Contributed equally
Lung cancer is the leading cause of cancer death, and it is widely accepted that chronic inflammation is an important risk for the development of lung cancer. Now, it is recognized that the nucleotide-binding and oligomerization domain (NOD) like receptors (NLRs)-containing inflammasomes are involved in cancer-related inflammation. This study was designed to investigate the effects of NLR family pyrin domain containing protein 3 (NLRP3) inflammasome on the proliferation and migration of lung adenocarcinoma cell line A549. Using 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, scratch assay, and Transwell migration assay, we showed that activation of the NLRP3 inflammasome by LPS+ATP enhanced the proliferation and migration of A549 cells. Western blot analysis showed that activation of phosphorylation of Akt, ERK1/2, CREB and the expression of Snail increased, while the expression of E-cadherin decreased after the activation of NLRP3 inflammasome. Moreover, these effects were inhibited by the following treatments: i) down-regulating the expression of NLRP3 by short hairpin RNA (shRNA) interference, ii) inhibiting the activation of NLRP3 inflammasome with a caspase-1 inhibitor, iii) blocking the interleukin-1β (IL-1β) and IL-18 signal transduction with IL-1 receptor antagonist (IL-1Ra) and IL-18 binding protein (IL-18BP). Collectively, these results indicate that NLRP3 inflammasome plays a vital role in regulating the proliferation and migration of A549 cells and it might be a potential target for the treatment of lung cancer.
Lung cancer is the leading cause of cancer death worldwide. Only 16.6% of lung cancer patients live 5 years or more after diagnosis (
Inflammasome, an intracellular multi-protein complex, switches on the inflammatory response of tissues to various danger signals. Among the inflammasomes, NLRP3 inflammasome is the most characterized activated by a diverse range of 'danger signals'. It is formed by the assembly of NOD-like receptor NLRP3, adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Once activated, NLRP3 inflammasome triggers the proteolytic processing of pro-caspase-1 into its active form, caspase-1 (p10 or p20), which subsequently cleaves pro-IL-1β and pro-IL-18 to mature bioactive forms. It has been well documented that two signals are required for the formation and activation of NLRP3 inflammasome. Firstly, pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) including viruses, bacteria, or lipopolysaccharide (LPS), prime the expressions of NLRP3, pro-IL-1β and pro-IL-18. Then, a second stimulus such as adenosine 5′-triphosphate (ATP), silica or monosodium urate (MSU) activates the NLRP3 inflammasome (
Mounting evidence indicates that NLRP3 inflammasome plays an important role in the development and progression of gastrointestinal cancer, skin cancer, breast cancer and hepatocellular carcinoma (
The A549 human alveolar epithelial adenocarcinoma cell line was purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (ScienCell, CA, USA) at 37°C in a humidified atmosphere of 5% CO2. LPS and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA). The caspase-1 inhibitor benzyloxycar-bonyl-tyrosine-valine-alanine-aspartate-fluoromethyl ketone (Z-YVAD-FMK) was purchased from PromoCell (Heidelberg, Germany). IL-18 binding protein (IL-18BP) was purchased from GenScript (Nanjing, China), and IL-1 receptor antagonist (IL-1Ra) was obtained from Fitzgerald Industries (MA, USA). Cells were stimulated by 1
Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 15 min, and incubated with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature (RT). Subsequently, cells were incubated with primary antibodies against NLRP3 (1:200, Abcam, London, UK) and ASC (1:200, Cell Signaling Technologies, MA, USA) overnight at 4°C. After three washes with PBS (5 min per wash), cells were incubated with Alexa Fluor 488-conjugated donkey anti-goat (1:1,000, Invitrogen, CA, USA) and Alexa Fluor 555-conjugated donkey anti-rabbit (1:500, Invitrogen) for 1 h at RT. After three washes with PBS, nuclei were stained with Hoechst 33342 (Beyotime, Nantong, China) for 30 min. Immunofluorescence images were captured using a Zeiss LSM5 Live confocal laser scanning microscope (Carl Zeiss, Jena, Germany).
Cells were seeded in 24-well plates overnight. For NLRP3 inflammasome activation, cells were stimulated by 1
5-ethynyl-2′-deoxyuridine (EdU) incorporation proliferation assay was performed to investigate the proliferation of A549 cells using a Cell-Light™ EdU Imaging detecting kit (RiboBio, Guangzhou, China). Cells were seeded in 6-well plates, and incubated for 24 h after different treatments. All of the EdU incorporation experiments were performed according to the manufacturer's protocol (
Cells were harvested after different treatments and washed with PBS. Then cells were resuspended in 500
Cell migration was examined using scratch assay and Transwell chamber migration assay. For scratch assay, cells were seeded in 6-well plates. Until the cells reached 100% confluence forming a monolayer, a sterile 50-
To knock down the NLRP3 expression, the A549 cells were transfected with lentiviral-mediated shRNA. The cells were transfected by lentiviral particles at a multiplicity of infection of 20–30 with control shRNA (shCtrl) or NLRP3 shRNAs (shNLRP3) (GenePharma, Shanghai, China), according to the manufacturer's instructions. Downregulation of NLRP3 expression was verified by reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting (data not shown).
Total RNA was extracted from cells using TRIzol (Invitrogen). Reverse transcription was performed using a reverse transcription kit (Takara, Shiga, Japan) according to the manufacturer's instructions. Standard real-time quantitative PCR was performed using the following primers: NLRP3: forward 5′-AAGGGCCATGGACTATTTCC-3′ and reverse 5′-GACTCCACCCGATGACAGTT-3′; GAPDH: forward 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse 5′-GAAGATGGTGATGGGATTTC-3′. All primers were synthesized by Invitrogen Life Technologies. The mRNA levels were quantified by SYBR Green Technology. Fold changes in gene expression were calculated using the 2−ΔΔCT method (
Cells were lysed on ice in lysis buffer with 1% EDTA and 1% protease inhibitor cocktail (Thermo Fisher Scientific, MA, USA). The total protein concentrations were quantified by BCA method (Beyotime). Proteins (60–100
All the data are expressed as the mean ± SEM. Differences between means were analyzed using one-way analysis of variance (ANOVA) or two-way ANOVA followed by Student-Newman-Keuls tests for multiple comparisons. Statistical significance was defined as P<0.05. All analyses were performed using the Statistical Package for Social Sciences statistical software (SPSS), version 20.0 (SPSS Inc., Chicago, IL, USA).
To investigate whether LPS and ATP trigger the formation of NLRP3 inflammasome in A549 cells, the association between NLRP3 and ASC was determined by immunofluorescence. As shown in
EdU incorporation assay was used to determine the cell proliferation. As shown in
Cell migration was investigated by using scratch assay and Transwell chamber migration assay. As shown in
To investigate the potential molecular mechanisms of activated NLRP3 inflammasome on A549 cell proliferation, the phosphorylation of two intracellular kinases (Akt and ERK1/2) and a transcription factor (CREB) involved in cell proliferation was determined by western blotting. As shown in
The cellular adhesion molecule E-cadherin and the transcription factor Snail are two important factors involved in inflammation-induced cancer cell migration (
Cancer-related inflammation supplies cytokines, chemokines, and extracellular matrix, to promote tumor growth and metastasis. Currently, inflammasomes, which sense exogenous and endogenous dangers and initiate inflammatory responses, are considered to contribute the development of cancer-related inflammation and have complex functions in carcinogenesis (
Activation of NLRP3 inflammasome commonly includes priming with a TLR agonist (such as LPS) and activating with a second stimulus (such as ATP) (
In the present study, blockage of IL-18 or IL-1β signaling alleviated the effects of activated NLRP3 inflammasome on A549 cell proliferation and migration. This result indicates that both IL-18 and IL-1β could contribute to the progression of lung adenocarcinoma. In a mouse model, Lewis lung carcinoma cells engineered to produce IL-1β exhibited increased tumor growth and expression of angiogenic factors (
Uncontrolled tumor cell growth and metastasis mainly result from the disruptions of regulatory signaling pathways. The Akt and ERK1/2 are two important signaling molecules in tumor proliferation. Our data demonstrated that activation of NLRP3 inflammasome could enhance the phosphorylation of Akt, ERK1/2 and CREB. The Akt pathway plays a pivotal role in fundamental cellular functions by phosphorylating a variety of substrates. Another important signal transduction pathway involves ERK1/2, which forms major cell-proliferation signaling pathways from the cell surface to the nucleus. Recent studies have demonstrated crosstalk between Akt and ERK1/2 signaling (
Tumor metastasis is one of the most common events that lead to mortality of cancer. There are several molecules involved in tumor metastasis, such as E-cadherin (
The final purpose of experimental research is to provide useful information for the clinical application. Literature shows that caspase-1 inhibitor, IL-1Ra and IL-18BP, which inhibit the NLRP3 inflammasome, are used in the clinical or in animal model research. For instance, the human recombinant IL1ra (hrIL1ra) has been used in phase II or III randomized control trial for stroke, severe sepsis and traumatic brain injury (
In conclusion, our data show that NLRP3 inflammasome activation can accelerate the proliferation and migration of A549 lung cancer cells by releasing IL-1β and IL-18 in an autocrine or paracrine manner. These results suggests that molecules participating in NLRP3 inflammasome signaling may be promising therapeutic targets for the treatment of lung adenocarcinoma.
This study was supported by the National Natural Science Foundation of China (NSFC) under grant 81273571, the National Technological Special Project for 'Significant New Drugs Development' (2011ZX09302-003-02), a Jiangsu Clinical Research Center for Respiratory Diseases project under grant BL2012012 and a project funded by the Priority Academic Program Development of Jiangsu Higher education Institutions (PAPD).
Effects of LPS and ATP on the activation of NLRP3 inflammasome in A549 cells. (A) Subcellular localization of inflammasome proteins was observed by immunofluorescence. Intracellular NLRP3 and ASC colocalized in LPS+ATP-treated cells but rarely in cells treated with LPS or ATP alone. The experiment was performed 3 times. (B) The expression of active caspase-1 p10 was significantly higher in LPS+ATP group than in control group. (C) The concentration of cleaved IL-18 and IL-1β in culture supernatants were significantly elevated in LPS+ATP group, not in LPS or ATP alone group. The values are presented as means ± SEM of five replicates. Scale bar, 20
Effects of NLRP3 inflammasome activation on LPS+ATP-induced proliferation. (A) Activated NLRP3 inflammasome by LPS+ATP enhanced cell proliferation. (B) NLRP3 protein expression was downregulated by shNLRP3 interference to 18% of that in shCtrl. (C) NLRP3 downregulation abolished LPS+ATP-induced cell proliferation. (D) Caspase-1 inhibitor Z-YVAD-FMK attenuated LPS+ATP-induced proliferation. (E) IL-18BP, or IL-1Ra also inhibited LPS+ATP-induced proliferation, and combined use of IL-18BP and IL-1Ra abolished LPS+ATP-induced proliferation. (F) LPS, ATP, and LPS+ATP had no effect on the apoptosis of A549 cells. Scale bar, 50
Effects of NLRP3 inflammasome activation on LPS+ATP-induced migration. (A) Scratch assay and (B) Transwell chamber migration assay showed that LPS+ATP-induced NLRP3 inflammasome activation enhanced A549 cell migration. (C and D) NLRP3 downregulation abolished LPS+ATP-induced cell migration. (E and F) Caspase-1 inhibitor Z-YVAD-FMK suppressed LPS+ATP-induced cell migration. (G and H) IL-18BP or IL-1Ra inhibited LPS+ATP-induced cell migration, and the combined use of IL-18BP and IL-1Ra reversed LPS+ATP-induced cell migration. Scale bar, 100
Effects of NLRP3 inflammasome activation on Akt, ERK1/2, and CREB phosphorylation. (A) Downregulation of NLRP3 reversed the LPS+ATP-induced phosphorylation of Akt, ERK1/2 and CREB. (B) Caspase-1 inhibitor Z-YVAD-FMK abolished LPS+ATP-induced Akt phosphorylation and inhibited ERK1/2 and CREB phosphorylation. (C) L-18BP, or IL-1Ra inhibited LPS+ATP-induced phosphorylation of Akt, ERK1/2 and CREB, and combined use of IL-18BP and IL-1Ra abolished the LPS+ATP-induced phosphorylation of Akt, ERK1/2 and CREB. L, LPS; A, ATP; FMK, Z-YVAD-FMK. Four independent experiments were examined. *P<0.05 compared with the control group; #P<0.05 compared with the LPS+ATP group.
Effects of NLRP3 inflammasome activation on the expression of Snail and E-cadherin. (A) Downregulation of NLRP3 reversed the LPS+ATP-induced downregulation of E-cadherin and upregulation of Snail. (B) Caspase-1 inhibitor Z-YVAD-FMK abolished LPS+ATP-induced downregulation of E-cadherin and upregulation of Snail. (C) L-18BP, or IL-1Ra inhibited LPS+ATP-induced downregulation of E-cadherin and upregulation of Snail, while the combined use of IL-18BP and IL-1Ra abolished LPS+ATP-induced downregulation of E-cadherin and upregulation of Snail. L, LPS; A, ATP; FMK, Z-YVAD-FMK. Four independent experiments were examined. *P<0.05 compared with the control group; #P<0.05 compared with the LPS+ATP group.
A schematic model to illustrate the mechanisms. Caspase-1 inhibitor Z-YVAD-FMK suppressed the LPS+ATP-induced phosphorylation of Akt more effectively than that of ERK1/2. IL-18BP functioned more effectively on the phosphorylation of Akt while IL-1Ra functioned more effectively on the phosphorylation of ERK1/2. Snail, which represses E-cadherin transcription, and can be mediated by Akt and ERK1/2.