a in rotenone treated cells compared with bafilomycin-a alone further confirmed that increase in LC3 levels was not due to the increase in expression, instead due to the accumulation caused by impairment in autophagic flux (Fig. 3A). Previous studies have established the role of NOX2- dependnet ROS generation in activation of PI3K/Akt pathway (1,2) and the role of rotenone-dependent oxidative stress on activation of Akt (13). We investigated whether rotenonemediated activation of NOX2-complex induces activation of Akt and thereby leads to the defective autophagy by activating mtorc1 signaling. We observed that rotenone treated cells showed a significant increase in PI3K (data not shown) and Akt phosphorylation (Fig. 3B) compared with untreated cells, which would suggest subsequent downstream activation of mtorc1. Of note, pretreatment with gp91 ds did not inhibit Akt phosphorylation mediated by rotenone (Fig. 3B). Cumulatively, the data indicated that acute activation of NOX2- dependent oxidative stress regulates a PI3K-Akt-mTORdependent pathway, which leads to the impairment of autophagic flux. Chronic-Exposure Increases NOX2-Dependent ROS Generation We then investigated whether chronic exposure of rotenone (500 nm for 24 h) further increases NOX2-dependnet oxidative stress and worsen the autophagic machinery. We found that prolonged activation of NOX2-activity further increases ROS generation compared with shorter rotenone-exposure (Figs. 1A and 4A). Inhibition of Nox2-activity by gp91 ds dramatically 390 Rotenone Impairs Autophagy in Human Cancer

4 FIG 3 Chronic activation of Nox2-mediated ROS generation impairs autophagic flux, potentially in a mtor-dependent fashion. (A) A549 cells were treated or untreated with 500 nm rotenone for 6 h. Some cells were preincubated with 5 mm gp91 ds tat or 1 h before the 6 h of rotenone treatment. Bafilomycin A was added to the cells 4 h before the end of each treatment or no treatment. LC3 and p62 levels were analyzed by immunoblotting. GAPDH served as a loading control. (B) Lysates obtained from A549 cells treated under similar condition as in (A) were probed for P-Akt and total Akt, as indicated. GAPDH served as a loading control. FIG 4 Chronic treatment with rotenone increases Nox2-dependent ROS generation and increases mtor-dependent autophagy. (A) A549 cells were treated or untreated with 500 nm rotenone for 24 h. Some cells were preincubated with 5 mm gp91 ds for 1 h before the 24 h of rotenone treatment. For control experiments, some cells were treated with 5 mm gp91 ds for 25 h and hydrogen peroxide (H 2 O 2 ) for 15 min. ROS generation was measured by monitoring DCF-DA fluorescence intensity. (B) Proteins were extracted from the A549 cells treated as described in (A) and LC3 levels were analyzed by immunoblotting. GAPDH served as a loading control. (C, D) To determine the autophagic flux, cells were treated with gp91 ds and rotenone as described in (A). Bafilomycin A was added to the cells 4 h before the end of each treatment or no treatment. LC3 and p62 levels were analyzed by immunoblotting. GAPDH served as a loading control. HU et al. 391

5 IUBMB LIFE decreased the rotenone-dependent increase in ROS generation (Fig. 4A). Surprisingly, in this condition of NOX2-activation we found a significant increase in LC3II level, but no changes in LC3I level (Fig. 4B). Inhibition of NOX2-activity significantly rescued the level LC3II (Fig. 4B). To determine whether the observed increase in LC3II level was due to an increase in lipidated form of LC3 production or accumulation of LC3II, we monitored autophagic flux by using p62 (Fig. 4C). We observed a significant decrease in p62 level upon prolonged NOX2- activation with low dose of rotenone compared with untreated cells, which was almost completely rescued upon NOX2- inhibition (Fig. 4C). Four hours of post-treatment with bafilomycin-a in rotenone-treated cells showed a decrease in LC3II and p62 levels compared with bafilomycin-a alone treated cell (Fig. 4C). This indicated that the rate of autophagic flux overwhelmed the rate of autophagosome maturation upon rotenone treatment, thereby post-treatment with bafilomycin A in rotenone treated cells failed to restore the level of LC3II and p62 similar to the cells treated with bafilomycin A alone. However, cells preincubated with NOX2-inhibitor before rotenone treatment showed a significant increase in LC3 level upon bafilomycin-a treatment compared with the cells without NOX2-inhibitor (Fig. 4D). These results cumulatively indicated an increase in autophagic flux upon prolonged rotenone treatment, which could be partially prevented upon inhibition of NOX2-dependent oxidative stress. Discussion In this study, we have determined that oxidative stress, caused by acute rotenone-dependent activation of NOX2-complex, can suppress autophagic flux and promote accumulation of autophagic vacuoles, resulting in accumulation of protein aggregates and cancer cell death. Our results show that chronic activation of NOX2-complex with similar dose of rotenone further increases ROS generation, triggers PI3K-Akt-mTORC1- dependent autophagy signaling pathway and significantly increases autophagic flux. Our data indicates that induction of autophagy signaling pathways by prolonged activation of NOX2-complex may be due to the compensatory mechanism to protect cancer cells against sudden onset of protein aggregation at early stage of rotenone treatment, and highlights the need for a chemotherapeutic regimen with sustained administration at a suboptimal dose for more efficacious outcome. Our findings provide the first demonstration of the molecular mechanistic link between NOX2-dependent oxidative stress and impairment of autophagic flux following treatment of cancer cells with rotenone. NOX2-dependnet increase in LC3 level could possibly be either by increase in expression level due to activation of autophagic stimuli or by impairment in autophagic flux, resulting in accumulation of these proteins. We did not find any significant effect of rotenone on transcript level of lysosomal associated membrane protein 1 (LAMP1) or its transcriptional regulator, transcription factor EB (TFEB), thus ruling out autophagic stimuli. We thus tested autophagic flux by monitoring p62/sqstm1 level. Bafilomycin-A was used as a negative regulator of autophagic flux in these experiments. Rapamycin induces LC3I and LC3II conjugation, but LC3II is also a target of autophagic degradation and normally will disappear if autophagolysosomal degradation is not blocked. Bafilomycin-A prevents autophagolysosomal degradation and hence induces accumulation of LC3II by preventing turn-over of LC3II. So, (a) increase in p62 level upon rotenone treatment and suppression of this increment by preincubation with gp91 ds indicated impairment of autophagic flux by Nox2-activation, and (b) failure to increase in LC3II, and p62 level by bafilomycin-a in rotenone treated cells compared with bafilomycin-a alone confirmed that increase in LC3 levels was not due to the increase in expression, instead due to the accumulation caused by impairment in autophagic flux. Chronic exposure of rotenone (500 nm for 24 h) leads to increase detection of LC3 II. Evaluation of p62 levels and comparison of effect of bafilomycin-a alone or in combination with rotenone indicates that the rate of autophagic flux overwhelmed the rate of autophagosome maturation upon rotenone treatment. Thus post-treatment with bafilomycin A in rotenone treated cells failed to restore the level of LC3II and p62 similar to the cells treated with bafilomycin A alone. Taken together, our study highlights pending further investigation in in vivo models, that proper modulation of NADPH oxidase through optimal, but sustained rotenone treatment might hold a potential therapeutic benefit for treatment of cancer and other diseases associated with accumulation of toxic cellular constituents. Acknowledgement This study was support by Natural Science Foundation of Shandong Province [ZR2013HM089]. References [1] Sosa, V., Moline, T., Somoza, R., Paciucci, R., Kondoh, H., et al. (2013) Oxidative stress and cancer: an overview. Ageing Res. Rev. 12, [2] Visconti, R. and Grieco, D. (2009) New insights on oxidative stress in cancer. Curr. Opin. Drug Discov. Dev. 12, [3] Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., et al. (2003) Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J. Biol. Chem. 278, [4] Li, S. Z., Hu, Y. Y., Zhao, J., Zhao, Y. B., Sun, J. D., et al. 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