Introduction
Lung cancer is the largest proportion of cancer deaths, which accounts for more than 1.8 million people every year in the world [34]. Lung cancers are largely classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC is more aggressive than NSCLC and the survival rate after 5 years is only 7% [7, 33]. Chemotherapy drugs such as cisplatin and doxorubicin, commonly used in cancer treatment, induce cell death through reactive oxygen species (ROS) production and DNA damages [36]. However, it affects both cancer cells and normal cells, causing side effects such as diarrhea, nausea, hearing loss, pancytopenia, neutropenic fever and resistance to anti-cancer drugs [30]. Chemotherapy burdens the patient's health status, resulting in difficulties in long-term treatment. Therefore, it is necessary to develop a treatment that selectively inhibits the cancer cells without any effect on other normal cells.
Apoptosis is a type of programmed cell death, the process of removing abnormal or dead cells that occurs in multicellular organisms. It is characterized by nuclear fragmentation and cell shrinkage [26]. Apoptosis is divided into intrinsic and extrinsic pathways. In extrinsic pathway, tumor necrosis factor α (TNFα) and Fas ligand (FasL) trigger activation of caspase-8 and Fas-associated protein with death domain (FADD) [8, 20]. Activation of caspase-8 results in caspase cascade, which also activates caspase-3 and caspase-7. In addition, activated caspase-8 cleaves Bid into tBid [35]. Then, tBid blocks B-cell lymphoma 2 (Bcl-2) and initiates Bcl-2 associated X protein (Bax) that associated with intrinsic pathway [25]. Activated Bax/Bak leads to mitochondrial dysfunction and releases apoptogenic proteins to cytosol. After the activation of apoptogenic proteins such as cytochrome c and second mitochondria-derived activator of caspase (Smac), caspases activate the cascade and inhibit the inhibitor of apoptosis protein (IAP) [17]. As a result of this process, caspase is activated, leading to cell death by apoptosis.
Autophagy is the process of removing unnecessary cell components or degrading proteins and reusing them as energy sources [22]. In this process, the autophagosome, which is double-membrane vesicles is formed for subsequent degradation by various autophagy genes such as autophagy-related (Atg) genes, UNC51-like kinase (ULK), mammalian target of rapamycin (mTOR) and microtuble-associated protein light chain 3 (LC3) [15]. However, under normal conditions, mTORC1 inhibits their binding and activation, thereby inhibiting autophagy [4]. Moreover, in the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, AKT) signaling pathway, AKT activates mTOR to inhibit autophagy [14]. After ULK1 is activated, it phosphorylates Ambra1 interacting with Beclin-1. When Beclin-1 moves to endoplasmic reticulum (ER), it forms a complex with Atg14L, Vps34 and Vps15 to initiate autophagosome formation. Vsp34, a PI3K, produces phosphatidylinositol-3-phosphate (PI3P) and initiates autophagosome formation using Atgs. Atg12/Atg5 complex activates LC3 protein to induce LC3 complex and the formed LC3 complex binds to phosphatidylethanolamine (PE) to form LC3-II. Finally, autophagosome binds to lysosome to form autophagolysosome, which decomposes organelles or proteins by internal enzymes [16].
Natural products have long been used as medicine, which gives a great help in improving human health [23]. In addition, these have fewer side effects than chemotherapy, resulting in being used more freely with safety in the treatment of diseases [37]. Curcumin is one of the yellow-colored phenols extracted from Curcuma longa, which has been mainly used as herbal medicine in India. Moreover, it has been reported to have effects such as anti-cancer, anti-oxidant, anti-inflammatory, anti-angiogenic, anti-microbial, anti-Alzheimer, anti-parasitic, and anti-viral activities [13, 19]. In particular, its anti-cancer activity was expressed through regulation of ROS without any significant effect on normal cells [21]. Therefore, curcumin is very valuable to be studied as an anti-cancer drug, with which many experiments are still being conducted. In this experiment, the effects of curcumin were compared with subtypes of human lung cancer cells, focusing on cell deaths.
Materials and Methods
Reagents
3-(4,5-Dimethyl-thiazol-2yl)-2,5-diphenyltertrazolium bromide (MTT), acridine orange (AO), propidium iodide (PI), and curcumin were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2',7'-Dichlorofluorescein diacetate (DCFH-DA) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Diphenyleneiodonium (DPI), a ROS inhibitor, and 3-methyladenine (3-MA), an autophagy inhibitor, were purchased from Calbiochem (Merck, Darmstadt, Germany) and Sigma-Aldrich, respectively. Annexin V/PI Apoptosis Detection Kit was purchased from BD Bioscience (San Jose, CA, USA). Antibodies against pro-PARP and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and those against cleaved-PARP, Bcl-2, Bax, pro-caspase-3, AKT, p-AKT, p62, and LC3B were purchased from Cell Signaling Technology (Beverly, MA, USA). And goat-anti-rabbit or -mouse IgG secondary antibodies were purchased from Enzo Life Science (Farmingdale, NY, USA). PRO-PREPTM Protein Extraction Solution and SMARTTM BCA Assay Kit were purchased from iNtRON Biotechnology (Seongnam, Korea).
Cell lines and cell culture
Human non-small cell lung cancer (NSCLC) cells, A549 and NCI-H23, and human small cell lung cancer (SCLC) cells, DMS53 and SW1271, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The human lung cancer cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich) medium with 10% FBS and 1% penicillin-streptomycin solution at 37℃ with 5% CO2.
Cell viability
Human lung cancer cells were seeded into 96-well cell culture plates. Cultured cells were treated with curcumin at 10, 30, and 50 μM for 24 hr. After incubation, 0.5 mg/ml of MTT was treated for 3 hr at 37℃. Then the precipitated formazan complexes were dissolved in DMSO. Optical density was measured at 570 nm using by VersaMax microplate reader (Molecular Devices, Toronto, Canada).
Annexin V/propidium iodide (PI) double staining
Apoptotic cells were analyzed using Annexin V/PI mixture. Human lung cancer cells were cultured in a 6-well cell culture plate and treated with 10, 30, and 50 μM of curcumin for 24 hr. Then, cultured cells were harvested by trypsinization and washed with 1x PBS. The harvested cells were resuspended in 1x binding buffer and incubated with Annexin V/PI mixture at room temperature for 20 min. The fluorescence intensity was measured by flow cytometry (FACS Canto II, BD Bioscience)
Measurement of reactive oxygen species (ROS) generation
ROS generation level was measured using DCFH-DA solution. Human lung cancer cells were cultured in a 6-well cell culture plate and treated with 10, 30, and 50 μM of curcumin for 24 hr. Then cultured cells were harvested by trypsinization, then treated with 10 μM DCFH-DA onto cells at room temperature for 20 min, and replaced with a fresh serum-free RPMI. After incubation, cells were resuspended with 1x PBS. The fluorescence intensity was measured by flow cytometry (FACS Canto II, BD Bioscience).
Acridine orange (AO) staining
Autophagic cells were analyzed using AO. Human lung cancer cells were cultured in a 6-well cell culture plate and treated with 10, 30, and 50 μM of curcumin for 24 hr. Then, cultured cells were harvested by trypsinization and washed with 1x PBS. The harvested cells were resuspended in 1x binding buffer and incubated with 1 μg/ml AO solution at 37℃ for 20 min. The fluorescence intensity was measured by flow cytometry (FACS Canto II, BD Bioscience).
Western blotting
A549 and DMS53 were cultured in a 6-well cell culture plate and treated with 10, 30, and 50 μM of curcumin for 24 hr. The cells were harvested and lysed with PRO-PREP solution. Ten μg of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12-15% gels. Then, proteins were transferred to polyvinylidene fluoride (PVDF) membrane. Next, the membrane was blocked with 5% skim milk in TBS-T buffer at room temperature for 2 hr. The membranes were incubated with primary antibodies at 4℃ overnight. And then the membrane was washed 3 times with TBS-T buffer for 15 min and incubated with secondary antibodies at room temperature for 3 hr. After washing 3 times with TBS-T buffer for 15 min, the proteins were detected using ECL buffer (iNtRON Biotechnology) and quantified by Amersham ImageQuant 800 (Cytiva, Marlborough, MA, USA).
Statistical analysis
Data were expressed as mean ± SD. ANOVA was used to compare experimental groups to control group. Results were statistically significant at *p<0.05, **p<0.01, and ***p<0.001.
Results
Curcumin reduces cell viability of human lung cancer cells
To determine the anti-cancer activity of curcumin, two types of human lung cancer cell lines were used with A549 and NCI-H23 as non-small cell lung cancer (NSCLC) cells and DMS53 and SW1271 as small cell lung cancer (SCLC) cells. Treatment of curcumin at 10, 30, and 50 μM for 24 hr reduced cell viability of A549 cells to 78.32±3.33, 64.30±1.27, and 42.34±.30%, NCI-H23 to 87.31±1.67, 64.83±1.41, and 44.20±1.43%, DMS53 to 81.26±3.46, 52.79±1.41, and 34.98±2.46%, and SW1271 to 81.66±3.66, 57.97±2.05, and 39.11±5.19%, respectively (Fig. 1). According to the results, the cytotoxicity of curcumin showed a dose-dependent manner and similar pattern in each cell lines. For further experiments, A549 and DMS53 were selected as a cell type of NSCLC and SCLC, respectively.
Fig. 1. Cell cytotoxicities of curcumin in human lung cancer cells.A549 and NCI-H23 as non-small cell lung cancer (NSCLS) cells and DMS53 and SW1271 as small cell lung cancer (SCLS) cells were used. To measure cell viability, MTT assay was conducted. Lung cancer cells were treated curcumin at 10, 30, and 50 μM for 24 hr. After incubation, 0.5 mg/ml of MTT was treated for 3 hr at 37℃. Then the precipitated formazan was dissolved in DMSO. Optical density was measured by microplate reader at 570 nm. Data were represented as mean ± SD (n=3) in three separate experiments. **p<0.01 and ***p<0.001, compared to control group.
Curcumin induces apoptosis of human lung cancer cells
When apoptosis progresses, phosphatidylserine (PS) present in the cell membrane moves to the outer cell membrane. Annexin V then recognizes and combines with externally exposed PS. In addition, during late apoptosis or necrosis, the integrity of plasma and nuclear membrane decreases and propidium iodide (PI) enters the cell to stain the nucleus. Therefore, curcumin-induced apoptosis on human lung cancer cells was analyzed by using Annexin V/PI double staining. Treatment of curcumin at 10, 30, and 50 μM for 24 hr increased apoptosis of A549 cells to 10.50±1.15, 23.10±1.83, and 26.73±3.18% and DMS53 to 18.03±2.60, 37.40±5.50, and 45.37±2.75%, respectively. As a result, it was shown a dose-pendent manner and higher apoptotic activity in DMS53 than in A549 (Fig. 2).
Fig. 2. Effect of curcumin on apoptosis of human lung cancer cells. (A) A549 and (B) DMS53 cells were treated curcumin at 10, 30, and 50 μM for 24 hr. The cells were washed with 1x PBS and harvested by trypsinization. The harvested cells were stained with Annexin V/PI mixture at room temperature for 20 min. Stained cells were analyzed by flow cytometry. Data were represented as mean ± SD (n=3) in three separate experiments. *p<0.05, **p<0.01, and ***p<0.001, compared to control group.
Curcumin regulates the expression of apoptosis-related proteins in human lung cancer cells
Bcl-2 and Bax belong to Bcl family proteins that regulate apoptosis and play a key role in the regulation of cytochrome c release into cytosol and mitochondrial function [31]. Also poly ADP-ribose polymerase (PARP) is regulated by caspase-3 and repairs DNA damage. Cleaved-PARP inhibits the ability of PARP to repair DNA to be considered a marker of apoptosis [1]. Western blotting was performed to measure the expression of apoptosis-related proteins, Bcl-2, Bax, procaspase-3, cleaved-PARP, and PARP, specially quantifying to ratio of Bax/Bcl-2 as an apoptosis indicator. Treatment of curcumin at 10, 30, and 50 μM for 24 hr increased the expression of Bax and cleaved-PARP but reduced pro-caspase-3 and Bcl-2 in both A549 (Fig. 3A) and DMS53 cells (Fig. 3B). The expression of apoptosis-related proteins was dose-dependently regulated by curcumin and it seemed that the ratios of Bax/Bcl-2 were shown higher in DMS53 than in A549, coincident with results of previous cell viability.
Fig. 3. Effect of curcumin on expression of apoptosis-related proteins in human lung cancer cells. (A) A549 and (B) DMS53 cells were treated with curcumin at 10, 30, and 50 μM and incubated at 37℃ with 5% CO2 for 24 hr. After incubation, cells were harvested and lysed by PRO-PREP. Western blotting was performed to measure level of expression of Bcl-2, Bax, pro-caspase-3, cleaved-PARP, and PARP. Relative Bax/Bcl-2 ratio was obtained as an indicator of apoptosis. Data were represented as mean ± SD (n=3) in three separate experiments. *p<0.05, **p<0.01, and ***p<0.001, compared to control group.
Curcumin triggers reactive oxygen species (ROS) production in human lung cancer cells
ROS is an important regulator of signaling pathways, which greatly affects cell metabolism and growth. However, excessive intracellular ROS levels damage proteins, lipids, and DNA, working as an anti-cancer agent to trigger apoptosis-related signaling pathway [27, 28]. Flow cytometry was applied to measure intracellular ROS generation induced by curcumin. Treatment of curcumin at 10, 30, and 50 μM for 24 hr increased ROS generation of A549 cells and DMS53 cells, which was similarly shown in a dose-dependent manner (data not shown). Diphenyleneiodonium (DPI), a ROS inhibitor, was used to confirm the intracellular ROS production. Curcumin at 30 μM was treated for 24 hr in the presence and absence of 0.5 μM DPI in A549 (Fig. 4A) and DMS53 (Fig. 4B) cells. DPI decreased ROS generation of A549 cells induced by curcumin from 28.43±1.75 to 23.37±2.39% and DMS53 from 13.13±0.95 to 8.47±2.71%, respectively. Taken together, curcumin triggered the ROS production in both lung cancer cells, which was regulated by ROS inhibitor, DPI.
Fig. 4. Effect of diphenyleneiodonium (DPI) on intracellular reactive oxygen species (ROS) induced by curcumin in human lung cancer cells. (A) A549 and (B) DMS53 cells were treated with curcumin at 30 μM for 24 hr in the presence and absence of 0.5 μMDPI. Then the cells were washed with 1x PBS and harvested by trypsinization. The harvested cells were stained with DCFH-DA solution at 37℃ for 20 min. Stained cells were analyzed by flow cytometry. Data were represented as mean ± SD (n=3) in three separate experiments. *p<0.05 and **p<0.01, compared to control group.
Curcumin regulates autophagy and the expression its related proteins in human lung cancer cells
Curcumin has been studied to be involved in not only apoptosis but also autophagy-related signaling pathway [38]. Acidic vesicular organelles (AVOs), the marker of autophagy, are formed when autophagy progresses. Acridine orange (AO) is a fluorescent dye that penetrates into AVOs. Autophagy in human lung cancer cells was analyzed by AO staining. Treatment of curcumin at 10, 30, and 50 μM for 24 hr increased autophagy of A549 cells and DMS53, which was similarly shown in a dose-dependent manner (data not shown). In the PI3K/AKT signaling pathway, which is the main pathway of autophagy, the expression of p-AKT was dose-dependently reduced by curcumin in both lung cancer cells (Fig. 5). On the other hand, expression of p62 and LC3B as an indicators of autophagy was increased (Fig. 5).
Fig. 5. Effect of curcumin on the expression of autophagy related proteins in human lung cancer cells. (A) A549 and (B) DMS53 cells were treated with curcumin at 10, 30 and 50 μM and incubated at 37℃ with 5% CO2 for 24 hr. Then cultured cells were harvested by trypsinization and lysed by PRO-PREP. Western blotting was performed to measured level of expression of autophagy-related proteins (p-AKT/AKT, p62, and LC3B).
3-Methyladenine (3-MA), an autophagy inhibitor to regulate the production of autophagosomes, was used to confirm autophagy phenotype. Curcumin at 30 μM was treated for 24 hr in the presence and absence of 1 mM 3-MA in A549 (Fig. 6A) and DMS53 (Fig. 6B) cells. 3-MA decreased autophagy of A549 cells induced by curcumin from 9.77±4.10 to 6.10± 3.66% and DMS53 from 15.83±1.46 to 9.27±2.76%. 3-MA restored induction of autophagy by curcumin in human lung cancer cells. However, autophagy inhibition by 3-MA was shown no significant effect on apoptosis induced by curcumin at 30 μM in A549 (Fig. 7A) and DMS53 (Fig. 7B) human lung cancer cells.
Fig. 6. Effect of 3-MA on autophagy induced by curcumin in human lung cancer cells. (A) A549 and (B) DMS53 cells were treated with curcumin at 30 μM for 24 hr in the presence and absence of 1 mM 3-MA. Then the cells were washed with 1x PBS and harvested by trypsinization. The harvested cells were stained with AO solution at 37℃ for 20 min. Stained cells were analyzed by flow cytometry. Data were represented as mean ± SD (n=3) in three separate experiments. *p<0.05 and **p<0.01, compared to control group.
Fig. 7. Effect of 3-MA on apoptosis induced by curcumin in human lung cancer cells. (A) A549 and (B) DMS53 cells were treated with curcumin at 30 μM for 24 hr in the presence and absence of 1 mM 3-MA. Cultured cells were washed with 1x PBS and harvested by trypsinization. The harvested cells were stained with Annexin V/PI mixture at room temperature for 20 min. Stained cells were analyzed by flow cytometry. Data were represented as mean ± SD (n=3) in three separate experiments. *p<0.05, compared to control group.
Diphenyleneiodonium (DPI) recoveries induction of apoptosis and autophagy by curcumin in human lung cancer cells
As shown in Fig. 4, DPI, a NADPH oxidase inhibitor, reduced intracellular ROS production induced by curcumin in human lung cancer cells. It was determined whether ROS inhibition by DPI affects apoptosis and autophagy induced by curcumin. After curcumin at 30 μM was treated for 24 hr in the presence and absence of 0.5 μM DPI in A549 and DMS53 cells, apoptosis and autophagy were determined by flow cytometry. DPI decreased apoptosis of A549 cells induced by curcumin from 28.23±2.84 to 22.67±1.34% (Fig. 8A) and DMS53 from 43.60±2.86 to 36.33±0.59% (Fig. 8B), which were confirmed as reduced expression of pro-caspase-3 and cleaved-PARP by western blotting. In addition, DPI decreased autophagy of A549 cells induced by curcumin from 10.83±1.46 to 5.50±1.91% (Fig. 9A) and DMS53 from 14.23 ±2.22 to 8.13±1.80% (Fig. 9B). Taken together, these results suggested that apoptosis and autophagy induced by curcumin is mediated through ROS generation.
Fig. 8. Effect of DPI on apoptosis induced by curcumin in human lung cancer cells. (A) A549 and (B) DMS53 cells were treated with curcumin at 30 μM for 24 hr in the presence and absence of 0.5 μM DPI. Cultured cells were washed with 1x PBS and harvested by trypsinization. The harvested cells were stained with Annexin V/PI mixture at room temperature for 20 min. Stained cells were analyzed by flow cytometry. Data were represented as mean ± SD (n=3) in three separate experiments. *p<0.05, compared to control group.
Fig. 9. Effect of DPI on autophagy induced by curcumin in human lung cancer cells. (A) A549 and (B) DMS53 cells were treated with curcumin at 30 μM for 24 hr in the presence and absence of 0.5 μM DPI. Cultured cells were-washed with 1x PBS and harvested by trypsinization. The harvested cells were stained with AO solution. Stained cells were analyzed by flow cytometry. Data were represented as mean ± SD (n=3) in three separate experiments. *p<0.05 and **p<0.01, compared to control group.
Discussion
Turmeric (Curcuma longa) is a spice and herb traditionally used in India and other Asian countries. Curcumin, a major substance extracted from turmeric, has anti-inflammatory, anti-oxidant, and anti-cancer activity. Especially, it has been shown to be effective in Alzheimer's and common malignancies such as lung, colon, stomach, skin and breast cancers [2, 6, 10]. Lung cancer is classified as small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [11]. Treatment of lung cancer chemotherapy has been widely used, especially to treat SCLC [29]. However, chemotherapy causes side effects such as asthenia, nausea, vaping, and alopecia, so long-term usage can be a burden on patients [12].
In this study, we tried to determine the anti-cancer activity of curcumin, a natural product extract with guaranteed safety, on human lung cancer cell lines. Curcumin of 10, 30, and 50 μM showed the cytotoxicity on all cell lines in a dose-dependent manner (Fig. 1). When observed the apoptotic process with Annexin V/PI double staining, apoptosis by curcumin was increased in A549 and DMS53 cells (Fig. 2). Apoptosis is regulated by the expression of apoptosis-related proteins, caspase cascade, cleavage of PARP, and Bcl-2/Bax interaction [3, 9]. In western blotting, the relative Bax/Bcl-2 ratio and the expression of cleaved caspase-3 and PARP as apoptosis markers were increased as concentration of curcumin increased (Fig. 3). ROS production, which induces apoptosis, was measured through flow cytometry analysis of DCFH-DA staining [32]
As a result, ROS production in A549 cells and DMS53 cells was increased in a dose-dependent manner of curcumin (data not shown). Furthermore, the ROS efficiency of curcumin was measured by treating diphenyleneiodonium (DPI) that suppresses ROS [24]. Treatment of 30 μM curcumin with 0.5 μM DPI decreased ROS production in A549 and DMS53 cells (Fig. 4). To determine autophagy, A549 and DMS53 cells were stained with 1 μg/ml acridine orange and analyzed by flow cytometry. Treatment of 10, 30, and 50 μM curcumin showed higher autophagy activity in DMS53 (data not shown). It was expected that cell death can be controlled in various ways such as the expression of autophagy-related proteins. The expression of proteins, such as p-AKT, p62, and LC3B, were regulated by curcumin treatment (Fig. 5). 3-Methyladenine (3-MA) is known to inhibit autophagy by suppressing the production of autophagosomes. Treatment of 30 μM curcumin with 1 mM 3-MA, restored autophagy induced by curcumin in A549 and DMS53 cells (Fig. 6). However, 3-MA was not shown any effect on apoptosis induced by curcumin (Fig. 7). Intracellular calcium (Ca2+) signals support life processes such as proliferation, differentiation, migration, and secretion. But, when calcium homeostasis collapses, apoptosis is induced [5]. The intracellular Ca2+ using Fluo-3/AM, a fluorescent dye that specifically binds to Ca2+, was analyzed by flow cytometry [18]. Treatment of curcumin at 10, 30, and 50 μM increased intracellular Ca2+ influx of A549 and DMS53 cells in a dose-dependent manner but slightly higher in DMS53 (data not shown). DPI was used to determine whether ROS production affects apoptosis and autophagy induced by curcumin. After treatment of 30 μM curcumin with 0.5 μM DPI, apoptosis was decreased in A549 from 28.23±2.84 to 22.67±1.34% and DMS53 from 43.60±2.86 to 36.33±0.59%, which were confirmed by expression of apoptosis-related proteins (Fig. 8). Moreover, treatment of 30 μM curcumin with 0.5 μM DPI also decreased autophagy in A549 cells from 10.83±1.46 to 5.50±1.91% and DMS53 from 14.23±2.22 to 8.13±1.80%(Fig. 9).
As a consequent, it was suggested that curcumin triggers ROS-mediated apoptosis and autophagy, leading to cellular death in human lung cancer cells. It was seemed that apoptosis and autophagy in DMS53 were more sensitive than in A549, when curcumin was treated. Finally, it was expected that curcumin as natural product might be established as an effective solution for the remedy of NSCLC and SCLC.
Acknowledgement
This work was supported by a 2-year Research Grant of Pusan National University.
The Conflict of Interest Statement
The authors declare that they have no conflicts of interest with the contents of this article.
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