Abstract
ANIMAL CELLS AND SYSTEMS 2020, VOL. 24, NO. 1, 44–52 https://doi.org/10.1080/19768354.2020.1726811 Decursin negatively regulates LPS-induced upregulation of the TLR4 and JNK signaling stimulated by the expression of PRP4 in vitro Muhammad Bilal Ahmed, Salman Ul Islam and Young Sup Lee School of Life Sciences, College of Natural Sciences, Kyungpook National University, Daegu, Korea ABSTRACT ARTICLE HISTORY Received 8 November 2019 The current investigation was carried out to analyze the correlation of bacterial lipopolysaccharide Revised 9 January 2020 (LPS) and pre-mRNA processing factor 4B (PRP4) in inducing inflammatory response and cell actin Accepted 3 February 2020 cytoskeleton rearrangement in macrophages (Raw 264.7) and colorectal (HCT116) as well as skin cancer (B16-F10) cells. Cell lines were stimulated with LPS, and the expression of PRP4 as well as KEYWORDS pro-inflammatory cytokines and proteins like IL-6, IL-1β, TLR4, and NF-κB were assayed. The LPS; PRP4; inflammation; results demonstrated that LPS markedly increased the expression of PRP4, IL-6, IL-1β, TLR4, and decursin; TLR4; JNK NF-κB in the cells. LPS and PRP4 concomitantly altered the morphology of cells from an aggregated, flattened shape to a round shape. Decursin, a pyranocoumarin from Angelica gigas, inhibited the LPS and PRP4-induced inflammatory response, and reversed the induction of morphological changes. Finally, we established a possible link of LPS with TLR4 and JNK signaling, through which it activated PRP4. Our study provides molecular insights for LPS and PRP4-related pathogenesis and a basis for developing new strategies against metastasis in colorectal cancer and skin melanoma. Our study emphasizes that decursin may be an effective treatment strategy for various cancers in which LPS and PRP4 perform a critical role in inducing inflammatory response and morphological changes leading to cell survival and protection against anti-cancer drugs. Introduction Ruoslahti 2004; Liu et al. 2010). A study reported that LPS upregulated the levels of metadherin, which in Lipopolysaccharide (LPS) is the major constituent of the turn induced lung metastasis of 4T1 mammary tumor outer membrane of Gram-negative bacteria which cells (Zhao et al. 2011; Sethi et al. 2012). It is thus postu- potently induces inflammatory response through produ- lated that LPS may promote angiogenesis and metasta- cing various cytokines, inflammatory mediators, and sis; however, the underlying mechanisms remain elusive. growth factors (Harmey et al. 2002; He et al. 2007; Gass- Angelica gigas, an important medicinal plant of mann et al. 2009; Ikebe et al. 2009). It has been shown Umbelliferae family, has been reported to possess that LPS may lead to the induction of systemic inflam- various compounds such as coumarins, polyacetylenes, mation and increases hepatic recruitment of cancer and essential oils (Chi and Kim 1988; Choi et al. 2000; cells in vivo (He et al. 2009; Ikebe et al. 2009; Wang Lee et al. 2002). Among the coumarins, pyranocoumarins et al. 2010). Moreover, it has been shown that LPS- such as decursin and decursinol angelate have got con- induced inflammation increased the growth of exper- siderable attention due to their potent pharmacological imental metastases in a murine tumor model, and led characteristics (Ahn et al. 1997; Shehzad et al. 2018). to increased angiogenesis in vitro and in vivo (Wang Decursin and its isomer have been shown to exhibit et al. 2010). In addition to these changes, increased anti-cancer, anti-inflammatory, antiangiogenic, and expression of vascular endothelial growth factor, higher anti-amnesic activities (Yim et al. 2005; Choi et al. vascular permeability and tumor cell invasion/migration 2012). It has been reported that decursin and DA inhib- were also noted (He et al. 2007; Killeen et al. 2009; Yan ited pro-inflammatory molecules, such as cytokines, che- et al. 2013). Multiple investigations have revealed that mokines, and enzymes such as cyclooxygenase-2 and activated Toll-like receptor 4 (TLR4) and the nuclear matrix metalloproteinases. factor-κB (NF-κB) signaling pathways are involved in Pre-mRNA processing factor 4B (PRP4), a transcription elevations of LPS-induced metastasis in each process, factor involved in pre-mRNA splicing, was first identified including tumor cell adhesion and invasion (Brown and in Schizosaccharomyces pombe (Kuhn and Käufer 2003). CONTACT Young Sup Lee yselee@knu.ac.kr School of Life Sciences, College of Natural Sciences, Kyungpook National University, Daegu, 41566, Korea © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MOLECULAR CELLULAR BIOLOGY ANIMAL CELLS AND SYSTEMS 45 Previously, it has been reported that PRP4 is involved in respectively. Both media were supplemented with 10% reversing anticancer drug-induced cell death in human Fetal Bovine Serum (Gibco #16000-044) and 1% penicil- cancer cell lines through actin cytoskeleton rearrange- lin–streptomycin (Gibco #15140-122). Cell cultures were ment and epithelial–mesenchymal transition (EMT) maintained in a humidified incubator containing 5% (Islam et al. 2017; Islam, Ahmed, et al. 2018). Herein, we CO2 at 37°C. Decursin was dissolved in dimethyl sulfoxide report that LPS induced the activation of PRP4 which and cells were treated with 10 μM curcumin for 24 h resulted in the activation of various cytokines and inflam- (Islam, Lee, et al. 2018). matory proteins. LPS and PRP4 concomitantly altered cell morphology, which was related to the rearrangement of F-actin staining the actin cytoskeleton. Decursin blocked the LPS and Alexa Fluor 488 phalloidin was used for F-actin visualiza- PRP4-induced inflammatory response, and reversed the tion. Briefly, after removal of full growth medium, cells induction of cell morphological changes. We also were washed twice with 1X phosphate-buffered saline struggled to elucidate the underlying mechanism for (PBS) and fixed with 4% paraformaldehyde for 15 min at LPS activating the PRP4. room temperature. Then, cells were permeabilized with 0.2% Triton X-100 for 5 min, and washed 2–3 times with 1X PBS. Alexa Fluor 488 phalloidin stock solution (6.6 μM Material and methods in methanol) was diluted to 1:40 with 1% bovine serum Chemicals and reagents albumin (BSA), and added to the cells for 50 min at room temperature in the dark. Cells were washed 5–6 LPS (cat# L 2630) and decursin were obtained from Sigma- times with 1X PBS and actin cytoskeleton was observed Aldrich (St. Louis, MO, USA). Dulbecco’smodified Eagle’s using a ZEISS LSM 800 confocal microscope. medium (DMEM), fetal bovine serum (FBS), and penicil- lin/streptomycin were obtained from Gibco (Carlsbad, CA, USA). PRP4 cDNA open reading frame (ORF) clone Western blotting HG10835-ACG was purchased from Sino Biological Total cell lysates were prepared using cell lysis buffer, (Wayne, PA, USA), and a PRP8 clone was obtained from and the protein concentration was determined using Origene (Rockville, MD, USA). Antibodies against PRP4, the Bio-Rad Protein Assay. Samples (20–40 μg) were pre- PRP8, TLR4, NF-κB, I-κBα, E-cadherin, Vimentin, AKT, JNK, pared in sodium dodecyl sulfate (SDS) sample buffer, ERK, and β-actin were obtained from Santa Cruz Biotech- separated via 10% SDS-polyacrylamide gel electrophor- nology (Santa Cruz, CA, USA). A Bradford protein assay kit esis, and transferred onto a nitrocellulose membrane. and electrophoresis reagents were purchased from Bio- The membranes were blocked with 2% albumin Rad Laboratories (Irvine, CA, USA). ECL Prime detection (Gendept, USA) solution for 2 h at 4°C. Chemilumines- reagent and nitrocellulose membrane were purchased cent signals were developed with Clarity™ ECL from Amersham (Little Chalfont, Buckinghamshire, UK). Western Blotting Substrate (Bio-Rad) according to the Vectashield mounting medium with DAPI (4 ,6-diami- manufacturer’s instructions (Islam et al. 2015). dino-2-phenylindole) from Vector Laboratories Inc. (Burlin- game, CA, USA) was used for staining nuclei. PRP4 siRNA Reverse-transcription polymerase chain reaction was obtained from Santa Cruz Biotechnology (SC- (RT-PCR) 76257). Lipofectamine® LTX with Plus™ Reagent (Cat# 15338100) and SuperScript III Reverse Transcriptase Total RNA (5 µg) was reverse-transcribed using the (Cat# 18080093) were obtained from Invitrogen (Carlsbad, SuperScript III First-strand synthesis kit, as has been CA, USA). Xfect transfection reagent was purchased from described previously (Islam et al. 2015; Islam, Ahmed Takara Bio USA, Inc. (Mountain View, CA, USA). JNK inhibi- et al. 2018). The synthesized cDNA was incubated with tor SP600125 (Cat# tlrl-sp60), and TLR4 signaling inhibitor RNase H at 37°C for 1 h. PCR was performed using 2 μL CLI-095 (Cat# tlrl-cli95) were obtained from InvivoGen of cDNA and the following primers: following primers: ′ ′ (California 92121 USA). All chemicals and products were IL-6 Forward 5 -GGTACATCCTCGACGGCATCT-3 and ′ ′ used as prescribed by the manufacturers. Reverse 5 -GTGCCTCTTTGCTGCTTTCAC-3 and IL-1β ′ ′ Forward 5 -ACAGATGAAGTGCTCCTTCCA-3 and Reverse ′ ′ 5 -GTCGGAGATTCGTAGCTGGAT-3 and glyceraldehyde- Cells culture and treatment 3-phosphate dehydrogenase (GAPDH) forward, 5ʹ- RAW 264.7 cells (ATCC #TIB-71), HCT 116 (ATCC #CCL-24), AGGGCTGCTTTTAACTCTGGT-3ʹ and GAPDH reverse, 5ʹ- and B16-F10 (ATCC #CRL-6475) were cultured in Dulbec- CCCCACTTGATTTTGGAGGGA-3ʹ. PCR was performed còs Modified Eagle Medium (DMEM, Gibco #11995-065), under the following conditions: one cycle at 98°C for 46 M. B. AHMED ET AL. 3 min followed by 30–35 cycles at 95°C for 30 s, 55°C for both at the mRNA and protein levels by RT–PCR and 30 s, and 72°C for 30 s, with a final extension step at 72°C western blotting, respectively (Figure 1(a)). It has been for 5 min. The amplified PCR products were analyzed via shown that when Gram-negative bacteria multiply in 2% agarose gel electrophoresis and EcoDye™ Nucleic the host, LPS is released into the blood stream, where Acid Staining Solution (Biofact Co., Ltd.); the relative it triggers the induction of NF-κB-dependent proinflam- intensities of the detected bands were measured on a matory cytokines such as prostaglandins, nitric oxide, Gel Doc2000 scanner (Bio-Rad, Hercules, CA, USA). tumor necrosis factor-α, and interleukin (IL)-1 (Triantafi- lou and Triantafilou 2005; Liu et al. 2017; Wassenaar and Zimmermann 2018). LPS has also been shown to Results induce inflammatory response in vitro (Lund et al. 2006). PRP4 has been shown to induce NF-κB signaling LPS and PRP4 concomitantly induce cytokines which probably prevented cancer cells from undergoing expressions and rearrange cell actin cytoskeleton apoptosis (Islam et al. 2017). In order to investigate the HCT116 cells were transfected with a PRP4 expression relation between LPS and PRP4, we treated Raw 264.7 construct, and PRP4 overexpression was confirmed cells with 100 ng/mL LPS, and analyzed the expression Figure 1. LPS and PRP4 induces cytokines expression. (A) mRNA and protein levels of PRP4 in control and PRP4-transfected cells. GAPDH and actin were used as the loading control. (B) Western blot and PCR analysis of PRP4 and PRP8 in Raw264.7, HCT116, B16-F10 cells after stimulation with 100 ng/ml LPS. (C) Cells were pre-treated with LPS, followed by transfection with PRP4, and then incubated for 24 h. RT-PCR was performed to examine the mRNA levels of IL-6 and IL-1β. GAPDH was used as internal control. (D) HCT116 cells were transfected with si-RNA-PRP4 using Xfect RNA transfection reagent from Takara as described by the manufac- turer. Cells were then stimulated with 100 ng/ml LPS. RT-PCT and western blots were performed on control transfected cells. GAPDH was used as the loading control. ANIMAL CELLS AND SYSTEMS 47 of PRP4. We noted that LPS induced the expression of that inhibition of TLR4 expression by rapamycin blocks PRP4 both at protein and mRNA levels in Raw 264.7 TLR4/NF-κB signaling and promotes apoptosis of colon cells. Similar results were obtained in HCT116 and B16- cancer (Sun et al. 2008). In order to investigate the con- F10 cell lines. However, LPS did not induce the comitant effect of LPS and PRP4 on TLR4 and NF-κB sig- expression of PRP8 (Figure 1(b)). Next, we pre-treated naling pathways, we pre-treated the cells with LPS the cells with LPS followed by PRP4 transfection, and followed by PRP4 transfection. Western blot analyses observed that LPS and PRP4 concomitantly induced revealed that LPS and PRP4 upregulated the expression the expressions of pro-inflammatory cytokines IL-6 and of TLR4 and NF-κB, while decreased the expression of IL-1β (Figure 1(c)). In order to confirm that LPS-induced inhibitory subunit I-κBα (Figure 3(a)). However, decursin cytokines activation is mediated through PRP4, we per- treatment reversed the LPS and PRP4-induced activation formed siRNA-mediated knockdown of PRP4 using a of TLR4 and NF-κB(Figure 3(b)). These data suggested pool of three target-specific siRNAs, 19–25 nucleotides that LPS and PRP4 concomitantly induced the inflamma- in length. Interestingly, siRNA-PRP4 also inhibits the tory response via activating TLR4/NF-κB signaling in the LPS-induced overexpression of IL-6 and IL-1β (Figure 1 cells. (d)). Furthermore, by treating the cells with 10 µM decur- sin, it was revealed that LPS-induced PRP4 expression LPS stimulated-PRP4 through activation of TLR4 was blocked (Figure 2(a)). In order to confirm that LPS and JNK is inhibited by decursin has induced PRP4 expression, we treated the cells with 10 µg/ml polymyxin B (LPS inhibitor) for 24 h, and Through western blot analyses, we observed that LPS observed the decreased expression of PRP4. However, induced the expressions of Akt, JNK, ERK, and p-ERK in PRP8 remained unaffected (Figure 2(b)). Previously, it Raw 264.7, HCT116, B16-F10 cells, which were then has been shown that PRP4 altered cell morphology in blocked by concomitant treatment of 10 µM decursin cancer cell lines (Islam et al. 2017; Islam, Ahmed et al. (Figure 4(a)). In order to investigate the involvement of 2018). In order analyze the correlation of LPS with PRP4 TLR4 and JNK pathway in LPS induced-PRP4 expression, regarding cell cytoskeleton rearrangement, cells were we utilized the inhibitors for TLR4 (CLI-095) and JNK pre-treated with LPS and transiently transfected with a (SP600125) along with LPS and decursin. CLI-095 is a PRP4 expression construct. Fluorescence confocal cyclohexene derivative that specifically suppresses TLR4 microscopy revealed that LPS and PRP4 over-expression signaling, inhibiting the production of nitric oxide and induced actin filament redistribution and changed the pro-inflammatory cytokines (Ii et al. 2006). CLI-095 acts cell morphology from an aggregated, flattened shape by blocking the signaling mediated by the intracellular to a round shape, whereas cells incubation with 10 µM domain of TLR4, and has been shown to potently suppress decursin reversed the induction of morphological both ligand-dependent and -independent signaling of changes (Figure 2(c)). These data suggest that decursin TLR4 (Kawamoto et al. 2008). SP600125, an anthrapyrazo- inhibits LPS-induced PRP4 expression, and thus blocks lone, is a novel and selective inhibitor of JNK that com- inflammatory response as well as cell morphology petes with ATP to inhibit the phosphorylation of c-Jun. It alterations. prevents the activation of inflammatory genes such as COX-2, IL-2 IFN-γ and TNF-α (Bennett et al. 2001). Interest- ingly, we found that CLI-095 and SP600125 treatment Decursin blocks LPS and PRP4-induced remarkably blocked LPS-induced PRP4 expression, while inflammatory pathway proteins it did not affect PRP8 expression (Figure 4(b and c)). It has been well documented that LPS stimulates the acti- These results suggest that PRP4 activation by LPS is associ- vation of TLR4 in various cell lines (Guijarro-Muñoz et al. ated with upregulation of TLR-4 and JNK. 2014). TLR4 is thought to share the MyD88-dependent pathway that activates NF-κB and mitogen-activated Discussion protein (MAP) kinases, and elevates the expression of genes encoding inflammatory cytokines (Kawai and In this study, we have shown that LPS induced the Akira 2005; Lu et al. 2008). A recent study reported that expression of PRP4 in Raw 264.7, HCT116, and B16-F10 TLR4-induced inflammation acted as a key promoter cell lines. Both the LPS and PRP4 induced the expression for cancer progression (Ran et al. 2019). It was reported of pro-inflammatory cytokines like IL-6 and IL-1β. that TLR4 was involved in facilitating migration of Additionally, LPS and PRP4 increased the expressions of colon cancer cells and preserving them from immune inflammatory pathways proteins like TLR4 and NF-κB. surveillance and cell death (O’Leary et al. 2012; Tang Decursin blocked the LPS-induced PRP4 expression as and Zhu 2012). Additionally, it has been demonstrated well downregualted the TLR4 and NF-κB. Finally, we 48 M. B. AHMED ET AL. Figure 2. Decursin inhibits LPS-induced PRP4 expressions and cell morphological alterations. (A) Western blot analysis of PRP4 and PRP8 in Raw264.7, HCT116, B16-F10 cells after stimulation with 100 ng/ml LPS and treating with 10 µM decursin. β-actin was used as a loading control. (B) Cells were pre-treated with LPS and incubated with 10 µM decursin and/or 10 µg/ml polymyxin B for 24 h. Western blot was performed to analyze the expressions of PRP4 and PRP8. β-actin was used as a loading control. (C) Cells were stimu- lated with 100 ng/ml LPS, followed by transfection with PRP4, and treatment with 10 µM decursin. Cells were stained with phalloidin and observed under a ZEISS LSM 800 confocal microscope at 1000 × magnification. established a possible link of LPS with TLR4 and JNK, including cytokines, prostaglandins, and nitric oxide. through which it activated PRP4. MAPKs including JNKs, ERKs, p38 MAPKs and ERK5 LPS has been shown to stimulate host cells such as have been reported to be stimulated by LPS (Zhu et al. macrophages to produce endogenous mediators 2000). JNK has been shown to play a special role in ANIMAL CELLS AND SYSTEMS 49 Figure 3. LPS and PRP4 induces inflammatory pathways proteins. (A) Cells were pre-treated with LPS and then transfected with PRP4. Cellular proteins were extracted using cell lysis buffer. Proteins were quantified by Bradford assay, and equal amount of proteins were separated on 10% SDS-PAGE. Proteins were then transferred onto nitrocellulose (NC) membranes. NC membranes were then incubated with specific antibodies for NF-κB, and I-κBα overnight at 4°C. Chemiluminescent signals were developed with Clarity™ ECL Western Blotting Substrate. (B) Western blot analysis of TLR4, NF-κB, and I-κBα after stimulating cells with LPS, followed by transfection with PRP4 and treatment with decursin. β-actin served as a loading control. mediating LPS responses in macrophages by study reported that TLR4 was essential for LPS-mediated phosphorylating transcription factors including c-Jun JNK activation, as JNK activation was abrogated in mice and ATF-2, which then activate the transcription of with TLR4 null-functional mutation (Muzio et al. 1998). iNOS, COX-2, and various other inflammatory cytokines It was shown that MyD88, which directly associates (Rao 2001). with the cytoplasmic domain of TLRs, was needed for Studies have shown that most of the LPS signals are the early activation of JNK by LPS, because JNK activation mediated by TLR4 (Lu et al. 2008; Hsu et al. 2011). TLRs was delayed in macrophages of MyD88-deficient mice recognize molecular patterns of invading pathogens. A (Kawai et al. 1999). 50 M. B. AHMED ET AL. Figure 4. LPS stimulates PRP4 by activating TLR4 and JNK. (A) Protein levels of Akt, JNK, Erk, and p-Erk (B) PRP4 and PRP8 protein expressions were analyzed through western blot after incubating the cells with LPS, decursin and SP600125. (C) Cells were stimulated with 100 µg/ml LPS and incubated with or without 10 µM Decursin, and CLI-095 for 24 h. Protein levels of PRP4 and PRP8 were analyzed through western blotting. β-actin was used as a loading control. ANIMAL CELLS AND SYSTEMS 51 During our investigation, to confirm that PRP4 was References stimulated by LPS, we utilized polymyxin B for inhibiting Ahn K-S, Sim W-S, Lee I-K, Seu Y-B, Kim I-H. 1997. Decursinol LPS action. Polymyxin B is a potent decapeptide cyclic angelate: a cytotoxic and protein kinase C activating agent cationic antibiotic, containing lipophilic and hydrophilic from the root of Angelica gigas. Planta Med. 63:360–361. 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Journal
Animal Cells and Systems
– Taylor & Francis
Published: Jan 2, 2020
Keywords: LPS; PRP4; inflammation; decursin; TLR4; JNK
