Sophia P M Sok1, Norhafiza M Arshad1,2, Mohamad Nurul Azmi3, Khalijah Awang3, Bulent Ozpolat4, Noor Hasima Nagoor1,2. 1. Institute of Biological Sciences (Genetics and Molecular Biology), Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. 2. Centre for Research in Biotechnology for Agriculture (CEBAR), University of Malaya, Kuala Lumpur, Malaysia. 3. Centre of Natural Product Research and Drug Discovery (CENAR), Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. 4. Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
Abstract
Autophagy plays a role in deciding the fate of cells by inducing either survival or death. 1'S-1-acetoxychavicol acetate (ACA) is a phenylpropanoid isolated from rhizomes of Alpinia conchigera and has been reported previously on its apoptotic effects on various cancers. However, the effect of ACA on autophagy remains ambiguous. The aims of this study were to investigate the autophagy-inducing ability of ACA in human non-small cell lung cancer (NSCLC), and to determine its role as pro-survival or pro-death mechanism. Cell viability assay was conducted using MTT. The effect of autophagy was assessed by acridine orange staining, GFP-LC3 punctate formation assay, and protein level were analysed using western blot. Annexin V-FITC/PI staining was performed to detect percentage of cells undergoing apoptosis by using flow cytometry. ACA inhibits the cell viability and induced formation of cytoplasmic vacuoles in NSCLC cells. Acidic vesicular organelles and GFP-LC3 punctate formation were increased in response to ACA exposure in A549 and SK-LU-1 cell lines; implying occurrence of autophagy. In western blot, accumulation of LC3-II accompanied by degradation of p62 was observed, which further confirmed the full flux of autophagy induction by ACA. The reduction of Beclin-1 upon ACA treatment indicated the Beclin-1-independent autophagy pathway. An early autophagy inhibitor, 3-methyaldenine (3-MA), failed to suppress the autophagy triggered by ACA; validating the existence of Beclin-1-independent autophagy. Silencing of LC3-II using short interfering RNA (siRNA) abolished the autophagy effects, enhancing the cytotoxicity of ACA through apoptosis. This proposed ACA triggered a pro-survival autophagy in NSCLC cells. Consistently, co-treatment with lysosomal inhibitor, chloroquine (CQ), exerted a synergistic effect resulting in apoptosis. Our findings suggested ACA induced pro-survival autophagy through Beclin-1-independent pathway in NSCLC. Hence, targeting autophagy pathway using autophagy inhibitor such as CQ represented a novel promising approach to potentiate the cytotoxicity of ACA through apoptosis in NSCLC.
Autophagy plays a role in deciding the fate of cells by inducing either survival or death. 1'S-1-acetoxychavicol acetate (ACA) is a phenylpropanoid isolated from rhizomes of Alpinia conchigera and has been reported previously on its apoptotic effects on various cancers. However, the effect of ACA on autophagy remains ambiguous. The aims of this study were to investigate the autophagy-inducing ability of ACA in humannon-small cell lung cancer (NSCLC), and to determine its role as pro-survival or pro-death mechanism. Cell viability assay was conducted using MTT. The effect of autophagy was assessed by acridine orange staining, GFP-LC3 punctate formation assay, and protein level were analysed using western blot. Annexin V-FITC/PI staining was performed to detect percentage of cells undergoing apoptosis by using flow cytometry. ACA inhibits the cell viability and induced formation of cytoplasmic vacuoles in NSCLC cells. Acidic vesicular organelles and GFP-LC3 punctate formation were increased in response to ACA exposure in A549 and SK-LU-1 cell lines; implying occurrence of autophagy. In western blot, accumulation of LC3-II accompanied by degradation of p62 was observed, which further confirmed the full flux of autophagy induction by ACA. The reduction of Beclin-1 upon ACA treatment indicated the Beclin-1-independent autophagy pathway. An early autophagy inhibitor, 3-methyaldenine (3-MA), failed to suppress the autophagy triggered by ACA; validating the existence of Beclin-1-independent autophagy. Silencing of LC3-II using short interfering RNA (siRNA) abolished the autophagy effects, enhancing the cytotoxicity of ACA through apoptosis. This proposed ACA triggered a pro-survival autophagy in NSCLC cells. Consistently, co-treatment with lysosomal inhibitor, chloroquine (CQ), exerted a synergistic effect resulting in apoptosis. Our findings suggested ACA induced pro-survival autophagy through Beclin-1-independent pathway in NSCLC. Hence, targeting autophagy pathway using autophagy inhibitor such as CQ represented a novel promising approach to potentiate the cytotoxicity of ACA through apoptosis in NSCLC.
Lung cancer is the most common cancer worldwide; accounting for 1.82 million new cases and 1.6 million deaths in 2012 [1]. Among the lung cancer cases, non-small cell lung cancer (NSCLC) contributes to approximately 85% and has a low 5-year survival rate [2]. Conventional cancer therapies such as surgery, chemotherapy and radiotherapy were found to have limitation in maintaining its effectiveness during the course of therapy which lead to recurrence and acquired apoptosis resistance in long term treatment [3]. Hence, it is crucial to elucidate the underlying reason to improve the efficiency of the available therapeutic agents. Emerging evidences proposed that identifying the role played by autophagy in cancer could be a strategy to overcome resistance towards chemotherapy due to the fact its potential in eliciting a pro-survival or pro-death effect in response to metabolic and therapeutic stresses [4, 5].Autophagy is a self-eating mechanism that is highly regulated by a set of autophagy-related (atg) genes to maintain the homeostasis in the cells through recycling of cellular components upon lysosomal degradation [6, 7]. Unlike canonical autophagy, a non-canonical autophagy occurs when certain ATG proteins such as ULK1, Beclin-1, class III phosphotidylinositol-3-kinase (PI3K) and Atg conjugation system are being bypassed during initiation of autophagy and formation of autophagosome [6, 7]. It was believed that autophagy induction often occur prior apoptosis to provide energy to allow survival of cancerous cells in response to stress such as nutrient deprivation or chemotherapy stress [8, 9]. Conversely, natural bioactive compounds such as silvestrol and guttiferon K were proven to elicit autophagy that lead to cell death [10, 11]. To date, the role of autophagy in cancer remains ambiguous due to the complex cross-talk machinery with apoptosis. Understanding of this mechanism is crucial in developing effective cancer therapy as it is a key regulator in determining the cell fate.Natural products have attracted the attention of researchers due to their anti-cancer property. 1’S-1-acetoxychavicol acetate (ACA) is a phenylpropanoid isolated from rhizomes of Alpinia conchigera Griff. Our group had previously reported the anti-cancer effects of ACA in breast (MCF-7), oral (HSC-2 and HSC-4), liver (HepG2), cervical (CaSki), lung cancer (A549) and prostate carcinoma (PC-3) via inducing apoptosis with minimal cytotoxic effect on normal human mammary cells (HMEC) and no physiological alteration in in vivo model [12-14]. It was reported that ACA targets NF-κB signalling pathway to alter the pro-inflammatory microenvironment environment both in vitro and in vivo [12, 14]. Despite numerous reports on its direct interaction on signalling pathway, ACA can modulate epigenetic machinery in cancer by altering miRNA expression that eventually has an impact in the gene expression [15]. Moreover, a synergistic anti-cancer effect was further observed in combination treatment of ACA with cisplastin or recombinant human alpha fetoprotein [12, 14, 15]. These studies revealed ACA as a potential anti-cancer remedy. Although it was known that ACA induced cytotoxicity against wide range of cancer types, its effect on autophagy remains ambiguous.The aims of this study were to investigate the autophagy-inducing ability of ACA in humanNSCLC. Effect of ACA on cytotoxicity and apoptosis induced was assessed after administering autophagy inhibitors and small interfering RNA (siRNA). Role of ACA-induced autophagy as pro-survival or pro-death mechanism was determined. In present study, we have shown that ACA triggered autophagy through non-canonical pathway in NSCLC and autophagy inhibitors can potentiate the cytotoxicity of ACA through apoptosis. Our results suggested autophagy inhibitors as a potential strategy to improve the efficiency of the ACA in future.
Materials and methods
Plant material
Rhizomes of wild Alpinia conchigera Griff were collected from Jeli (5°42′N 101°50′E) province of Kelantan, east-coast of Penisular Malaysia. No specific permission was required for collecting the sample from this site as they are wildly grown and thi field study did not involve endangered or protected species. The sample was identified by Prof. Dr. Halijah Ibrahim from Institute of Biological Science, University of Malaya. A voucher specimen (KL5049) was deposited in Herbarium of Chemistry Department, Faculty of Sciece, University of Malaya.
Chemicals and antibodies
ACA was dissolved in dimethyl sulfoxide (DMSO; Fisher Scientific, USA) to serve as a stock solution. The autophagy inhibitors used were 3-methyladenine (3-MA; Calbiochem, USA) and chloroquine diphosphate (CQ; Sigma, USA). The 3-MA was dissolved directly in culture media and the stock solution CQ was prepared in distilled water. Acridine orange solution was obtained from Sigma. Roswell Park Memorial Institute-1640 (RPMI-1640) medium was purchased from GE Healthcare HyClone (USA) whereas alpha-Minimum Essential Medium (α-MEM) was obtained from Nacalai Tesque (Japan). Fetal bovine serum (FBS) was from Sigma (USA).Primary antibodies included rabbit antibodies against microtubule associated protein 1 light chain 3-I/II (LC3-I/II), p62, GAPDH and mouse antibodies against Beclin-1 were used. Anti-rabbit IgG and anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody were used. All the antibodies were purchased from Cell Signaling Technology (USA).
Cell culture
Two humanNSCLC cells lines A549 was purchased from America Type Culture Collection (ATCC, USA) and SK-LU-1 was purchased from AseaCycte Sdn. Bhd., Malaysia. A549 cells were cultured in RPMI-1640 medium while SK-LU-1 cells were cultured in α-MEM medium. The media were supplemented with 10% (v/v) FBS and cells maintained at 37°C incubator with 5% CO2. A non-tumourigenic human mammary epithelial cells, MCF 10A was purchased from ATCC and cultured in Mammary Epithelial Cell Growth Medium (MEGM) supplemented with 100 ng/ml cholera toxin.
MTT cell viability assay
Briefly, cells were seeded at a density of 1×104 cells/well in 96-well plate and treated with ACA (0–30 μM) for 24 h. Following incubation, MTT (5 mg/ml) (Merck, Germany) with a volume of 10 μl was added to each well and the cells were incubated at 37°C incubator for 1 h. The formazan crystal was dissolved in 200 μl of DMSO. The absorbance reading at 560 nm with background subtraction at 630 nm was measured by Tecan Sunrise microtitre plate reader using Magellan Software Version 6.3. The IC50 of ACA for each of the cell lines was then determined and this IC50 was used for the downstream experiments in this study. MCF 10A was used to determine the toxicity of ACA on non-tumourigenic cells. To investigate the time-dependent effect, the cell viability of A549 and SK-LU-1 cells at IC50 value was assessed from 0–24 h. To evaluate the effect of autophagy on ACA-induced cell death, the cells were pre-treated with siLC3 (20 nM) or CQ (20 μM) for 4 h, followed by addition of IC50 concentration of ACA for 24 h and the cell viability was measured.
Morphological observation
Cells were seeded and treated with ACA, 3-MA, CQ standalone or in combination of ACA with inhibitors. At 24 h, the morphology of cells was examined under a bright field Nikon Eclipse TS100 inverted fluorescent microscope (Nikon, Japan) at 200 × magnification.
Acidic Vesicular Organelles (AVO) staining
Cells were plated in 6-well plate and incubated overnight at 37°C incubator with 5% CO2 for adherence. The following day, cells were incubated with ACA for 0, 3, 6, 12 and 24 h or in presence of siLC3 or CQ for 24 h. At indicated time point, cells incubated with acridine orange (AO) (1μg/ml) (Sigma-Aldrich, USA) at 37°C for 15 minutes. The cells were observed under Nikon Eclipse TS100 inverted fluorescent microscope (Nikon, Japan) using a blue filter (B-2A) at 400 × magnification. The red relative fluorescence intensity (RFI) which represent the AVO was measured using Nikon NIS-BR Element software.
Analysis of GFP-LC3-II
Cells were seeded at in 6-well plate and incubate overnight in 37°C incubator with 5% CO2 for attachment. All cells were transduced with RFP-GFP-LC3-II reagent using commercially available Premo Autophagy Tandem Sensor RFP-GFP-LC3-II Kit (Life technologies, USA) and treated with ACA for 0, 3 and 6 h or in presence of siLC3 or CQ for 6 h. At indicated time point, the cells were visualized under Nikon Eclipse TS100 inverted fluorescent microscope (Nikon, Japan) using a blue filter at 400 × magnification to detect the neutral pH autophagosome with GFP emission. Cells with five GFP-LC3-II dots were regarded as positive cells. Quantitation of GFP-LC3-II was examined by counting the number of cells with positive punctate dots in a total of 200 GFP-LC3-II cells.
Western blot analysis
Cellular proteins were extracted using NE-PER® Nuclear & Cytoplasmic Extraction Kit (Pierce, USA) according to manufacturer’s protocol. The lysate proteins were quantitated using bicinchoninic acid (BCA) protein assay kit (Thermo, USA) according to manufacturer’s instruction. For western blot analysis, 20 μg of lysate proteins were resolved using 12% sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was incubated in relevant primary antibody overnight at 4°C at appropriate dilution. Anti-GAPDH antibody was used at a 1:10000 dilution whereas anti-LC3-I/II, anti-p62, and anti-Beclin-1 were used at a 1:1000 dilution. On the following day, membranes were incubated with anti-biotin and HRP-conjugated secondary antibody at a 1:1:1000 dilution at room temperature with agitation. Detection of bound antibody was done by subjecting the membrane to 1: 1 of Western Bright Quantum components (Advansta, USA) for 2 min in the dark. Protein bands were visualized using a chemiluminescent imaging system (Fusion FX7). GAPDH was used for normalization of band intensity by using ImageJ v1.48 (NIH, USA) densitometry software.
RNA interference
The cells were seeded 24 h before transfection. Small interfering RNA (siRNA) against LC3 (siLC3) (Origene, USA) or universal scrambled negative control siRNA (OriGene, USA) were transfected at a concentration of 20 nM using Dharmfect I tranfection reagent (Dharmacon, USA) according to the manufacturer’s manual. The knockdown efficiency of 3 unique siRNA duplexes was examined using western blot analysis and the one with best knockdown efficiency was selected for the subsequent experiments. Sequence of the 3 unique siRNA duplexes is shown in Table 1.
Table 1
Sequence of three unique 27 mer siRNA duplexes targeting LC3 (OriGene, USA).
siRNA
Duplex sequence
A
5’-CCUGUAUACGUUAGUGAAAGCUGTT-3’
B
5’-UACAGAUACUAAUGUCAAGAGUUAA-3’
C
5’-CGUUUAGACUGUAUACAUCAUAUCT-3’
Annexin V-FITC/PI apoptosis assay
Apoptosis was measured by annexin V-FITC apoptosis detection kit (Calbiochem, USA) according to the manufacturer’s instruction. Briefly, the seeded cells were pre-treated with siLC3 (20 nM) or CQ (20 μM) (Sigma-Aldrich, USA) for 4 h, followed by addition of IC50 concentration of ACA for 24 h. At indicated time point, both floating and attached cells were harvested, washed with cold PBS, and followed by incubation with 500 μl binding buffer containing 1.25 μl of annexin V-FITC and 10 μl of propidium iodide (PI) in the dark. The samples were analysed using BD FACSCantoII flow cytometer (BD Biosciences, USA) with BD FACSDiva (BD Biosciences, USA) software.
Statistical analysis
The data are presented as mean ± standard deviation (SD) of at least three independent experiments. The statistical data analysis was performed using one-way ANOVA, followed by post-hoc Turkey analysis, with a p value of < 0.05 indicating a statistically significant difference.
Results
ACA inhibited the cell viability in NSCLC cell lines
We have previously characterized the structure of ACA (Fig 1A) and reported on its cytotoxicity towards various cancer cell lines [13]. In current study, the cytotoxic effect of ACA treatment in NSCLC cell lines (A549 and SK-LU-1) was investigated by exposing the cells to increasing doses of ACA (0–30 μM) for 24 h. Results indicated ACA elicited a dose-dependent cytotoxicity in both A549 and SK-LU-1 cell lines. The IC50 value of ACA in A549 and SK-LU-1 cells were 29.2 ± 1.4 μM and 25.0 ± 1.0 μM respectively. This implied ACA is slightly more sensitive towards SK-LU-1 in comparison to A549 cell lines (Fig 1B). Based on the results from MTT assay, the IC50 values of respective cell lines at 24 h were used for subsequent experiments. On the other hand, the cell viability of MCF 10A maintains above 75% after treated with ACA at 30 μM for 24 h with undetermined IC50. We also conducted a time-dependent study (0–24 h) of ACA at IC50 value in A549 and SK-LU-1 cell line. In Fig 1C, the cell viability of A549 declined more rapidly in comparison to SK-LU-1 at their respective IC50 value for ACA (Fig 1C). Under microscopic examination, ACA induced morphological changes in cells as compared to the untreated group with increased cytoplasmic vacuoles in both NSCLC cell lines (Fig 1D); suggesting the possibility of autophagy induction.
Fig 1
ACA inhibited cell viability of A549 and SK-LU-1 cell lines.
(A) Chemical structure of 1’S-1’-acetoxychavicol acetate (ACA). (B) The cell viability of MCF 10A, A549 and SK-LU-1 cells lines after exposure to ACA (0–30 μM) for 24 h was assessed using MTT assay. Data represented as mean percentage of cell viability ± SD for three independent experiments. (C) The cell viability of A549 and SK-LU-1 cells lines after exposure to IC50 of ACA (25 μM for SK-LU-1 and 30 μM for A549 cells) on respective cell lines for 0–24 h was assessed using MTT assay. Data represented as mean percentage of cell viability ± SD for three independent experiments. * p < 0.05 and ** p < 0.01 statistically different in comparison to 0 h (D) Representative photomicrograph (200 × magnification) of A549 and SK-LU-1 cell lines upon ACA treatment. Arrow indicates the cytoplasmic vacuole.
ACA inhibited cell viability of A549 and SK-LU-1 cell lines.
(A) Chemical structure of 1’S-1’-acetoxychavicol acetate (ACA). (B) The cell viability of MCF 10A, A549 and SK-LU-1 cells lines after exposure to ACA (0–30 μM) for 24 h was assessed using MTT assay. Data represented as mean percentage of cell viability ± SD for three independent experiments. (C) The cell viability of A549 and SK-LU-1 cells lines after exposure to IC50 of ACA (25 μM for SK-LU-1 and 30 μM for A549 cells) on respective cell lines for 0–24 h was assessed using MTT assay. Data represented as mean percentage of cell viability ± SD for three independent experiments. * p < 0.05 and ** p < 0.01 statistically different in comparison to 0 h (D) Representative photomicrograph (200 × magnification) of A549 and SK-LU-1 cell lines upon ACA treatment. Arrow indicates the cytoplasmic vacuole.
ACA induced autophagy in NSCLC cells
To investigate the induction of autophagy, AO staining was used to detect the presence of acidic vesicular organelles (AVO). AO stain will emit red fluorescence in acidic vesicles organelles whereas green fluorescence in non-acidic compartment such as cytoplasm and nucleus [16]. After ACA treatment, both AO stained A549 and SK-LU-1 cells exhibited a significant increase in red fluorescence signal that indicated the presence of AVO in comparison to untreated cells (Fig 2A and S1 Fig).
Fig 2
Autophagy effect of ACA on A549 and SK-LU-1 cell lines.
(A) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treated with ACA for 0, 3, 6, 12, and 24 h. Data were presented as relative fluorescence intensity in comparison to untreated cells ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 statistically different in comparison to untreated. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to ACA. Data were represented as mean percentage of cells with GFP-LC3-II punctate ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 statistically different in comparison to untreated. (C) Protein expression of LC3-I/II and p62 after ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as mean normalized intensity ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 statistically different in comparison to untreated.
Autophagy effect of ACA on A549 and SK-LU-1 cell lines.
(A) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treated with ACA for 0, 3, 6, 12, and 24 h. Data were presented as relative fluorescence intensity in comparison to untreated cells ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 statistically different in comparison to untreated. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to ACA. Data were represented as mean percentage of cells with GFP-LC3-II punctate ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 statistically different in comparison to untreated. (C) Protein expression of LC3-I/II and p62 after ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as mean normalized intensity ± SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 statistically different in comparison to untreated.Autophagy activity of ACA was verified by localizing the LC3, a marker of autophagosome, by observing the formation of GFP-LC3-II punctate in the cells. ACA exerted an accumulation of GFP-LC3 punctate in comparison to the diffused pattern of green fluorescence seen in untreated cells (Fig 2B and S1 Fig). Consistent with the results of GFP-LC3-II punctate formation analysis, elevated LC3-II protein expression in A549 and SK-LU-1 cell lines was observed after ACA treatment (Fig 2C). The LC3-II protein expression upon ACA exposure peaked earlier at 6 h in A549 cells compared to 12 h in SK-LU-1 cells. This may indicate that ACA promotes the recruitment of LC3-II into autophagosomes earlier in A549 cells. In the untreated SK-LU-1 cells, a basal level of GFP-LC3-II punctate was seen. However. this level was significantly increased at 3 h (p < 0.05) and 6 h (p < 0.01) when treated with ACA.In order to monitor the autophagy flux, we further assessed the degradation of p62 protein expression. Impaired autophagy will lead to accumulation of p62; whereas reduction in expression of p62 indicates an effective autophagy flux [17]. In this study, we noted ACA down-regulated the protein expression of p62 in A549 and SK-LU-1 cells in a time-dependent manner (Fig 2C); indicating a functional autophagy machinery was activated by ACA in these cell lines.
ACA-induced autophagy is independent of Beclin-1/PI3K complex in NSCLC cell lines
Studies have shown initiation of autophagy can be Beclin-1-dependent (canonical) or Beclin-1-independent (non-canonical) [7]. To examine the involvement of Beclin-1 in ACA-induced autophagy, we first examined the expression of Beclin-1 protein level using western blot. Our data demonstrated that ACA treatment reduced the expression of Beclin-1 in a time-dependent manner in A549 and SK-LU-1 cells (Fig 3A). Reduction in Beclin-1 protein expression was reported to be representative characteristic of a non-canonical autophagy pathway [18, 19]. Hence, we hypothesized ACA triggered autophagy bypasses the Beclin-1/PI3K complex. In order to verify the non-involvement of Beclin-1/PI3K complex in ACA-induced autophagy, we blocked the Beclin-1/PI3K complex in early process of autophagy using a PI3K class III inhibitor, 3-methyladenine (3-MA). Previous studies showed that 3-MA with concentration of 5 mM can be used to inhibit the early autophagy in lung cancer cells [20, 21]. In these investigations, 3-MA standalone does not show a decrease in basal autophagy and LC3-I expression. Our current study demonstrated administration of 3-MA did not block the red relative fluorescence intensity (RFI) of AO induced by ACA (Fig 3B and S2 Fig), indicating non-involvement of Beclin-1/PI3K complex. Consistently, western blot and GFP-LC3-II revealed that pre-treatment with 3-MA prior to ACA treatment failed to suppress LC3-II protein expression (Fig 3C and 3D and S2 Fig). This further verified that ACA induced Beclin-1/PI3K-independent autophagy. MTT assay was conducted using 3-MA standalone, ACA standalone and combination of 3-MA with ACA to assess the effect of 3-MA on cell viability. The cell viability was maintained at similar level, which is from 55.4 ± 7.9% to 54.7 ± 9.4% and from 50.8 ± 4.2% to 47.0 ± 4.1% in A549 and SK-LU-1cell lines respectively with pre-treatment of 3-MA prior to ACA treatment (Fig 3E). This indicated that 3-MA has no significant effect on cell viability of A549 and SK-LU-1 cells; most probably attributed by the autophagy flux that could not be inhibited using 3-MA.
Fig 3
ACA-induced autophagy is independent of Beclin-1/PI3K complex in A549 and SK-LU-1 cell lines.
(A) Protein expression of Beclin-1 after ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as mean normalized intensity against GAPDH ± SD. *** p < 0.001 statistically different in comparison to untreated. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treatment with ACA in presence or absence of 3-MA. Data were presented as relative fluorescence intensity in comparison to untreated cells ± SD of three independent experiments * p < 0.05 and ** p < 0.01 statistically different in comparison to untreated. # p < 0.05 and ## p < 0.01 statistically different in comparison to 3-MA. (C) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to co-treatment of 3-MA and ACA. Data were represented as mean percentage of cells with GFP-LC3-II punctate ± SD of three independent experiments. *** p < 0.001 statistically different in comparison to untreated. ### p < 0.001 statistically different in comparison to 3-MA. (D) Effect of 3-MA on LC3-I/LC3-II protein expression after pre-treatment of 3-MA prior to ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as normalized intensity ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 statistically different in comparison to untreated. ## p < 0.01 and ### p < 0.001 statistically different in comparison to 3-MA. (E) Effect of 3-MA on the cell viability of ACA-treated A549 and SK-LU-1 cell lines. Data represented as mean percentage of cell viability ± SD for three independent experiments. * p < 0.05 and *** p < 0.001 statistically different in comparison to untreated.
ACA-induced autophagy is independent of Beclin-1/PI3K complex in A549 and SK-LU-1 cell lines.
(A) Protein expression of Beclin-1 after ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as mean normalized intensity against GAPDH ± SD. *** p < 0.001 statistically different in comparison to untreated. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treatment with ACA in presence or absence of 3-MA. Data were presented as relative fluorescence intensity in comparison to untreated cells ± SD of three independent experiments * p < 0.05 and ** p < 0.01 statistically different in comparison to untreated. # p < 0.05 and ## p < 0.01 statistically different in comparison to 3-MA. (C) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to co-treatment of 3-MA and ACA. Data were represented as mean percentage of cells with GFP-LC3-II punctate ± SD of three independent experiments. *** p < 0.001 statistically different in comparison to untreated. ### p < 0.001 statistically different in comparison to 3-MA. (D) Effect of 3-MA on LC3-I/LC3-II protein expression after pre-treatment of 3-MA prior to ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as normalized intensity ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 statistically different in comparison to untreated. ## p < 0.01 and ### p < 0.001 statistically different in comparison to 3-MA. (E) Effect of 3-MA on the cell viability of ACA-treated A549 and SK-LU-1 cell lines. Data represented as mean percentage of cell viability ± SD for three independent experiments. * p < 0.05 and *** p < 0.001 statistically different in comparison to untreated.
Knockdown of LC3 enhanced ACA-induced cytotoxicity through apoptosis
To clarify the role of ACA-induced Beclin-1/PI3K-independent autophagy in cell death, we inhibited the process of autophagy in NSCLC cells by reducing LC3 expression, which is the key player in forming autophagsome. First, the efficiency of the 3 unique siRNA duplexes targeting LC3, siRNA3 A, B and C were assessed using western blot. siRNA A targeting LC was selected for the downstream experiments due to its best knockdown ability in both A549 and SK-LU-1 cells (Fig 4A). Result showed siRNA A inhibited the protein expression of LC3 successfully in both NSCLC cell lines even when ACA was presented (Fig 4B). After transfected with siLC3, the cell viability upon ACA exposure was decreased from 40.8 ± 6.9% to 20.8 ± 1.7% in A549 cells, while 51.0 ± 6.3% to 35.1 ± 1.3% in SK-LU-1 cells (Fig 4C). Administration of ACA increased apoptosis in LC3 silenced NSCLC cells (Fig 4D). This implies that the autophagy mediated by ACA limits the death inducing effect in these cells and was taking on a pro-survival role.
Fig 4
Knockdown of LC3 enhanced ACA-induced cytotoxicity through apoptosis.
(A) Knockdown efficiency of the three unique 27 mer siRNA duplexes targeting LC3 in A549 and SK-LU-1. Data were represented as mean percentage of relative intensity ± SD of three independent experiments. * p < 0.05 and *** p < 0.001 statistically different in comparison to negative control (NC). (B) Effect of siLC3 on ACA-induced LC3-I/LC3-II protein expression in A549 and SK-LU-1 cell lines. Data were represented as normalized intensity ± SD of three independent experiments. *** p < 0.001 statistically different in comparison to negative control (NC). (C) Effect of siLC3 on the cell viability of ACA-treated A549 and SK-LU-1 cell lines. Data represented as mean percentage of cell viability ± SD for three independent experiments. ** p < 0.01 and *** p < 0.001 statistically different in comparison to negative control (NC). # p < 0.05 and ### p < 0.001 statistically different in comparison to NC + ACA. (D) Effect of siLC3 on ACA-induced apoptotic cells in A549 and SK-LU-1 cell lines. Representative annexin V-FITC/PI scatter plots of 1 × 104 cells after 24 h of treatment. Data represented as mean percentage of apoptotic cells ± SD for three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 statistically different in comparison to negative control (NC). ### p < 0.001 statistically different in comparison to NC + ACA.
Knockdown of LC3 enhanced ACA-induced cytotoxicity through apoptosis.
(A) Knockdown efficiency of the three unique 27 mer siRNA duplexes targeting LC3 in A549 and SK-LU-1. Data were represented as mean percentage of relative intensity ± SD of three independent experiments. * p < 0.05 and *** p < 0.001 statistically different in comparison to negative control (NC). (B) Effect of siLC3 on ACA-induced LC3-I/LC3-II protein expression in A549 and SK-LU-1 cell lines. Data were represented as normalized intensity ± SD of three independent experiments. *** p < 0.001 statistically different in comparison to negative control (NC). (C) Effect of siLC3 on the cell viability of ACA-treated A549 and SK-LU-1 cell lines. Data represented as mean percentage of cell viability ± SD for three independent experiments. ** p < 0.01 and *** p < 0.001 statistically different in comparison to negative control (NC). # p < 0.05 and ### p < 0.001 statistically different in comparison to NC + ACA. (D) Effect of siLC3 on ACA-induced apoptotic cells in A549 and SK-LU-1 cell lines. Representative annexin V-FITC/PI scatter plots of 1 × 104 cells after 24 h of treatment. Data represented as mean percentage of apoptotic cells ± SD for three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 statistically different in comparison to negative control (NC). ### p < 0.001 statistically different in comparison to NC + ACA.
Autophagy inhibitor chloroquine enhanced ACA-induced cytotoxicity through apoptosis
We further investigated the effect of the inhibitor, chloroquine (CQ) that inhibits lysosomal turnover of autophagosome contents during the ACA-induced autophagy. Administration of CQ (20 μM) alone blocked the fusion of autophagosome and lysosome; leading to accumulation of LC3-II formation and lysosomes. The RFI of ACA in combination with CQ was increased in NSCLC cells; indicating the increase in acidic compartments (Fig 5A and S3 Fig). GFP-LC3-II punctate and western blot showed that CQ further elevated the LC3-II protein level induced by ACA (Fig 5B and 5C and S3 Fig). These data confirmed that ACA stimulated autophagy. In the MTT assay, cell viability was reduced significantly from 53.3 ± 1.0% to 46 ± 2.4% and 53.6 ± 2.9% to 45 ± 2.9% in A549 and SK-LU-1cell lines respectively with pre-treatment of CQ prior to ACA treatment (Fig 5D). In addition, annexin V-FITC/PI staining disclosed that pre-treatment with CQ increased ACA-induced apoptotic cells (Fig 5E). These results proposed the pharmacological inhibition of autophagy using autophagy inhibitor such as CQ can potentiate the cytotoxic ability of ACA to induce apoptosis; thus giving a new perspective for improvement of chemotherapeutic outcome in the future.
Fig 5
Autophagy inhibitor chloroquine enhanced ACA-induced cytotoxicity through apoptosis.
(A) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treatment with ACA in presence or absence of CQ. Data were presented as relative fluorescence intensity in comparison to untreated cells ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 statistically different in comparison to untreated. # p < 0.05 statistically different in comparison to CQ. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to co-treatment of CQ and ACA. Data were represented as mean percentage of cells with GFP-LC3-II punctate ± SD of three independent experiments. *** p < 0.001 statistically different in comparison to untreated. # p < 0.05 and ### p < 0.001 statistically different in comparison to CQ. (C) Effect of CQ on LC3-I/LC3-II protein expression after pre-treated with CQ prior ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as normalized intensity ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 statistically different in comparison to untreated. # p < 0.05 and ## p < 0.01 statistically different in comparison to CQ. (D) Effect of CQ on the cell viability of ACA-treated A549 and SK-LU-1 cell lines. Data represented as mean percentage of cell viability ± SD for three independent experiments. *** p < 0.001 statistically different in comparison to untreated. ### p < 0.001 statistically different in comparison to ACA. (E) Effect of CQ on ACA-induced apoptotic cells in A549 and SK-LU-1 cell lines. Representative annexin V-FITC/PI scatter plots of 1 × 104 cells after 24 h of treatment. Data represented as mean percentage of apoptotic cells ± SD for three independent experiments. *** p < 0.001 statistically different in comparison to untreated. # p < 0.05 and ### p < 0.001 statistically different in comparison to ACA.
Autophagy inhibitor chloroquine enhanced ACA-induced cytotoxicity through apoptosis.
(A) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treatment with ACA in presence or absence of CQ. Data were presented as relative fluorescence intensity in comparison to untreated cells ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 statistically different in comparison to untreated. # p < 0.05 statistically different in comparison to CQ. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to co-treatment of CQ and ACA. Data were represented as mean percentage of cells with GFP-LC3-II punctate ± SD of three independent experiments. *** p < 0.001 statistically different in comparison to untreated. # p < 0.05 and ### p < 0.001 statistically different in comparison to CQ. (C) Effect of CQ on LC3-I/LC3-II protein expression after pre-treated with CQ prior ACA treatment in A549 and SK-LU-1 cell lines. Data were represented as normalized intensity ± SD of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 statistically different in comparison to untreated. # p < 0.05 and ## p < 0.01 statistically different in comparison to CQ. (D) Effect of CQ on the cell viability of ACA-treated A549 and SK-LU-1 cell lines. Data represented as mean percentage of cell viability ± SD for three independent experiments. *** p < 0.001 statistically different in comparison to untreated. ### p < 0.001 statistically different in comparison to ACA. (E) Effect of CQ on ACA-induced apoptotic cells in A549 and SK-LU-1 cell lines. Representative annexin V-FITC/PI scatter plots of 1 × 104 cells after 24 h of treatment. Data represented as mean percentage of apoptotic cells ± SD for three independent experiments. *** p < 0.001 statistically different in comparison to untreated. # p < 0.05 and ### p < 0.001 statistically different in comparison to ACA.
Discussion
The naturally occurring ginger compound, ACA, has been extensively studied which revealed its potential as an anti-cancer agent through activation of apoptosis [12-14]. However, the ability of ACA to induce autophagy in NSCLC remains unclear. Our current study revealed that ACA induced autophagy in NSCLC as evidenced by the accumulation of AVO and GFP-LC3-II along with elevated LC3-II protein level indicating the recruitment LC3-II on autophagosomes. Furthermore, degradation of p62 observed further implied that ACA induced autophagy flux. This is the first study reporting on autophagy inducing ability of ACA in NSCLC. Natural occurring compounds such as curcumin and resveratrol were also found to activate autophagy and/or apoptosis via reactive oxygen species (ROS) pathway in oral and colon carcinoma respectively [22-24]. Previous studies reported that ACA can stimulate the production of ROS in myeloid leukemia and hepatocellular carcinoma [25, 26]. This might be the possible mechanism of ACA regulating autophagy in both A549 and SK-LU-1 cells.Autophagy is governed by a series of ATG proteins for initiation and followed by formation of autophagosome. One of the major components during formation of autophagosome in canonical autophagy pathway is PI3K complex which consists of Beclin-1, Vps34 and UVRAG/Atg14. Substantial studies demonstrated activation of autophagy can be in a Beclin-1/PI3K-independent manner. For instance, gossypol can initiate autophagy in the absence of Beclin-1 in HeLa cells but dependent on Vps34 and Atg5; verifying Beclin-1-independence [27]. Earlier study also provided evidence on existence of Beclin-1-independent autophagy induced by resveratrol in humanbreast cancer cells when 3-MA was unable to suppress autophagy [28]. It was reported autophagy activated by proteasome inhibitors and arsenic trioxide is concomitant with reduction in Beclin-1 expression in ovarian carcinoma cells; suggesting a Beclin-1-independent pathway [29, 30]. These studies provided evidences on the existence of non-canonical autophagy. In the current study, we found that autophagy induced by ACA was independent of Beclin-1/PI3K complex which was reflected in the down regulation of Beclin-1. Furthermore, 3-MA, a PI3K inhibitor was unable to block ACA-induced autophagy and did not potentiate the cytotoxic effect of ACA. An earlier report has identified NF-κB p65/Rel A as a positive regulator of Beclin-1 expression [31]. Since ACA was found to inhibit p65/Rel A previously [14], it is possible that the reduction of Beclin-1 observed upon ACA treatment in this study was as a result of the low expression of p65/Rel A. However, the underlying mechanism for formation of autophagosome which bypass the Beclin-1/PI3K complex and the specific function of the non-canonical autophagy in cancer remains to be further investigated.Autophagy is a double-edged sword that plays a significant role in tumourigenesis and cancer therapeutics. Hence, anti-cancer agents that modulate autophagy may have an important implication in clinical application. Anti-cancer agents have been found to induce pro-survival autophagy which eventually contributes to the chemoresistance in NSCLC. Innate resistance to erlotinib in NSCLC with wild-type epidermal growth factor receptor (EGFR) was as a result of autophagy [32]. Likewise, gefitinib which is initially effective for inducing death in NSCLC, was seen to develop resistance due to the pro-survival autophagy [33]. Morover, a recent finding published by Wang et al. indicated knock down of LC3 uing siRNA has a significant impact on the cell viability of cancerous cells; suggesting LC3 plays an important role in controlling the cell death [34]. These implied that targeting genes responsible for autophagy can be a strategy for treating chemoresistance that resulted from protective autophagy. In the current study, we observed that the ACA-induced apoptosis was enhanced in A549 and SK-LU-1 cells when LC3 was silenced. It reflected that autophagy induced by ACA in NSCLC is limiting the apoptotic cell death.Studies have shown that gefitinib- and erlotinib-resistance in NSCLC can be resolved by combine therapy with CQ [32, 33]. For instance, Zou and coworkers reported that CQ has no growth inhibitory effect on NSCLC even in prolong erlotinib treatment. It also potentiated the anti-cancer effect of erlotinib [32]. Our present study demonstrated combination of CQ with ACA exhibited a synergistic anti-cancer effect of ACA as shown in the MTT assay and annexin V/PI apoptosis assay. Currently, autophagy inhibitors such as CQ and hydroxychloroquine (HCQ) are under investigation in clinical phase I/II for various cancers including lung cancer [17, 35]. Results from phase I study had provided a strong evidence that HCQ with or without erlotinib does not elicit adverse side effects in advance NSCLCpatients [36]. Although available data showed the autophagy inhibitors in combination with anti-cancer agents have been recognised as a potential strategy to improve the efficiency of the available therapeutic agents and avoid acquired resistance; the role of autophagy requires further elucidation in other cancer types due to the fact that the autophagy stimulated by a single compound could have different role in different cancer types at different stage of cancer development [35].
Conclusion
Collectively, our current study demonstrated for the first time that ACA induced autophagy is through Beclin-1-independent/non-canonical pathway in NSCLC. In addition, we showed autophagy inhibitor CQ or inhibition of autophagy gentically by knockdown of LC3 using siRNA promoted ACA-induced cell death through the apoptosis mechanism. These knowledge suggested targeting autophagy mechanism in cancer therapy may be a promising approach to sensitized NSCLC to ACA treatment.
Photomicrograph of A549 and SK-LU-1 upon ACA treatment.
(A) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treatment with ACA. Arrow indicates the acidic vesicular organelles. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines after ACA treatment. Arrow indicates the GFP-LC3-II punctate.(TIF)Click here for additional data file.
Photomicrograph of A549 and SK-LU-1 after treatment with ACA in presence or absence of 3-MA.
(A) Cells were treated with 3-MA in presence or absence of ACA. Arrow indicates the cytoplasmic vacuole. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treatment with ACA in presence or absence of 3-MA. Arrow indicates the acidic vesicular organelles. (C) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to co-treatment of 3-MA and ACA. Arrow indicates the GFP-LC3-II punctate.(TIF)Click here for additional data file.
Photomicrograph of A549 and SK-LU-1 after treatment with ACA in presence or absence of CQ.
(A) Cells were treated with CQ in presence or absence of ACA. Arrow indicates the cytoplasmic vacuole. (B) Representative fluorescence photomicrograph (400 × magnification) illustrating the acidic vesicular organelles in A549 and SK-LU-1 cell lines after treatment with ACA in presence or absence of CQ. Arrow indicates the acidic vesicular organelles. (C) Representative fluorescence photomicrograph (400 × magnification) illustrating the GFP-LC3-II punctate formation in A549 and SK-LU-1 cell lines upon exposure to co-treatment of CQ and ACA. Arrow indicates the GFP-LC3-II punctate.(TIF)Click here for additional data file.
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Authors: Jacques Ferlay; Isabelle Soerjomataram; Rajesh Dikshit; Sultan Eser; Colin Mathers; Marise Rebelo; Donald Maxwell Parkin; David Forman; Freddie Bray Journal: Int J Cancer Date: 2014-10-09 Impact factor: 7.396
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Audrey Esclatine; Mafalda Escobar-Henriques; Eeva-Liisa Eskelinen; Lucile Espert; Makandjou-Ola Eusebio; Gemma Fabrias; Cinzia Fabrizi; Antonio Facchiano; Francesco Facchiano; Bengt Fadeel; Claudio Fader; Alex C Faesen; W Douglas Fairlie; Alberto Falcó; Bjorn H Falkenburger; Daping Fan; Jie Fan; Yanbo Fan; Evandro F Fang; Yanshan Fang; Yognqi Fang; Manolis Fanto; Tamar Farfel-Becker; Mathias Faure; Gholamreza Fazeli; Anthony O Fedele; Arthur M Feldman; Du Feng; Jiachun Feng; Lifeng Feng; Yibin Feng; Yuchen Feng; Wei Feng; Thais Fenz Araujo; Thomas A Ferguson; Álvaro F Fernández; Jose C Fernandez-Checa; Sonia Fernández-Veledo; Alisdair R Fernie; Anthony W Ferrante; Alessandra Ferraresi; Merari F Ferrari; Julio C B Ferreira; Susan Ferro-Novick; Antonio Figueras; Riccardo Filadi; Nicoletta Filigheddu; Eduardo Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; Vittorio Fineschi; Francesca Finetti; Steven Finkbeiner; Edward A Fisher; Paul B Fisher; Flavio Flamigni; Steven J Fliesler; Trude H Flo; Ida Florance; Oliver Florey; Tullio Florio; Erika Fodor; Carlo Follo; Edward A Fon; Antonella Forlino; Francesco Fornai; Paola Fortini; Anna Fracassi; Alessandro Fraldi; Brunella Franco; Rodrigo Franco; Flavia Franconi; Lisa B Frankel; Scott L Friedman; Leopold F Fröhlich; Gema Frühbeck; Jose M Fuentes; Yukio Fujiki; Naonobu Fujita; Yuuki Fujiwara; Mitsunori Fukuda; Simone Fulda; Luc Furic; Norihiko Furuya; Carmela Fusco; Michaela U Gack; Lidia Gaffke; Sehamuddin Galadari; Alessia Galasso; Maria F Galindo; Sachith Gallolu Kankanamalage; Lorenzo Galluzzi; Vincent Galy; Noor Gammoh; Boyi Gan; Ian G Ganley; Feng Gao; Hui Gao; Minghui Gao; Ping Gao; Shou-Jiang Gao; Wentao Gao; Xiaobo Gao; Ana Garcera; Maria Noé Garcia; Verónica E Garcia; Francisco García-Del Portillo; Vega Garcia-Escudero; Aracely Garcia-Garcia; Marina Garcia-Macia; Diana García-Moreno; Carmen Garcia-Ruiz; Patricia García-Sanz; Abhishek D Garg; Ricardo Gargini; Tina Garofalo; Robert F Garry; Nils C Gassen; Damian Gatica; Liang Ge; Wanzhong Ge; Ruth Geiss-Friedlander; Cecilia Gelfi; Pascal Genschik; Ian E Gentle; Valeria Gerbino; Christoph Gerhardt; Kyla Germain; Marc Germain; David A Gewirtz; Elham Ghasemipour Afshar; Saeid Ghavami; Alessandra Ghigo; Manosij Ghosh; Georgios Giamas; Claudia Giampietri; Alexandra Giatromanolaki; Gary E Gibson; Spencer B Gibson; Vanessa Ginet; Edward Giniger; Carlotta Giorgi; Henrique Girao; Stephen E Girardin; Mridhula Giridharan; Sandy Giuliano; Cecilia Giulivi; Sylvie Giuriato; Julien Giustiniani; Alexander Gluschko; Veit Goder; Alexander Goginashvili; Jakub Golab; David C Goldstone; Anna Golebiewska; Luciana R Gomes; Rodrigo Gomez; Rubén Gómez-Sánchez; Maria Catalina Gomez-Puerto; Raquel Gomez-Sintes; Qingqiu Gong; Felix M Goni; Javier González-Gallego; Tomas Gonzalez-Hernandez; Rosa A Gonzalez-Polo; Jose A Gonzalez-Reyes; Patricia González-Rodríguez; Ing Swie Goping; Marina S Gorbatyuk; Nikolai V Gorbunov; Kıvanç Görgülü; Roxana M Gorojod; Sharon M Gorski; Sandro Goruppi; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Martin Graef; Markus H Gräler; Veronica Granatiero; Daniel Grasso; Joshua P Gray; Douglas R Green; Alexander Greenhough; Stephen L Gregory; Edward F Griffin; Mark W Grinstaff; Frederic Gros; Charles Grose; Angelina S Gross; Florian Gruber; Paolo Grumati; Tilman Grune; Xueyan Gu; Jun-Lin Guan; Carlos M Guardia; Kishore Guda; Flora Guerra; Consuelo Guerri; Prasun Guha; Carlos Guillén; Shashi Gujar; Anna Gukovskaya; Ilya Gukovsky; Jan Gunst; Andreas Günther; Anyonya R Guntur; Chuanyong Guo; Chun Guo; Hongqing Guo; Lian-Wang Guo; Ming Guo; Pawan Gupta; Shashi Kumar Gupta; Swapnil Gupta; Veer Bala Gupta; Vivek Gupta; Asa B Gustafsson; David D Gutterman; Ranjitha H B; Annakaisa Haapasalo; James E Haber; Aleksandra Hać; Shinji Hadano; Anders J Hafrén; Mansour Haidar; Belinda S Hall; Gunnel Halldén; Anne Hamacher-Brady; Andrea Hamann; Maho Hamasaki; Weidong Han; Malene Hansen; Phyllis I Hanson; Zijian Hao; Masaru Harada; Ljubica Harhaji-Trajkovic; Nirmala Hariharan; Nigil Haroon; James Harris; Takafumi Hasegawa; Noor Hasima Nagoor; Jeffrey A Haspel; Volker Haucke; Wayne D Hawkins; Bruce A Hay; Cole M Haynes; Soren B Hayrabedyan; Thomas S Hays; Congcong He; Qin He; Rong-Rong He; You-Wen He; Yu-Ying He; Yasser Heakal; Alexander M Heberle; J Fielding Hejtmancik; Gudmundur Vignir Helgason; Vanessa Henkel; Marc Herb; Alexander Hergovich; Anna Herman-Antosiewicz; Agustín Hernández; Carlos Hernandez; Sergio Hernandez-Diaz; Virginia Hernandez-Gea; Amaury Herpin; Judit Herreros; Javier H Hervás; Daniel Hesselson; Claudio Hetz; Volker T Heussler; Yujiro Higuchi; Sabine Hilfiker; Joseph A Hill; William S Hlavacek; Emmanuel A Ho; Idy H T Ho; Philip Wing-Lok Ho; Shu-Leong Ho; Wan Yun Ho; G Aaron Hobbs; Mark Hochstrasser; Peter H M Hoet; Daniel Hofius; Paul Hofman; Annika Höhn; Carina I Holmberg; Jose R Hombrebueno; Chang-Won Hong Yi-Ren Hong; Lora V Hooper; Thorsten Hoppe; Rastislav Horos; Yujin Hoshida; I-Lun Hsin; Hsin-Yun Hsu; Bing Hu; Dong Hu; Li-Fang Hu; Ming Chang Hu; Ronggui Hu; Wei Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Jinlian Hua; Yingqi Hua; Chongmin Huan; Canhua Huang; Chuanshu Huang; Chuanxin Huang; Chunling Huang; Haishan Huang; Kun Huang; Michael L H Huang; Rui Huang; Shan Huang; Tianzhi Huang; Xing Huang; Yuxiang Jack Huang; Tobias B Huber; Virginie Hubert; Christian A Hubner; Stephanie M Hughes; William E Hughes; Magali Humbert; Gerhard Hummer; James H Hurley; Sabah Hussain; Salik Hussain; Patrick J Hussey; Martina Hutabarat; Hui-Yun Hwang; Seungmin Hwang; Antonio Ieni; Fumiyo Ikeda; Yusuke Imagawa; Yuzuru Imai; Carol Imbriano; Masaya Imoto; Denise M Inman; Ken Inoki; Juan Iovanna; Renato V Iozzo; Giuseppe Ippolito; Javier E Irazoqui; Pablo Iribarren; Mohd Ishaq; Makoto Ishikawa; Nestor Ishimwe; Ciro Isidoro; Nahed Ismail; Shohreh Issazadeh-Navikas; Eisuke Itakura; Daisuke Ito; Davor Ivankovic; Saška Ivanova; Anand Krishnan V Iyer; José M Izquierdo; Masanori Izumi; Marja Jäättelä; Majid Sakhi Jabir; William T Jackson; Nadia Jacobo-Herrera; Anne-Claire Jacomin; Elise Jacquin; Pooja Jadiya; Hartmut Jaeschke; Chinnaswamy Jagannath; Arjen J Jakobi; Johan Jakobsson; Bassam Janji; Pidder Jansen-Dürr; Patric J Jansson; Jonathan Jantsch; Sławomir Januszewski; Alagie Jassey; Steve Jean; Hélène Jeltsch-David; Pavla Jendelova; Andreas Jenny; Thomas E Jensen; Niels Jessen; Jenna L Jewell; Jing Ji; Lijun Jia; Rui Jia; Liwen Jiang; Qing Jiang; Richeng Jiang; Teng Jiang; Xuejun Jiang; Yu Jiang; Maria Jimenez-Sanchez; Eun-Jung Jin; Fengyan Jin; Hongchuan Jin; Li Jin; Luqi Jin; Meiyan Jin; Si Jin; Eun-Kyeong Jo; Carine Joffre; Terje Johansen; Gail V W Johnson; Simon A Johnston; Eija Jokitalo; Mohit Kumar Jolly; Leo A B Joosten; Joaquin Jordan; Bertrand Joseph; Dianwen Ju; Jeong-Sun Ju; Jingfang Ju; Esmeralda Juárez; Delphine Judith; Gábor Juhász; Youngsoo Jun; Chang Hwa Jung; Sung-Chul Jung; Yong Keun Jung; Heinz Jungbluth; Johannes Jungverdorben; Steffen Just; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Daniel Kaganovich; Alon Kahana; Renate Kain; Shinjo Kajimura; Maria Kalamvoki; Manjula Kalia; Danuta S Kalinowski; Nina Kaludercic; Ioanna Kalvari; Joanna Kaminska; Vitaliy O Kaminskyy; Hiromitsu Kanamori; Keizo Kanasaki; Chanhee Kang; Rui Kang; Sang Sun Kang; Senthilvelrajan Kaniyappan; Tomotake Kanki; Thirumala-Devi Kanneganti; Anumantha G Kanthasamy; Arthi Kanthasamy; Marc Kantorow; Orsolya Kapuy; Michalis V Karamouzis; Md Razaul Karim; Parimal Karmakar; Rajesh G Katare; Masaru Kato; Stefan H E Kaufmann; Anu Kauppinen; Gur P Kaushal; Susmita Kaushik; Kiyoshi Kawasaki; Kemal Kazan; Po-Yuan Ke; Damien J Keating; Ursula Keber; John H Kehrl; Kate E Keller; Christian W Keller; Jongsook Kim Kemper; Candia M Kenific; Oliver Kepp; Stephanie Kermorgant; Andreas Kern; Robin Ketteler; Tom G Keulers; Boris Khalfin; Hany Khalil; Bilon Khambu; Shahid Y Khan; Vinoth Kumar Megraj Khandelwal; Rekha Khandia; Widuri Kho; Noopur V Khobrekar; Sataree Khuansuwan; Mukhran Khundadze; Samuel A Killackey; Dasol Kim; Deok Ryong Kim; Do-Hyung Kim; Dong-Eun Kim; Eun Young Kim; Eun-Kyoung Kim; Hak-Rim Kim; Hee-Sik Kim; Jeong Hun Kim; Jin Kyung Kim; Jin-Hoi Kim; Joungmok Kim; Ju Hwan Kim; Keun Il Kim; Peter K Kim; Seong-Jun Kim; Scot R Kimball; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Matthew A King; Kerri J Kinghorn; Conan G Kinsey; Vladimir Kirkin; Lorrie A Kirshenbaum; Sergey L Kiselev; Shuji Kishi; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Richard N Kitsis; Josef T Kittler; Ole Kjaerulff; Peter S Klein; Thomas Klopstock; Jochen Klucken; Helene Knævelsrud; Roland L Knorr; Ben C B Ko; Fred Ko; Jiunn-Liang Ko; Hotaka Kobayashi; Satoru Kobayashi; Ina Koch; Jan C Koch; Ulrich Koenig; Donat Kögel; Young Ho Koh; Masato Koike; Sepp D Kohlwein; Nur M Kocaturk; Masaaki Komatsu; Jeannette König; Toru Kono; Benjamin T Kopp; Tamas Korcsmaros; Gözde Korkmaz; Viktor I Korolchuk; Mónica Suárez Korsnes; Ali Koskela; Janaiah Kota; Yaichiro Kotake; Monica L Kotler; Yanjun Kou; Michael I Koukourakis; Evangelos Koustas; Attila L Kovacs; Tibor Kovács; Daisuke Koya; Tomohiro Kozako; Claudine Kraft; Dimitri Krainc; Helmut Krämer; Anna D Krasnodembskaya; Carole Kretz-Remy; Guido Kroemer; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Sabine Kuenen; Lars Kuerschner; Thomas Kukar; Ajay Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Sharad Kumar; Shinji Kume; Caroline Kumsta; Chanakya N Kundu; Mondira Kundu; Ajaikumar B Kunnumakkara; Lukasz Kurgan; Tatiana G Kutateladze; Ozlem Kutlu; SeongAe Kwak; Ho Jeong Kwon; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert La Spada; Patrick Labonté; Sylvain Ladoire; Ilaria Laface; Frank Lafont; Diane C Lagace; Vikramjit Lahiri; Zhibing Lai; Angela S Laird; Aparna Lakkaraju; Trond Lamark; Sheng-Hui Lan; Ane Landajuela; Darius J R Lane; Jon D Lane; Charles H Lang; Carsten Lange; Ülo Langel; Rupert Langer; Pierre Lapaquette; Jocelyn Laporte; Nicholas F LaRusso; Isabel Lastres-Becker; Wilson Chun Yu Lau; Gordon W Laurie; Sergio Lavandero; Betty Yuen Kwan Law; Helen Ka-Wai Law; Rob Layfield; Weidong Le; Herve Le Stunff; Alexandre Y Leary; Jean-Jacques Lebrun; Lionel Y W Leck; Jean-Philippe Leduc-Gaudet; Changwook Lee; Chung-Pei Lee; Da-Hye Lee; Edward B Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Heung Kyu Lee; Jae Man Lee; Jason S Lee; Jin-A Lee; Joo-Yong Lee; Jun Hee Lee; Michael Lee; Min Goo Lee; Min Jae Lee; Myung-Shik Lee; Sang Yoon Lee; Seung-Jae Lee; Stella Y Lee; Sung Bae Lee; Won Hee Lee; Ying-Ray Lee; Yong-Ho Lee; Youngil Lee; Christophe Lefebvre; Renaud Legouis; Yu L Lei; Yuchen Lei; Sergey Leikin; Gerd Leitinger; Leticia Lemus; Shuilong Leng; Olivia Lenoir; Guido Lenz; Heinz Josef Lenz; Paola Lenzi; Yolanda León; Andréia M Leopoldino; Christoph Leschczyk; Stina Leskelä; Elisabeth Letellier; Chi-Ting Leung; Po Sing Leung; Jeremy S Leventhal; Beth Levine; Patrick A Lewis; Klaus Ley; Bin Li; Da-Qiang Li; Jianming Li; Jing Li; Jiong Li; Ke Li; Liwu Li; Mei Li; Min Li; Min Li; Ming Li; Mingchuan Li; Pin-Lan Li; Ming-Qing Li; Qing Li; Sheng Li; Tiangang Li; Wei Li; Wenming Li; Xue Li; Yi-Ping Li; Yuan Li; Zhiqiang Li; Zhiyong Li; Zhiyuan Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Weicheng Liang; Yongheng Liang; YongTian Liang; Guanghong Liao; Lujian Liao; Mingzhi Liao; Yung-Feng Liao; Mariangela Librizzi; Pearl P Y Lie; Mary A Lilly; Hyunjung J Lim; Thania R R Lima; Federica Limana; Chao Lin; Chih-Wen Lin; Dar-Shong Lin; Fu-Cheng Lin; Jiandie D Lin; Kurt M Lin; Kwang-Huei Lin; Liang-Tzung Lin; Pei-Hui Lin; Qiong Lin; Shaofeng Lin; Su-Ju Lin; Wenyu Lin; Xueying Lin; Yao-Xin Lin; Yee-Shin Lin; Rafael Linden; Paula Lindner; Shuo-Chien Ling; Paul Lingor; Amelia K Linnemann; Yih-Cherng Liou; Marta M Lipinski; Saška Lipovšek; Vitor A Lira; Natalia Lisiak; Paloma B Liton; Chao Liu; Ching-Hsuan Liu; Chun-Feng Liu; Cui Hua Liu; Fang Liu; Hao Liu; Hsiao-Sheng Liu; Hua-Feng Liu; Huifang Liu; Jia Liu; Jing Liu; Julia Liu; Leyuan Liu; Longhua Liu; Meilian Liu; Qin Liu; Wei Liu; Wende Liu; Xiao-Hong Liu; Xiaodong Liu; Xingguo Liu; Xu Liu; Xuedong Liu; Yanfen Liu; Yang Liu; Yang Liu; Yueyang Liu; Yule Liu; J Andrew Livingston; Gerard Lizard; Jose M Lizcano; Senka Ljubojevic-Holzer; Matilde E LLeonart; David Llobet-Navàs; Alicia Llorente; Chih Hung Lo; Damián Lobato-Márquez; Qi Long; Yun Chau Long; Ben Loos; Julia A Loos; Manuela G López; Guillermo López-Doménech; José Antonio López-Guerrero; Ana T López-Jiménez; Óscar López-Pérez; Israel López-Valero; Magdalena J Lorenowicz; Mar Lorente; Peter Lorincz; Laura Lossi; Sophie Lotersztajn; Penny E Lovat; Jonathan F Lovell; Alenka Lovy; Péter Lőw; Guang Lu; Haocheng Lu; Jia-Hong Lu; Jin-Jian Lu; Mengji Lu; Shuyan Lu; Alessandro Luciani; John M Lucocq; Paula Ludovico; Micah A Luftig; Morten Luhr; Diego Luis-Ravelo; Julian J Lum; Liany Luna-Dulcey; Anders H Lund; Viktor K Lund; Jan D Lünemann; Patrick Lüningschrör; Honglin Luo; Rongcan Luo; Shouqing Luo; Zhi Luo; Claudio Luparello; Bernhard Lüscher; Luan Luu; Alex Lyakhovich; Konstantin G Lyamzaev; Alf Håkon Lystad; Lyubomyr Lytvynchuk; Alvin C Ma; Changle Ma; Mengxiao Ma; Ning-Fang Ma; Quan-Hong Ma; Xinliang Ma; Yueyun Ma; Zhenyi Ma; Ormond A MacDougald; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; Sandra Maday; Frank Madeo; Muniswamy Madesh; Tobias Madl; Julio Madrigal-Matute; Akiko Maeda; Yasuhiro Maejima; Marta Magarinos; Poornima Mahavadi; Emiliano Maiani; Kenneth Maiese; Panchanan Maiti; Maria Chiara Maiuri; Barbara Majello; Michael B Major; Elena Makareeva; Fayaz Malik; Karthik Mallilankaraman; Walter Malorni; Alina Maloyan; Najiba Mammadova; Gene Chi Wai Man; Federico Manai; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Masoud H Manjili; Ravi Manjithaya; Patricio Manque; Bella B Manshian; Raquel Manzano; Claudia Manzoni; Kai Mao; Cinzia Marchese; Sandrine Marchetti; Anna Maria Marconi; Fabrizio Marcucci; Stefania Mardente; Olga A Mareninova; Marta Margeta; Muriel Mari; Sara Marinelli; Oliviero Marinelli; Guillermo Mariño; Sofia Mariotto; Richard S Marshall; Mark R Marten; Sascha Martens; Alexandre P J Martin; Katie R Martin; Sara Martin; Shaun Martin; Adrián Martín-Segura; Miguel A Martín-Acebes; Inmaculada Martin-Burriel; Marcos Martin-Rincon; Paloma Martin-Sanz; José A Martina; Wim Martinet; Aitor Martinez; Ana Martinez; Jennifer Martinez; Moises Martinez Velazquez; Nuria Martinez-Lopez; Marta Martinez-Vicente; Daniel O Martins; Joilson O Martins; Waleska K Martins; Tania Martins-Marques; Emanuele Marzetti; Shashank Masaldan; Celine Masclaux-Daubresse; Douglas G Mashek; Valentina Massa; Lourdes Massieu; Glenn R Masson; Laura Masuelli; Anatoliy I Masyuk; Tetyana V Masyuk; Paola Matarrese; Ander Matheu; Satoaki Matoba; Sachiko Matsuzaki; Pamela Mattar; Alessandro Matte; Domenico Mattoscio; José L Mauriz; Mario Mauthe; Caroline Mauvezin; Emanual Maverakis; Paola Maycotte; Johanna Mayer; Gianluigi Mazzoccoli; Cristina Mazzoni; Joseph R Mazzulli; Nami McCarty; Christine McDonald; Mitchell R McGill; Sharon L McKenna; BethAnn McLaughlin; Fionn McLoughlin; Mark A McNiven; Thomas G McWilliams; Fatima Mechta-Grigoriou; Tania Catarina Medeiros; Diego L Medina; Lynn A Megeney; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Alfred J Meijer; Annemarie H Meijer; Jakob Mejlvang; Alicia Meléndez; Annette Melk; Gonen Memisoglu; Alexandrina F Mendes; Delong Meng; Fei Meng; Tian Meng; Rubem Menna-Barreto; Manoj B Menon; Carol Mercer; Anne E Mercier; Jean-Louis Mergny; Adalberto Merighi; Seth D Merkley; Giuseppe Merla; Volker Meske; Ana Cecilia Mestre; Shree Padma Metur; Christian Meyer; Hemmo Meyer; Wenyi Mi; Jeanne Mialet-Perez; Junying Miao; Lucia Micale; Yasuo Miki; Enrico Milan; Małgorzata Milczarek; Dana L Miller; Samuel I Miller; Silke Miller; Steven W Millward; Ira Milosevic; Elena A Minina; Hamed Mirzaei; Hamid Reza Mirzaei; Mehdi Mirzaei; Amit Mishra; Nandita Mishra; Paras Kumar Mishra; Maja Misirkic Marjanovic; Roberta Misasi; Amit Misra; Gabriella Misso; Claire Mitchell; Geraldine Mitou; Tetsuji Miura; Shigeki Miyamoto; Makoto Miyazaki; Mitsunori Miyazaki; 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Authors: Seung Wan Son; Han Yeoung Lee; Sokviseth Moeng; Hyo Jeong Kuh; Soo Young Choi; Jong Kook Park Journal: Molecules Date: 2020-10-14 Impact factor: 4.411
Authors: Yasir Osman Ali Abdalla; Bavani Subramaniam; Shaik Nyamathulla; Noorasyikin Shamsuddin; Norhafiza M Arshad; Kein Seong Mun; Khalijah Awang; Noor Hasima Nagoor Journal: J Trop Med Date: 2022-03-11
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