Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Curcumin-induced Cell Death of Human Lung Cancer Cells

Curcumin에 의해 유도되는 인간 폐암 세포주의 세포사멸

  • Hwasin Lee (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Bobae Park (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Sun-Nyoung Yu (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Ho-Yeon Jeon (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Bu Kyung Kim (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Ae-Li Kim (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Dong Hyun Sohn (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Ye-Rin Kim (Department of Biochemistry, Pusan National University School of Medicine) ;
  • Sang-Yull Lee (Department of Biochemistry, Pusan National University School of Medicine) ;
  • Dong-Seob Kim (Department of Food Science& Technology, College of Natural Resources & Life Science, Pusan National University) ;
  • Soon-Cheol Ahn (Department of Microbiology & Immunology, Pusan National University School of Medicine)
  • 이화신 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 박보배 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 유선녕 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 전호연 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 김부경 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 김애리 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 손동현 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 김예린 (부산대학교 의과대학 생화학 교실) ;
  • 이상율 (부산대학교 의과대학 생화학 교실) ;
  • 김동섭 (부산대학교 생명자원과학대학 식품공학과) ;
  • 안순철 (부산대학교 의과대학 미생물학 및 면역학 교실)
  • Received : 2023.02.17
  • Accepted : 2023.06.30
  • Published : 2023.09.30
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Abstract

Lung cancer is a type of cancer that has the highest mortality rate. It is mainly classified into small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). Chemotherapy is used to treat lung cancer, but long-term treatment causes side effects and drug resistances. Curcumin is a bright yellow polyphenol extracted from the root of turmeric. It has biological activities, such as anti-oxidant, anti-cancer, and anti-inflammatory effects. In this study, we observed differential cell death in human lung cancer cells. Based on the results, curcumin at 10, 30, and 50 μM exhibited a dose-dependent inhibition on the cell survival of several lung cancer cells, with minor differential phenotypes. In addition, apoptosis, autophagy, and reactive oxygen species (ROS) regeneration were observed through flow cytometry. Curcumin dose-dependently increased these phenotypes in A549 (NSCLC) and DMS53 (SCLC), which were restored by corresponding inhibitors. Western blotting was performed to measure the level of expression of apoptosis- and autophagy-related proteins. The results indicate that Bax, PARP, pro-caspase-3, and Bcl-2 were dose-dependently regulated by curcumin, with seemingly higher Bax/Bcl-2 ratios in DMS53. In addition, autophagic proteins, p-AKT, p62, and LC3B, were dose-dependently regulated by curcumin. ROS inhibition by diphenyleneiodonium reduced the induction of apoptosis and autophagy generated by curcumin. Taken together, it is suggested that curcumin induces apoptosis and autophagy via ROS generation, leading to cell death, with minor differences between human lung cancer cells.

폐암은 전 세계적으로 사망률이 높은 암으로서 소세포성 폐암(small cell lung cancer, SCLC)과 비소세포성 폐암(non-small cell lung cancer, NSCLC)으로 분류된다. 오늘날까지 폐암 치료를 위해 많은 화학요법이 사용되어 왔으나 장기간 화학요법에 의한 부작용 및 항암제 내성 유발로 인해 항암 치료에 한계가 있으므로, 이와 같은 문제점을 해결하기 위해 현재는 천연물로부터 유래한 항암물질을 탐색하는 추세이다. Curcumin은 강황의 뿌리에서 추출된 polyphenol 화합물로서 항균, 항암, 항산화 및 항염증 작용이 보고되어 현재까지 꾸준히 항암 연구가 진행되어 왔으나 인간 폐암 세포주의 종류에 따른 항암 효과는 거의 연구되지 않았다. 따라서 본 연구에서는 여러 종류의 인간 폐암 세포주에 대한 curcumin의 항암 효과를 조사하고자 하였다. 그 결과, 10, 30, 50 μM 농도의 curcumin은 폐암 세포주에 따라 경미한 차이가 나타내면서 농도 의존적으로 세포 생존을 저해하였다. 또한 A549 (NSCLC) 및 DMS53 (SCLC) 세포주를 대상으로 apoptosis, autophagy, reactive oxygen species (ROS)를 flow cytometry로 측정한 결과, 농도 의존적으로 이러한 현상들이 증가되었으며 각각의 저해제를 처리했을 때 이러한 현상들이 회복되는 것이 관찰되었다. Apoptosis와 관련된 단백질인 Bax, PARP, pro-caspase-3 및 Bcl-2의 발현이 curcumin 농도 의존적으로 조절되었으며, 특히 DMS53에서 Bax/Bcl-2 비율이 더 높았다. 또한, autophagy 단백질인 p-AKT, p62 및 LC3B도 curcumin 농도 의존적으로 조절되었다. 한편, ROS 저해제인 diphenyleneiodonium 처리 시, curcumin에 의해 유도된 apoptosis와 autophagy 수준이 저해되었으며 관련 단백질의 발현도 조절되었다. 이를 통해 curcumin의 항암 작용은 curcumin에 의해 생산된 ROS를 매개로 하여 폐암 세포주의 apoptosis 및 autophagy의 유도를 통해 나타나는 것으로 확인되었다.

Keywords

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.

SMGHBM_2023_v33n9_713_f0001.png 이미지

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).

SMGHBM_2023_v33n9_713_f0002.png 이미지

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.

SMGHBM_2023_v33n9_713_f0003.png 이미지

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.

SMGHBM_2023_v33n9_713_f0004.png 이미지

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).

SMGHBM_2023_v33n9_713_f0005.png 이미지

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.

SMGHBM_2023_v33n9_713_f0006.png 이미지

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.

SMGHBM_2023_v33n9_713_f0007.png 이미지

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.

SMGHBM_2023_v33n9_713_f0008.png 이미지

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.

SMGHBM_2023_v33n9_713_f0009.png 이미지

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.

References

  1. Agarwal, A., Mahfouz, R. Z., Sharma, R. K., Sarkar, O., Mangrola, D. and Mathur, P. P. 2009. Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes. Reprod. Biol. Endocrinol. 7, 143. 
  2. Akaberi, M., Sahebkar, A. and Emami, S. A. 2021. Turmeric and curcumin: from traditional to modern medicine. Adv. Exp. Med. Biol. 1291, 15-39.  https://doi.org/10.1007/978-3-030-56153-6_2
  3. Brentnall, M., Rodriguez-Menocal, L., De Guevara, R. L., Cepero, E. and Boise, L. H. 2013. Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. BMC Cell Biol. 14, 32. 
  4. Chen, C., Gao, H. and Su, X. 2021. Autophagy-related signaling pathways are involved in cancer (Review). Exp. Ther. Med. 22, 710. 
  5. Dubois, C., Vanden Abeele, F. and Prevarskaya, N. 2013, Targeting apoptosis by the remodelling of calcium-transporting proteins in cancerogenesis. FEBS J. 280, 5500-5510.  https://doi.org/10.1111/febs.12246
  6. Fadus, M. C., Lau, C., Bikhchandani, J. and Lynch, H. T. 2016. Curcumin: an age-old anti-inflammatory and antineoplastic agent. J. Tradit. Complement. Med. 7, 339-346.  https://doi.org/10.1016/j.jtcme.2016.08.002
  7. Gazdar, A., Bunn, P. and Minna, J. 2017. Small-cell lung cancer: what we know, what we need to know and the path forward. Nat. Rev. Cancer 17, 725-737.  https://doi.org/10.1038/nrc.2017.87
  8. Gupta, S., Kim, C., Yel, L. and Gollapudi, S. 2004. A Role of fas-associated death domain (FADD) in increased apoptosis in aged humans. J. Clin. Immunol. 24, 24-29.  https://doi.org/10.1023/B:JOCI.0000018059.56924.99
  9. Hassan, M., Watari, H., AbuAlmaaty, A., Ohba, Y. and Sakuragi, N. 2014. Apoptosis and molecular targeting therapy in cancer. Biomed. Res. Int. 2014, 150845. 
  10. Hatcher, H., Planalp, R., Cho, J., Torti, F. M. and Torti, S. V. 2008. Curcumin: from ancient medicine to current clinical trials. Cell. Mol. Life Sci. 65, 1631-1652.  https://doi.org/10.1007/s00018-008-7452-4
  11. Howlader, N., Forjaz, G., Mooradian, M. J., Meza, R., Kong, C. Y., Cronin, K. A., Mariotto, A. B., Lowy, D. R. and Feuer, E. J. 2020. The effect of advances in lung-cancer treatment on population mortality. N. Engl. J. Med. 383, 640-649.  https://doi.org/10.1056/NEJMoa1916623
  12. Joly, F., Ahmed-Lecheheb, D., Thiery-Vuillemin, A., Orillard, E. and Coquan, E. 2019. Side effects of chemotherapy for testicular cancers and post-cancer follow-up. Bull. Cancer 106, 805-811.  https://doi.org/10.1016/j.bulcan.2019.04.004
  13. Khandelwal, P., Alam, A., Choksi, A., Chattopadhyay, S. and Poddar, P. 2018. Retention of anticancer activity of curcumin after conjugation with fluorescent gold quantum clusters: an in vitro and in vivo xenograft study. ACS Omega 3, 4776-4785  https://doi.org/10.1021/acsomega.8b00113
  14. Kma, L. and Baruah, T. J. 2022. The interplay of ROS and the PI3K/Akt pathway in autophagy regulation. Biotechnol. Appl. Biochem. 2022, 248-264.  https://doi.org/10.1002/bab.2104
  15. Kobayashi, S. 2015. Choose delicately and reuse adequately: the newly revealed process of autophagy. Biol. Pharm. Bull. 38, 1098-1103.  https://doi.org/10.1248/bpb.b15-00096
  16. Lee, M. S. 2014. Role of islet a cell autophagy in the pathogenesis of diabetes. Trends Endocrinol. Metab. 25, 620-627.  https://doi.org/10.1016/j.tem.2014.08.005
  17. Leibowitz, B. and Yu, J. 2010. Mitochondrial signaling in cell death via the Bcl-2 family. Cancer Biol. Ther. 9, 417-422.  https://doi.org/10.4161/cbt.9.6.11392
  18. Li, L., Yang, J., Wang, J. and Kopeeek, J. 2018. Drug-free macromolecular therapeutics induce apoptosis via calcium influx and mitochondrial signaling pathway. Macromol. Biosci. 18, 1700196. 
  19. Manoharan, Y., Haridas, V., Vasanthakumar, K. C., Muthu, S., Thavoorullah, F. F. and Shetty, P. 2020. Curcumin: a wonder drug as a preventive measure for COVID19 management. Ind. J. Clin. Biochem. 35, 373-375.  https://doi.org/10.1007/s12291-020-00902-9
  20. Meier, P. and Vousden, K. H. 2007. Lucifer's labyrinth Ten years of path finding in cell death. Mol. Cell 28, 746-754.  https://doi.org/10.1016/j.molcel.2007.11.016
  21. Nakamae, I., Morimoto, T., Shima, H., Shionyu, M., Fujiki, H., Yoneda-Kato, N., Yokoyama, T., Kanaya, S., Kakiuchi, K., Shirai, T., Meiyanto, E. and Kato, J. Y. 2019. Curcumin derivatives verify the essentiality of ROS upregulation in tumor suppression. Molecules 24, 4067. 
  22. Nakatogawa, H. 2020. Mechanisms governing autophagosome biogenesis. Nat. Rev. Mol. Cell. Biol. 21, 439-458.  https://doi.org/10.1038/s41580-020-0241-0
  23. Oglah, M. K., Mustafa, Y. F., Bashir,M. K. and Jasim, M. H. 2020. Curcumin and its derivatives: A review of their biological activities. Sys. Rev. Pharm. 11, 472-481. 
  24. Osaki, T., Uchida, Y., Hirayama, J. and Nishina, H. 2011. Diphenyleneiodonium chloride, an inhibitor of reduced nicotinamide adenine dinucleotide phosphate oxidase, suppresses light-dependent induction of clock and DNA repair genes in zebrafish. Biol. Pharm. Bull. 34, 1343-1347.  https://doi.org/10.1248/bpb.34.1343
  25. Ott, M., Norberg, E., Zhivotovsky, B. and Orrenius, S. 2009. Mitochondrial targeting of tBid/Bax: a role for the TOM complex?. Cell Death Differ. 16, 1075-1082.  https://doi.org/10.1038/cdd.2009.61
  26. Ou, L., Lin, S., Song, B., Liu, J., Lai, R. and Shao, L. 2017. The mechanisms of graphene-based materials-induced programmed cell death: a review of apoptosis, autophagy, and programmed necrosis. Int. J. Nanomedicine 12, 6633-6646. https://doi.org/10.2147/IJN.S140526
  27. Perillo, B., Di Donato, M., Pezone, A., Zazzo, E. D., Giovannelli, P., Galasso, G., Castoria, G. and Migliaccio, A. 2020. ROS in cancer therapy: the bright side of the moon. Exp. Mol. Med. 52, 192-203.  https://doi.org/10.1038/s12276-020-0384-2
  28. Redza-Dutordoir, M. and Averill-Bates, D. A. 2016. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim. Biophys. Acta 1863, 2977-2992.  https://doi.org/10.1016/j.bbamcr.2016.09.012
  29. Saltos, A., Shafique, M. and Chiappori, A. 2020. Update on the biology, management, and treatment of small cell lung cancer (SCLC). Front. Oncol. 10, 1074. 
  30. Steenaard, R. V., Rutjens, M., Ettaieb, M. H. T., Noesel, M. M. and Haak, H. R. 2022. EDP-mitotane in children: reassuring evidence of reversible side-effects and neurotoxicity. Discov. Onc. 13, 25. 
  31. Wang, Q., Zhang, L., Yuan, X., Ou, Y., Zhu, X., Cheng, Z., Zhang, P., Wu, X., Meng, Y. and Zhang, L. 2016. The relationship between the Bcl-2/Bax proteins and the mitochondria-mediated apoptosis pathway in the differentiation of adipose-derived stromal cells into neurons. PLoS One 11, e0163327. 
  32. Wang, X., Wei, L., Li, Q. and Lai, Y. 2022. HIF-1a protects osteoblasts from ROS-induced apoptosis. Free Radic. Res. 56, 143-153.  https://doi.org/10.1080/10715762.2022.2037581
  33. Wang, X. D., Hu, R., Ding, Q., Savage, T. K., Huffman, K. E., Williams, N., Cobb, M. H., Minna, J. D., Johnson, J. E. and Yu, Y. 2019. Subtype-specific secretomic characterization of pulmonary neuroendocrine tumor cells. Nat. Commun. 10, 3201. 
  34. World Health Organization. February 3, 2022. Cancer. https://www.who.int/en/news-room/fact-sheets/detail/cancer 
  35. Wu, Y., Zhao, D., Zhuang, J., Zhang, F. and Xu, C. 2016. Caspase-8 and caspase-9 functioned differently at different stages of the cyclic stretch-induced apoptosis in human periodontal ligament cells. PLoS One 11, e0168268. 
  36. Yang, H., Villani, R. M., Wang, H., Wang, H., Simpson, M. J., Roberts, M. S., Tang, M. and Liang, X. 2018. The role of cellular reactive oxygen species in cancer chemotherapy. J. Exp. Clin. Cancer Res. 37, 266. 
  37. Zhang, Q. Y., Wang, F. X., Jia, K. K. and Kong, L. D. 2018. Natural product interventions for chemotherapy and radiotherapy-induced side effects. Front. Pharmacol. 9, 1253. 
  38. Zhu, Y. and Bu, S. 2017. Curcumin induces autophagy, apoptosis, and cell cycle arrest in human pancreatic cancer cells. Evid. Based Complement. Alternat. Med. 2017, 5787218.