A wide array of conventional and experimental chemotherapeutic agents has been shown to stimulate oxidative stress along with apoptosis in cancer cells (Pelicano et al., 2004). The damage caused by lysosomes during oxidative stress has been the primary focus of interest for decades. Lysosomes have been classically considered one of the main targets of the reactive oxygen species (ROS). Nevertheless, recent data have shown that molecules relative to oxidative stress, for example ROS, play role as signaling molecules in the activation of autophagy (Scherz-Shouval et al., 2007; Essick and Sam, 2010). A growing body of evidence shows that autophagy is involved in adaptation to stress and help maintaining cellular homeostasis in response to oxidative stress (Azad et al., 2009; Rouschop et al., 2009). Whereas in other cellular settings, autophagy can trigger a form of cell death known as Type II programmed cell death (Mizushima, 2007; Ye et al., 2012). The functional role of autophagy is complex, and the exact role of autophagy in oxidative stress-induced tumor cell death remains to be elucidated.
Autophagy is a strictly regulated lysosomal pathway that degrades cytoplasmic material and organelles. It involves the formation of double-layered membrane structures called autophagosomes. The fusion of the outer membrane of the autophagosome and the lysosome leads to the formation of autolysosome, where the contents are degraded and recycled. This dynamic process is termed the “autophagic flux” (Eskelinen and Saftig, 2009). Autophagy is upregulated in response to various stress conditions such as, nutrient deprivation, growth factor withdrawal, and anticancer treatment. More importantly, autophagy is essential in maintaining homeostasis, which requires protein degradation for energy needs, by removing damaged substrates for recycling (Mizushima et al., 2010).
Oxidative stress can also lead to protein misfolding. Although recent evidence has shown that endoplasmic reticulum (ER) stress may trigger ROS production (Malhotra et al., 2008) and redox deviation (Merksamer et al., 2008) in the ER, another possibility is that ROS may cause ER stress through generation and accumulation of oxidized proteins (van der Vlies et al., 2003; Malhotra and Kaufman, 2007). When misfolded proteins accumulate in the ER, the resulting stress activates the unfolded protein response (UPR) to induce the expression of chaperons and proteins involved in the recovery process. Severe ER stress can cause cell death, usually by activating intrinsic apoptosis (Szegezdi et al., 2006). Moreover, to clear accumulated terminally misfolded protein aggregates in the ER lumen that cannot be degraded by the proteasome, the UPR may upregulate the autophagy machinery (Ogata et al., 2006; Ding et al., 2007).
Here, by treating Hela cells with menadione, we have tested the hypothesis that autophagy activation can alleviate misfolded proteins, thus enabling the cell to avoid ER stress while downregulating mitochondrial pathway-induced apoptosis. We found that menadione could induce autophagy. The lysosomal inhibitor, NH4Cl inhibited the autophagic flux and increased the level of misfolded proteins, which elevated ER stress and resulted in a higher apoptotic rate of these cells when they were treated with menadione. These findings thus may provide useful insight into the role of lysosome through autophagy during oxidative stress and also in the design of effective cancer therapy where autophagy inhibitors are used.
MATERIALS AND METHODS
Human Hela cells were purchased from the American Type Culture Collection (Rockville, MD). Cells were cultured at 37°C with 5% CO2 in a humidified atmosphere, in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin (all from Gibco BRL).
The viability of the Hela cells was determined by MTT assays. Briefly, cells were plated for 24 hr in a 96-well plate at a density of 1 × 104 cells per well in 200 μL of complete medium containing different concentrations of menadione 0, 15, 30, and 60 μM (10-mM stock solution, Sigma) for 6, 12, and 24 hr, or treated with 30 μM menadione and/or the lysosomal inhibitor 10 mM NH4Cl (Sigma) 1 hr before menadione. Each treatment was repeated in three wells. The cells were incubated for 20 hr at 37°C in a humidified chamber. MTT reagent (20 μL, 5 mg/mL in PBS, Sigma) was added to each well and incubated for 4 hr. The microtiter plate containing the cells was centrifuged at 1,800 rpm for 5 min at 4°C. The MTT solution was removed from the wells by aspiration and the formazan crystals were dissolved in 150 μL DMSO (Beijing Chemical Industry, China). Absorbance was recorded at 570 nm wavelength using a Microplate Reader (Bio-Tek Instruments). Cell viability was calculated as follows: Cell viability (100%) = absorbance of experiments group/absorbance of control group × 100%.
For protein analysis, the cells were harvested 12 hr following different treatment as described above, washed with cold PBS, then incubated in ice-cold RIPA buffer. Cell lysates were sonicated for 30 sec on ice, and then lysed at 4°C for 60 min. The cell lysates were centrifuged for 30 min at 12,000g. Protein concentrations in the supernatants were determined by the BCA reagent (Pierce, IL). Protein concentrations were measured using BIO-RAD kit (Pierce). For western blot analysis, lysate proteins (30–50 μg) were resolved over 12, 15% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose transfer membranes (Whatman, UK). The membranes were blocked with 5% nonfat dry milk in buffer (10 mM Tris–HCl (pH 7.6), 100 mM NaCl, and 0.1% Tween 20) for 1 hr at room temperature and then incubated with the desired primary antibody (anti-LC3, anti-caspase-3, anti-p62, anti-Ubiquitin, and anti-GRP78 from Santa Cruz Biotechnology, CA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Thermo, Waltham, MA) at 1:2,000 dilution for 1 hr at room temperature. The immunoreactive bands were visualized by the DAB (Sigma, St Louis, MO) coloration method. The representative bands of proteins were measured with Quantity one and analyzed as described previously. The levels of proteins were normalized to that of β-actin, and the ratios to β-actin were presented as a mean ± SD from three independent experiments.
Immunofluorescence Staining and Confocal Laser Microscopy
Autophagy is characterized by development of autophagic vacuoles. Monodansylcadaverine (MDC; Invitrogen, Carlsbad, CA) has been proposed as a tracer for autophagic vacuoles (Biederbick et al., 1995). Hela cells were cultured on coverslips overnight, then treated with different doses of stimuli for 12 hr as described above, and rinsed with PBS. They were then stained with 50 μM MDC at 37°C for 1 hr. After incubation, the cells were fixed for 15 min with ice-cold 4% paraformaldehyde at 4°C, washed twice with PBS, and followed by excitation at 420 nm with the confocal laser microscope. Acridine orange (AO; 5 μg/mL, Sigma) excited by blue light at 405 nm for 15 min at 37°C and then analyzed by fluorescence microscopy. When excited by blue light, AO shows red fluorescence at high (lysosomal) concentrations and green fluorescence at low (nuclear and cytosolic) concentrations (Erdal et al., 2005; Boya and Kroemer, 2008; Xu et al., 2011). For the assessment of ROS in situ, cells grown on cover slips were incubated with 2,7-dichlorodihydro-fluorescein diacetate (DCFH-DA, 10 μM, Sigma) staining 20 min at 37°C (Huang et al., 2008). The fluorescence emitted was measured at 525 nm. Cells were treated for 3 hr with various compounds as indicated, fixed for 20 min with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 for 5 min. After blocking with BSA, cells were incubated with the following primary antibodies (Santa Cruz; 1:100 dilution). Cells were incubated with primary antibodies Cathepsin D, LC3, LAMP1 overnight at 4°C and FITC/Texas-conjugated secondary antibodies (Santa Cruz; 1:400 dilution) for 1 hr. The cells were examined by confocal fluorescence microscopy.
Cells were fixed for 30 min with ice-cold 3% glutaraldehyde in 0.1 M cacodylate buffer, embedded in Epon, and processed for transmission electron microscopy by standard procedures. Representative areas were chosen for ultrathin sectioning and examined with a transmission electron microscope.
Flow Cytometry Assays
We used Rhodamine 123 (Rh123, 10 μM, Sigma) to quantify the mitochondrial membrane potential (ΔΨm) as described previously (Ferlini and Scambia, 2007). The samples were then analyzed by a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
Data are representative of at least three independent experiments each in triplicate determination. Statistical analysis of the data was performed using one-way ANOVA. The Tukey post-hoc test was used to determine the significance for all pairwise comparisons of interest. P values of less than 0.05 were considered to represent a statistically significant difference.
Menadione Treatment Activates Autophagy in Hela Cells
Based on MTT assay and previous studies, we treated Hela cells with 30 μM menadione for 6, 12, and 24 hr, and the cell viability was found to be 87%, 71%, and 53%, respectively (Fig. 1A). Using western blotting analysis, we found that compared with controls, the expression levels of microtubule-associated protein light chain 3-II (LC3-II) was increased in menadione-treated cells for 6 and 12 hr, especially for 12 hr (Fig. 1B,C). Under the phase microscope, we found many vacuoles in Hela cells treated with menadione combined with NH4Cl (Fig. 1D) and by western blotting analysis, we found that the expression of LC3-II was upregulated (Fig. 1E,F). These results indicate that menadione inhibited the proliferation of Hela cells, and can lead to autophagy activation.
Inhibition of Autolysosomes Formation in Hela Cells Treated with Menadione and NH4Cl with Lysosomal Membrane Permeabilization
Using electron microscopy, we observed the ultrastructure changes in Hela cells treated with menadione alone and in combination with NH4Cl. Compared with cells treated only with menadione, cells treated with menadione and NH4Cl had enlarged autophagosomes containing a large number of undegraded materials (Fig. 2A). Consistent with this, compared with menadione alone, we observed the appearance of enlarged vacuoles brightly stained by MDC in Hela cells treated with menadione and NH4Cl (Fig. 2B).
To directly address the fusion of autophagosomes with lysosomes, we labeled autophagosomes and lysosomes using antibodies against LC3 and lysosomal-associated membrane protein 1 (LAMP1), respectively. Using confocal microscopy, we found that LC3 colocalized with LAMP1 after menadione treatment, whereas colocalization of LC3 with LAMP1 was significantly reduced in Hela cells treated with menadione and NH4Cl (Fig. 2C). Using western blotting analysis, we observed that the level of p62 was reduced in Hela cells with menadione treatment alone, whereas cells treated with menadione combined with NH4Cl showed high expression of p62 (Fig. 2D,E).
Using AO staining, we found that combination treatment with menadione and NH4Cl caused a decline in the red staining of lysosomes and increase in green staining of the cytoplasm, whereas no such changes were observed after treatment with menadione alone or NH4Cl alone (Fig. 3A). As shown in Fig. 3B, cathepsin D-specific immunostaining revealed punctuate cytoplasmic structures in controls. Interestingly, these studies also showed diffuse staining throughout the entire cell, indicating release from the lysosome to the cytosol, after treatment with menadione and NH4Cl, although menadione alone or NH4Cl alone did not have this effect (Fig. 3B). These results demonstrated that inhibition of lysosome can block the formation of autolysosomes in Hela cells treated with menadione, and is associated with lysosomal membrane permeabilization.
Inhibition of Autolysosomes Formation Enhances Menadione-Induced Apoptosis
We measured the intracellular levels of ROS using a fluorescent probe, 2′,7′-dichlorofluorescin diacetate (DCF-DA). We observed that ROS level was increased in menadione-treated cells, and significantly enhanced in Hela cells treated with menadione and NH4Cl (Fig. 4A). MTT assays indicated that, compared with menadione treatment, the viability of Hela cells treated with menadione and NH4Cl was decreased (Fig. 4B). Rhodamine123 revealed that, compared with menadione treatment, menadione and NH4Cl induced a drop in Rhodamine123 staining (Fig. 4C). The latter was accompanied by caspase-3 activation, as revealed by the production of 17-kDa active caspase fragments (Fig. 4D,E). These results demonstrated that inhibition of lysosome by NH4Cl resulted in accumulation of ROS, and enhanced menadione-induced apoptosis of Hela cells through the mitochondrial pathway.
Inhibition of Autolysosomes Formation Increases Ubiquitinated Proteins and ER Stress
Using western blotting analysis, we found that ubiquitinated proteins accumulated after menadione treatment. Compared with menadione alone, cells treated with menadione combined with NH4Cl enhanced ubiquitinated proteins accumulation (Fig. 5A,B). Electron microscopy revealed that, compared with menadione alone, cells treated with menadione and NH4Cl had a significant degree of cellular vacuolization (Fig. 2C, arrowheads). In addition, levels of GRP78 increased in cells treated with menadione and NH4Cl (Fig. 5C,D). These results demonstrated that menadione could induce ER stress. When autolysosomes formation was blocked, misfolded proteins accumulated and ER stress enhanced.
Many anticancer drugs have been found to induce formation of ROS, and several studies have demonstrated that ROS formation is essential for anticancer drugs cytotoxicity (Scherz-Shouval and Elazar, 2007; Ayyanathan et al., 2012). For example, As2O3 and evodiamine induce ROS accumulation in U251 and Hela cells, respectively, and are capable of inducing apoptosis that involves the mitochondria (Engel and Evens, 2006; Yang et al., 2008). ROS have been identified as signaling molecules stimulating the autophagy process. Although As2O3 can induce autophagic cell death in U251 cells, evodiamine triggered autophagy that mediates cell survival effects. However, the exact role of autophagy in oxidative stress-induced cell death is unclear. Zhang et al. have reported that H2O2 triggered apoptosis and autophagy in U251 in parallel with an increase in the protein levels of LC3-II, and with accumulation of autophagic vacuoles in the cytoplasm, while pretreatment with autophagic inhibitor 3-MA increased apoptosis (Zhang et al., 2009). Lysosomotropic reagent NH4Cl potentiated the neurotoxin's cytotoxicity in K562 cells (Yan et al., 2006). Recent data have shown that lysosomal inhibitors, by increasing the lysosomal pH inside the lysosome, inhibited the autophagosome-lysosome fusion resulting in reduced autophagic flux (Kawai et al., 2007). Our results indicate that autophagy occurred with the increase in LC3-II and accumulation of autophagosomes after menadione treatment. When pretreated with NH4Cl, menadione-mediated cell viability was decreased and the mitochondrial pathway apoptosis was increased.
Autophagy is a dynamic process during which isolation membranes package substrates to form autophagosomes that are fused with lysosomes to form autolysosomes for degradation. The degradation of autophagic substrates inside the lysosomes has been reported in different diseases and during cell stress (Boland et al., 2008; Ambegaokar and Jackson, 2012; Fouillet et al., 2012). Recent studies have indicated that disrupting the fusion of autophagosomes with lysosomes caused autophagic vacuoles (Avs) accumulation in neuronal cells and impaired clearance of Avs resulted in neurodegenerative disorders. Kawai et al. showed that when the pH in acidic compartments was increased by bafilomycin A1, NH4Cl, and chroloquine in CHO cells, no colocalization of lysosomes and mitochondria was observed (Kawai et al., 2007). Inhibiting fusion between autophagosomes and lysosomes by bafilomycin A1 and chroloquine triggered apoptosis, which depend on mitochondrial outer membrane permeabilization and subsequent caspase activation in tumor cells under nutrient-free (Boya et al., 2005) and anticancer drug (Shingu et al., 2009; Lotze et al., 2012) conditions. LAMP1 is known to be one of the major protein components of the lysosomal membrane. It is reported that LAMP1, a membrane protein expressed in lysosomes induced during macrophagy (Biederbick et al., 1999; Dennemarker et al., 2010), shows expression during the late autophagosome and autolysosome period and was conjunctively used with LC3 immunostaining to improve the accuracy of detecting the fusion of autophagosomes with lysosomes (Natsumeda et al., 2011). In the present study, colocalization of LAMP1 and LC3 was decreased in Hela cells treated with menadione and NH4Cl, suggesting a failure to form autolysosomes. We demonstrated that when menadione was combined with NH4Cl, lysosomal permeability occurred, and the protein levels of LC3-II and p62, which used to monitor autophagic flux was increased (Klionsky et al., 2008; Mizushima et al., 2010). Simultaneously, accumulation of Avs and ROS were also increased. These data indicate that inhibition of fusion of lysosome with Avs containing damaged components and reduced autophagic flux may be resulted from lysosomal permeability by NH4Cl in menadione-treated Hela cells, which enhances oxidative stress-mediated cytotoxicity through accumulation of ROS.
Our previous studies found that autophagy efficiently transports cisplatin-induced misfolded proteins for degradation in cisplatin-resistant HOCCs and Hela cells, allowing cells to avoid ER stress mediated apoptosis, and thus maintaining cell homeostasis and survival (Xu et al., 2012; Yu et al., 2011). In addition, some researchers suggested that ER stress and mitochondrial dysfunction might cooperatively regulate apoptotic-signaling cascades (Lee et al., 2010; Zhao et al., 2010; Zhong et al., 2012). In the present study, we found that menadione could lead to the accumulation of ubiquitinated proteins, increase GRP78 protein expression, and expand the ER lumen, which reflects ER stress. When autophagy was blocked with NH4Cl, ubiquitinated proteins accumulation, GRP78 protein expression and the ER lumen dilation were enhanced in Hela cells treated with menadione. Simultaneously, the mitochondrial pathway of apoptosis was also increased. These data indicate that impaired autophagy enhanced oxidative stress-induced cytotoxicity through exacerbating ER stress. Activation of autophagy clears ubiquitinated proteins, which alleviates ER stress and subsequently the mitochondrial pathway of apoptosis.
In summary, we found that menadione treatment induces apoptosis, autophagy, and ER stress in human cervical cancer Hela cells. Lysosomal system efficiently eliminates menadione-induced misfolded proteins for degradation through autophagy, allowing cells to avoid ER stress and the mitochondrial pathway of apoptosis. In addition, reduced autophagic flux by NH4Cl increased the sensitivity of Hela cells to menadione. This evidence indicates that the degradation of misfolded proteins by autophagy allows Hela cells to effectively avoid ER stress and the mitochondrial pathway of apoptosis, thus protecting cells from oxidative stress-mediated apoptosis and contributing to survival. Lysosome inhibition or autophagy inhibition could be therapeutically targeted for improvement of menadione efficacy.