Abstract
Background/Aim: We investigated the effects of chloroquine, an autophagy inhibitor, on doxorubicin-induced apoptosis in A549 cells. Materials and Methods: A549 cells were treated with doxorubicin, chloroquine, or both. Then, cytotoxicity was measured. The expression levels of caspases and mitogen-activated protein kinases were also quantified. In addition, the levels of doxorubicin-derived reactive oxygen species were measured. Results: Chloroquine enhanced doxorubicin-induced apoptosis and oxidative stress and suppressed the doxorubicin-induced extracellular-signal-regulated kinase activation. Conclusion: Chloroquine enhances doxorubicin-induced and oxidative stress-mediated apoptosis. This mechanism may involve the dephosphorylation of extracellular-signal-regulated kinases.
- Chloroquine
- doxorubicin
- A549 cells
- autophagy
- apoptosis
- oxidative stress
- caspase
- extracellular-signal-regulated kinase
Lung cancer is a leading cause of cancer-related deaths in men and women, causing approximately 1.4 million deaths every year worldwide. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for approximately 80% of all lung cancer cases (1). It is commonly treated through surgery, radiation therapy, and chemotherapy with anticancer drugs. Various types of anticancer drugs, such as cytotoxic anticancer agents and molecular-targeted drugs, are used to treat NSCLC (2).
Doxorubicin (DOX), an anthracycline anticancer drug, is widely used to treat different cancers, including NSCLC (3). However, cumulative and dose-related cardiotoxicity induced by DOX impedes its clinical use (4). DOX induces apoptosis via oxidative stress in several cancer cells (5, 6). The oxidative stress is induced by generating excessive reactive oxygen species (ROS) (7). Mitogen-activated protein kinase (MAPK) cascade is a key signaling pathway that regulates several cellular processes, including proliferation, differentiation, apoptosis, and stress response (8). The MAPK signaling pathway is also responsible for DOX-mediated toxicity (9, 10).
Autophagy is a cellular process in which aggregated proteins and damaged organelles in the cytoplasm are degraded by the lysosomes. It is a type of programmed cell death but is generally considered as a stress-responsive survival mechanism (11). It is induced under ROS-mediated oxidative stress and protects cells from apoptosis (12, 13). Autophagy is associated with many diseases such as cancer, metabolic dysfunction, neurodegenerative diseases, and inflammatory diseases (14-16). Autophagy plays a dual role in cancer development depending on the type and stage of cancer (17-19). In the early stages of cancer progression, autophagy acts as a tumor suppressor. However, once the tumors progress to advanced stages, autophagy supplies nutrients and metabolic intermediates to cancer cells under nutrient-depleted conditions to promote their survival (20, 21). This study focused on the effect of autophagy in cancer cells treated with anticancer drugs. Specifically, the study aimed to determine the effect of autophagy inhibition on DOX-induced apoptosis in NSCLC cells.
Materials and Methods
Cell culture. A549 cells were grown to 80-90% confluency in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, MA, USA) containing 10% fetal bovine serum, L-glutamine (4 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified chamber with 5% CO2.
Cyto-ID staining. A549 cells (1×105 cells/well) were seeded in a 24-well plate and treated with DOX (Merck, Darmstadt, Germany) for 24 h, followed by staining using the Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA) as per the manufacturer’s protocol. Images were captured using a confocal microscope (Carl Zeiss, Germany).
Measurement of cytotoxicity. Cell viability was measured using the Cell Titer 96AQueous One Solution Cell Proliferation Assay (MTS assay; Promega, Madison, WI, USA). Briefly, A549 cells in 96-well plates were treated with 2 μM DOX, 20 μM chloroquine (CQ, an autophagy inhibitor), or both for 4-24 h. Then, the media were discarded, cells were washed with serum-free DMEM, and incubated with complete DMEM (100 μl) and MTS assay solution (10 μl) at 37°C for 60 min. The MTS formazan produced was measured at 490 nm using a Bio-Rad Model 680 microplate reader (Hercules, CA, USA).
Cell death was assessed by measuring the lactate dehydrogenase (LDH) release. The A549 cells in 96-well plates were treated with 2 μM DOX, 20 μM CQ, or both for 4-24 h. LDH release was measured using the Cytotoxicity LDH Assay Kit-WST (DOJINDO, Kumamoto, Japan) according to the manufacturer’s protocol.
Measurement of protein levels. LC3, p62, caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, p38 mitogen-activated protein kinase (p38), phospho-p38 (p-p38), c-Jun N-terminal kinase (JNK), phospho-JNK (p-JNK), extracellular-signal-regulated kinase (ERK), and phospho-ERK (p-ERK) protein levels were analyzed using western blotting. Briefly, cells treated with 2 μM DOX alone, 20 μM CQ, or MAPK inhibitor. Then, they were washed with Dulbecco’s phosphate-buffered saline (DPBS) and lysed in lysis buffer [50 mM HEPES (pH 7.4), 5 mM EDTA, 120 mM NaCl, 1% Triton X-100, protease inhibitors (10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin), and phosphatase inhibitors (50 mM sodium fluoride, 1 mM sodium orthovanadate, and 10 mM sodium pyrophosphate)]. The lysate was centrifuged at 10,000 × g for 15 min, and 20 μg protein from the supernatant was resolved using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were blotted onto polyvinylidene difluoride membranes. To detect proteins, membranes were incubated with the corresponding primary antibodies: anti-rabbit caspase-3, anti-rabbit cleaved caspase-3, anti-mouse caspase-8, anti-rabbit caspase-9, anti-rabbit p38, anti-rabbit phospho-p38, anti-rabbit JNK, anti-rabbit phospho-JNK, anti-rabbit ERK, anti-rabbit phospho-ERK, anti-rabbit p62 (Cell Signaling Technology, Danvers, MA, USA), anti-rabbit LC3 polyclonal antibody (Novus Biologicals, Centennial, CO, USA), or anti-mouse β-actin polyclonal antibody (Sigma-Aldrich Co., St. Louis, MO, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence was detected using an Immobilon block (Merck KGaA, Darmstadt, Germany).
Measurement of glutathione (GSH) levels. Intracellular GSH levels were measured using previously described spectrophotometric methods (22). A549 cells, seeded in a 12-well plate, were treated with DOX. To measure GSH levels, the samples were mixed with 0.6 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), 0.2 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH), and 5 mM ethylenediaminetetraacetic acid (EDTA) in 0.1 M sodium phosphate buffer (pH 7.5). The reaction was initiated by the addition of glutathione reductase.
Measurement of ROS. Mitosox (Thermo Fisher Scientific) was used to estimate intracellular ROS. A549 cells were treated with DOX, 20 μM CQ, or both and incubated in a medium containing Mitosox for 20 min. Then, the cells were washed with DPBS. Changes in the intracellular ROS levels were visualized as red fluorescence using confocal laser scanning microscopy.
Statistical analysis. All experiments were performed independently in triplicates. Results are expressed as mean±standard deviation (SD). Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. All analyses were performed using the GraphPad Prism 7 software. Differences were considered significant at p-value <0.05.
Results
DOX-induced autophagy. To measure DOX-induced autophagy, we evaluated p62 levels (a marker of autophagic activity) and autophagic flux (a measure of autophagic degradation) (23). We used LC3 as a model substrate to measure the autophagic flux. In A549 cells, DOX decreased p62 protein levels and increased autophagic flux (Figure 1A).
Doxorubicin (DOX)-induced autophagy. (A) Western blots of p62 and LC3 proteins detected in A549 cells treated with 2 μM DOX, 20 μM chloroquine (CQ), or both for 16 h. (B) Autophagosomes visualized under a fluorescence microscope, in A549 cells treated with 2 μM DOX for 24 h.
The Cyto-ID autophagy detection kit was used to measure autophagic activity. This kit measures the number of autophagic vacuoles and monitors autophagic flux by using a dye (Cyto-ID) that selectively labels the accumulated autophagic vacuoles. DOX increased the Cyto-ID fluorescence (Figure 1B). The results suggested that DOX induces autophagy in A549 cells.
CQ enhanced DOX-induced apoptosis. To investigate the effect of an autophagy inhibitor on DOX-induced cytotoxicity, we analyzed MTS reduction as an indicator of cell viability, and LDH release as an indicator of cell death. The combination of DOX and CQ decreased cell viability and induced cell death in A549 cells (Figure 2A and B). These results suggested that autophagy inhibitors enhance DOX-induced cytotoxicity.
CQ enhanced DOX-induced apoptosis. A549 cells were treated with 2 μM DOX, 20 μM CQ, or both for 24 h. Graphs depict (A) cell viability and (B) cell death as estimated by MTS assay and LDH release, respectively; (C) western blot results for expression of Caspase-3. (D) Cell viability analysis of A549 cells treated with 2 μM DOX, 20 μM CQ, or both, with or without QVD for 24 h. Values are represented as means±SD of six experiments (n=6). *Statistical significance was set at p<0.05. DOX: Doxorubicin; LDH: lactate dehydrogenase; CQ: chloroquine.
Next, we focused on apoptosis, a type of programmed cell death. We measured the levels of caspase-3 as an apoptosis indicator. Figure 2C shows that DOX slightly increased the levels of cleaved caspase-3, the activated form of caspase-3. In addition, CQ remarkably enhanced the levels of active caspase-3 in DOX-treated cells. Moreover, Quinoline-Val-Asp-Difluorophenoxymethylketone (QVD), an apoptosis inhibitor, decreased the combined cytotoxic effects of DOX and CQ. These results suggested that autophagy inhibitors enhance DOX-induced apoptosis.
Mechanism of apoptosis induced by the combination of DOX and CQ. We investigated the mechanism of apoptosis induced by the combination of DOX and CQ. Apoptosis is induced via two main routes: the mitochondrial pathway or the death receptor pathway. First, we focused on the mitochondria-mediated apoptosis that is mainly induced by oxidative stress (24). Mitosox when oxidized by superoxide, a ROS, exhibits red fluorescence. DOX increased the mitosox-derived fluorescence, and CQ further enhanced this fluorescence (Figure 3A). A549 cells treated with DOX showed a decrease in GSH (a major antioxidant) levels. CQ augmented this decrease in GSH levels (Figure 3B). These results suggested that DOX-induced oxidative stress is intensified by CQ. Caspase-9 is located downstream of the mitochondria-mediated apoptotic pathway. DOX increased the levels of cleaved caspase-9 (Figure 3C), and CQ enhanced the Dox-mediated increase in cleaved caspase-9 levels. The results suggest that CQ enhances DOX-induced mitochondrial-mediated apoptosis.
Mechanism of apoptosis induced by the combination of DOX and CQ. (A) Mitosox fluorescence depicting ROS production in A549 cells treated with 2 μM DOX, 20 μM CQ, or both for 24 h. (B) GSH levels in A549 cells treated with 2 μM DOX, 20 μM CQ, or both for 8 h, as estimated by DTNB recycling assay; western blot results for the expression of (C) Caspase-9, (D) Fas, and (E) Caspase-8 proteins. Values are represented as means±SD of six experiments (n=6). *Statistical significance was set at p<0.05. DOX: Doxorubicin; CQ: chloroquine; GSH: glutathione.
Next, we focused on apoptosis mediated by the death receptor (caspase-8) pathway. DOX increased Fas and cleaved caspase-8 levels (Figure 3D and E). However, CQ did not affect the DOX-induced increase in Fas and caspase-8 levels. These results suggested that CQ does not influence the DOX-induced apoptosis that is mediated by the death receptor pathway.
Effects of MAPK on DOX and CQ-induced cytotoxicity. We evaluated the expression of various members of the MAPK cascade. DOX increased phospho-p38, phospho-JNK, and phospho-ERK levels in A549 cells (Figure 4A, C and E). CQ enhanced DOX-mediated phosphorylation of p38 and JNK. In contrast, CQ inhibited DOX-mediated phosphorylation of ERK. These results suggested that autophagy inhibitors enhanced DOX-induced p38 and JNK activation and suppressed DOX-induced ERK activation.
Effects of MAPK on DOX and CQ-induced cytotoxicity. (A) p-p38 and p38, (C) p-JNK and JNK, and (E) p-ERK and ERK protein levels in A549 cells treated with 2 μM DOX, 20 μM CQ, or both for 24 h, as measured by western blot. (B, D, F) Cell viability of A549 cells treated with 2 μM DOX, 20 μM CQ, or both, with or without MAPK inhibitor for 24 h. Values are represented as means±SD of six experiments (n=6). *Statistical significance was set at p<0.05. DOX: Doxorubicin; CQ: chloroquine; MAPK: mitogen-activated protein kinase; JNK: c-Jun N-terminal kinase; ERK: extracellular-signal-regulated kinase.
Moreover, SB203580 (a p38 inhibitor) and SP600125 (a JNK inhibitor) did not affect DOX- and CQ-mediated decrease in cell viability. In contrast, SCH772984 (an ERK inhibitor) promoted the decrease in cell viability. These results suggested that the ERK pathway is involved in the DOX- and CQ-induced cytotoxicity.
Effect of ERK inhibitor on DOX- and CQ-induced apoptosis. Finally, we investigated the effects of SCH772984 on the cytotoxicity induced by the combination of DOX and CQ. SCH772984 enhanced ROS production in cells treated with both DOX and CQ (Figure 5A). As shown in Figure 2C and Figure 3C, it also enhanced the DOX-mediated increase in the levels of cleaved caspase-3 and cleaved caspase-9. Therefore, we investigated whether SCH772984 affects the DOX-and CQ-induced increase in the levels of cleaved caspase-3 and cleaved caspase-9 and found that SCH772984 further enhanced these levels (Figure 5B and C). These results demonstrated that SCH772984 enhances the effect of the DOX and CQ combination on mitochondria-mediated apoptosis induced by oxidative stress.
Effect of ERK inhibitor on DOX and CQ-induced apoptosis. (A) Mitosox fluorescence to detect ROS production; and western blots for the expression of (B) caspase-9 and (C) caspase-3 proteins in A549 cells treated with 2 μM DOX, 20 μM CQ, or both with or without the ERK inhibitor SCH772984 for 24 h. DOX: Doxorubicin; CQ: chloroquine; ERK: extracellular-signal-regulated kinase.
Discussion
The present study demonstrated that CQ, an autophagy inhibitor, enhances DOX-induced and oxidative stress-mediated apoptosis. Moreover, we found that ERK dephosphorylation was involved in this mechanism. These findings provide a more detailed understanding of the mechanisms underlying DOX-induced cytotoxicity in cancer cell lines.
NSCLC is the most common type of lung cancer. The A549 cell line used in the present study is a type of NSCLC-derived cell line. DOX, an anthracycline anticancer drug, is widely used in chemotherapy to treat various cancers, including NSCLC (3). However, various epidemiological and case reports on DOX-induced cardiotoxicity have been published (25). Although various factors, such as oxidative stress, have been reported to affect the mechanism of DOX-induced cardiotoxicity, the exact molecular mechanism has not been elucidated (26). The cardiotoxicity of DOX is dose-dependent. Therefore, safe dose limits of DOX have been established (4). The use of DOX within the defined doses is required to avoid DOX-induced cardiotoxicity.
Several reports have shown that DOX induces oxidative stress, which is a crucial factor in cardiotoxicity (27-29). Autophagy acts as a stress response mechanism to digest and remove proteins and organelles denatured by oxidative stress (30). CQ, an inhibitor of autophagy, enhanced DOX-induced oxidative stress (Figure 3A and B), supporting the findings of the previous reports. CQ also dramatically enhanced DOX-induced apoptosis (Figure 2). Besides inhibition of autophagy, other factors could also have enhanced the oxidative stress.
DOX induces apoptosis in several cell lines (5, 6, 31, 32). It activates caspase-3, -8, and -9 in HSC-2 and HL-60 cells (6). As shown in Figure 3C and E, the combination of DOX and CQ remarkably increased the cleaved caspase-9 levels compared to cleaved caspase-8 levels. These results suggested that CQ enhances DOX-induced apoptosis via the mitochondria-mediated apoptotic pathway. Moreover, CQ also augmented mitochondrial ROS production induced by DOX (Figure 3A). DOX induces mitochondrial toxicity and oxidative stress (33). One role of autophagy is to protect against oxidative stress. The p62 molecule promotes the antioxidant defense system by activating the Nrf2 pathway (34). We demonstrated that the autophagy inhibitor CQ enhances apoptosis via DOX-induced caspase-9 activation and ROS production.
As shown in Figure 4E, CQ inhibited DOX-mediated phosphorylation of ERK. Moreover, SCH772984, an ERK inhibitor, increased the DOX-and CQ-induced cytotoxicity and the levels of cleaved caspase-3 and cleaved caspase-9 (Figure 4F, and Figure 5B and C). These results suggested that ERK protects against cytotoxicity and apoptosis induced by the combination of DOX and CQ. ERK activation is widely associated with anti-apoptotic functions. ERK regulates cell proliferation and differentiation (35, 36). It can exert an anti-apoptotic effect by down-regulating and up-regulating the expression of pro-apoptotic and anti-apoptotic proteins, respectively, through both transcriptional and post-translational mechanisms (36). In contrast, several studies have reported that ERK signaling can be a pro-apoptotic pathway (37). In the present study, ERK was found to have anti-apoptotic effects that inhibited DOX- and CQ-induced apoptosis in A549 cells. Furthermore, CQ promoted the DOX-induced phosphorylation of p38 and JNK (Figure 4A and C). SB203580, a p38 inhibitor, and SP600125, a JNK inhibitor, did not affect DOX- and CQ-induced cytotoxicity (Figure 4B and D). These results indicated that compared to ERK, p38 and JNK do not contribute to DOX- and CQ-induced cytotoxicity enhancement. However, the reason behind this remains unclear and requires further investigation. Moreover, it is well known that the MAPK pathway is activated by stress responses such as oxidative stress. In the present study, we found that the inhibition of ERK activation by CQ enhanced DOX-induced oxidative stress (Figure 4E and F, and Figure 5A). This may be due to feedback inhibition of MAPK activation by oxidative stress.
Attempts to use autophagy as a therapeutic target have already been initiated. Clinical trials using CQ for cancer treatment are underway (38). In this study, we proposed a mechanism by which CQ augmented doxorubicin-induced apoptosis (Figure 6). DOX-induced autophagy protected against DOX-induced apoptosis. CQ dramatically enhanced oxidative stress-derived apoptosis potentially by inhibiting DOX-induced autophagy as well as ERK phosphorylation. These findings could contribute to the development of novel anticancer drugs in the future.
CQ enhances DOX-induced apoptosis via the dephosphorylation of ERK. DOX: Doxorubicin; CQ: chloroquine; ERK: extracellular-signal-regulated kinase.
Acknowledgements
The Authors would like to thank Dr. Andrew Thorburn for his helpful suggestions. This work was supported by JSPS KAKENHI (grant no. JP 20K16084).
Footnotes
Authors’ Contributions
K Sato and R Tatsunami conceived and designed the experiments. K Sato, N Ota and S Endo performed the experiments and analyzed the data. A Nakata and H Yamashita analyzed and interpreted the data. K Sato and R Tatsunami wrote the manuscript. A Nakata and H Yamashita reviewed and edited the manuscript. All Authors read and approved the final version of the manuscript.
Conflicts of Interest
The Authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this article.
- Received May 20, 2022.
- Revision received June 3, 2022.
- Accepted June 6, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.