recombinant human globular adiponectin (gacrp; #450-21)  (PeproTech)

Journal: Journal of Translational Medicine

Article Title: Adiponectin suppresses tumor growth of nasopharyngeal carcinoma through activating AMPK signaling pathway

doi: 10.1186/s12967-022-03283-0

Figure Lengend Snippet: Circulating adiponectin levels and risk of nasopharyngeal carcinoma in retrospective and prospective cohorts

Article Snippet: Recombination human full-length adiponectin was dissolved in deionized water to prepare a working stock solution of approximately 0.5 mg/mL (BioVendor, Brno, Czech Republic).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Adiponectin suppresses tumor growth of nasopharyngeal carcinoma through activating AMPK signaling pathway

doi: 10.1186/s12967-022-03283-0

Figure Lengend Snippet: Adiponectin suppresses nasopharyngeal carcinoma growth. A , B 1 × 10 6 CNE-2 cells were injected subcutaneously into 5- to 6- week-old adiponectin-deficient nude mice, or the control nude mice ( n = 6 per group). Tumor growth were monitored by measuring the tumor volume for 10 days. Next, the mice were sacrificed, and tumors were collected, measured, weighed. C The plate colony assay was performed to determine colony-formation ability. CNE-2 and C666-1 cells were treated with various concentrations of adiponectin for 7 days. Graphs show the number of colonies. D EdU incorporation assay was performed to determine cell proliferation. CNE-2 and C666-1 cells were treated with various concentrations of adiponectin for 48 h. Bars: 50 μm. Graphs show the relative cell proliferation percentage. E , F CNE-2 and C666-1 cells were incubated with adiponectin (40 μg/mL) for 48 h. At the end of incubation, the cells were collected for FACS analysis. G Western blot analysis of p-AMPKα (T172), p-LKB1 and p-ERk1/2 in cultured CNE-2 and C666-1 cells after the treatment with adiponectin (40 μg/mL) for 30 min. Results are presented as mean ± SD of three independent experiments performed in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.0001

Article Snippet: Recombination human full-length adiponectin was dissolved in deionized water to prepare a working stock solution of approximately 0.5 mg/mL (BioVendor, Brno, Czech Republic).

Techniques: Injection, Colony Assay, Incubation, Western Blot, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Adiponectin suppresses tumor growth of nasopharyngeal carcinoma through activating AMPK signaling pathway

doi: 10.1186/s12967-022-03283-0

Figure Lengend Snippet: Adiponectin suppresses proliferation of NPC cells via AMPK activation. A , B CNE-2 and C666-1 cells were pretreated with compound C (10 mM) followed by treatment with adiponectin (40 μg/mL) for 24 h. Cell cycle was then analyzed using flow cytometer. # P < 0.05 and ## P < 0.01 compared to cells treated with adiponectin but not ComC; ** P < 0.01 compared with cells treated without adiponectin and ComC. C CNE-2 and C666-1 cells viability was determined after treatment with or without adiponectin for 48 h in the presence or absence of ComC (10 μM). ** P < 0.01, *** P < 0.001. D CNE-2 and C666-1 cells were pretreated with compound C (10 mM), p-AMPKα (Thr172) protein level was then determined by Western blot analysis after the treatment with adiponectin (40 μg/mL) for 30 min; cyclin D1, p21, and p27 protein level was then determined by Western blot analysis after the cells were exposed to adiponectin (40 μg/mL) for 48 h. E Quantitative analysis of p-AMPKα level was performed by densitometric analysis. Results are presented as mean ± SD of three independent experiments performed in triplicate. ** P < 0.01, *** P < 0.001

Article Snippet: Recombination human full-length adiponectin was dissolved in deionized water to prepare a working stock solution of approximately 0.5 mg/mL (BioVendor, Brno, Czech Republic).

Techniques: Activation Assay, Flow Cytometry, Western Blot

Journal: Journal of Translational Medicine

Article Title: Adiponectin suppresses tumor growth of nasopharyngeal carcinoma through activating AMPK signaling pathway

doi: 10.1186/s12967-022-03283-0

Figure Lengend Snippet: AdipoR1 and AdipoR2 mediate the anti-proliferative effect of adiponectin in NPC cells. A , B Expression of AdipoR1 and AdipoR2 was determined by qRT-PCR and Western blot in NPC cell lines. C , D CNE-2 and C666-1 cells were transfected with 50 μM siRNAs of NC, AdipoR1, or AdipoR2. The relative amounts of each AdipoR1/R2 mRNA against β-actin were measured with qRT-PCR. E Effect of the knockdown of AdipoR1 and AdipoR2 expression on the cell viability of CNE-2 and C666-1 cells treated with or without 40 μg/mL adiponectin for 48 h. F CNE-2 and C666-1 cells were transfected with AdipoR1, AdipoR2 siRNA or NC siRNA and treated with 40 μg/mL adiponectin for 30 min. AMPKα and p-AMPKα (T172) protein levels were determined by Western blot analysis. Quantitative analysis of p-AMPKα level was performed by densitometric analysis and shown in the below part. Results are presented as mean ± SD of three independent experiments performed in triplicate. ** P < 0.01, *** P < 0.001

Article Snippet: Recombination human full-length adiponectin was dissolved in deionized water to prepare a working stock solution of approximately 0.5 mg/mL (BioVendor, Brno, Czech Republic).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Suppression of the inflammasome activation by globular adiponectin in breast and hepatic cancer cells. ( A – H ) MCF-7 breast cancer cells were treated with gAcrp (1 µg/mL) for the indicated time duration ( A , C , E , G ) or treated with gAcrp at different concentrations for 24 h ( B , D , F , H ). Total protein lysates were immunoblotted for IL-1β ( A , B ), Caspase-1 ( C , D ), NOD-like receptor pyrin domain-containing protein 3 (NLRP3) ( E , F ), or the apoptosis-associated speck-like protein containing a CARD (ASC) ( G , H ). ( I ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time periods before sequentially incubated with antibodies for ASC (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). ASC speck formation was indicated by white arrows. Representative images from three independent experiments were presented along with the quantitation of ASC speck in the right panel. Values are expressed as percentage of the cells presenting the ASC dots with respect to DAPI by using Image Inside Software version 2.32. Scale bar: 5 µm. ( J – M ) HepG2 hepatic cancer cells were incubated with gAcrp (1 µg/mL) for time durations as indicated. Western blot analyses were performed for the measurement of IL-1β (J), caspase-1 (K), NLRP3 (L), and ASC (M). For Western blot experiments, representative images from three independent experiments are presented. β-actin was used as a loading control. Bar diagrams show the relative band intensity of the target proteins compared to β-actin or the respective proform, determined by densitometric analysis. Values are presented as the fold change compared with the control cells and are expressed as mean ± standard error of mean (SEM). * denotes p < 0.05 compared with control cells.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Incubation, Quantitation Assay, Software, Western Blot, Control

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Suppression of ER stress by globular adiponectin and its implication in the modulation of inflammasomes activation in breast cancer cells. ( A – C ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time duration. Expression levels of phospho- and total protein kinase RNA-like endoplasmic reticulum kinase (PERK) ( A ), phospho- and total eukaryotic translation initiation factor 2A (eIF2α) (B), and C/EBP homologous protein (CHOP) were determined by Western blot analysis. ( D – G ) MCF-7 cells were incubated with the indicated concentrations of tauroursodeoxycholic acid (TUDCA) ( D , E ) or tunicamycin ( F , G ) for 24 h or 12 h, respectively. Immunoblot analysis was carried out for determining the levels of interleukin-1β (IL-1β) and caspase-1. For all the Western blot analyses, the expression level of the target genes was estimated by densitometric analysis and is shown in the lower panel. Values represent fold change in comparison to the control group after being normalized to β-actin and are expressed as mean ± standard error of mean (SEM), n = 3. * denotes p < 0.05 compared with control cells.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Expressing, Western Blot, Incubation, Comparison, Control

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Crucial role of AMP-activated protein kinase (AMPK) signaling in the modulation of endoplasmic reticulum (ER) stress and inflammasomes by globular adiponectin. ( A ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time periods. The expression levels of AMPKα were measured by Western blot analysis. ( B , C ) MCF-7 cells were pretreated with compound C (1 µM), a pharmacological inhibitor of AMPK, for 1h followed by further treatment with gAcrp (1 µg/mL) for additional 24 h. Total protein lysates were prepared and used for immunoblotting analysis for the measurement of interleukin-1β (IL-1β) ( B ) and Caspase-1 ( C ). ( D ) After pretreatment with compound C for 1 h, MCF-7 cells were incubated with gAcrp (1 µg/mL) for additional 1 h and further incubated with antibodies for specific ASC (green) and DAPI (blue). Scale bar: 5µm. ( E – I ) MCF-7 cells were transfected with siRNA targeting AMPKα or control scrambled siRNA. Gene silencing efficiency of AMPKαwas monitored by Western blot analysis (Upper panel in E). After transient gene silencing of AMPKα, cells were treated with gAcrp for 24h ( E , F ) or 1 h ( G – I ). IL-1β (E), Caspase-1 ( F ), protein kinase RNA-like endoplasmic reticulum kinase (PERK) (G), Eukaryotic translation initiation factor 2A (eIF2α) ( H ), and C/EBP homologous protein (CHOP) ( I ) expression levels were determined by Western blot analysis. Values are presented as the fold change compared with the control cells and are expressed as mean± standard error of mean (SEM), n = 3. * denotes p < 0.05 compared to control cells, # denotes p < 0.05 compared with the cells treated with globular adiponectin but not pretreated with compound C ( B , C ) or not transfected with siRNA ( E – I ).

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Expressing, Western Blot, Incubation, Transfection, Control

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Role of sestrin2 (SESN2) induction in AMP-activated protein kinase (AMPK) phosphorylation, endoplasmic reticulum (ER) stress amelioration, and inflammasome inhibition by globular adiponectin in breast cancer cells. ( A ) MCF-7 cells were treated with gAcrp (1 µg/mL) for the indicated time periods. The expression level of SESN2 was determined by Western blot analysis. ( B ) MCF-7 cells were transiently transfected with siRNA targeting AMPKα and the gene silencing efficiency was monitored after 36 h using immunoblotting analysis (upper panel). After transfection, MCF-7 cells were further incubated with gAcrp (1 µg/mL) for 1 h and the expression of AMPK was examined (lower panel). ( C ) MCF-7 cells were treated with gAcrp (1 µg/mL) for 30 min. The SESN2-associated protein levels of AMPK and LKB-1 were determined by Immunoprecipitation assay. ( D ) After transfection with SESN2 siRNA, MCF-7 cells were further incubated with gAcrp (1 µg/mL) for 30 min. AMPKα amount associated with liver kinase B1 (LKB-1) was measured by immunoprecipitation assay. ( E – I ) MCF-7 cells were transfected with siRNA targeting SESN2 or control scrambled siRNA followed by treatment with gAcrp (1 µg/mL) for 1 h (G,H) or 24 h ( E , F , I ). Total protein lysates were immunoblotted for interleukin-1β (IL-1β) (E), Caspase-1 ( F ), the protein kinase RNA-like endoplasmic reticulum kinase (PERK) ( G ), Eukaryotic translation initiation factor 2A (eIF2α) ( H ), and C/EBP Homologous Protein (CHOP) ( I ). Values represent the fold change relative to the control cells and are expressed as mean± standard error of mean, n = 3. * denotes p < 0.05 compared to control cells, # denotes p < 0.05 compared with the cells treated with gAcrp but not transfected with siRNA.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Phospho-proteomics, Inhibition, Expressing, Western Blot, Transfection, Incubation, Immunoprecipitation, Control

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Inhibition of the inflammasome activation mediates modulation of breast cancer cell growth. ( A – D ) MCF-7 cells were treated with pharmacological inhibitors of inflammasomes ( A , C ), including Ac-YVAD-cmk (10 µM), a caspase-1 inhibitor, MCC950 (10 µM), a small molecule inhibitor of NLRP3, or interleukin-1 receptor antagonist (1 µg/mL), or gAcrp (1 µg/mL) ( B , D ) for the indicated time duration. Cell viability ( A , B ) and Caspase-7 enzyme activity ( C , D ) were then determined as described in the Materials and Methods. ( E ) MCF-7 cells were incubated with gAcrp (1 µg/mL) or pharmacological inhibitors of inflammasomes for 48 h followed by staining with propidium iodide and flow cytometric analysis. ( F – O ) MCF-7 cells were incubated with gAcrp (1 µg/mL) ( F – J ) or pharmacological inhibitors of inflammasomes ( L – O ) for the indicated time periods. Immunoblot analysis was performed to determine the expression levels of Bcl2-associated X protein (Bax) ( F , K ), B-cell lymphoma 2 (Bcl-2) ( G , L ), cyclin D1 ( H , M ), p27 ( I , N ), and p53 ( J , O ). Images are the representative for three independent experiments that showed similar results. Values represent the fold change relative to the control cells and are expressed as mean± standard error of mean, n =3. * denotes p < 0.05 compared to control cells.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Inhibition, Activation Assay, Activity Assay, Incubation, Staining, Western Blot, Expressing, Control

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Suppressive effects of globular adiponectin on NLRP3 inflammasomes activation in MCF-7 tumor xenograft model and its potential role in the modulation of growth of breast cancer cells. After xenograft tumors were generated in BALB/c nude male mice, the animals were randomly divided into four groups of 5 mice and intratumorally administered with gAcrp (2µg/mouse), MCC950 (40µg/mouse), IL-1Ra (2µg/mouse) or phosphate-buffered saline (control group) every 48 h for 3 weeks. ( A ) Representative images of mice bearing xenografted tumors from each group at the end of the treatment period. ( B ) Tumor tissues were isolated from each mouse after 3 weeks of treatment. ( C ) Tumor growth rate were monitored every three days during treatment. ( D ) After 3 weeks treatment, tumor tissues from each mouse were collected and weighed. ( E ) Tissue sections were prepared from tumor of each mouse and subjected to immunohistochemistry (IHC) staining for Ki-67. Scale bar: 5µm. ( F – H ) The expression levels of the proliferative and apoptotic markers, such as cyclin D1, p27, B-cell lymphoma 2 (Bcl-2), and Bcl2-associated X protein (Bax) ( F), and genes related to inflammasomes, including IL-1β and caspase-1 ( G), NLRP3, and the apoptosis-associated speck-like protein containing a CARD (ASC) ( H), were determined by Western blot. ( I ) The expression levels of NLRP3 and ASC were further confirmed by IHC. For IHC experiments, five mice were used in each group and statistical analysis was made with 3 different tumor sections of each animal. The percentage of staining positive cells (nuclear staining) or staining positive area (cytoplasmic staining) was determined using Image J Software version 1.52. Magnification 20×. Scale bar: 5µm. ( J ) Expression levels of AMP-activated protein kinase (AMPK), sestrin2 (SESN2), and C/EBP homologous protein (CHOP) were examined by Western blot analysis. For Western blot analyses, 5 mice were used in each group and the results from each mouse were included in the statistical analysis. Of the five samples, representative images for three samples in each group were presented. Values are expressed as mean± standard error of mean, n = 5. * denotes p < 0.05 compared with control group.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Generated, Saline, Control, Isolation, Immunohistochemistry, Expressing, Western Blot, Staining, Software

Journal: Cancers

Article Title: Globular Adiponectin Inhibits Breast Cancer Cell Growth through Modulation of Inflammasome Activation: Critical Role of Sestrin2 and AMPK Signaling

doi: 10.3390/cancers12030613

Figure Lengend Snippet: Proposed model for the modulation of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasomes activation by globular adiponectin in breast cancer cells and its role in the suppression of tumor growth. Inflammasomes modulate tumor growth by complicated manners and its physiological roles would be depending on specific context. Herein, we clearly demonstrated that inflammasomes activation contributes to growth of breast cancer cells via activation of cell cycle and suppression of apoptosis. The physiological actions of adiponectin are initiated by binding with its specific transmembrane receptor (adiponectin receptor type 1 and type 2 (adipoR1 and adipoR2)). Binding of globular adiponectin with adipoR1/R2 generates signaling, which suppresses NLRP3 inflammasomes activation leading to the prevention of interleukin-1β (IL-1β) maturation and caspase-1 activation in both in vitro and in vivo xenograft models, implying that suppression of tumor growth by globular adiponectin is mediated via modulation of the inflammasome. Modulation of the inflammasome by globular adiponectin is mediated by sestrin2 (SESN2)/AMP-activated protein kinase (AMPK)/Endoplasmic reticulum (ER) stress axis. SESN2 induction leads to AMPK phosphorylation by promoting association of AMPK with its upstream kinase, LKB-1. Activation of AMPK signaling plays a pivotal role in reduction in ER stress, which subsequently leads to prevention of inflammasomes activation. In here, inflammasomes activation and IL-1β signaling might be required for survival and growth of breast tumor growth. Therefore, negative modulation of inflammasomes activation by globular adiponectin, MCC950, Ac-YVAD, and IL-1 receptor antagonist promotes apoptosis and impedes cancer cell proliferation. Therefore, modulation of inflammasomes and various molecules involved would be potential therapeutic targets for the treatment of breast cancer. The molecular mechanisms underlying SESN2 induction by globular adiponectin remain elusive. In addition, the molecular mechanisms by which ER stress induces inflammasomes activation and IL-1 signaling modulates apoptosis and cell cycle remain to be elucidated.

Article Snippet: Recombinant human globular adiponectin (gAcrp) was purchased from PeproTech Inc. (Rocky Hill, NJ, USA).

Techniques: Activation Assay, Binding Assay, In Vitro, In Vivo, Phospho-proteomics, Biomarker Discovery