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Verteporfin inhibits lipopolysaccharide-induced inflammation by multiple functions in RAW 264.7 cells
Journal Pre-proof Verteporfin inhibits lipopolysaccharide-induced inflammation by multiple functions in RAW 264.7 cells
Yuting Wang, Lei Wang, James T.F. Wise, Xianglin Shi, Zhimin Chen PII:
S0041-008X(19)30460-0
DOI:
https://doi.org/10.1016/j.taap.2019.114852
Reference:
YTAAP 114852
To appear in:
Toxicology and Applied Pharmacology
Received date:
23 May 2019
Revised date:
3 December 2019
Accepted date:
4 December 2019
Please cite this article as: Y. Wang, L. Wang, J.T.F. Wise, et al., Verteporfin inhibits lipopolysaccharide-induced inflammation by multiple functions in RAW 264.7 cells, Toxicology and Applied Pharmacology (2019), https://doi.org/10.1016/j.taap.2019.114852
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
Verteporfin inhibits lipopolysaccharide-induced inflammation by multiple functions in RAW 264.7 cells
Department of Pulmonology, Children's Hospital, Zhejiang University School of Medicine,
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a
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Yuting Wanga,b , Lei Wangb, James T.F. Wiseb,c, Xianglin Shib,⁎, Zhimin Chena,⁎⁎
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Hangzhou, Zhejiang 310052, People's Republic of China b
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Center for Research on Environmental Disease, College of Medicine, University of Kentucky,
Department of Pharmacology and Nutritional Sciences, College of Medicine, University of
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c
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1095 VA Drive, Lexington, KY 40536, USA
Correspondence to: X. Shi, Center for Research on Environmental Disease, College of
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⁎
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Kentucky, 1095 VA Drive, Lexington, KY 40536, USA
Medicine, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USA. [email protected] ⁎⁎
Correspondence to: Z. Chen, Department of Pulmonology, Children's Hospital, Zhejiang
University School of Medicine, Hangzhou, Zhejiang 310052, People's Republic of China. [email protected]
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Journal Pre-proof Abstract: Inflammation is a physiologic response to damage triggered by infection, injury or chemical irritation. Chronic inflammation produces repeated damage to cells and tissues, which can induce a variety of human diseases including cancer. Verteporfin, an FDA approved drug, is used for the treatment of age-related macular degeneration. The anti-tumor effects of verteporfin have been
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demonstrated by a number of studies. However, fewer studies focus on the anti-inflammatory functions of this drug. In this study, we investigated the anti-inflammatory effects and potential
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mechanisms of verteporfin. The classic lipopolysaccharide (LPS)-induced inflammation cell
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model was used. RAW 264.7 cells were pre-treated with verteporfin or vehicle control, followed
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by LPS stimulation. Verteporfin inhibited IL-6 and TNF-α at mRNA and protein expression
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levels. This effect was mediated through inhibition of the NF-κB and JAK/STAT pathways. Finally, verteporfin exhibited an anti-inflammation effect by crosslinking of protein such as NF-
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κB p65, JAK1, JAK2, STAT1, or STAT3 leading to inflammation. Taken together, these results
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inflammatory diseases.
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indicate that verteporfin has the potential to be an effective therapeutic agent against
Key words: inflammation; verteporfin; LPS; RAW 264.7
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Journal Pre-proof 1. INTRODUCTION
Inflammation is a physiologic response to damage triggered by infection, injury or chemical irritation (Philip et al., 2004). A successful acute inflammatory response results in recovery from the damage with subsequent healing. However, chronic inflammation will lead to persistent
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damage to cells and tissues and generate a variety of human diseases, such as cancer,
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autoimmune disease, cardiovascular disease, diabetes, and neuro degenerative disease (Nathan,
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2002; Coussens et al., 2002; Kim and Moudgil, 2017; Ruparelia et al., 2017; Wellen and Hotamisligil, 2005; Heneka et al., 2015). Macrophages play an important role in the
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inflammatory process. Macrophages are recruited to primary inflammatory sites and secrete a
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variety of inflammatory mediators, including TNF-a, IL-6, CCL2, and CCL5 (Wynn et al.,
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2013). Targeting macrophages could be consequential as a therapeutic approach against inflammatory diseases. Lipopolysaccharide (LPS), derived from gram-negative bacteria, is one
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of the common inflammatory inducers. LPS-induced macrophages are exceptionally adept at
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producing a cascade of inflammatory cytokines and mediators (Hamidzadeh et al., 2017). Thus, the LPS-induced macrophage cell model is widely used in the study of inflammation. Increasing evidences show that NF-κB and JAK/STAT signaling pathways play an important role in LPS-induced inflammation for macrophages. The transcription factor NF-κB is one of the major regulators in inflammatory process (DiDonato et al., 2012). LPS is able to combine with toll-like receptors (TLRs), leading to activation of the IκB kinase (IKK) complex and phosphorylation of IκBα. The activated NF-κB is then released from its inhibitory subunit (IκBα) and translocated to the nucleus to stimulate transcription of a number of target genes, including 3
Journal Pre-proof TNF-α and IL-6 (Hoesel and Schmid, 2013). These pro-inflammatory cytokines contribute to the overall inflammatory process. When IL-6 combines with its receptor (IL-6R), JAK family tyrosine kinases (JAK1, JAK2, and Tyk2) are phosphorylated and activated. Signal transducer and activator of transcription 3 (STAT3) and signal transducer and activator of transcription 1 (STAT1) are recruited and
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phosphorylated by JAK kinases. The activated STAT3 and STAT1 proteins dimerize to create STAT3 or STAT1 homodimers and STAT3/STAT1 heterodimers. These activated STAT dimers
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genes to promote expression (Kojima et al., 2013).
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enter the nucleus and bind to specific DNA sequences in the regulatory regions of their target
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Verteporfin (VT) is a photosensitizing protoporphyrin derivative, which is an FDA approved
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drug used for the photodynamic treatment of age-related macular degeneration (Dasari et al.,
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2017). Verteporfin has been identified as a YAP/TEAD inhibitor (Wei et al., 2017) as well as autophagosome inhibitor by promoting oligomerization of p62 (Gibault et al., 2016). Recent
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studies show that verteporfin has the potential for the treatment of various types of cancers, such
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as pancreatic ductal adenocarcinoma (Donohue et al., 2013), ovarian cancer (Feng et al., 2016), colon cancer (Zhang et al., 2015), and prostate cancer (Wang et al., 2018). Inflammatory process plays an important role in different stages of tumor development (Elinav et al., 2013). There are only limited studies available about the effect of verteporfin on inflammatory process. We hypothesized that verteporfin has the property to inhibit inflammation. In this study we tested this hypothesis and explored the possible mechanisms responsible for this process. 2. METHODS 2.1. Reagents 4
Journal Pre-proof Verteporfin was purchased from AdooQ Bioscience (Irvine, CA). Lipopolysaccharide (LPS) from Escherichia coli O111:B4 and InSolution™ NF-κB Activation Inhibitor - Calbiochem was purchased from Sigma‐ Aldrich (#481407) (St. Louis, MO). The antibodies against IL-6, TNF-α, p-IKKα/β, p-NF-κB p65, NF-κB p65 (rabbit), HDAC1, JAK1, JAK2, p-STAT1 (S727), STAT1, p-STAT3 (Y705), and STAT3 were purchased from Cell Signaling Technology (Danvers, MA). NF-κB p65 (mouse) antibody was purchased from Santa Cruz Biotechnology, Inc (Dallas,
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Texas). β-actin antibody was purchased from Sigma‐ Aldrich.
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2.2. Cell culture
Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased
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from Gibco (Thermo Fisher Scientific, Waltham, MA). RAW 264.7 and U-937 cells were
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obtained from American Type Culture Collection (ATCC, Manassas, VA). RAW 264.7 cells
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were cultured in DMEM supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml) and 10% FBS. U-937 cells were cultured in RPMI-1640 (Thermo Fisher Scientific)
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supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml) and 10% FBS. Primary
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murine peritoneal macrophages were isolated from new euthanasia C3H mouse and cultured in DMEM medium. Cells were incubated at 37 °C in 5% carbon dioxide (CO 2 ) with a humidified atmosphere of 95% air. RAW 264.7 cells were stimulated by LPS (1 μg/ml) for the indicated time period(s) after pretreated with different concentrations of verteporfin (0, 0.5, 1, or 2 μM) for 2 h. All verteporfin treatments are in darkness unless otherwise stated. 2.3. Cell viability Cell viability was evaluated by MTT assay. Briefly, RAW 264.7 cells were seeded at a density of 5 103 cells/well in 96-well plates. After 24 h, cells were treated with different 5
Journal Pre-proof concentrations of verteporfin (0-12 μM) for 24 h, followed by the addition of 20 μl 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT, Sigma‐ Aldrich) solution (5 mg/ml) to each well for 2 h. The formazan product was solubilized with 200 μl DMSO/well. The absorbance was measured using a microplate reader at a test wavelength of 560 nm. Results are presented as a calculated percentage of absorbance in control groups.
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2.4. Live and dead assay and live cell counting assay
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Live and dead assay was performed using LIVE/DEAD™ Viability/Cytotoxicity Kit (Thermo Fisher Scientific). Briefly, RAW 264.7 cells were seeded into 6 well plates and treated with
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different concentrations of verteporfin (0~10 μM) for 24 h with or without light stimulation.
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Light stimulation groups were stimulated with ambient light 30 min before cell staining. Then
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culture medium were removed and 1 ml of the staining solution (20 µL ethidium homodimer-1 and 5 µL calcein AM mixed in 10 mL PBS) were added directly to cells. After 15 min
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incubation, cells were imaged using a fluorescent microscope.
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Live cell counting was performed to obtain live cell percentage. Briefly, RAW 264.7 cells
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were seeded into 6 well plates and treated with different concentrations of verteporfin (0~10 μM) for 24 h. Then 10 μl of cell suspension were mixed with 10 μl of trypan blue (Bio-Rad, Hercules, CA). Live cells were counted using TC20™ Automated Cell Counter (Bio-Rad). 2.5. Cytokine array Cytokine array analyses were performed using RayBio C-Series Mouse Cytokine Antibody Array C1, which was purchased from RayBiotech Life (Norcross, GA). RAW 264.7 cells were pretreated with 2 μM verteporfin for 2 h, then stimulated with LPS (1 μg/ml) for 22 h. Cell pellets were obtained by centrifugation and the associated cell lysates were analyzed according 6
Journal Pre-proof to the manufacturer's instructions. Briefly, cytokine array membranes were incubated with blocking buffer for 30 min, replaced with 500 μg of total protein in 1 ml, and incubated overnight at 4 °C. The next day, the sample was aspirated and the membrane washed. Next, 1 ml of the biotinylated antibody cocktail was added to the membrane and incubated for 2 h at room temperature. Washed membranes were incubated with 2 ml 1 HRP-Streptavidin and washed. The membrane were developed using the kit's enhanced chemiluminescence system and data
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2.6. Real-Time PCR
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were analyzed with the Azure Biosystems imager.
Total RNA was isolated using the TRIzol Reagent (Thermo Fisher Scientific) according to the
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manufacturer’s protocol. Reverse transcription reactions were performed by the following
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process. For a 20 μl reaction, 1 μl oligo (dT) (500 μg/ml), and 2 μg total RNA were added to a nuclease-free micro-centrifuge tube, then adjusted to 12 μl with nuclease-free water. The mixture
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was heated to 65 °C for 5 min and quick chilled on ice. Next 4 μl of 5 First-Strand Buffer, 2 μl
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0.1 M DTT, and 1 μl RNaseOUT Recombinant Ribonuclease Inhibitor (40 units/μl) were added
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and mixed gently. The mixture was incubated at 37 °C for 2 min followed by the addition of 1 μl (200 units) M-MLV and again incubated at 37 °C for 50 min. The reaction was inactivated by heating at 70 °C for 15 min. The cDNA obtained was used for real-time PCR. Real-time PCR was performed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). Primer for IL-6 (Mm00446190_m1), TNF-α (Mm00443258_m1), and β-actin (Mm02619580_g1) (endogenous control) were purchased from Thermo Fisher Scientific. 2.7. Western blot analysis 7
Journal Pre-proof RAW 264.7 cells treated with indicated reagent were lysed in RIPA buffer (Cell Signaling Technology) with protease inhibitor cocktail. Protein concentration was detected by Braford protein assay (Bio-Rad). Samples with approximately 20 μg protein were separated by SDSPAGE electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% milk for 1 h, membranes were probed with the indicated primary antibodies overnight at 4 °C and then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Pierce,
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Rockford, IL). After proteins of interest were visualized using a Clarity Western ECL Substrate
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(Bio-Rad), the photographic images were obtained with the Azure Biosystems imager.
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2.8. Nuclear and Cytoplasmic Extraction
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Nuclear and cytoplasmic extractions were performed using NE-PER Nuclear and Cytoplasmic
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Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol.
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2.9. Immunoprecipitation
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Cells were washed with cold PBS and 1 ml pre-chilled PBS added to each plate. Using cell scraper, cells were collected and centrifuged at 5000 rpm for 5 min. After removal of the
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supernatant, RIPA buffer containing protease inhibitor cocktail was added to each tube and incubated on ice for 20 min. The mixture was centrifuged for 10 min at 13200 rpm, 4 °C to obtain the cell lysate. A volume of primary antibody was added so that the appropriate dilution was contained in a total volume of 800 μl along with 1000 μg (1 mg) of the cell lysate. The mixture was incubated with gentle rocking overnight at 4 °C. The next day protein G agarose (20 μl of 50% bead slurry) was added with an additional incubation of 1 h at 4 °C with gentle rocking. The samples were micro-centrifuged for 1 min at 4 °C and the pellets washed 3 times
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Journal Pre-proof with RIPA buffer, and then resuspended with SDS sample buffer. Finally the samples were vortexed, microcentrifuged, heated, and detected by western blot analysis. 2.10. Immunofluorescence microscopy Immunofluorescence microscopy was performed using the Alexa Fluor 488 Tyramide SuperBoost Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s protocol.
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Briefly, RAW 264.7 cells were seeded to the chamber slides (Corning Falcon, NY, USA). After
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treatment with indicated reagents for 24 h, cells were fixed by 4 % formaldehyde and
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permeabilized with 0.1% Triton 100 at room temperature for 15 min. Endogenous peroxidase activity was quenched by the addition of 3% hydrogen peroxide solution. After blocking the
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sample with blocking buffer for 60 min at room temperature, cells were incubated with primary
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antibody overnight at 4 °C. Next the cells were incubated with poly-HRP-conjugated secondary
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antibody for 60 min at room temperature followed by the addition of tyramide working solution and then reaction stop reagent. Slides were overlain with coverslips using antifade mounting
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microscope.
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medium with DAPI (Vectashield, Burlingame, CA). Cells were visualized using a fluorescent
2.11. Luciferase reporter assay The luciferase reporter assay was performed to measure NF-κB activation. Briefly, 1 × 106 cells were seeded into a 10 cm cell culture dish to reach 60% confluence. Reporter vectors were transfected with 8 μg luciferase vector/plate using lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. After transfection, cells were re-seeded into 24-well plates, and then pretreated with the indicated concentration of verteporfin for 2 h before stimulation with LPS for 22 h. Cells were lysed by luciferase lysis buffer (Promega). Renilla luciferase 9
Journal Pre-proof reporter was used as a transfection efficiency control. The luciferase activity of lysates was measured according to the manufacturer’s protocol using a GloMax® 96 Microplate Luminometer (Promega). The NF-κB luciferase reporter and Renilla vector were kept in our lab. 2.12. Statistical analysis All experiments were performed by three independent studies. All the data were expressed as
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mean ± SE. Statistical comparisons between groups were performed by T.TEST. P value ≤ 0 .05
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was considered statistically significant.
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3. RESULTS
3.1. Cytotoxicity of verteporfin in RAW 264.7 cells
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MTT assay was performed to examine the cytotoxicity of verteporfin in RAW 264.7 cells.
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Cell viability was measured after RAW 264.7 cells were treated with different concentrations of
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verteporfin (Fig. 1A), ranging from 1 to 12 μM for 24h. As shown in Fig. 1B, no significant difference was found in any verteporfin-treated group when compared to values from the control group (0 μM verteporfin). Live and dead assay (Fig. 1C) and live cell counting (Fig. 1D) confirmed that low cytotoxicity in RAW 264.7 cells when exposed to verteporfin. These data indicate that verteporfin exhibit low cytotoxicity in RAW 264.7 cells at the indicated concentrations. Nevertheless, the lower concentrations of verteporfin (0, 0.5, 1, and 2 μM) were selected for use in the current study.
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Journal Pre-proof 3.2. Verteporfin inhibits LPS-induced inflammation cytokine expression in RAW 264.7 cells To examine the potential of verteporfin to inhibit LPS-induced inflammation, an inflammatory cytokine array was used to assess cytokine release. As shown in Fig. 2, compared with control group (0 μM VT), LPS stimulation dramatically increased the production of GCSF,
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IL-6, CCL2, MCP-5, CCL5, and TNF-α. When RAW 264.7 cells were treated with 2 μM verteporfin, these cytokines were significantly inhibited compared with cells treated with LPS
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alone. These results suggest that verteporfin is able to inhibit LPS-induced inflammatory
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cytokine production such as GCSF, IL-6, CCL2, MCP-5, CCL5, and TNF-α, and indicate that
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verteporfin can be used as potential anti-inflammation drug.
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protein level
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3.3. Verteporfin inhibits LPS- induced IL-6 and TNF-α expression at both mRNA and
IL-6 and TNF-α play an important role in both the inflammation and carcinogenesis
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processes. To support the results obtained with the cytokine analysis, IL-6 and TNF-α levels were measured by real-time PCR. Fig. 3A shows that compared with non-treated group, IL-6 mRNA levels were significantly increased when stimulated with LPS. When cells were cotreated with LPS and verteporfin, IL-6 mRNA expression was decreased in a dose-dependent manner. In addition, the western blot analysis verified that the L-6 protein level was decreased by verteporfin in a dose-dependent manner (Fig. 3B). These results indicate that verteporfin inhibited LPS-induced IL-6 expression at both the mRNA and protein levels. Similarly, Figs. 3C and 3D show that compared with non-treated group, TNF-α mRNA and protein levels were 11
Journal Pre-proof significantly increased when stimulated with LPS. When cells were treated with verteporfin and LPS, both TNF-α mRNA and protein levels decreased. Another two cell lines, primary murine peritoneal macrophages (Figs. 3E and 3F) and human monocyte cell U-937 (Figs. 3G and 3H), were used to test IL-6 and TNF-α mRNA levels. The results show the same trend with RAW 264.7 cells in both cell lines. These data indicate that verteporfin inhibited LPS-induced TNF-α expression at both the mRNA and protein levels. Together these results indicate that verteporfin
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significantly downregulates the productions of IL-6 and TNF-α in LPS-induced cells.
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3.4. Verteporfin negatively regulates NF-κB activation in LPS-stimulated RAW 264.7 cells The NF-κB pathway is involved in a variety of inflammatory processes. To evaluate the
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potential molecular mechanism of vertrporfin against LPS-induced inflammation, NF-κB
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activation was examined. As shown in Fig. 4A, compared with control group (non-treated group), p-IKKα/β and p-NF-κB expressions were increased when cells were treated with LPS
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alone, indicating that the NF-κB pathway was activated in LPS-induced RAW 264.7 cells. When
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cells were co-treated with both verteporfin and LPS, the level of p-IKKα/β, p-NF-κB p65, and NF-κB p65 were decreased in a dose-dependent manner. These results indicated that verteporfin inhibited NF-κB activation in LPS-induced RAW 264.7 cells. Since NF-κB activates its target gene transcription through translocating into nucleus, p65 translocation was examined by western blot and cell immunofluorescence staining. Cytoplasmic and nuclear extractions were obtained for western blots. Results show that the nuclear NF-κB p65 level was increased in cells treated with LPS alone and decreased when cells were co-treated with both verteporfin and LPS (Fig. 4B). Similarly, immunofluorescence shows that NF-κB p65 translocation was inhibited by
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Journal Pre-proof verteporfin (Fig. 4C). NF-κB activation inhibitor（InSolution™ NF-κB Activation Inhibitor Calbiochem ）was used to further demonstrate that verteporfin inhibits LPS induced inflammation in RAW 264.7 cells through negative regulation of NF-κB activation. Cells were pretreated with 1 µM NF-κB activation inhibitor for 2 h before stimulated with LPS for 22 h and DMSO was used as vehicle control. Results show that NF-κB activation inhibitor could inhibit LPS induced TNF-α and IL-6 production. When in combination with verteporfin, it shows an
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additive inhibitory effect on LPS induced TNF-α and IL-6 mRNA expression (Fig. 4D). Taken
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together, these results support the hypothesis that verteporfin inhibits NF-κB activation in LPS-
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induced RAW 264.7 cells.
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3.5. Verteporfin inhibits the JAK/STAT pathway in LPS-stimulated RAW 264.7 cells
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Since IL-6 was downregulated by verteporfin in LPS-stimulated RAW 264.7 cells, we
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hypothesize that the IL-6/JAK/STAT pathway participates in the anti-inflammatory process of verteporfin. Therefore, the JAK/STAT signaling pathway was examined. As shown in Fig. 5A,
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JAK1 and JAK2 levels were decreased in a dose-dependent manner when RAW 264.7 cells were co-treated with both verteporfin and LPS, compared with cells stimulated with LPS alone. Moreover, p-STAT1 (S727), STAT1, p-STAT3 (Y705), and total STAT3 productions were inhibited by verteporfin in a dose-dependent manner (Fig. 5B). These results suggest that verteporfin inhibits the JAK/STAT pathway in LPS-induced RAW 264.7 cells.
3.6. Verteporfin induces NF-κB p65, JAK1, JAK2, STAT1, and STAT3 crosslinkages
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Journal Pre-proof Since verteporfin was reported to induce the oligomerization of many proteins, we hypothesize that verteporfin exhibits its anti-inflammatory effect through crosslinks of proteins in the signaling pathways. As expected, Fig. 6A shows a basal level of NF-κB p65 at the molecular weight of 65 kDa in non-treated RAW 264.7 cells. Verteporfin treatment induced crosslink of NF-κB p65 at a molecular weight greater than 250 kDa with a concomitant decrease in the non-crosslinked NF-κB p65 protein. In addition, in cells co-treated with both LPS and
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verteporfin, crosslinked NF-κB p65, at the higher molecular weights, increased in a dose-
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dependent manner. Similarly, verteporfin caused crosslink of JAK1 and JAK2 proteins,
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respectively, generating products at molecular weights greater than 250 kDa (Fig. 6B). Fig. 6C
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shows that verteporfin also induced crosslinks of STAT1 at molecular weight higher than 150 kDa with a decrease in original STAT1. The crosslinked STAT1 increased in a dose-dependent
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manner. In addition, Fig. 6D shows a similar crosslink of STAT3 in a dose-dependent manner.
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To demonstrate that the higher molecular weight proteins were crosslinked signaling proteins,
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immunoprecipitation was performed. As shown in Fig. 7A, NF-κB p65 was immunoprecipitated from cells stimulated with or without LPS after co-treated with or without verteporfin using an
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NF-κB p65 antibody (anti-p65 antibody #1) and immunobloted with a different NF-κB p65 antibody (anti-p65 antibody #2). High molecular weight products of NF-κB p65 were only detected in immunoprecipitates from verteporfin-treated cells. Similarly, STAT3 was immunoprecipitated from cells stimulated with or without LPS after co-treated with or without verteporfin using STAT3 rabbit antibody (anti-STAT3 antibody #1). A different antibody, STAT3 mouse antibody (anti-STAT3 antibody #2) was used for the immunoblot. Again, the high molecular weight products of STAT3 were detected only in the verteporfin treated group. These results further demonstrate that the high molecular weight oligomerization products contain NF14
Journal Pre-proof κB p65 and STAT3. NF-κB luciferase reporter assay was performed to measure the activation of NF-κB. As shown in Fig. 7C, the NF-κB transcriptional activation was inhibited by verteporfin. The western blot results also show that verteporfin induced crosslink of NF-κB p65 but not the crosslink of activated form of NF-κB p65 (p-p65) (Fig. 7D). These results suggest that the crosslinked p65 lost its signaling transduction function. The findings presented above support the concept that verteporfin causes key signaling proteins to crosslink and that these crosslinked
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proteins may have a diminished capacity to activate target inflammatory cytokines expression,
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leading to inhibition of inflammation in LPS-induced RAW 264.7 cells.
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4. DISCUSSION
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A number of widespread and devastating chronic diseases have a pathophysiological
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important inflammatory component which remains to be investigated (Tabas and glass, 2013). Full understanding about the mechanism of the inflammatory response will provide an important
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approach for the treatment of chronic diseases. Demand for potent, non-toxic and effective
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therapeutics for the treatment of inflammatory response is increasing. Verteporfin is clinically used for the photodynamic treatment of age-related macular degeneration. A phase Ⅰ/Ⅱ study shows that verteporfin photodynamic therapy in locally advanced pancreatic cancer is feasible and safe (Huggett et al., 2014). Upon the identification of verteporfin as an inhibitor of YAP/TEAD and as an autophagy inhibitor, new functions for verteporfin as an anti-tumor therapy approach without photoactivation have been published. Most reports focus on its anticancer activities for different types of tumors, with a dearth of reports about its relationship with inflammatory diseases. Our present studies demonstrate that verteporfin has a potent anti-
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Journal Pre-proof inflammatory effect in the LPS-induced RAW 264.7 cell model through inhibition of the NF-κB and JAK/STAT pathways by inducing crosslink of signaling proteins in these pathways. These findings demonstrate that verteporfin could serve as a therapeutic agent for inflammatory diseases. IL-6 and TNF-α are important and widely studied pro-inflammatory cytokines which are
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produced by many cell types including macrophages (Browning et al., 2018; Popa et al., 2007). Dysregulation of IL-6 expression has been implicated in the pathogenesis of various disorders.
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IL-6 is the major stimulator for the production of most acute phase proteins (Gauldie et al., 1987).
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High levels of acute phase proteins are observed in both acute and chronic inflammatory diseases,
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contributing to the disease pathology. These proteins have long been used as a clinical guide for
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diagnosis and management. IL-6 was initially considered to be a marker for a variety of inflammatory diseases. In addition, IL-6 was demonstrated to be a target for inflammatory
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diseases (Yao et al., 2014). The level of IL-6 in serum was elevated in a number of
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inflammation-related diseases, such as rheumatoid arthritis, Castleman disease, and Crohn's disease (Kishimoto, 2010). TNF-α also plays a major role in the pathogenesis of inflammation
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and serves as therapeutic target in numerous autoimmune and inflammatory system diseases (Jarrot and Kaplanski, 2014). Elevated serum and tissue levels of TNF-α were found in inflammatory and infectious conditions and serum levels were correlated with the severity of infections (Bradley, 2008). The important roles of IL-6 and TNF-α in inflammatory diseases and pathology led to the development of therapies that target TNF-α and IL-6. Our current study shows that verteporfin inhibited IL-6 and TNF-α at both mRNA and protein levels, indicating that verteporfin, which is already in clinical use, can serve as a therapeutic agent against inflammatory diseases. 16
Journal Pre-proof TNF-α is a potent inducer of NF-κB (Schütze, 1995) and IL-6 is important in inflammatory processes by activating the JAK/STAT pathway (Rincon and Irvin, 2012). NF-κB activation also increases the production of TNF-α (Liu et al., 2017) and IL-6 (Libermann and Baltimore, 1990), which forms a positive feedback loop. Targeting NF-κB and JAK/STAT pathways with the eventual down regulation of TNF-α and IL-6 plays an important role in therapeutic strategies against inflammatory diseases (Lin TH et al., 2017; Gao Q et al., 2018). In present study, our
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data demonstrate that the NF-κB and JAK/STAT pathways participate in this process. Our results
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show that NF-κB activation and JAK/STAT expression were inhibited by verteporfin. Those
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results demonstrate that verteporfin inhibits inflammatory processes through the NF-κB and
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JAK/STAT pathways in LPS-induced RAW 264.7 cells.
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Beyond the traditional signaling inhibition, verteporfin is also known as a powerful inducer for the function of protein oligomers. Previous studies showed that verteporfin could induce high
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molecular oligomers, such as p62 (Donohue et al., 2014) and STAT3 (Zhang et al., 2015).
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Oxidized and crosslinked proteins can lead to loss of function of these proteins depending on the sites and extent of modifications (Luo J et al., 2006; Kim et al., 2001). Verteporfin causes high
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molecular weight crosslinked p62 oligomers by a mechanism that involved low-level singlet oxygen production. These crosslinked p62 oligomers have a defective binding to polyubiquitinated proteins (Donohue et al., 2014). In present study, we observed verteporfininduced crosslinks of NF-κB p65, JAK1, JAK2, STAT1, and STAT3. Immunoprecipitation verified the presence of high molecular weight proteins, documenting that the crosslinks were induced by verteporfin. Although there is less direct evidence, the crosslinked proteins at high molecular weights have the potential to lose their transcriptional activities and thus inhibit the inflammatory process. 17
Journal Pre-proof In summary, this study demonstrates that: (1) verteporfin inhibits the pro-inflammatory cytokines IL-6 and TNF-α expression at both mRNA and protein levels; (2) verteporfin inhibits the inflammatory process by negatively regulating NF-κB and JAK/STAT pathways; (3) verteporfin induces crosslinks with proteins including NF-κB p65, JAK1, JAK2, STAT1, and STAT3, which may impair the function of these proteins; and (4) the anti-inflammation ability of
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verteporfin may in part be due to its ability to generate crosslinked protein products.
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CONFLICT OF INTEREST
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The authors declare no conflicts of interest.
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Journal Pre-proof REFERENCES Bradley J.R., 2008. TNF‐ mediated inflammatory disease. J Pathol. 214, 149-160. Browning L., Patel M.R., Horvath E.B., Tawara K., Jorcyk C.L., 2018. IL-6 and ovarian cancer: inflammatory cytokines in promotion of metastasis. Cancer Manag. Res. 10, 6685-6693.
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Ruparelia N., Chai J.T., Fisher E.A., Choudhury R.P., 2017. Inflammatory processes in cardiovascular disease: a route to targeted therapies. Nat. Rev. Cardiol. 14, 133-144. Schütze S., Wiegmann K., Machleidt T., Krönke M., 1995. TNF-Induced Activation of NF-κB. Immunobiology 193, 193-203. Tabas I., Glass C.K., 2013. Anti-inflammatory therapy in chronic disease: challenges and opportunities. Science 339, 166-172.
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Journal Pre-proof FIGURE LEGENDS Fig. 1. Verteporfin has low cytotoxicity in RAW 264.7 cells. A) The chemical structure of verteporfin. RAW 264.7 cells were treated with different concentrations of verteporfin (0-12 μM) for 24 h. B) Cell viability was quantified by MTT assay. The data are expressed as the mean ± SE of six repetitions. C) Cell cytotoxicity was detected by live and dead assay. Live and dead assay shows live cells stained with Calcein-AM (green) and dead cells with EthD-1 (red). Scale
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bar = 100 µm. D) Live cell percentage was quantified by live cell counting assay. Cell
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suspension was mixed with trypan blue and live cells were counted using Bio-Rad cell counter.
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The data are expressed as the mean ± SE of three repetitions.
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Fig. 2. Verteporfin inhibits LPS-induced cytokines in RAW 264.7 cells. A) Raw 264.7 cells
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were pre-incubated with 2 μM verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. Cytokines in cell lysates were detected. Red boxes indicate inhibited cytokines. B)
(BLANK).
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Cytokine array map. Positive Control Spots (POS), Negative Control Spots (NEG), Blank Spots
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Fig. 3. Verteporfin inhibits LPS-induced IL-6 and TNF-α expression. RAW 264.7 cells were
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pre-incubated with different concentration of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. A) The graph shows IL-6 mRNA levels measured by real-time RCR. B) The blot shows IL-6 protein level was measured by western blot analysis. C) The graph shows TNF-α mRNA levels measured by real-time PCR. D) The blot shows TNF-α protein level was measured by western blot analysis. The primary murine peritoneal macrophages cells were pre-incubated with 0.5 μM verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. E) The graph shows IL-6 mRNA levels measured by real-time RCR. F) The graph shows TNF-α mRNA levels measured by real-time PCR. Human monocyte cells U-937 were pre-incubated with 1 μM 24
Journal Pre-proof verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. G) The graph shows IL-6 mRNA levels measured by real-time RCR. H) The graph shows TNF-α mRNA levels measured by real-time PCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05 versus control on cells treated with LPS alone. Fig. 4. Verteporfin negatively regulates NF-κB activation in LPS-stimulated RAW 264.7
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Cells. RAW 264.7 cells were pre-incubated with different concentrations of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. A) Protein levels of p-IKKα/β, p-NF-κB
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p65, and NF-κB p65 were determined by western blot analysis. B) Protein levels of NF-κB p65
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in the cytoplasmic extracts (CE) and nuclear extracts (NE) were determined by western blot
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analysis. β-actin and HDAC1 were used as cytoplasm and nuclei loading controls respectively.
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C) Images of immunofluorescence show inhibition of NF-κB p65 (green) translocation with verteporfin treatment. Nuclei were stained with DAPI (blue). Scale bar = 50 µm. RAW 264.7
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cells were pre-incubated with different concentration of verteporfin or vehicle control and 1 μM
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NF-κB activation inhibitor for 2 h, and then stimulated with 1 μg/ml LPS for 22 h. D) The graph shows IL-6 mRNA levels measured by real-time RCR. E) The graph shows TNF-α mRNA levels
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measured by real-time PCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05 versus control on cells treated with LPS alone. Fig. 5. Verteporfin inhibits JAK/STAT pathway in LPS-stimulated RAW 264.7 cells. Raw 264.7 cells were pre-incubated with different concentrations of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. A) Protein levels of JAK1 and JAK2 were determined by western blot analysis. B) Protein levels of p-STAT1 (S727), STAT1, p-STAT3 (Y705), and STAT3 were determined by western blot analysis.
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Journal Pre-proof Fig. 6. Verteporfin causes protein crosslinkages in LPS-stimulated RAW 264.7 cells. Raw 264.7 cells were pre-incubated with different concentrations of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. A) The crosslinked NF-κB p65 was determined by western blot analysis. B) The crosslinked JAK1 and JAK2 were determined by western blot analysis. C) The crosslinked STAT1 was determined by western blot analysis. D) The crosslinked STAT3 was determined by western blot analysis. Different exposure times were
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showed in A, C, and D.
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Fig. 7. Verteporfin induces protein high molecular weight oligomerization. Raw 264.7 cells
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were pre-incubated with different concentrations of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. Cell lysates were prepared for immunoprecipitation experiments.
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A) Proteins were immunoprecipitated with NF-κB p65 antibody #1, and identified on the
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immunoblot using a different NF-κB p65 antibody (antibody #2). B) Proteins were
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immunoprecipitated with STAT3 antibody #1, and then identified on the immunoblot using STAT3 antibody #2. C) RAW 264.7 cells were transfected with NF-κB luciferase reporter and
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Renila vector, and then treated as indicated conditions. The graph shows luciferase-reported NF-
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κB transcriptional activity. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05 versus control on cells treated with LPS alone. D) The crosslinked NF-κB p65 and activated p65 form p-p65 were determined by western blot analysis.
SUPPLEMENTARY DATA Supplementary Fig. 1. Verteporfin inhibits LPS-induced CCL5 mRNA in RAW 264.7 cells. RAW 264.7 cells were pre-incubated with different concentration of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. The graph shows CCL5 mRNA levels measured 26
Journal Pre-proof by real-time RCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤ 0.05 versus control on cells treated with LPS alone. Supplementary Fig. 2. Light stimulation enhances inhibition effect of verteporfin on IL-6 and TNF-a mRNA expression in RAW 264.7 cells. RAW 264.7 cells were pre-incubated with 2 μM verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. Light stimulation
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groups were stimulated with light 30 min before collected cells. A) The graph shows IL-6
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mRNA levels measured by real-time RCR. B) The graph shows TNF-α mRNA levels measured
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by real-time PCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05
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versus control on cells treated with LPS alone.
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Supplementary Fig. 3. The effect of LPS dose in RAW264.7 cells. RAW 264.7 cells were treated with different concentration of LPS (0~3 μg/ml). A) The graph shows IL-6 mRNA levels
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measured by real-time RCR. B) The graph shows TNF-α mRNA levels measured by real-time
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PCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05 versus
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control on cells treated with 0.5 µg/ml LPS.
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Author Contribution Statement
Yuting Wang: Conceptualization, Investigation, Writing- Original draft preparation, Writing review & editing. Lei Wang: Conceptualization, Writing - review & editing James T.F. Wise: Writing - review & editing
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Xianglin Shi: Supervision, Project administration, Writing - review & editing
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Zhimin Chen: Supervision, Project administration, Writing - review & editing
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Journal Pre-proof Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Highlights
Verteporfin inhibits inflammation in RAW 264.7 cells.
Verteporfin inhibits IL-6 and TNF-α expression at both mRNA and protein levels in RAW 264.7 cells.
Verteporfin inhibits NF-κB and JAK/STAT pathways in RAW 264.7 cells.
Verteporfin induces crosslinks with proteins including NF-κB p65, JAK1, JAK2, STAT1,
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and STAT3 in RAW 264.7 cells.
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Figure 1
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Yuting Wang, Lei Wang, James T.F. Wise, Xianglin Shi, Zhimin Chen PII:
S0041-008X(19)30460-0
DOI:
https://doi.org/10.1016/j.taap.2019.114852
Reference:
YTAAP 114852
To appear in:
Toxicology and Applied Pharmacology
Received date:
23 May 2019
Revised date:
3 December 2019
Accepted date:
4 December 2019
Please cite this article as: Y. Wang, L. Wang, J.T.F. Wise, et al., Verteporfin inhibits lipopolysaccharide-induced inflammation by multiple functions in RAW 264.7 cells, Toxicology and Applied Pharmacology (2019), https://doi.org/10.1016/j.taap.2019.114852
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
Verteporfin inhibits lipopolysaccharide-induced inflammation by multiple functions in RAW 264.7 cells
Department of Pulmonology, Children's Hospital, Zhejiang University School of Medicine,
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a
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Yuting Wanga,b , Lei Wangb, James T.F. Wiseb,c, Xianglin Shib,⁎, Zhimin Chena,⁎⁎
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Hangzhou, Zhejiang 310052, People's Republic of China b
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Center for Research on Environmental Disease, College of Medicine, University of Kentucky,
Department of Pharmacology and Nutritional Sciences, College of Medicine, University of
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c
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1095 VA Drive, Lexington, KY 40536, USA
Correspondence to: X. Shi, Center for Research on Environmental Disease, College of
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⁎
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Kentucky, 1095 VA Drive, Lexington, KY 40536, USA
Medicine, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USA. [email protected] ⁎⁎
Correspondence to: Z. Chen, Department of Pulmonology, Children's Hospital, Zhejiang
University School of Medicine, Hangzhou, Zhejiang 310052, People's Republic of China. [email protected]
1
Journal Pre-proof Abstract: Inflammation is a physiologic response to damage triggered by infection, injury or chemical irritation. Chronic inflammation produces repeated damage to cells and tissues, which can induce a variety of human diseases including cancer. Verteporfin, an FDA approved drug, is used for the treatment of age-related macular degeneration. The anti-tumor effects of verteporfin have been
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demonstrated by a number of studies. However, fewer studies focus on the anti-inflammatory functions of this drug. In this study, we investigated the anti-inflammatory effects and potential
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mechanisms of verteporfin. The classic lipopolysaccharide (LPS)-induced inflammation cell
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model was used. RAW 264.7 cells were pre-treated with verteporfin or vehicle control, followed
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by LPS stimulation. Verteporfin inhibited IL-6 and TNF-α at mRNA and protein expression
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levels. This effect was mediated through inhibition of the NF-κB and JAK/STAT pathways. Finally, verteporfin exhibited an anti-inflammation effect by crosslinking of protein such as NF-
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κB p65, JAK1, JAK2, STAT1, or STAT3 leading to inflammation. Taken together, these results
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inflammatory diseases.
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indicate that verteporfin has the potential to be an effective therapeutic agent against
Key words: inflammation; verteporfin; LPS; RAW 264.7
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Journal Pre-proof 1. INTRODUCTION
Inflammation is a physiologic response to damage triggered by infection, injury or chemical irritation (Philip et al., 2004). A successful acute inflammatory response results in recovery from the damage with subsequent healing. However, chronic inflammation will lead to persistent
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damage to cells and tissues and generate a variety of human diseases, such as cancer,
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autoimmune disease, cardiovascular disease, diabetes, and neuro degenerative disease (Nathan,
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2002; Coussens et al., 2002; Kim and Moudgil, 2017; Ruparelia et al., 2017; Wellen and Hotamisligil, 2005; Heneka et al., 2015). Macrophages play an important role in the
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inflammatory process. Macrophages are recruited to primary inflammatory sites and secrete a
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variety of inflammatory mediators, including TNF-a, IL-6, CCL2, and CCL5 (Wynn et al.,
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2013). Targeting macrophages could be consequential as a therapeutic approach against inflammatory diseases. Lipopolysaccharide (LPS), derived from gram-negative bacteria, is one
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of the common inflammatory inducers. LPS-induced macrophages are exceptionally adept at
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producing a cascade of inflammatory cytokines and mediators (Hamidzadeh et al., 2017). Thus, the LPS-induced macrophage cell model is widely used in the study of inflammation. Increasing evidences show that NF-κB and JAK/STAT signaling pathways play an important role in LPS-induced inflammation for macrophages. The transcription factor NF-κB is one of the major regulators in inflammatory process (DiDonato et al., 2012). LPS is able to combine with toll-like receptors (TLRs), leading to activation of the IκB kinase (IKK) complex and phosphorylation of IκBα. The activated NF-κB is then released from its inhibitory subunit (IκBα) and translocated to the nucleus to stimulate transcription of a number of target genes, including 3
Journal Pre-proof TNF-α and IL-6 (Hoesel and Schmid, 2013). These pro-inflammatory cytokines contribute to the overall inflammatory process. When IL-6 combines with its receptor (IL-6R), JAK family tyrosine kinases (JAK1, JAK2, and Tyk2) are phosphorylated and activated. Signal transducer and activator of transcription 3 (STAT3) and signal transducer and activator of transcription 1 (STAT1) are recruited and
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phosphorylated by JAK kinases. The activated STAT3 and STAT1 proteins dimerize to create STAT3 or STAT1 homodimers and STAT3/STAT1 heterodimers. These activated STAT dimers
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genes to promote expression (Kojima et al., 2013).
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enter the nucleus and bind to specific DNA sequences in the regulatory regions of their target
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Verteporfin (VT) is a photosensitizing protoporphyrin derivative, which is an FDA approved
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drug used for the photodynamic treatment of age-related macular degeneration (Dasari et al.,
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2017). Verteporfin has been identified as a YAP/TEAD inhibitor (Wei et al., 2017) as well as autophagosome inhibitor by promoting oligomerization of p62 (Gibault et al., 2016). Recent
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studies show that verteporfin has the potential for the treatment of various types of cancers, such
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as pancreatic ductal adenocarcinoma (Donohue et al., 2013), ovarian cancer (Feng et al., 2016), colon cancer (Zhang et al., 2015), and prostate cancer (Wang et al., 2018). Inflammatory process plays an important role in different stages of tumor development (Elinav et al., 2013). There are only limited studies available about the effect of verteporfin on inflammatory process. We hypothesized that verteporfin has the property to inhibit inflammation. In this study we tested this hypothesis and explored the possible mechanisms responsible for this process. 2. METHODS 2.1. Reagents 4
Journal Pre-proof Verteporfin was purchased from AdooQ Bioscience (Irvine, CA). Lipopolysaccharide (LPS) from Escherichia coli O111:B4 and InSolution™ NF-κB Activation Inhibitor - Calbiochem was purchased from Sigma‐ Aldrich (#481407) (St. Louis, MO). The antibodies against IL-6, TNF-α, p-IKKα/β, p-NF-κB p65, NF-κB p65 (rabbit), HDAC1, JAK1, JAK2, p-STAT1 (S727), STAT1, p-STAT3 (Y705), and STAT3 were purchased from Cell Signaling Technology (Danvers, MA). NF-κB p65 (mouse) antibody was purchased from Santa Cruz Biotechnology, Inc (Dallas,
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Texas). β-actin antibody was purchased from Sigma‐ Aldrich.
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2.2. Cell culture
Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased
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from Gibco (Thermo Fisher Scientific, Waltham, MA). RAW 264.7 and U-937 cells were
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obtained from American Type Culture Collection (ATCC, Manassas, VA). RAW 264.7 cells
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were cultured in DMEM supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml) and 10% FBS. U-937 cells were cultured in RPMI-1640 (Thermo Fisher Scientific)
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supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml) and 10% FBS. Primary
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murine peritoneal macrophages were isolated from new euthanasia C3H mouse and cultured in DMEM medium. Cells were incubated at 37 °C in 5% carbon dioxide (CO 2 ) with a humidified atmosphere of 95% air. RAW 264.7 cells were stimulated by LPS (1 μg/ml) for the indicated time period(s) after pretreated with different concentrations of verteporfin (0, 0.5, 1, or 2 μM) for 2 h. All verteporfin treatments are in darkness unless otherwise stated. 2.3. Cell viability Cell viability was evaluated by MTT assay. Briefly, RAW 264.7 cells were seeded at a density of 5 103 cells/well in 96-well plates. After 24 h, cells were treated with different 5
Journal Pre-proof concentrations of verteporfin (0-12 μM) for 24 h, followed by the addition of 20 μl 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT, Sigma‐ Aldrich) solution (5 mg/ml) to each well for 2 h. The formazan product was solubilized with 200 μl DMSO/well. The absorbance was measured using a microplate reader at a test wavelength of 560 nm. Results are presented as a calculated percentage of absorbance in control groups.
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2.4. Live and dead assay and live cell counting assay
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Live and dead assay was performed using LIVE/DEAD™ Viability/Cytotoxicity Kit (Thermo Fisher Scientific). Briefly, RAW 264.7 cells were seeded into 6 well plates and treated with
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different concentrations of verteporfin (0~10 μM) for 24 h with or without light stimulation.
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Light stimulation groups were stimulated with ambient light 30 min before cell staining. Then
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culture medium were removed and 1 ml of the staining solution (20 µL ethidium homodimer-1 and 5 µL calcein AM mixed in 10 mL PBS) were added directly to cells. After 15 min
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incubation, cells were imaged using a fluorescent microscope.
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Live cell counting was performed to obtain live cell percentage. Briefly, RAW 264.7 cells
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were seeded into 6 well plates and treated with different concentrations of verteporfin (0~10 μM) for 24 h. Then 10 μl of cell suspension were mixed with 10 μl of trypan blue (Bio-Rad, Hercules, CA). Live cells were counted using TC20™ Automated Cell Counter (Bio-Rad). 2.5. Cytokine array Cytokine array analyses were performed using RayBio C-Series Mouse Cytokine Antibody Array C1, which was purchased from RayBiotech Life (Norcross, GA). RAW 264.7 cells were pretreated with 2 μM verteporfin for 2 h, then stimulated with LPS (1 μg/ml) for 22 h. Cell pellets were obtained by centrifugation and the associated cell lysates were analyzed according 6
Journal Pre-proof to the manufacturer's instructions. Briefly, cytokine array membranes were incubated with blocking buffer for 30 min, replaced with 500 μg of total protein in 1 ml, and incubated overnight at 4 °C. The next day, the sample was aspirated and the membrane washed. Next, 1 ml of the biotinylated antibody cocktail was added to the membrane and incubated for 2 h at room temperature. Washed membranes were incubated with 2 ml 1 HRP-Streptavidin and washed. The membrane were developed using the kit's enhanced chemiluminescence system and data
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2.6. Real-Time PCR
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were analyzed with the Azure Biosystems imager.
Total RNA was isolated using the TRIzol Reagent (Thermo Fisher Scientific) according to the
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manufacturer’s protocol. Reverse transcription reactions were performed by the following
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process. For a 20 μl reaction, 1 μl oligo (dT) (500 μg/ml), and 2 μg total RNA were added to a nuclease-free micro-centrifuge tube, then adjusted to 12 μl with nuclease-free water. The mixture
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was heated to 65 °C for 5 min and quick chilled on ice. Next 4 μl of 5 First-Strand Buffer, 2 μl
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0.1 M DTT, and 1 μl RNaseOUT Recombinant Ribonuclease Inhibitor (40 units/μl) were added
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and mixed gently. The mixture was incubated at 37 °C for 2 min followed by the addition of 1 μl (200 units) M-MLV and again incubated at 37 °C for 50 min. The reaction was inactivated by heating at 70 °C for 15 min. The cDNA obtained was used for real-time PCR. Real-time PCR was performed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). Primer for IL-6 (Mm00446190_m1), TNF-α (Mm00443258_m1), and β-actin (Mm02619580_g1) (endogenous control) were purchased from Thermo Fisher Scientific. 2.7. Western blot analysis 7
Journal Pre-proof RAW 264.7 cells treated with indicated reagent were lysed in RIPA buffer (Cell Signaling Technology) with protease inhibitor cocktail. Protein concentration was detected by Braford protein assay (Bio-Rad). Samples with approximately 20 μg protein were separated by SDSPAGE electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% milk for 1 h, membranes were probed with the indicated primary antibodies overnight at 4 °C and then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Pierce,
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Rockford, IL). After proteins of interest were visualized using a Clarity Western ECL Substrate
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(Bio-Rad), the photographic images were obtained with the Azure Biosystems imager.
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2.8. Nuclear and Cytoplasmic Extraction
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Nuclear and cytoplasmic extractions were performed using NE-PER Nuclear and Cytoplasmic
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Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol.
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2.9. Immunoprecipitation
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Cells were washed with cold PBS and 1 ml pre-chilled PBS added to each plate. Using cell scraper, cells were collected and centrifuged at 5000 rpm for 5 min. After removal of the
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supernatant, RIPA buffer containing protease inhibitor cocktail was added to each tube and incubated on ice for 20 min. The mixture was centrifuged for 10 min at 13200 rpm, 4 °C to obtain the cell lysate. A volume of primary antibody was added so that the appropriate dilution was contained in a total volume of 800 μl along with 1000 μg (1 mg) of the cell lysate. The mixture was incubated with gentle rocking overnight at 4 °C. The next day protein G agarose (20 μl of 50% bead slurry) was added with an additional incubation of 1 h at 4 °C with gentle rocking. The samples were micro-centrifuged for 1 min at 4 °C and the pellets washed 3 times
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Journal Pre-proof with RIPA buffer, and then resuspended with SDS sample buffer. Finally the samples were vortexed, microcentrifuged, heated, and detected by western blot analysis. 2.10. Immunofluorescence microscopy Immunofluorescence microscopy was performed using the Alexa Fluor 488 Tyramide SuperBoost Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s protocol.
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Briefly, RAW 264.7 cells were seeded to the chamber slides (Corning Falcon, NY, USA). After
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treatment with indicated reagents for 24 h, cells were fixed by 4 % formaldehyde and
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permeabilized with 0.1% Triton 100 at room temperature for 15 min. Endogenous peroxidase activity was quenched by the addition of 3% hydrogen peroxide solution. After blocking the
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sample with blocking buffer for 60 min at room temperature, cells were incubated with primary
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antibody overnight at 4 °C. Next the cells were incubated with poly-HRP-conjugated secondary
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antibody for 60 min at room temperature followed by the addition of tyramide working solution and then reaction stop reagent. Slides were overlain with coverslips using antifade mounting
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microscope.
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medium with DAPI (Vectashield, Burlingame, CA). Cells were visualized using a fluorescent
2.11. Luciferase reporter assay The luciferase reporter assay was performed to measure NF-κB activation. Briefly, 1 × 106 cells were seeded into a 10 cm cell culture dish to reach 60% confluence. Reporter vectors were transfected with 8 μg luciferase vector/plate using lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. After transfection, cells were re-seeded into 24-well plates, and then pretreated with the indicated concentration of verteporfin for 2 h before stimulation with LPS for 22 h. Cells were lysed by luciferase lysis buffer (Promega). Renilla luciferase 9
Journal Pre-proof reporter was used as a transfection efficiency control. The luciferase activity of lysates was measured according to the manufacturer’s protocol using a GloMax® 96 Microplate Luminometer (Promega). The NF-κB luciferase reporter and Renilla vector were kept in our lab. 2.12. Statistical analysis All experiments were performed by three independent studies. All the data were expressed as
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mean ± SE. Statistical comparisons between groups were performed by T.TEST. P value ≤ 0 .05
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was considered statistically significant.
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3. RESULTS
3.1. Cytotoxicity of verteporfin in RAW 264.7 cells
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MTT assay was performed to examine the cytotoxicity of verteporfin in RAW 264.7 cells.
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Cell viability was measured after RAW 264.7 cells were treated with different concentrations of
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verteporfin (Fig. 1A), ranging from 1 to 12 μM for 24h. As shown in Fig. 1B, no significant difference was found in any verteporfin-treated group when compared to values from the control group (0 μM verteporfin). Live and dead assay (Fig. 1C) and live cell counting (Fig. 1D) confirmed that low cytotoxicity in RAW 264.7 cells when exposed to verteporfin. These data indicate that verteporfin exhibit low cytotoxicity in RAW 264.7 cells at the indicated concentrations. Nevertheless, the lower concentrations of verteporfin (0, 0.5, 1, and 2 μM) were selected for use in the current study.
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Journal Pre-proof 3.2. Verteporfin inhibits LPS-induced inflammation cytokine expression in RAW 264.7 cells To examine the potential of verteporfin to inhibit LPS-induced inflammation, an inflammatory cytokine array was used to assess cytokine release. As shown in Fig. 2, compared with control group (0 μM VT), LPS stimulation dramatically increased the production of GCSF,
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IL-6, CCL2, MCP-5, CCL5, and TNF-α. When RAW 264.7 cells were treated with 2 μM verteporfin, these cytokines were significantly inhibited compared with cells treated with LPS
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alone. These results suggest that verteporfin is able to inhibit LPS-induced inflammatory
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cytokine production such as GCSF, IL-6, CCL2, MCP-5, CCL5, and TNF-α, and indicate that
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verteporfin can be used as potential anti-inflammation drug.
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protein level
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3.3. Verteporfin inhibits LPS- induced IL-6 and TNF-α expression at both mRNA and
IL-6 and TNF-α play an important role in both the inflammation and carcinogenesis
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processes. To support the results obtained with the cytokine analysis, IL-6 and TNF-α levels were measured by real-time PCR. Fig. 3A shows that compared with non-treated group, IL-6 mRNA levels were significantly increased when stimulated with LPS. When cells were cotreated with LPS and verteporfin, IL-6 mRNA expression was decreased in a dose-dependent manner. In addition, the western blot analysis verified that the L-6 protein level was decreased by verteporfin in a dose-dependent manner (Fig. 3B). These results indicate that verteporfin inhibited LPS-induced IL-6 expression at both the mRNA and protein levels. Similarly, Figs. 3C and 3D show that compared with non-treated group, TNF-α mRNA and protein levels were 11
Journal Pre-proof significantly increased when stimulated with LPS. When cells were treated with verteporfin and LPS, both TNF-α mRNA and protein levels decreased. Another two cell lines, primary murine peritoneal macrophages (Figs. 3E and 3F) and human monocyte cell U-937 (Figs. 3G and 3H), were used to test IL-6 and TNF-α mRNA levels. The results show the same trend with RAW 264.7 cells in both cell lines. These data indicate that verteporfin inhibited LPS-induced TNF-α expression at both the mRNA and protein levels. Together these results indicate that verteporfin
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significantly downregulates the productions of IL-6 and TNF-α in LPS-induced cells.
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3.4. Verteporfin negatively regulates NF-κB activation in LPS-stimulated RAW 264.7 cells The NF-κB pathway is involved in a variety of inflammatory processes. To evaluate the
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potential molecular mechanism of vertrporfin against LPS-induced inflammation, NF-κB
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activation was examined. As shown in Fig. 4A, compared with control group (non-treated group), p-IKKα/β and p-NF-κB expressions were increased when cells were treated with LPS
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alone, indicating that the NF-κB pathway was activated in LPS-induced RAW 264.7 cells. When
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cells were co-treated with both verteporfin and LPS, the level of p-IKKα/β, p-NF-κB p65, and NF-κB p65 were decreased in a dose-dependent manner. These results indicated that verteporfin inhibited NF-κB activation in LPS-induced RAW 264.7 cells. Since NF-κB activates its target gene transcription through translocating into nucleus, p65 translocation was examined by western blot and cell immunofluorescence staining. Cytoplasmic and nuclear extractions were obtained for western blots. Results show that the nuclear NF-κB p65 level was increased in cells treated with LPS alone and decreased when cells were co-treated with both verteporfin and LPS (Fig. 4B). Similarly, immunofluorescence shows that NF-κB p65 translocation was inhibited by
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Journal Pre-proof verteporfin (Fig. 4C). NF-κB activation inhibitor（InSolution™ NF-κB Activation Inhibitor Calbiochem ）was used to further demonstrate that verteporfin inhibits LPS induced inflammation in RAW 264.7 cells through negative regulation of NF-κB activation. Cells were pretreated with 1 µM NF-κB activation inhibitor for 2 h before stimulated with LPS for 22 h and DMSO was used as vehicle control. Results show that NF-κB activation inhibitor could inhibit LPS induced TNF-α and IL-6 production. When in combination with verteporfin, it shows an
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additive inhibitory effect on LPS induced TNF-α and IL-6 mRNA expression (Fig. 4D). Taken
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together, these results support the hypothesis that verteporfin inhibits NF-κB activation in LPS-
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induced RAW 264.7 cells.
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3.5. Verteporfin inhibits the JAK/STAT pathway in LPS-stimulated RAW 264.7 cells
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Since IL-6 was downregulated by verteporfin in LPS-stimulated RAW 264.7 cells, we
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hypothesize that the IL-6/JAK/STAT pathway participates in the anti-inflammatory process of verteporfin. Therefore, the JAK/STAT signaling pathway was examined. As shown in Fig. 5A,
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JAK1 and JAK2 levels were decreased in a dose-dependent manner when RAW 264.7 cells were co-treated with both verteporfin and LPS, compared with cells stimulated with LPS alone. Moreover, p-STAT1 (S727), STAT1, p-STAT3 (Y705), and total STAT3 productions were inhibited by verteporfin in a dose-dependent manner (Fig. 5B). These results suggest that verteporfin inhibits the JAK/STAT pathway in LPS-induced RAW 264.7 cells.
3.6. Verteporfin induces NF-κB p65, JAK1, JAK2, STAT1, and STAT3 crosslinkages
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Journal Pre-proof Since verteporfin was reported to induce the oligomerization of many proteins, we hypothesize that verteporfin exhibits its anti-inflammatory effect through crosslinks of proteins in the signaling pathways. As expected, Fig. 6A shows a basal level of NF-κB p65 at the molecular weight of 65 kDa in non-treated RAW 264.7 cells. Verteporfin treatment induced crosslink of NF-κB p65 at a molecular weight greater than 250 kDa with a concomitant decrease in the non-crosslinked NF-κB p65 protein. In addition, in cells co-treated with both LPS and
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verteporfin, crosslinked NF-κB p65, at the higher molecular weights, increased in a dose-
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dependent manner. Similarly, verteporfin caused crosslink of JAK1 and JAK2 proteins,
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respectively, generating products at molecular weights greater than 250 kDa (Fig. 6B). Fig. 6C
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shows that verteporfin also induced crosslinks of STAT1 at molecular weight higher than 150 kDa with a decrease in original STAT1. The crosslinked STAT1 increased in a dose-dependent
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manner. In addition, Fig. 6D shows a similar crosslink of STAT3 in a dose-dependent manner.
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To demonstrate that the higher molecular weight proteins were crosslinked signaling proteins,
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immunoprecipitation was performed. As shown in Fig. 7A, NF-κB p65 was immunoprecipitated from cells stimulated with or without LPS after co-treated with or without verteporfin using an
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NF-κB p65 antibody (anti-p65 antibody #1) and immunobloted with a different NF-κB p65 antibody (anti-p65 antibody #2). High molecular weight products of NF-κB p65 were only detected in immunoprecipitates from verteporfin-treated cells. Similarly, STAT3 was immunoprecipitated from cells stimulated with or without LPS after co-treated with or without verteporfin using STAT3 rabbit antibody (anti-STAT3 antibody #1). A different antibody, STAT3 mouse antibody (anti-STAT3 antibody #2) was used for the immunoblot. Again, the high molecular weight products of STAT3 were detected only in the verteporfin treated group. These results further demonstrate that the high molecular weight oligomerization products contain NF14
Journal Pre-proof κB p65 and STAT3. NF-κB luciferase reporter assay was performed to measure the activation of NF-κB. As shown in Fig. 7C, the NF-κB transcriptional activation was inhibited by verteporfin. The western blot results also show that verteporfin induced crosslink of NF-κB p65 but not the crosslink of activated form of NF-κB p65 (p-p65) (Fig. 7D). These results suggest that the crosslinked p65 lost its signaling transduction function. The findings presented above support the concept that verteporfin causes key signaling proteins to crosslink and that these crosslinked
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proteins may have a diminished capacity to activate target inflammatory cytokines expression,
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leading to inhibition of inflammation in LPS-induced RAW 264.7 cells.
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4. DISCUSSION
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A number of widespread and devastating chronic diseases have a pathophysiological
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important inflammatory component which remains to be investigated (Tabas and glass, 2013). Full understanding about the mechanism of the inflammatory response will provide an important
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approach for the treatment of chronic diseases. Demand for potent, non-toxic and effective
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therapeutics for the treatment of inflammatory response is increasing. Verteporfin is clinically used for the photodynamic treatment of age-related macular degeneration. A phase Ⅰ/Ⅱ study shows that verteporfin photodynamic therapy in locally advanced pancreatic cancer is feasible and safe (Huggett et al., 2014). Upon the identification of verteporfin as an inhibitor of YAP/TEAD and as an autophagy inhibitor, new functions for verteporfin as an anti-tumor therapy approach without photoactivation have been published. Most reports focus on its anticancer activities for different types of tumors, with a dearth of reports about its relationship with inflammatory diseases. Our present studies demonstrate that verteporfin has a potent anti-
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Journal Pre-proof inflammatory effect in the LPS-induced RAW 264.7 cell model through inhibition of the NF-κB and JAK/STAT pathways by inducing crosslink of signaling proteins in these pathways. These findings demonstrate that verteporfin could serve as a therapeutic agent for inflammatory diseases. IL-6 and TNF-α are important and widely studied pro-inflammatory cytokines which are
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produced by many cell types including macrophages (Browning et al., 2018; Popa et al., 2007). Dysregulation of IL-6 expression has been implicated in the pathogenesis of various disorders.
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IL-6 is the major stimulator for the production of most acute phase proteins (Gauldie et al., 1987).
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High levels of acute phase proteins are observed in both acute and chronic inflammatory diseases,
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contributing to the disease pathology. These proteins have long been used as a clinical guide for
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diagnosis and management. IL-6 was initially considered to be a marker for a variety of inflammatory diseases. In addition, IL-6 was demonstrated to be a target for inflammatory
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diseases (Yao et al., 2014). The level of IL-6 in serum was elevated in a number of
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inflammation-related diseases, such as rheumatoid arthritis, Castleman disease, and Crohn's disease (Kishimoto, 2010). TNF-α also plays a major role in the pathogenesis of inflammation
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and serves as therapeutic target in numerous autoimmune and inflammatory system diseases (Jarrot and Kaplanski, 2014). Elevated serum and tissue levels of TNF-α were found in inflammatory and infectious conditions and serum levels were correlated with the severity of infections (Bradley, 2008). The important roles of IL-6 and TNF-α in inflammatory diseases and pathology led to the development of therapies that target TNF-α and IL-6. Our current study shows that verteporfin inhibited IL-6 and TNF-α at both mRNA and protein levels, indicating that verteporfin, which is already in clinical use, can serve as a therapeutic agent against inflammatory diseases. 16
Journal Pre-proof TNF-α is a potent inducer of NF-κB (Schütze, 1995) and IL-6 is important in inflammatory processes by activating the JAK/STAT pathway (Rincon and Irvin, 2012). NF-κB activation also increases the production of TNF-α (Liu et al., 2017) and IL-6 (Libermann and Baltimore, 1990), which forms a positive feedback loop. Targeting NF-κB and JAK/STAT pathways with the eventual down regulation of TNF-α and IL-6 plays an important role in therapeutic strategies against inflammatory diseases (Lin TH et al., 2017; Gao Q et al., 2018). In present study, our
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data demonstrate that the NF-κB and JAK/STAT pathways participate in this process. Our results
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show that NF-κB activation and JAK/STAT expression were inhibited by verteporfin. Those
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results demonstrate that verteporfin inhibits inflammatory processes through the NF-κB and
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JAK/STAT pathways in LPS-induced RAW 264.7 cells.
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Beyond the traditional signaling inhibition, verteporfin is also known as a powerful inducer for the function of protein oligomers. Previous studies showed that verteporfin could induce high
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molecular oligomers, such as p62 (Donohue et al., 2014) and STAT3 (Zhang et al., 2015).
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Oxidized and crosslinked proteins can lead to loss of function of these proteins depending on the sites and extent of modifications (Luo J et al., 2006; Kim et al., 2001). Verteporfin causes high
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molecular weight crosslinked p62 oligomers by a mechanism that involved low-level singlet oxygen production. These crosslinked p62 oligomers have a defective binding to polyubiquitinated proteins (Donohue et al., 2014). In present study, we observed verteporfininduced crosslinks of NF-κB p65, JAK1, JAK2, STAT1, and STAT3. Immunoprecipitation verified the presence of high molecular weight proteins, documenting that the crosslinks were induced by verteporfin. Although there is less direct evidence, the crosslinked proteins at high molecular weights have the potential to lose their transcriptional activities and thus inhibit the inflammatory process. 17
Journal Pre-proof In summary, this study demonstrates that: (1) verteporfin inhibits the pro-inflammatory cytokines IL-6 and TNF-α expression at both mRNA and protein levels; (2) verteporfin inhibits the inflammatory process by negatively regulating NF-κB and JAK/STAT pathways; (3) verteporfin induces crosslinks with proteins including NF-κB p65, JAK1, JAK2, STAT1, and STAT3, which may impair the function of these proteins; and (4) the anti-inflammation ability of
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verteporfin may in part be due to its ability to generate crosslinked protein products.
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CONFLICT OF INTEREST
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The authors declare no conflicts of interest.
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Journal Pre-proof FIGURE LEGENDS Fig. 1. Verteporfin has low cytotoxicity in RAW 264.7 cells. A) The chemical structure of verteporfin. RAW 264.7 cells were treated with different concentrations of verteporfin (0-12 μM) for 24 h. B) Cell viability was quantified by MTT assay. The data are expressed as the mean ± SE of six repetitions. C) Cell cytotoxicity was detected by live and dead assay. Live and dead assay shows live cells stained with Calcein-AM (green) and dead cells with EthD-1 (red). Scale
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bar = 100 µm. D) Live cell percentage was quantified by live cell counting assay. Cell
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suspension was mixed with trypan blue and live cells were counted using Bio-Rad cell counter.
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The data are expressed as the mean ± SE of three repetitions.
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Fig. 2. Verteporfin inhibits LPS-induced cytokines in RAW 264.7 cells. A) Raw 264.7 cells
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were pre-incubated with 2 μM verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. Cytokines in cell lysates were detected. Red boxes indicate inhibited cytokines. B)
(BLANK).
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Cytokine array map. Positive Control Spots (POS), Negative Control Spots (NEG), Blank Spots
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Fig. 3. Verteporfin inhibits LPS-induced IL-6 and TNF-α expression. RAW 264.7 cells were
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pre-incubated with different concentration of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. A) The graph shows IL-6 mRNA levels measured by real-time RCR. B) The blot shows IL-6 protein level was measured by western blot analysis. C) The graph shows TNF-α mRNA levels measured by real-time PCR. D) The blot shows TNF-α protein level was measured by western blot analysis. The primary murine peritoneal macrophages cells were pre-incubated with 0.5 μM verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. E) The graph shows IL-6 mRNA levels measured by real-time RCR. F) The graph shows TNF-α mRNA levels measured by real-time PCR. Human monocyte cells U-937 were pre-incubated with 1 μM 24
Journal Pre-proof verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. G) The graph shows IL-6 mRNA levels measured by real-time RCR. H) The graph shows TNF-α mRNA levels measured by real-time PCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05 versus control on cells treated with LPS alone. Fig. 4. Verteporfin negatively regulates NF-κB activation in LPS-stimulated RAW 264.7
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Cells. RAW 264.7 cells were pre-incubated with different concentrations of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. A) Protein levels of p-IKKα/β, p-NF-κB
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p65, and NF-κB p65 were determined by western blot analysis. B) Protein levels of NF-κB p65
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in the cytoplasmic extracts (CE) and nuclear extracts (NE) were determined by western blot
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analysis. β-actin and HDAC1 were used as cytoplasm and nuclei loading controls respectively.
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C) Images of immunofluorescence show inhibition of NF-κB p65 (green) translocation with verteporfin treatment. Nuclei were stained with DAPI (blue). Scale bar = 50 µm. RAW 264.7
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cells were pre-incubated with different concentration of verteporfin or vehicle control and 1 μM
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NF-κB activation inhibitor for 2 h, and then stimulated with 1 μg/ml LPS for 22 h. D) The graph shows IL-6 mRNA levels measured by real-time RCR. E) The graph shows TNF-α mRNA levels
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measured by real-time PCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05 versus control on cells treated with LPS alone. Fig. 5. Verteporfin inhibits JAK/STAT pathway in LPS-stimulated RAW 264.7 cells. Raw 264.7 cells were pre-incubated with different concentrations of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. A) Protein levels of JAK1 and JAK2 were determined by western blot analysis. B) Protein levels of p-STAT1 (S727), STAT1, p-STAT3 (Y705), and STAT3 were determined by western blot analysis.
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Journal Pre-proof Fig. 6. Verteporfin causes protein crosslinkages in LPS-stimulated RAW 264.7 cells. Raw 264.7 cells were pre-incubated with different concentrations of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. A) The crosslinked NF-κB p65 was determined by western blot analysis. B) The crosslinked JAK1 and JAK2 were determined by western blot analysis. C) The crosslinked STAT1 was determined by western blot analysis. D) The crosslinked STAT3 was determined by western blot analysis. Different exposure times were
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showed in A, C, and D.
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Fig. 7. Verteporfin induces protein high molecular weight oligomerization. Raw 264.7 cells
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were pre-incubated with different concentrations of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. Cell lysates were prepared for immunoprecipitation experiments.
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A) Proteins were immunoprecipitated with NF-κB p65 antibody #1, and identified on the
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immunoblot using a different NF-κB p65 antibody (antibody #2). B) Proteins were
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immunoprecipitated with STAT3 antibody #1, and then identified on the immunoblot using STAT3 antibody #2. C) RAW 264.7 cells were transfected with NF-κB luciferase reporter and
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Renila vector, and then treated as indicated conditions. The graph shows luciferase-reported NF-
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κB transcriptional activity. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05 versus control on cells treated with LPS alone. D) The crosslinked NF-κB p65 and activated p65 form p-p65 were determined by western blot analysis.
SUPPLEMENTARY DATA Supplementary Fig. 1. Verteporfin inhibits LPS-induced CCL5 mRNA in RAW 264.7 cells. RAW 264.7 cells were pre-incubated with different concentration of verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. The graph shows CCL5 mRNA levels measured 26
Journal Pre-proof by real-time RCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤ 0.05 versus control on cells treated with LPS alone. Supplementary Fig. 2. Light stimulation enhances inhibition effect of verteporfin on IL-6 and TNF-a mRNA expression in RAW 264.7 cells. RAW 264.7 cells were pre-incubated with 2 μM verteporfin or vehicle control, and then stimulated with 1 μg/ml LPS. Light stimulation
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groups were stimulated with light 30 min before collected cells. A) The graph shows IL-6
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mRNA levels measured by real-time RCR. B) The graph shows TNF-α mRNA levels measured
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by real-time PCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05
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versus control on cells treated with LPS alone.
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Supplementary Fig. 3. The effect of LPS dose in RAW264.7 cells. RAW 264.7 cells were treated with different concentration of LPS (0~3 μg/ml). A) The graph shows IL-6 mRNA levels
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measured by real-time RCR. B) The graph shows TNF-α mRNA levels measured by real-time
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PCR. Presented graphic data represent the mean ± SE of three repetitions. *, P≤0.05 versus
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control on cells treated with 0.5 µg/ml LPS.
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Author Contribution Statement
Yuting Wang: Conceptualization, Investigation, Writing- Original draft preparation, Writing review & editing. Lei Wang: Conceptualization, Writing - review & editing James T.F. Wise: Writing - review & editing
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Xianglin Shi: Supervision, Project administration, Writing - review & editing
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Zhimin Chen: Supervision, Project administration, Writing - review & editing
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Journal Pre-proof Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Highlights
Verteporfin inhibits inflammation in RAW 264.7 cells.
Verteporfin inhibits IL-6 and TNF-α expression at both mRNA and protein levels in RAW 264.7 cells.
Verteporfin inhibits NF-κB and JAK/STAT pathways in RAW 264.7 cells.
Verteporfin induces crosslinks with proteins including NF-κB p65, JAK1, JAK2, STAT1,
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and STAT3 in RAW 264.7 cells.
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