- Open Access
1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation
- Jinseon Jeong†1, 2, 3,
- Yong-Jae Kim†3,
- Do Young Lee3,
- Byoung-Gon Moon3,
- Ki-Young Sohn3,
- Sun Young Yoon3 and
- Jae Wha Kim1, 2Email authorView ORCID ID profile
© The Author(s) 2019
- Received: 4 October 2018
- Accepted: 24 December 2018
- Published: 3 January 2019
Cancer patients treated with chemotherapy often experience a rapid decline of blood neutrophils, a dose-limiting side effect called chemotherapy-induced neutropenia. This complication brings about dose reductions or cessation of chemotherapy during treatment of cancer patients because a rapid decline of neutrophil counts increases susceptibility to infection. Here, we found that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation via the inhibition of neutrophil-attracting chemokine production in macrophages using in vivo and in vitro approaches. A single intraperitoneal administration of gemcitabine induced the migration of circulating neutrophils into the peritoneal cavity in normal mice, and PLAG effectively decreased neutrophil migration by inhibiting the expression of adhesion molecules, L-selectin and LFA-1. Inhibition of CXCR2 by its antagonist, reparixin, abrogated gemcitabine-induced neutrophil migration, indicating that chemokines produced by gemcitabine mainly support neutrophil activation. In vitro experiments demonstrated that PLAG inhibited NADPH oxidase 2 (NOX2)-mediated reactive oxygen species production induced by gemcitabine, which is the upstream of MIP-2 and/or CXCL8. Importantly, PLAG down-regulated gemcitabine-induced membrane translocation of the cytosolic NOX subunit, Rac1, and phosphorylation of p47phox. The activation of upstream signaling molecules of p47phox phosphorylation, phospholipase C β3 and protein kinase C, were effectively regulated by PLAG. We also demonstrated that 1-palmitoyl-2-linoleic-3-hydroxyl-rac-glycerol (PLH), the natural form of diacylglycerol, has no effects on gemcitabine-induced CXCL8 production and dHL-60 migration, suggesting that an acetyl group at the third position of the glycerol backbone may have a key role in the regulation of neutrophil activation. Altogether, this study suggests the potential of PLAG as a therapeutic strategy to modulate chemotherapy-induced neutrophil activation for cancer patients undergoing chemotherapeutic treatment.
- Chemotherapy induced neutropenia (CIN)
- Reactive oxygen species
- NADPH oxidase 2
Neutrophils are the most abundant white blood cell in circulation and play a key role in the first-line of defense against foreign substances and/or invading pathogens [1, 2]. Cancer patients undergoing treatment with chemotherapeutics often suffer from a rapid decline of circulating neutrophils . This complication, which is called chemotherapy-induced neutropenia (CIN), increases the patient’s susceptibility to infection, which necessitates dose reduction or cessation of chemotherapy . Unfortunately, exact mechanisms of CIN remains unknown, for that reason, current treatment options rely mostly on promoting production of granulocytes and precursor cells in the bone marrow. Filgrastim, the recombinant human granulocyte-colony stimulating factor (rhG-CSF), is one of the few treatment options since its natural form is a key regulator of neutrophil differentiation and proliferation [5–7]. Therefore, a better understanding of how neutropenia arises in response to chemotherapy would be the key for the development of new pharmaceuticals.
The immunosuppressive effect of chemotherapeutics has long been considered as an immediate cause of neutrophil depletion due to its high susceptibility to cytotoxic activity [8, 9]. However, increasing evidences suggest that the immune systems are activated by many chemotherapeutics . For example, paclitaxel activates mouse macrophages and dendritic cells (DCs) by binding to mouse toll like receptor 4 (TLR4) and mimicking bacterial lipopolysaccharides (LPS) . In animal models with chemotherapy-induced peripheral neuropathy (CIPN), there are several reports of increased neutrophil activity and infiltration in the peripheral nervous system (PNS) and dorsal root ganglion (DRG) following injection with anti-cancer agents [12, 13].
Gemcitabine is a nucleoside deoxycytidine analogue, which is a commonly used anti-cancer agent with a high incidence of CIN . It is an antimetabolite that induces cell death by interfering with enzymes that are necessary for nucleic acid production during S phase of the cell cycle [15, 16]. Gemcitabine also acts by generating reactive oxygen species (ROS) through upregulation of NADPH oxidase (NOX) activity . In the previous study, gemcitabine enhanced pancreatic cancer stemness through NOX-mediated ROS production . Gemcitabine also enhances the production of CXC motif chemokine ligand 8 (CXCL8) and/or macrophage inflammatory protein 2 (MIP-2; a murine homologue of CXCL8), which are the primary chemokines that recruit neutrophils to damaged tissue at early phases [19, 20]. However, a direct association between NOX-mediated ROS and the chemokine production in gemcitabine—treated condition remains unclear.
1-Palmitoryl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) was originally isolated from the antlers of sika deer and chemically synthesized as a single compound that is identical to the natural form . A previous study showed that PLAG exerts a synergistic therapeutic effect with long acting G-CSF (pegfilgrastim) to treat CIN by modulating neutrophil migration . Also, PLAG reduced the infiltration of neutrophils into the arthritic joints by inhibiting IL-6/STAT3/MIP-2 signaling cascade in rheumatoid arthritis . In this study, we aimed to demonstrate low neutrophil counts in blood after gemcitabine treatment partially due to neutrophil chemotaxis from blood to tissues along a MIP-2 gradient, and investigated the protective effect of PLAG on the maintenance of circulating neutrophil counts using a murine model of gemcitabine-induced neutropenia. We further demonstrated the molecular mechanism in which gemcitabine promotes CIN by inducing the chemokine gradient through the activation of PLCβ3/PKC/NOX2/ROS/MAPK signaling cascade. Together, we show that PLAG has a therapeutic effect against CIN via modulating NOX2-mediated ROS generation and downstream ROS signaling pathways, which leads to chemokine production.
PLAG and PLH were obtained from Enzychem Lifesciences Corporation (Daejeon, South Korea). Gemcitabine hydrochloride was purchased from Dong-A ST (Seoul, South Korea). Reparixin was purchased from MedChem Express (NJ, USA). N-acetyl-l-cycteine, diphenyleneiodonium were purchased from Sigma-Aldrich (MO, USA). U73122 and Rottlerin were purchased from ENZO Life Sciences (NY, USA).
Male BALB/c mice (6–8 weeks of age, 20–22 g) were purchased from Koatech Corporation (Pyeongtaek, South Korea), and male BALB/c Slc-nu mice (5 weeks of age, 17–21 g) were purchased from Central Lab Animal Inc. (Seoul, South Korea). The mice were maintained in a specific pathogen-free facility under consistent temperature and 12-h light/dark cycles. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (Daejeon, South Korea) and performed in compliance with the National Institutes of Health Guidelines for the care and use of laboratory animals and Korean national laws for animal welfare.
For gemcitabine-induced neutropenia mice model, male BALB/c mice were intraperitoneally (i.p.) injected with 50 mg/kg gemcitabine to induce neutropenia. PLAG was diluted in phosphate buffered saline (PBS) and then orally administrated at a dose of 50 or 250 mg/kg/day. The normal control group was administered PBS only during the experiment. At 15 h after gemcitabine treatment, the whole blood was collected from the orbital sinuses using capillary tubes (Kimble Chase Life Science and Research Products LLC, FL, USA) and collection tubes containing K3E-K3EDTA (Greiner Bio-One International, Kremsmünster, Austria). To obtain peritoneal cells, 5 ml of cold PBS was injected to the left side of peritoneal wall using a 5 ml syringe, and the fluid was aspirated from the peritoneum. The collected cells were counted by complete blood count (CBC) analysis using Mindray BC-5300 auto-hematology analyzer (Shenzhen Mindray Biomedical Electronics, Guangdong Sheng, China).
For a human tumor xenograft model, athymic nude mice (n = 10 per treatment group) were inoculated subcutaneously on the right flank 107 RPMI8226 cells in 0.2 ml PBS on day 0. Chemotherapy and PLAG administration were begun when tumor volume reached between 81 and 118 mm3 on day 12. Gemcitabine was administered (i.p.) twice weekly for 4 weeks, and PLAG was orally administered once per day for 4 weeks. On day 40, all mice were sacrificed, and tumor weights were measured.
The collected whole blood was incubated with ACK Lysing Buffer (Life Technologies, Karlsruhe, Germany) for 5 min to remove erythrocytes, and washed with PBS containing 1% fetal bovine serum (FBS) (Welgene, Kyungsan, South Korea) three times. The cells were subsequently stained with FITC-conjugated anti-Ly6G and PE-Cy7-conjugated anti-CD11b antibodies (BD biosciences, NJ, USA) to determine the circulating neutrophil population. To investigate the expression of adhesion molecules on Ly6G+/CD11b+ cells, the cells were further stained with APC-conjugated anti-L-selectin and APC-conjugated anti-LFA-1 antibodies (BD biosciences). The stained cells were analyzed with a FACSVerse flow cytometer (BD biosciences), and the data were processed with FlowJo software (TreeStar, OR, USA).
A human promyelocytic leukemia cell line, HL-60, and a human monocytic cell line, THP-1, were obtained from American Cell Culture Collection (ATCC, USA). The cells were maintained in RPMI-1640 (Welgene) supplemented with 10% heat-inactivated FBS (Welgene), 2 mM l-glutamate, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Welgene) and cultured at 37 °C in a humidified atmosphere with 5% CO2.
For bone marrow-derived macrophages (BMDMs), the femur and tibia were collected from balb/c mice, and bone marrow cells were flushed with complete DMEM supplemented with 10% FBS and 0.1 mg/ml streptomycin. Red blood cells were removed using ACK lysing buffer (Gibco, NY, USA), and the cell suspension were filtered through a 40-μm cell strainer (Corning, NY, USA) to remove any cell clumps. The single cell suspensions were cultured for 7d in complete DMEM supplemented with 30% conditioned medium of L929 mouse fibroblasts in order to fully differentiate the cells into BMDMs. The culture medium was replaced every 2d, and more than 95% of the differentiated cells were F4/80+ when analyzed by flow cytometry (Additional file 1: Fig. S1A).
HL-60 differentiation and migration assay
HL-60 cells were differentiated (dHL-60) to neutrophil-like cells in the culture medium with the addition of 1.3% DMSO (Sigma) for 5 days in a humidified atmosphere at 37 °C without changing the medium. The differentiated cells were stained with anti-CD11b-PE and were analyzed by flow cytometry (Additional file 1: Fig. S1B). Migration assay was performed as described previously with minor modifications . Briefly, THP-1 cells were pretreated with various concentrations of PLAG for 1 h and stimulated with gemcitabine for 24 h. The THP-1 supernatant was placed on the bottom chamber as chemoattractant. The differentiated HL-60 cells (1 × 106 cells/100 μL) were transferred to the upper chamber of Transwell® 24-well plates with 5 μm polycarbonate membrane filters (Corning) and were incubated for overnight. The total number of migrated cells was counted by trypan blue staining using a hemocytometer.
Enzyme-linked immunosorbent assay (ELISA)
The concentration of murine MIP-2 in serum and cell supernatants was measured using MIP-2 ELISA kit (R&D systems, MN, USA), and the level of human CXCL8 from cell supernatants was detected by human CXCL8 ELISA kit (BD biosciences) according to the manufacturer’s instructions. Optical densities were measured at 450 nm using a Bio-Rad Model 550 microplate reader (Bio-Rad Laboratories, CA, USA). The chemokine levels were calculated from a standard curve generated by a curve-fitting program.
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNAs from cells were isolated using TRIzol® reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. RT-PCR was performed using PCR reagent (Bioassay, Daejeon, South Korea). Complementary DNA (cDNA) was synthesized from total RNA using a RT kit (Bioassay), followed by conventional PCR. The primers used in this study are as follows: human CXCL8, 5′-AGGGTTGCCAGATGCAATAC-3′ and 5′-GTGGATCCTGGCTAGCAGAC-3′; mouse MIP-2, 5′-AGTGAACTGCGCTGTCAATG-3′ and 5′-CTTTGGTTCTTCCGTTGAGG-3′; GAPDH, 5′-CCATCACCATCTTCCAGGAG-3′ and 5′-ACAGTCTTCTGGGTGGCAGT-3′.
Membrane fractionation and immunoblotting
Total cells were lysed on ice for 30 min in RIPA buffer composed of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium fluoride, 2 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 10 mM sodium orthovanadate. Membrane-and-cytoplasmic protein fractions of cultured cells were obtained with Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scientific™, MA, USA) according to the manufacturer’s instructions. The lysates were centrifuged at 13,000 rpm for 20 min at 4 °C and protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories). Denatured samples were mixed with a 5× SDS-PAGE loading buffer and heated to 100 °C for 15 min. The samples were separated on the 10% of SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore Corporation, MA, USA). Membranes were blocked with 5% non-fat milk in PBS (10 mM Tris-HCl, pH7.5, 150 mM NaCl) for 1 h and probed with primary antibodies against ERK1/2, phospho-ERK1/2, P38, phospho-P38, SAPK/JNK, phospho-SAPK/JNK, Na,K-ATPase, α-Tubulin, phospho-PLCβ3, PLCβ3, phospho-PKCα/βII, phospho-PKCδ, PKCδ and β-actin from Cell Signaling Technology (MA, USA), Rac1 from Merck Millipore Corporation), p47phox and phospho-p47phox from Invitrogen™ for overnight at 4 °C. The blots were washed and incubated with appropriate secondary antibodies and visualized using Pierce™ ECL Western Blotting Substrate (Thermo Scientific™).
Intracellular ROS measurement
A total of 1 × 106 BMDMs and THP-1 were seeded, cultured, and subsequently exposed to various concentrations of PLAG with gemcitabine (10 μg/mL) for 3 h. The cells were then incubated with the ROS-sensitive probe CM-H2DCFDA (Invitrogen™) for 30 min at 37 °C in the dark. After incubation, the cells were washed 3 times with PBS and immediately analyzed using FACS verse (BD biosciences) with an excitation/emission peak at 495/527 nm. A total of 10,000 cells were counted in each determination, and results presented are mean ± S.E. of three independent experiments. Intracellular ROS production was also measured with a confocal laser scanning microscope (Zeiss LSM 800, Oberkochen, Germany). After incubating CM-H2DCFDA as above, the cells were fixed with 4% paraformaldehyde for 30 min and washed 3 times with PBS before photographing. The excitation and emission wavelengths were identical as described above and a minimum of five random fields was captured for each culture.
Cells were washed in PBS, fixed at 4% paraformaldehyde, and permeabilized with methanol. Subsequently, they were blocked with 1% BSA for 1 h and incubated overnight with antibody against monoclonal human Rac1 at 4 °C. Alexa Fluor 488-conjugated goat antibody to mouse (Thermo Fisher Scientific) for Rac1 was used as a second antibody at 1:1000. The samples were washed, dried and mounted using Prolong Gold Antifade Reagent with DAPI (Roche, Basel, Switzerland) and visualized by Zeiss LSM 800.
All experiments were performed in triplicate and the results were expressed as the mean ± standard deviation (SD). For comparison of the statistical differences of more than two groups, one-way ANOVA test was used and p values < 0.05 were considered statistically significant.
PLAG maintains circulating neutrophils by down-regulating cell surface expression of adhesion molecules in gemcitabine-induced neutropenia model
Since the peritoneal neutrophil counts increased while the blood neutrophil counts decreased following gemcitabine treatment, we next investigated whether gemcitabine provokes neutrophil extravasation by up-regulating cell surface expression of adhesion molecules. First, we confirmed that PLAG maintained the population of Gr-1-positive (Gr-1+) and CD11b-positive (CD11b+) neutrophils in the blood, which was decreased by gemcitabine (Fig. 1c, d). We further examined cell surface expression of the adhesion molecules, L-selectin and LFA-1, which mediate extravasation of Gr-1+/CD11b+ cells. As a result, we found that PLAG effectively inhibited gemcitabine-induced cell surface expression of these adhesion molecules (Fig. 1e, f). These observations suggest that PLAG has a significant effect on preventing gemcitabine-induced neutrophil migration by down-regulating the surface expression of adhesion molecules.
PLAG attenuates gemcitabine-induced expression of neutrophil-attracting chemokines MIP-2 (CXCL8) by down-regulating MAPK activation
PLAG inhibits gemcitabine-induced ROS production
PLAG attenuates NOX2 activation by inhibiting membrane translocation of the cytosolic subunits, Rac1 and phosphorylation of p47phox
PLAG inhibits gemcitabine-induced phosphorylation of PLCβ3, PKCα/βII and PKCδ, which are upstream signaling regulators of p47phox phosphorylation
The acetylated form of PLAG stimulates dHL-60 transmigration
PLAG does not interfere with the anti-cancer effect of gemcitabine
It is well known that chemotherapeutic agents often elevate the intracellular level of ROS , which plays a doubled-edged sword role in both normal and cancer cells . Gemcitabine-induced ROS is also an important mechanism of action for tumor killing . For this reason, there is a concern whether the inhibition effect of PLAG on ROS generation might reduce the anti-tumor effect of gemcitabine. Therefore, we investigated how PLAG affects tumor growth when combined with gemcitabine treatment in athymic nude mice implanted with a human multiple myeloma cell line, RPMI8226 cells. Additional file 1: Fig. S6a shows a schematic diagram of the experiment. As a result, we observed that PLAG 500 mg/kg-treated group exhibited significantly less tumor growth compared to the negative control group at day 40. Also, gemcitabine 80 or 120 mg/kg-treated group showed significantly less tumor growth compared to the negative control group. Addition of PLAG to gemcitabine, however, did not exhibit differences in tumor weight compared to gemcitabine only treatment (Additional file 1: Fig. 6b). This result indicated that the administration PLAG does not interfere with the tumor killing effect of gemcitabine.
Gemcitabine is a chemotherapeutic agent used widely for various malignant tumor types including pancreatic cancer . During chemotherapy, genotoxic agent—like gemcitabine—generally induce neutropenia, which occurs when circulating neutrophil number falls below the absolute neutrophil count in the blood, necessitating dose reduction or cessation of chemotherapy . Neutropenic patients are more vulnerable to infection because circulating neutrophils play a key role in the first-line of defense against invading pathogens . In spite of its importance, the mechanism for CIN has not been well elucidated. In this study, we demonstrate that extravasation of circulating neutrophils is promoted by chemokine gradients produced by gemcitabine treatment, which may be one of the major causes of CIN. Importantly, we also show that PLAG blocks excessive neutrophil egress and maintains circulating neutrophils in the blood, making it a potential therapeutic option for patients with CIN.
Several studies demonstrated that gemcitabine-induced ROS acted as an upstream signaling molecule in the activation of mitogen-activated protein kinase (MAPK) signaling pathways [33–35]. We also observed that gemcitabine induced the phosphorylation of ERK1/2, P38, and JNK, which is effectively inhibited by PLAG and a NADPH inhibitor, DPI. One discrepancy between PLAG and DPI is that gemcitabine-induced JNK phosphorylation is inhibited by DPI but not by PLAG. While PLAG specifically inhibits NOX2-mediated ROS production, DPI is a potent NOX inhibitor that inactivates flavoenzymes of all NOX isoforms . In addition, DPI is known to potently reduce mitochondrial ROS production through inhibiting NADH-ubiquinone oxidoreductase . We presume that other sources of ROS besides NOX2 contribute to gemcitabine-induced JNK activation.
Excessive ROS production by neutrophils causes the vascular endothelium to open inter-endothelial junctions and to promote the extravasation of inflammatory cells across the endothelial vessels . In addition, ROS, especially hydrogen peroxide (H2O2), not only relay signals from the immune cell surface to activate adhesion molecules for cell movement, but also act in non-migrating cells to influence the behavior of migrating cells . We demonstrated the effect of PLAG on down-regulation of adhesion molecules of neutrophils mainly attributed to the reduced amount of MIP-2 and/or CXCL8 chemokines produced by resident macrophages. However, we acknowledge that this would not be the only mechanism regulating the migration of neutrophils. Therefore, it is necessary to study how other different types of cells, such as endothelial cells, respond to chemotherapeutic agents and influence neutrophil migration and activation.
PLAG is a synthetic form of 1,2-acyl-3-acetylglycerol, which naturally occurs in trace amounts in a broad range of organisms, including the antlers of sika deer (Cervus Nippon), the udders of domestic cows (Bos primigenius), and the seeds of the burning bush plant (Euonymus alatus) [40–42]. The third position in the glycerol backbone of PLAG is esterified with an acetyl group, which may confer distinct features on this form compared to the three-chain fatty acid esterified triacylglycerol or diacylglycerol with two-chain fatty acid esterified. Many studies have demonstrated the therapeutic efficacy of PLAG in various disease models. Our previous study showed that PLAG exerts a synergistic effect with pegfilgrastim to treat CIN without promoting neutrophil release from bone marrow or inducing surface expression of CXCR2, implying that the mechanism of PLAG is different from that of pegfilgrastim . We also identified that PLAG has an anti-inflammatory effect in an OVA-induced allergic asthma model by attenuating CCL26 expression from lung epithelial cells and eosinophil infiltration into the respiratory tract . In a 5-fluorouracil-induced oral mucositis model, PLAG enhanced the formation of newly differentiated epidermis and blood vessels in the cheek pouch by decreasing infiltration of inflammatory cells and mucositis-associated weight loss . Taken together, PLAG has a remarkable effect on regulating excessive immune activation in a variety of inflammation-associated diseases.
Here, we show that CIN is the consequence of excessive circulating neutrophil transmigration in response to chemotactic gradients produced by resident macrophages, and that PLAG has remarkable effects on CIN prevention through the regulation of PLCβ3/PKC/NOX2/ROS/MAPK signaling cascades. Therefore, we expect that PLAG could be a new potential therapeutic strategy for CIN.
Conception and design: JWK and SYY. Development of methodology: JJ and YJK. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): JWK, SYY, JJ and YJK. Writing and review of the manuscript: JJ, YJK, JWK and SYY. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): DYL, BGM and KYS. Study supervision: JWK. All authors read and approved the final manuscript.
The authors thank to Su-Hyun Shin from KRIBB for assisting to establish the gemcitabine-induced neutropenia mice model.
This study was supported by Enzychem Lifesciences Corporation, Seoul, South Korea as a grant for an investigator-sponsored study between Enzychem Lifesciences Corporation and Korea Research Institute of Bioscience and Biotechnology (KRIBB). The patent “Method for treating leukopenia and thrombocytopenia: US20160128966A1” was invented by Ki-Young Sohn and Jae Wha Kim and applied by Enzychem Lifesciences Coporation.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this article.
Consent for publication
This work was supported by the KRIBB Research Initiative Program (KGM5251712), Grants (IGM0021711) from ENZYCHEM Lifesciences, and a Grant (KDDF-201410-10 and KDDF-201606-08) by Korean Drug Development Fund (KDDF).
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