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Protective Effects of a Synthesized Butyrolactone Derivative against Chloroquine-Induced Autophagic Vesicle Accumulation and the Disturbance of Mitochondrial Membrane Potential and Na+,K+-ATPase Activity in Vascular Endothelial Cells Bin Huang,†,† Ning Meng,†,† BaoXiang Zhao,*,‡ Jing Zhao,†,† Yun Zhang,† ShangLi Zhang,†,† and JunYing Miao*,†,† Institute of DeVelopmental Biology, School of Life Science, Shandong UniVersity, Jinan 250100, China, Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong UniVersity, Jinan 250100, China, and The Key Laboratory of CardioVascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong UniVersity Qilu Hospital, Jinan 250100, China ReceiVed July 31, 2008
We previously found a butyrolactone derivative, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran2(3H)-one (3BDO), could inhibit vascular endothelial cell (VEC) apoptosis and senescence induced by a deprivation of serum and FGF-2. In this study, we aimed to investigate its actions in VEC autophagy induced by chloroquine (CQ). The measurement on the volume of acidic compartments (VAC) and autophagy analysis by acridine orange (AO) staining and microtubule-associated protein 1 light chain 3 (MAP1LC3) process revealed that 3BDO was an effective inhibitor of autophagic vesicle accumulation (vacuolation) induced by CQ in VECs. 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was used for mitochondrial membrane potential (MMP) measurement. The results showed that CQ elevated MMP significantly and that 3BDO could significantly inhibit CQ-induced MMP increase. Na+,K+-ATPase activity assay showed that CQ inhibited this enzyme activity significantly and that 3BDO attenuated the alteration of Na+,K+-ATPase activity caused by CQ. We concluded that 3BDO was a promising inhibitor of CQ-induced accumulation of autophagic vesicles in VECs and could weaken the alterations of MMP and Na+,K+-ATPase activity induced by CQ. The data indicate that 3BDO will be a potential tool for investigating the mechanism of autophagy. Introduction Under normal conditions, autophagy is a process for the turnover and recycling of long-lived macromolecules and organelles via the lysosomal degradative pathway, involved in maintaining cellular homeostasis, differentiation, and tissue remodeling. However, under pathophysiological conditions, autophagy may serve a protective role or contribute to cell damage. For example, nutrient depletion classically induces autophagy in order to provide amino acids for the synthesis of essential proteins, thus prolonging cell survival (1). Moreover, autophagy may counteract apoptotic stimuli (2, 3). Interestingly, autophagy shares common molecular regulators with apoptosis (4), indicating a probable cross-talk between the two pathways. We previously found that a butyrolactone derivative, 3-benzyl5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO1), * Corresponding author. Tel: + 86 531 88364929. Fax: + 86 531 88565610. E-mail:
[email protected] (J.M.);
[email protected] (B.Z.). † Institute of Developmental Biology, Shandong University. ‡ Institute of Organic Chemistry, Shandong University. † Shandong University Qilu Hospital. 1 Abbreviations: 3BDO, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one; VEC, vascular endothelial cell; CQ, chloroquine; VAC, volume of acidic compartments; AO, acridine orange; MAP1LC3, microtubule-associated protein 1 light chain 3; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolylcarbocyanine iodide; MMP, mitochondrial membrane potential; LC3-I, light chain 3-I; LC3-II, light chain 3-II; FBS, fetal bovine serum; FGF-2, fibroblast growth factor-2; DMSO, dimethyl sulfoxide; HUVECs, human umbilical vein endothelial cells; PBS, phosphatebuffered saline; OD, optical density; BSA, bovine serum albumin; PVDF, polyvinylidene fluoride; DAB, 3,3-diaminobenzidine tetrahydrochloride.
could inhibit vascular endothelial cell (VEC) apoptosis and senescence induced by a deprivation of serum and FGF-2 (5, 6). Our recent study showed that 3BDO maintained endothelial cell functions through selectively affecting Na+,K+-ATPase activity and mitochondria membrane potential during in vitro angiogenesis (7). Because nutrient depletion and growth-factor deprivation classically induce autophagy, and autophagy is related to angiogenesis (8), we supposed 3BDO might modulate autophagy in VECs. Chloroquine (CQ) is a potent autophagic drug that may lead to cellular degradation of hepatocytes in the liver with the concurrent production of vacuoles (9). Moreover, CQ has the ability to disrupt lysosomal function, inhibiting the last critical step in autophagy, the acid-dependent degradation of autophagosome contents, which results in the accumulation of autophagic vesicles that cannot be cleared (10, 11). To understand whether 3BDO disturbs the process of autophagy in VECs, we investigated the effect of this small molecule on the accumulation of autophagic vesicles induced by CQ. Microtubule-associated protein 1 light chain 3 (MAP1LC3) is the mammalian equivalent of yeast Atg8. It exists in two forms, LC3-I (light chain3-I) and its proteolytic and lipidated derivative LC3-II (light chain 3-II), which are localized in the cytosol or in autophagosomal membranes, respectively. LC3II can thus be used to estimate the abundance of autophagosomes (2). The mitochondrion has been noted as a switch between apoptosis and autophagy (12). This could be due to the loss of mitochondrial membrane potential, which could result from bioenergetic failure due to the absence of nutrients and the
10.1021/tx8002824 CCC: $40.75 2009 American Chemical Society Published on Web 02/09/2009
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impossibility of recruiting endogenous nutrients by means of autophagy-dependent catabolic reactions. Moreover, autophagy leads to the removal of damaged mitochondria. If autophagy is blocked and the damaged mitochondria are not removed, the mitochondria undergoing mitochondrial membrane potential loss can prime apoptosis. Therefore, we investigated the effect of 3BDO on mitochondrial membrane potential in VECs treated with CQ. Chloroquine has been found to inhibit Na+,K+-ATPase activity in vitro and in vivo in the microsomal membranes of different organs (13, 14) in rats, and it could induce vascular endothelial cell apoptosis (15). Moreover, it has been shown that the apoptosis-associated Na+,K+-ATPase inactivation is related to mitochondria membrane potential changes in an ATPand reactive-oxygen-species-dependent manner in neural cells (16). By contrast, whether autophagy is related to the Na+,K+-ATPase activity is largely undefined. Therefore, we further examined the effect of 3BDO on Na+,K+-ATPase activity of VECs treated with CQ.
Experimental Procedures Reagents, Chemicals, and the Preparation of Drugs. Fetal bovine serum (FBS) and M199 medium were obtained from Hycolon Co. (USA). Fibroblast growth factor-2 (FGF-2) was purchased from EssexBio Group (China). Acridine orange (AO) was purchased from Fluka (USA). Rabbit polyclonal MAP1LC3 primary antibody and HRP-conjugated goat anti-rabbit secondary antibody were obtained from Santa Cruz Biotechnology (USA). JC-1 was purchased from Invitrogen (USA). Na+,K+-ATPase detection kit was obtained from Nanjing Jiancheng Biotechnology Institute (China). Chloroquine diphosphate from Sigma (USA) was dissolved in distilled water as a stock solution at a concentration of 100 mM. 3BDO (3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one) was synthesized as described by Sha et al. (17). 3BDO was dissolved in dimethyl sulfoxide (DMSO) as a stock solution at a concentration of 200 mM. The final concentration of DMSO used in the culture medium was below 0.1% (v/v) and did not affect cell viability. Cell Culture. Human umbilical vein endothelial cells (HUVECs) were gained as described previously (18). Cells were grown in M199 medium supplemented with 10% (v/v) FBS and 2 ng/mL FGF-2. All experiments were performed on cells from 10 to 20 passages. Volume of Acidic Compartments (VAC) Assay. The VAC assay was carried out as described previously (19, 20) and was appropriately modified. The 0.5% neutral red stock solution was prepared in 0.9% normal saline and filtered. Staining solutions were prepared before each experiment by diluting the stock solution (1: 10) in 1% PBS (phosphate-buffered saline). Cells had been seeded in 60 mm dishes 24 h before treatment. After treatment, cells were washed twice with PBS and incubated for 4 min with 4 mL of staining solution. Then cells were washed twice with PBS, and the neutral red sample was extracted from cells by adding 3 mL of acidified alcohol (50% alcohol, 1% acetic acid, and 49% water) per dish. The optical density (OD) at 540 nm of samples was determined by using a Cintra 5 UV-vis spectrometer. The OD value of each sample was subtracted from the OD of the dish without cells to yield a net OD. The neutral red uptake readings for each dish would be normalized for total protein. Then cell monolayers were washed once with 1% PBS and lysed with 100 µL of lysis buffer per dish. The total protein concentration was determined with the Coomassie brilliant blue protein assay method by using BSA (bovine serum albumin) as a standard. The value of the VAC for each dish was normalized by dividing the neutral red uptake data by the total protein concentration. Autophagy Detection with Acridine Orange Staining. It has been reported that the volume of the cellular acidic compartment is a marker of autophagy that can be visualized by acridine orange
Huang et al. staining (21, 22). Briefly, the cells were cultured in fresh medium and treated as described, and were stained with 5 µg/mL of AO at room temperature. Then the cells were observed and photographed under an Olympus (Japan) BH-2 fluorescence microscope. Western Blot. The Western blot analysis was performed as described by Georgia Gliki (23). Cells with various treatments were lysed in protein lysis buffer (1% SDS in 25 mM Tris-HCl, pH 7.5, 4 mM EDTA, 100 mM NaCl, 1 mM PMSF, 10 µg/mL leupeptin, and 10 µg/mL soybean trypsin inhibitor). The protein concentration of the cell was determined with the Coomassie brilliant blue protein assay method. Equal amounts of protein in each lane were loaded on a 12% SDS polyacrylamide gel. After separation, the protein was electrophoretically transferred to a PVDF (polyvinylidene fluoride) membrane. The membrane was incubated in TBST (10 mM Tris-HCl, pH 7.6, 150 mM Nacl, and 0.1% Tween 20) containing 5% (w/v) nonfat milk at room temperature for 1 h. Subsequently, the membrane was probed with the primary antibody overnight at 4 °C and then washed three times for 5 min in TBST. The membrane was subsequently incubated with the secondary antibody for 1 h at room temperature and washed with TBST. Then the membrane was soaked in Ni-enhanced 3,3-diaminobenzidine tetrahydrochloride (DAB) solution (18 mL of 0.1 M Ttris-HCl (pH 7.6), 12 mg of DAB, 2 mL of 0.3% CoCl2, 20 µL of 30% H2O2) until the protein strip could be visualized; afterward, the dyeing reaction was ended in double distilled water. Intensity of the immunoreactive bands was quantified using Quantity-One software (Bio-Rad). Mitochondrial Membrane Potential (MMP) Measurement. Mitochondrial membrane potential was estimated by fluorescence of JC-1 aggregates that are formed as a function of inner mitochondrial membrane potential (24, 25). The formation of JC-1 aggregates and their fluorescence responds linearly to an increase in membrane potential (24). After treatment, the cells plated on 24-well plates were incubated with 4 µg/mL JC-1 for 15 min at 37 °C in a humidified incubator. Then the cells were washed 2 times with PBS and subjected to fluorescence (for red fluorescence, excitation, 543 nm; emission, 600 nm; for green fluorescence, excitation, 488 nm; emission, 535 nm) ratio detection. We randomly selected the region of interest (ROI) and then zoomed in the same frames. The relative ratio of red/green fluorescence intensity values was used for data presentation. Na+,K+-ATPase Activity Assay. The cells were trypsinized after washing twice with PBS. Then, the cells were homogenized with ultrasonic (400 W, 12 min) in 3 mL of buffer (20 mM Tris-HCl, pH 7.0, 10 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 0.34 mM sucrose) on ice. The enzyme samples were used for the Na+,K+-ATPase activity assay as described in the instructions of the detection kit. ATPase may hydrolyze ATP and provide both ADP and inorganic phosphate (Pi); therefore, the enzyme activity was determined by measuring the amount of inorganic Pi liberated from ATP during the incubation of the reaction mixture. The optical density was measured at 636 nm (wavelength). The enzyme activity was expressed as U per mg of protein (U/mgprot; 1 U ) 1 µmol Pi/mgprot/h). The experiment was repeated at least four times independently. Statistical Analysis. Data were presented as the means ( SE and analyzed by SPSS software (v11.5, SPSS Inc., Chicago, IL). Differences at P values < 0.05 were considered statistically significant. Mean values were derived from four or five independent experiments.
Results Effects of 3BDO on the Volume of Acidic Compartments (VAC). Previously, we synthesized 11 kinds of butyrolactone derivatives (17). In this study, the effects of these 11 kinds of butyrolactone derivatives on CQ-induced accumulation of autophagic vesicles were observed under a phase contrast microscope. We found that the most effective one was 3-benzyl5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO)
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Figure 1. Chemical structure of 3BDO.
Figure 4. Effects of 3BDO on acidic vacuoles of VECs treated with CQ at 6 h. The cells were stained by AO to show the acidic vacuoles in VECs. (A) Cells cultured in normal condition. (B) Cells treated with 16 µM CQ. (C) Cells treated with 16 µM CQ and 120 µM 3BDO (400 × magnification).
Figure 2. Effects of 3BOD on VAC of VECs treated with CQ at 6 h. The increase of VAC induced by CQ was inhibited by 3BDO in a dose-dependent manner. Control, VAC of the cells cultured in normal condition. CQ, VAC of the cells treated with 16 µM CQ. CQ+3BDO 60, CQ+3BDO 120, CQ+3BDO 180, VAC of the cells treated with 16 µM CQ and 3BDO 60, 120, and 180 µM, respectively. (&&, P < 0.01 vs control group; **, P < 0.01 vs CQ group, n ) 5).
Figure 5. Effects of CQ and 3BDO on the LC3 process of VECs at 6 h. (A) Representative Western blot by which we measured the process of LC3 in control samples or CQ (16 µM), 3BDO (120 µM), CQ+3BDO (16 µM CQ and 120 µM 3BDO)-treated VECs. Equal protein loading was verified by detecting the protein level of β-Actin. (B) The relative quantity of the ratio of LC3-II to LC3-I is depicted as a bar chart. (&&, P < 0.01 vs control group; **, P < 0.01 vs CQ group, n ) 4).
Figure 3. Inhibitory effect of 3BDO on CQ-induced accumulation of autophagic vesicles in VECs at 6, 12, and 24 h, respectively. (A,D,G) Cells treated with 16 µM CQ. (B,E,H) Cells treated with 16 µM CQ and 120 µM 3BDO. (C,F,I) Cells treated with 16 µM CQ and 180 µM 3BDO (400 × magnification).
(Figure 1). Neutral Red was traditionally used to stain lysosomes and quantify the VAC in cells (19, 20). The results showed that the VAC of CQ-treated groups was dramatically increased and that 3BDO (from 60 µM to 180 µM) could inhibit this change in VECs (Figure 2). On the basis of these results, we chose 16 µM CQ and 120 µM 3BDO as the most appropriate concentrations for the following study. Effect of the Butyrolactone Derivative on CQ-Induced Accumulation of Autophagic Vesicles in Morphology. When VECs were exposed to CQ (from 4 µM to 32 µM) for 24 h, autophagic vesicles were accumulated gradually as the time continued, and a representative concentration of CQ was 16 µM (Figure 3A, D, and G). When the cells were treated with 16 µM CQ and 3BDO (from 30 µM to 180 µM), the accumulation was dramatically inhibited, and the most effective concentration
of 3BDO for morphological observation under a phase contrast microscope was 120 µM (Figure 3). Effects of 3BDO on the Accumulation of Autophagic Vesicles Induced by CQ. As a marker of autophagy, the volume of the cellular acidic compartment could be visualized by acridine orange staining (21, 22). As shown in Figure 4B, after treatment with CQ (16 µM) for 6 h, many acidic vacuoles appeared in the cytoplasm. Compared to the CQ group, the amount of acidic vacuoles obviously decreased when the cells were treated with both CQ and 3BDO (Figure 4C). Effects of 3BDO on the CQ-Induced LC3 Process. Microtubule-associated protein 1 light chain 3 (MAP1LC3), a mammalian homologue of the yeast autophagic protein Atg8, has been used as an autophagosomal marker (2). To determine the effects of chloroquine and 3BDO on autophagosomes, the processing of LC3 was assessed via Western blot. The increase in LC3-II induced by CQ was significantly inhibited by 3BDO, which was reflected by the ratio of autophagosome-bound LC3II to that of cytosolic LC3-I (Figure 5). Effects of CQ and 3BDO on Mitochondrial Membrane Potential (MMP). The mitochondrial membrane potential (MMP) is an important mediator and monitor of key cellular processes. It is a highly sensitive indicator of the energetic state of mitochondria and the health of cells (26). Therefore, we investigated the effects of CQ and 3BDO on MMP in VECs. As shown in Figure 6, when VECs were treated with CQ for 6, 12, and 24 h, the MMP was much higher than that in the control
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Figure 7. Effects of CQ and 3BDO on Na+,K+-ATPase activity in VECs at 6 h. Control, the enzyme activity of the cells cultured in normal condition. 3BDO, the enzyme activity of the cells treated with 120 µM 3BDO. CQ, the enzyme activity of the cells treated with 16 µM CQ. CQ+3BDO, the enzyme activity of the cells treated with 16 µM CQ and 120 µM 3BDO. (&, P < 0.05 vs control group; &&, P < 0.01 vs control group; *, P < 0.05 vs CQ group, n ) 5).
Figure 6. Effects of CQ and 3BDO on mitochondrial membrane potential (MMP) at 6, 12, and 24 h, respectively. The cells were incubated with 4 µg/mL JC-1 for 15 min at 37 °C in a humidified incubator to reveal MMP change. (A,E,I) Control, cells cultured in normal condition. (B,F,J) 3BDO cells treated with 120 µM 3BDO. (C,G,K) CQ cells treated with 16 µM CQ. (D,H,L) CQ+3BDO, cells treated with 16 µM CQ and 120 µM 3BDO. (M) Relative level of MMP was quantified by the relative ratio of red/green fluorescence intensity values per cell (&, P < 0.05 vs control group; &&, P < 0.01 vs control group; **, P < 0.01 vs CQ group, n ) 4).
group (P < 0.01). When VECs were treated with both CQ and 3BDO, MMP was decreased significantly (P < 0.01). Our results showed that the inhibitory effect of 3BDO on CQ-induced accumulation of autophagic vesicles was accompanied by a recovery decrease in MMP. Effects of CQ and 3BDO on the Activity of Na+,K+ATPase. It has been reported that the sodium- and potassiumactivated adenosinetriphosphatase (Na+,K+-ATPase) is inhibited by CQ (9). Therefore, we investigated the effects of 3BDO and CQ on the activity of this enzyme. In VECs treated with 16 µM CQ or with 120 µM 3BDO for 6 h, the activity of Na+,K+ATPase was significantly or obviously decreased, respectively (P < 0.01 or P < 0.05, Figure 7). But, when VECs were treated with both CQ and 3BDO, the activity of Na+,K+-ATPase was obviously elevated compared with that of the only CQ-treated group (P < 0.05).
Discussion Autophagy can promote cell survival not only by recycling cellular constituents to support bioenergetics but also by clearing damaged organelles, whose accumulation following cellular stress can further damage cells. Inhibition of autophagy can trigger apoptosis (2), and upregulation of autophagy protects against apoptotic stimuli (3). However, by genetic methods, it has recently been demonstrated that autophagy is involved in both cell survival and cell death in mammalian cells. Boya et al. (2) and Lum et al. (27) for the first time showed that in mammalian cells autophagy proteins were needed for survival under nutrient and growth-factor removal. Yu, Pyo, and Shimizu,
by contrast, first showed that autophagic genes were needed for autophagic cell death (28-30). In view of these points, despite having autophagy’s pro-survival role clearly demonstrated by numerous studies, its opposite role in cell death requires further investigations. It is well known that growth-factor deprivation induces autophagy, but it has also been demonstrated that deprivation of growth factors induces apoptosis in VECs (31), indicating that the relationship between the two processes is very complex when VECs were deprived of growth factors. To understand the effects of 3BDO on autophagy in VECs, in this study, we blocked autophagy by CQ in the presence of survival factors. The results showed that 3BDO could effectively inhibit the action of CQ, suggesting that 3BDO was a useful tool for investigating the mechanism of autophagy in VECs. Recently, autophagy-related publications in cancer and cardiovascular research have increased considerably, indicating that autophagy is becoming a topic of major importance. Despite the increasing interest in autophagy, the process is still unclear as compared with apoptosis. The molecular basis and the regulation of autophagy in cell death need to be clarified along with the relationship between autophagy and the apoptotic machinery (32). In the decision of cellular fate, mitochondria could have a determining role as the switch between apoptosis and autophagy. Therefore, we examined the effect of 3BDO on mitochondrial membrane potential in VECs treated with CQ. Previous studies have shown that, in some cancer cells and fibroblasts, CQ induces cell apoptosis accompanied with a disruption in mitochondrial membrane potential (33). In contrast, our results showed that CQ elevated the MMP of VECs while autophagic vesicles accumulated, and 3BDO could attenuate these changes. The data suggested that 3BDO had mitochondrial protective effects against CQ in VECs. It was reported that CQ inhibited Na+,K+-ATPase activity and induced VEC apoptosis (9, 15). Consistent with these reports, the present results showed that CQ dramatically suppressed the activity of Na+,K+-ATPase in VECs, indicative of the importance of Na+,K+-ATPase in autophagy. When 3BDO inhibited CQ-induced accumulation of autophagic vesicles, it correspondingly blocked the decrease of Na+,K+-ATPase activity induced by CQ. In summary, we found that 3BDO effectively inhibited the accumulation of autophagic vesicles induced by CQ in VECs. The results showed that CQ significantly depressed mitochondrial membrane potential and Na+,K+-ATPase activity. 3BDO could inhibit these changes. The data suggested that 3BDO could protect VECs against CQ. Our data indicated that cell-permeable
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3BDO with an antivacuolation role in VECs is an exciting tool for investigating the mechanism of autophagy. Acknowledgment. This work was financially supported by the National 973 Research Project (No.2006CB503803), National Natural Science Foundation of China (No.90813022), and Natural Science Foundation of Shandong Province (Z2006D02).
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