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Chemotherapy induces tumor cell death and inhibits tumor progression, but the accompanying immune responses in the surrounding dying tissue cause significant inflammation. These responses, such as excessive neutrophil infiltration into tumor tissue, are the main causes of resistance to anticancer treatment. The development of drugs that reduce neutrophil infiltration into tumors is necessary to increase the anticancer effect of chemotherapy. Here, we show that the antitumor effect of the chemotherapy AC regimen (Adriamycin and cyclophosphamide) was increased by 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) cotreatment in the MDA-MB-231 triple-negative breast cancer xenograft mouse model. Tumor growth was inhibited up to 56% in mice treated with AC and inhibited up to 94% in mice cotreated with AC and PLAG. Side effects of chemotherapy, such as a reduction in body weight, were alleviated in mice cotreated with AC and PLAG. Excessive neutrophil infiltration caused by the AC regimen was successfully cleared in mice cotreated with AC and PLAG. We conclude that PLAG inhibits excessive neutrophil infiltration that aids tumor growth. Reduced neutrophils and increased lymphocytes in PLAG-treated mice can maximize the antitumor effect of the AC regimen and inhibit tumor growth.

Supplementary Figure 3 from Functional and Clinical Evidence for NDRG2 as a Candidate Suppressor of Liver Cancer Metastasis

Supplementary Figure 1 from Functional and Clinical Evidence for NDRG2 as a Candidate Suppressor of Liver Cancer Metastasis

We searched for potential suppressors of tumor metastasis by identifying the genes that are frequently down-regulated in hepatocellular carcinomas (HCC) while being negatively correlated with clinical parameters relevant to tumor metastasis, and we report here on the identification of N-myc downstream regulated gene 2 (NDRG2) as a promising candidate. NDRG2 expression was significantly reduced in HCC compared with nontumor or normal liver tissues [87.5% (35 of 40) and 62% (62 of 100) at RNA and protein levels, respectively]. Reduction of NDRG2 expression was intimately associated with promoter hypermethylation because its promoter region was found to carry extensively methylated CpG sites in HCC cell lines and primary tumors. Immunohistochemical analysis of NDRG2 protein in 100 HCC patient tissues indicated that NDRG2 expression loss is significantly correlated with aggressive tumor behaviors such as late tumor-node-metastasis (TNM) stage (P = 0.012), differentiation grade (P = 0.024), portal vein thrombi (P = 0.011), infiltrative growth pattern (P = 0.015), nodal/distant metastasis (P = 0.027), and recurrent tumor (P = 0.021), as well as shorter patient survival rates. Ectopically expressed NDRG2 suppressed invasion and migration of a highly invasive cell line, SK-Hep-1, and experimental tumor metastasis in vivo, whereas small interfering RNA–mediated knockdown resulted in increased invasion and migration of a weakly invasive cell line, PLC/PRF/5. In addition, NDRG2 could antagonize transforming growth factor β1–mediated tumor cell invasion by specifically down-regulating the expression of matrix metalloproteinase 2 and laminin 332 pathway components, with concomitant suppression of Rho GTPase activity. These results suggest that NDRG2 can inhibit extracellular matrix–based, Rho-driven tumor cell invasion and migration and thereby play important roles in suppressing tumor metastasis in HCC. [Cancer Res 2008;68(11):4210–20]

Supplementary Methods and Materials, Tables 1-4, Figure Legends 1-3 from Functional and Clinical Evidence for NDRG2 as a Candidate Suppressor of Liver Cancer Metastasis

Background: Immune checkpoint inhibitors (ICIs) have been proven to be very effective and have fewer side effects than conventional anticancer drugs by responding specifically to tumors. However, tumors developed resistance through various tumor-friendly factors, gradually weakening the effects of ICI. These factors suppress the activation of cytotoxic T-lymphocytes (CTLs), thereby inhibiting the ICI efficacy on tumors. Therefore, it is very important to develop a combination therapy that induces the anticancer efficacy of ICI by suppressing resistance factors produced by tumors. Our previous results demonstrated that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) effectively controls the tumor-friendly factors (neutrophils infiltration, adenosine, etc.) while increasing CTL infiltration to suppress tumor progression. We suggest that PLAG is one of the effective alternatives to enhance the antitumor efficacy of ICI. Method: CT26 colorectal carcinoma (CRC) cell was inoculated into the syngeneic Balb/c mice SC and maintained for 4 days. PLAG (25/50/100 mpk) was daily administered orally for 3 weeks w/wo 5 mpk aPD-1 (RMP1-14) via IP once/week. AZD4635 was daily administered orally at 30 mpk, and anti-CD73 by IP at 10 mpk twice/week. Tumor growth was measured in 3-day intervals. Result: aPD-1 treatment inhibited tumor growth by 39%, whereas the aPD-1/PLAG treatment reduced tumor growth by 85% in the CT26 ICI low-sensitivity CRC model. Tumor weight was reduced by 17.5% in aPD-1, whereas aPD-1/PLAG treatment decreased by 68%. PLAG treatments (PLAG only group: 4 out of 6, PLAG/aPD-1 combination group: 5 out of 6) significantly improved the survival rate of tumor-bearing mice compared to control or aPD-1 group (2 out of 6) (p Conclusion: Our findings show that PLAG inhibits tumor growth by suppressing massive adenosine production, which may increase the antitumor efficacy of aPD-1 through improved CTLs infiltration. We propose that PLAG could be a novel therapeutic strategy for patients with ICI-resistant tumors. Citation Format: Jae Sam Lee, Guen Tae Kim, Eun Young Kim, Su-Hyun Shin, Hyowon Lee, Se Hee Lee, Ji Won Choi, Dong Sung Kim, Ki-Young Sohn, Jae Wha Kim. A synergistic antitumor effect of PLAG with aPD-1 by modulating the adenosine signaling pathway in ICI low-sensitivity CRC model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1835.

Activated epidermal growth factor receptors (EGFRs) are crucial for inducing metastasis in cancer cells by promoting matrix metalloproteinase (MMP) expression. The present study was designed to investigate the effects of 1‑palmitoyl‑2‑linoleoyl‑3‑acetyl‑rac‑glycerol (PLAG) on MMP expression in epidermal growth factor (EGF)‑stimulated breast cancer cells in vitro. EGF stimulation induced internalization of its cognate receptor, EGFR, for stimulus‑desensitization. These internalized receptors, complexed with the ubiquitin ligase c‑Cbl and EGFR pathway substrate 15 (EPS15) (for degradation), were evaluated by confocal microscopy at 5‑90 min time intervals. During intracellular trafficking of EGFRs, EGF‑induced signaling cascades were analyzed by examining EGFR and SHC phosphorylation. Modulation of MMP expression was assessed by evaluating the activity of transcription factor AP‑1 using a luciferase assay. PLAG accelerated the assembly of EGFRs with c‑Cbl and EPS15 and promoted receptor degradation. This faster intracellular EGFR degradation reduced AP‑1‑mediated MMP expression. PLAG stimulation upregulated thioredoxin‑interacting protein (TXNIP) expression, and this mediated the accelerated receptor internalization. This PLAG‑induced increase in EGFR trafficking was blocked in TXNIP‑silenced cells. By downregulating MMP expression, PLAG effectively attenuated EGF‑induced mobility and invasiveness in these cancer cells. These data suggest that PLAG may be a potential therapeutic agent for blocking metastasis.

Neutrophil activity, including trapping of damage-associated molecular patterns by neutrophil extracellular traps (NETs), is an important response to microbial infection. Most activated neutrophils commit to apoptosis and are removed by activated macrophages in the process of efferocytosis. Improper clearance of apoptotic neutrophils often causes an unnecessary and exaggerated immune response and subsequent chronic inflammation. Effective macrophage mobility toward activated neutrophils, which is triggered by binding of 'find-me' signals to receptors such as P2Y2, is a crucial step for the timely clearance of apoptotic neutrophils. In this paper, we investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on efferocytosis and the underlying molecular mechanisms. In a coculture of apoptotic neutrophils with macrophages, PLAG treatment increased levels of efferocytosis of apoptotic neutrophils. PLAG induced faster translocation of P2Y2 from lipid rafts to nonlipid raft plasma membrane domains in macrophages. This repositioning of P2Y2 enables the polarization of the cytoskeleton by association of the receptor with cytoskeletal proteins such as α-tubulin and actin to improve the mobility of macrophages. The formation of vesicular, chylomicron-like structures by PLAG was a prerequisite for the induction of this macrophage activity, as none of these effects was seen when the vesicle receptor GPIHBP1 was absent. Taken together, these data showed that PLAG is a powerful immune resolvent that triggers the prompt clearance of apoptotic neutrophils by enhanced efferocytosis activity. PLAG could therefore be an effective lipid-based efferocytosis enhancer for use as a therapeutic drug to prevent inflammatory disease caused by uncontrolled immune responses.

Objective This study was designed to investigate whether necroptosis is involved in the pathogenesis of chemoradiation-induced oral mucositis in a murine model and whether 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) ameliorates this disorder. Materials and methods A chemoradiation-induced oral mucositis model was established by treating mice with concurrent 5-fluorouracil (100 mg/kg, i.p.) and head and neck X-irradiation (20 Gy). Phosphate-buffered saline or PLAG (100 mg/kg or 250 mg/kg, p.o.) was administered daily. Body weights were recorded daily, and mice were sacrificed on Day 9 for tongue tissue analysis. Results On Day 9, chemoradiotherapy-treated (ChemoRT) mice had tongue ulcerations and experienced significant weight loss (Day 0:26.18 ± 1.41 g; Day 9:19.44 ± 3.26 g). They also had elevated serum macrophage inhibitory protein 2 (MIP-2) (control: 5.57 ± 3.49 pg/ml; ChemoRT: 130.14 ± 114.54 pg/ml) and interleukin (IL)-6 (control: 198.25 ± 16.91 pg/ml; ChemoRT: 467.25 ± 108.12 pg/ml) levels. ChemoRT-treated mice who received PLAG exhibited no weight loss (Day 0:25.78 ± 1.04 g; Day 9:26.46 ± 1.68 g) and had lower serum MIP-2 (4.42 ± 4.04 pg/ml) and IL-6 (205.75 ± 30.41 pg/ml) levels than ChemoRT-treated mice who did not receive PLAG. Tongue tissues of mice who received PLAG also displayed lower phosphorylation levels of necroptotic signalling proteins. Conclusion 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol mitigated chemoradiation-induced oral mucositis by modulating necroptosis.

Introduction: Head and neck squamous cell carcinomas (HNSCC) are very unfavorable carcinoma that lowers the quality of life. In addition, side effects associated with treatment such as oral mucositis also deteriorate enough to be classified as a disease. This study verified the synergistic antitumor effect of EC-18 (PLAG, 1-Palmitoyl-2-linoleoyl-3-acetyl-roc-glycerol) with or without cisplatin as a chemotherapy and side effects alleviation effects in the metastatic mouse oral squamous carcinoma (MOSCC) orthotopic model. Method: After inserting mouse-derived squamous oral carcinoma into the right side of the tongue (n=12), it was treated with EC-18 alone or with cisplatin for three weeks. The changes in feed rate and body weight were quantitatively verified on a 2-day interval. Changes in tumor size and oral mucositis symptoms (toluidine-blue positive) were analyzed on the sacrifice day. In addition, changes in the amount of damage-associated molecular pattern (DAMP) increased by tumor and cisplatin, and changes in related active factors were quantitatively analyzed. Result: Compared to the positive control group, the tumor size was reduced by 42% in the group treated with EC-18 alone (P Conclusion: EC-18 can effectively inhibit the growth of HNSCC and alleviate the side effects caused by existing anticancer drugs at the same time. Therefore, it can be a desirable therapy for HNSCC patients. Citation Format: Guentae KIM, Eun Young Kim, Su-Hyun Shin, Hyowon Lee, Sun Young Yoon, Kaapjoo Park, Ki-young Sohn, Jae Wha Kim. EC-18 suppressed tumor growth and alleviated side effects caused by cisplatin in the HNSCC implantation model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5478.

The growing risk of accidental radiation exposure due to increased usage of ionizing radiation, such as in nuclear power, industries and medicine, has increased the necessity for the development of radiation countermeasures. Previously, we demonstrated the therapeutic potential of the acetylated diacylglycerol, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), as a radiation countermeasure by mitigating radiation-associated mortality and hematopoietic acute radiation syndrome (H-ARS) in BALB/c mice after a lethal dose (LD70/30) of gamma-ray total-body irradiation (TBI). In this study, we show that PLAG mitigates symptoms of H-ARS, as characterized by mature blood cell recovery and restoration of bone marrow cellularity, by regulating systemic inflammation. Log-rank test demonstrated that high levels of WBCs, lymphocytes and neutrophils on day 10 post-TBI resulted in significantly improved survival rate. PLAG significantly enhanced the nadir values of all major blood cell types as well as bone marrow cellularity. A single TBI at LD70/30 induced an immediate increase in the blood levels of CXCL1 (12.5 fold), CXCL2 (1.5 fold), IL-6 (86.9 fold), C-reactive protein (CRP; 1.3 fold) and G-CSF (15.7 fold) at 6 h post-TBI, but the cytokine levels returned to baseline level afterward. When the irradiated mice started to die around 15 days post-TBI, they exhibited a second surge in blood levels of CXCL1 (49.3 fold), CXCL2 (87.1 fold), IL-6 (208 fold), CRP (3.6 fold) and G-CSF (265.7 fold). However, PLAG-treated groups showed a significant decrease in these same blood levels (P < 0.001). Considering the inverse correlation between inflammatory cytokine levels and hematological nadirs, PLAG exerts its therapeutic effects on H-ARS by regulating inflammatory cytokine production. These data suggest that PLAG has high potential as a radiation countermeasure to mitigate H-ARS after accidental exposure to radiation.

The effectiveness of chemotherapy and radiotherapy to treat lung cancer is limited because of highly metastatic nature. Novel strategies and drugs to attenuate metastatic activity are urgently required. In this study, red fluorescence proteins (RFP)-labeled A549 human lung cancer cells were orthotopically implantation, where they developed primary tumors. Metastasis in brain and intestines were reduced by up to 80% by treatment with 100 mpk 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) compared with that in control mice. PLAG treatment also reduced the migration of the primary tumors. Interestingly, substantial neutrophil infiltration was observed in the tumors in control mice. The neutrophil contribution to A549 cell metastatic activity was examined in in vitro co-culture system. Metastatic activity could be achieved in the A549 cells through epidermal growth factor receptor (EGFR) transactivation mediated by protease activating receptor 2 (PAR2) receptor. Neutrophil elastase secreted from tumor-infiltrating neutrophils stimulated PAR2 and induced EGFR transactivation. However, this transactivation was inhibited by inducing PAR2 degradation following PLAG treatment and metastatic activity was effectively inhibited. PLAG attenuated cancer metastatic activity via modulated PAR2/EGFR transactivation by accelerating PAR2 degradation. These results suggest PLAG as potential therapeutic agent to combat tumor metastasis via regulating the activation signal pathway of PAR2 by tumor infiltrate-neutrophils.

Streptozotocin (STZ) is widely used to induce diabetic rodent models. It is specifically toxic to pancreatic beta cells and causes severe destruction and dysfunction. We investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on an STZ-induced diabetic mouse model. PLAG attenuated the glucose increase and maintained serum insulin at levels similar to those seen with control mice. In pancreatic beta cell line INS-1, STZ-induced cell apoptosis and intracellular reactive oxygen species (ROS) generation were significantly reduced to nearly normal levels after PLAG treatment. Glucose transporter 2 (GLUT2) localization analyses and glucose uptake assays showed that PLAG accelerated GLUT2 internalization, which ameliorated excessive entry of glucose, as well as STZ. STZ-induced cytotoxic effects were significantly reduced in PLAG-treated groups. The biological activity of PLAG was further confirmed in GLUT2-silenced cells, and the specificity of PLAG was verified using its derivative 1-palmitoyl-2-linoleoyl-3-hydroxyl-rac-glycerol (PLH). Our results suggest that PLAG may be a useful agent for protecting beta cells in the setting of excessive glucose influx.

Background: There are limited side effects with ICI therapy such as not responding or reduced therapeutic effect caused by its tolerance. In particular, adenosine-induced tumor progression and drug adaptability are the obstacles in use of ICI therapy. Removing adenosine around the tumor is a prerequisite factor of improving the effect of ICI therapy along with suppression of tumor progression. Methods: To investigate the anti-tumor effect of PLAG with the PD-1 antibody (aPD-1), the syngeneic model was used (n=6/group). LLC-1 lung carcinoma cells were implanted into the C57BL/6 mice via subcutaneous. PLAG was daily administrated for 4 weeks with or without 5 mpk of aPD-1 (RMP1-14). it was delivered via IP injection biweekly. The adenosine levels and infiltrated immune cell populations in the tumor and blood were analyzed weekly until the sacrifice day. Results: The tumor size decreased by 23%/65%/81% in a dose-dependently compared to the positive control. Especially, the tumor completely disappeared in 1 animal in treated with aPD-1 and 50 mpk of PLAG, and the 2 animals in treated with 100 mpk. The adenosine levels of blood and tumor burden in the PLAG-treated group decreased by more than 50% compared to the positive control group. It was also confirmed that the adenosine level was about 25% lower in the PLAG-treated group compared to the aPD-1 alone. We found that the expression of A2B receptor in the tumor was significantly reduced in PLAG-treated group. Interestingly, the tumor growth inhibitory effect of PLAG was about 23% better than MRS1754, an A2B receptor antagonist. MRS1754-treated group showed significantly high adenosine level in the blood and tumor, whereas, in the PLAG-treated group, similar adenosine level in blood was continuously maintained as the negative control and its level in the tumor was decreased compared to the positive control. Increasing cancer cell growth and adenosine secretion were shown at 8 h post-treatment of adenosine. PLAG co-treatment decreased the adenosine level by half rapidly and then reduced continuously. In particular, degradation of the A2B receptor was observed after 4 h in PLAG-treated cells. Also intracellular ROS generated during the trafficking of the receptor was elevated within a short period by PLAG and then rapidly decreased at the same time as the signal protein change. PLAG may induce intracellular ROS regulation and A2B receptor degradation through overexpression of αARR. The effect of PLAG disappeared when αARR was knock-down. Conclusion: Extracellular adenosine level of tumor in ICI therapy could be the critical factor to be a successful anticancer treatment. To attenuate adenosine levels, a number of targeted therapies are being developed. Unlike these targeted therapeutics, PLAG fundamentally blocks cancer growth by adenosine clearance. Therefore, PLAG has its own anti-cancer effect and can maximize the effect of ICI therapy. Citation Format: Guen Tae Kim, Sun Young Yoon, Kaapjoo Park, Heesoo Kim, Ki-Young Sohn, Jae Wha Kim. Suppressive effect of PLAG on tumor progression and its synergistic therapeutic effect with ICI therapy through adenosine clearance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1447.

// Young-Jun Kim 1,* , Jae Min Shin 1,* , Su-Hyun Shin 1 , Joo Heon Kim 2 , Ki-Young Sohn 3 , Heung-Jae Kim 3 , Jong-Koo Kang 4 , Sun Young Yoon 3 and Jae Wha Kim 1 1 Cell Factory Research Center, Division of Systems Biology and Bioengineering, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 2 Department of Pathology, EulJi University School of Medicine, Daejeon, Republic of Korea 3 Enzychem Lifesciences, Daejeon, Republic of Korea 4 Department of Laboratory Animal medicine, College of Veterinary medicine, Chungbuk National University, Cheongju, Republic of Korea * These authors have contributed equally to this work Correspondence to: Jae Wha Kim, email: // Sun Young Yoon, email: // Keywords : PLAG, collagen-induced arthritis, IL-6, STAT3, neutrophil, Immunology and Microbiology Section, Immune response, Immunity Received : December 05, 2016 Accepted : July 06, 2017 Published : July 19, 2017 Abstract The pathogenesis of rheumatoid arthritis (RA) has been implicated neutrophil extracellular traps (NETs) formation which could generate autoantigen. Neutrophil contributes to initiate and maintain the inflammatory process in the joint. In this study, we show that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) decreases neutrophil migration by regulating the activity of STAT3, a regulator of IL-6 and MIP-2 expression. PLAG caused a decrease in IL-6 production in the RAW264.7 macrophage cell line and in rheumatoid arthritis–fibroblast-like synoviocytes via the regulation of STAT3 signaling without affecting NF-κB signaling. In a mouse model of collagen-induced arthritis (CIA), arthritic symptoms were recapitulated, with increased IL-6 level in the synovium, and PLAG treatment restored IL-6 to a level comparable to that achieved with commercial therapeutics (such as Remicade or methotrexate). Staining of joint tissue with neutrophil-specific antibody showed that PLAG significantly reduced the infiltration of neutrophils into the joint synovium of CIA mice. The inhibitory effect of PLAG on IL-6/STAT3 or MIP-2 signaling also reduced the migration of differentiated neutrophils in vitro . Therefore, PLAG inhibits the infiltration of destructive neutrophils into inflammatory sites, and can be utilized as a potent therapeutic agent for the treatment of sustained inflammation and joint destruction.

Streptozotocin (STZ) is widely used to induce diabetic rodent models. It is specifically toxic to pancreatic beta cells and causes severe destruction and dysfunction. We investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on an STZ-induced diabetic mouse model. PLAG attenuated the glucose increase and maintained serum insulin at levels similar to those seen with control mice., Streptozotocin (STZ) is widely used to induce diabetic rodent models. It is specifically toxic to pancreatic beta cells and causes severe destruction and dysfunction. We investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on an STZ-induced diabetic mouse model. PLAG attenuated the glucose increase and maintained serum insulin at levels similar to those seen with control mice. In pancreatic beta cell line INS-1, STZ-induced cell apoptosis and intracellular reactive oxygen species (ROS) generation were significantly reduced to nearly normal levels after PLAG treatment. Glucose transporter 2 (GLUT2) localization analyses and glucose uptake assays showed that PLAG accelerated GLUT2 internalization, which ameliorated excessive entry of glucose, as well as STZ. STZ-induced cytotoxic effects were significantly reduced in PLAG-treated groups. The biological activity of PLAG was further confirmed in GLUT2-silenced cells, and the specificity of PLAG was verified using its derivative 1-palmitoyl-2-linoleoyl-3-hydroxyl-rac-glycerol (PLH). Our results suggest that PLAG may be a useful agent for protecting beta cells in the setting of excessive glucose influx.

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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. Electronic supplementary material The online version of this article (10.1186/s13578-018-0266-7) contains supplementary material, which is available to authorized users.

α-Lactalbumin and β-lactoglobulin are two major whey proteins that specifically bind immunoglobulin E and are suspected as major allergens causing cow's milk allergy (CMA). Recent studies have shown that thymic stromal lymphopoietin is a critical factor linking at the interface of the body and environment to the T-helper 2 response. However, it is not known whether thymic stromal lymphopoietin expression is changed by α-lactalbumin and β-lactoglobulin in immune cells. Using RT-PCR and ELISA, the present study was conducted to examine if intravenous injection of α-lactalbumin and β-lactoglobulin increased pro-inflammatory cytokines, T-helper 2 cytokines, and thymic stromal lymphopoietin expression in several immune cells, including macrophages, mast cells, and keratinocytes. Results showed that α-lactalbumin and β-lactoglobulin induced thymic stromal lymphopoietin, interleukin-6, and tumor necrosis factor-α expression. It was concluded that the allergenicity of α-lactalbumin and β-lactoglobulin may be attributed to thymic stromal lymphopoietin induction, T-helper 2 cytokines, and pro-inflammatory cytokines.

Genkwadaphnin (GD-1) is isolated from the flower buds of Daphne genkwa Siebold et Zuccarini (Thymelaeaceae), and it has been used as a traditional Korean and Chinese medicine. In this study, the authors observe that GD-1 inhibits the growth of the colon cancer cell line, SW620, through the up-regulation of p21 expression in a PRDM1-dependent manner. After treatment with GD-1, the transcriptional repressor PRDM1 is prominently induced in SW620 cells. Furthermore, GD-1 induce the phosphorylation of PKD1 and MEK and subsequently provide PRDM1 enhancement, resulting in the suppression of c-Myc expression and the up-regulation of p21. PKD1 knockdown using siRNA abrogates PRDM1 expression by GD-1 and subsequently disrupts the regulation of c-Myc and p21 expression. Treating SW620 cells with GD-1 inhibits cell-cycle progression and is characterized by the down-regulation of c-Myc followed by the up-regulation of p21 expression. The up-regulation of p21 by GD-1 induces the growth arrest of the SW620 colon cancer cell line. Based on these data, the authors propose that GD-1 has tumor-suppressor activity that may contribute to the anti-tumor effects of PRDM1 in colon cancer.

Background: Currently, no approved drug exist to treat oral mucositis (OM) in patients receiving chemoradiation for the treatment of head and neck cancer. To address this limitation, we examined the therapeutic effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), which is an immune resolution accelerator that can effectively modulate the neutrophil recruitment, against chemoradiation-induced oral mucositis (CRIOM) in a murine model. Additionally, we investigated whether necroptosis is involved in the pathogenesis of CRIOM in a murine model and the therapeutic effect of PLAG against the disease. Methods: To investigate the improved effect of PLAG on CRIOM, we established the CRIOM-bearing BALB/c mouse model using concurrent treatments of 5-fluorouracil (100 mg/kg, i.p.) and X-radiation (20 Gy) on the heads and necks of mice. Phosphate buffer saline or PLAG (100 and 250 mg/kg, p.o.) was administered daily. The body weights of mice were observed daily and on Day 9, the mice were sacrificed for extracting their tongues for further tissue analyses. The histopathological grading of OM was analyzed in the collected tongues. Human keratinocytes (HaCaT) were utilized to identify the mechanism of PLAG on regulating the necroptosis signals. Results: On Day 9, chemoradiotherapy-treated (ChemoRT) mice had tongue ulcerations and experienced significant weight loss (Day 0: 26.18 ± 1.41 g; Day 9: 19.44 ± 3.26 g). They also had elevated serum macrophage inhibitory protein 2 (MIP-2) (control: 5.57 ± 3.49 pg/ml; ChemoRT: 130.14 ± 114.54 pg/ml) and interleukin (IL)-6 (control: 198.25 ± 16.91 pg/ml; ChemoRT: 467.25 ± 108.12 pg/ml) levels. ChemoRT-treated mice who received PLAG exhibited no weight loss (Day 0: 25.78 ± 1.04 g; Day 9: 26.46 ± 1.68 g) and had lower serum MIP-2 (4.42 ± 4.04 pg/ ml) and IL-6 (205.75 ± 30.41 pg/ml) levels than ChemoRT-treated mice without the PLAG treatment. Tongue tissues of mice who received PLAG also displayed lower phosphorylation levels of necroptotic signaling proteins (RIPK1/3 and MLKL). Chemoradiation induced the release of damaged-associated molecular patterns (DAMPs), which activates the necroptosis signaling pathway if not promptly eliminated intracellularly in HaCaT cells. PLAG promoted the effective removal of DAMPs, thereby downregulating the activation of necroptosis signaling pathway. Conclusion: On day 9, the tongues of mice-treated with chemoradiotherapy activated the necroptosis signaling pathway. As a result, cells from oral mucosa released DAMPs and pro-inflammatory cytokines to induce neutrophil migration to the inflamed oral epithelium. PLAG ameliorated the degree of OM by terminating the necroptosis signaling pathway. These data suggest that PLAG may be a useful therapeutic agent for the treatment of CRIOM. Citation Format: Solji Choi, Su-Hyun Shin, Sun Young Yoon, Ji Sun Park, Ki-Young Sohn, Jae Wha Kim. 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol ameliorates chemoradiation-induced oral mucositis in a mouse model by regulating necroptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6175.

Background: The development of immune-checkpoint drugs, a fourth-generation anticancer drug, is a significant breakthrough in anticancer research. Cancer therapy using immune-checkpoint drugs are still enlarged. To improve the efficacy of immune-checkpoint drugs, modulation of the tumor microenvironment is essentially required. In this experiment, we examined the elevated anti-cancer efficacy of 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG), which has been demonstrated to attenuate tumor infiltrated neutrophils (TINs) in the tumor, along with the PD-L1 immune checkpoint inhibitor treatment. Methods: The syngeneic model was used (n=6) to investigate the enhanced anti-tumor effect of PD-L1 antibody with the addition of PLAG. MB49 murine bladder cancer cells were implanted into the C57BL/6 mice subcutaneously and bred for 5 weeks. After a week from tumor implantation, PLAG at different dosages (25/50/100 mpk) were daily administered orally for another 4 weeks with or without 5 mpk PD-L1 antibody (10F.9G2). PD-L1 antibody was delivered via IP injection twice a week. Tumor growth was calculated in 3-day intervals. Neutrophil chemotaxis-related chemokines and neutrophil-to-lymphocyte rate (NLR) were also evaluated with a 2-week interval. The degree of neutrophil infiltration in the tumor on the day of sacrifice and the degree of infiltration between lymphocytes and neutrophils were analyzed. Results: The PLAG treatment groups demonstrated that the tumor burden decreased in a concentration-dependent manner. In 50 and 100 mpk of PLAG treated mice, the tumor burden was decreased to a significant value compared to a positive control (p < 0.05). In the group treated with PD-L1 antibody alone, the growth rate of the tumor decreased until about 2 weeks. In the group treated simultaneously with PLAG and PD-L1 antibody, the growth of the tumor was significantly reduced compared to the group treated with PD-L1 antibody alone. As a result of calculating neutrophils and lymphocytes every two weeks, NLR level in the group treated with PLAG and PD-L1 was significantly decreased compared to that treated with PD-L1 antibody alone. Besides, the number of TINs were effectively reduced by PLAG treatment alone. The interruption of tumor growth and reduced chemokine secretion were also observed in the PLAG treated groups. In summary, our data suggest that PLAG provides an enhanced PD-L1 antibody effect on the regression of tumor burden in the syngeneic mice model via reducing the number of TINs. Conclusion: PLAG may be utilized for improving the efficacy of PD-L1 antibody on reducing the tumor burden at the devastating tumor microenvironment. Citation Format: Guen Tae Kim, Sun Young Yoon, Ji Sun Park, Ki-Young Sohn, Jae Wha Kim. The effect of PLAG on the PD-L1 immune-checkpoint drug therapy in the MB49 bladder cancer syngeneic model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5553.

Sialyllactose (SL) is a representative human milk oligosaccharide (HMO) of human breast milk. The roles of SL in infant brain development and immunity have been reported in previous studies. In this study, we identified the impact of SL on innate immunity. Our results showed that the administration of SL had significant efficacy on bacterial clearance in Pseudomonas aeruginosa K-infected mice. We also examined the role of SL in the human THP-1 macrophage-like cell line. SL effectively promoted receptor-mediated endocytosis and phagocytosis. Furthermore, SL accelerated the recruitment of Rac1 to the cell membrane, leading to the generation of reactive oxygen species for the elimination of phagocytosed bacteria. Our findings provide a new perspective on the role of SL in breast milk and suggest its potential application as a therapeutic agent to treat bacterial and viral infections.

Sialyllactose (SL) is a representative human milk oligosaccharide (HMO) of human breast milk. The roles of SL in infant brain development and immunity have been reported in previous studies., Sialyllactose (SL) is a representative human milk oligosaccharide (HMO) of human breast milk. The roles of SL in infant brain development and immunity have been reported in previous studies. In this study, we identified the impact of SL on innate immunity. Our results showed that the administration of SL had significant efficacy on bacterial clearance in Pseudomonas aeruginosa K-infected mice. We also examined the role of SL in the human THP-1 macrophage-like cell line. SL effectively promoted receptor-mediated endocytosis and phagocytosis. Furthermore, SL accelerated the recruitment of Rac1 to the cell membrane, leading to the generation of reactive oxygen species for the elimination of phagocytosed bacteria. Our findings provide a new perspective on the role of SL in breast milk and suggest its application as a therapeutic agent to treat bacterial and viral infections.

Background: A recent study reported that tumor-infiltrating neutrophil (TIN) has potential on malignant tumor progression leading to metastasis. In our study, to control the effect of TIN, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) was used. PLAG has shown its efficacy in modulating the neutrophil counts in chemotherapy-induced neutropenia by attenuating neutrophil extravasation via down-regulation of adhesion molecules, inflammatory cytokines, and chemokines. In this study, we investigated whether PLAG has an anti-metastatic effect on A549 lung cancer orthotopic implantation model via modulating TIN. Methods: To investigate the anti-metastatic effect of PLAG on lung cancer metastasis, RFP-labeled A549 cells were implanted into the lungs of BALB/c nude mice via intratracheal injection and bred for 12 weeks. After 6 weeks from tumor implantation, PLAG with three concentrations (25/50/100 mpk) were daily administrated for another 6 weeks. Each image of the growth of the primary tumor and colonization of metastatic tumor to other sites were acquired using CT and IVIS. TIN was analyzed in the primary tumor using immunohistochemistry (IHC). To identify the mechanism of PLAG on preventing the neutrophil-associated metastasis of cancer cells, direct or non-direct co-culture methods were used. Results: In the orthotopic implantation model, metastatic tumors to GI-track and brain were detected by IVIS. It was confirmed that metastasis to GI-track was decreased by 73% in 100 mpk PLAG-treated group, while metastasis to the brain was decreased by about 92% in PLAG-diet compared with positive control. Primary lung tumor growth was retarded by PLAG in a dose-dependent manner. The size of the primary tumor was much smaller than the positive control and alveolar tissue was similar to that of normal mice in 100 mpk PLAG-diet. In contrast, massive neutrophil infiltrations in positive and delivery groups, but significantly reduced upon PLAG administration. Moreover, p-EGFR was concomitantly detected with a similar pattern of neutrophil infiltration. During the direct contact in vitro assay, the A549 lung cancer spheroid cells were scattered by neutrophils, but it was effectively hindered by PLAG treatment. Neutrophil-stimulated cancer in the non-direct contact method showed enhanced metastatic activity. However, such a phenomenon was significantly reduced with the PLAG-treated group. Moreover, neutrophil stimulated the expression of EMT markers, whereas it was effectively down-regulated by PLAG administration. Transactivation between tumor cells and neutrophil was mediated by neutrophil elastase (NE). NE binds to and activates protease-activated receptor 2 (PAR2) on cancer cells and subsequently activated EGFR for metastasis to occur by cleaving HB-EGF through the ARR2/clathrin complex. On the other hand, the suppression of metastasis by PLAG was mediated through the regulation of the NE-PAR2 pathway and acceleration of intracellular trafficking and degradation of PAR2. Conclusion: In this study, TIN was observed in the primary lung tumor and metastasis into GI-track and brain, but PLAG dramatically reduced infiltration of neutrophil. The collective results suggest that TIN educates tumor cells for enhancing metastasis by EGFR transactivation. However, PLAG effectively interfered the transactivation. Having not met the medical needs for treating metastasis, PLAG may be a promising candidate as an anti-metastatic drug by modulating the TIN. Citation Format: GUEN TAE KIM, SUN YOUNG YOON, KI-YOUNG SOHN, MYUNG-HWAN KIM, JAE WHA KIM. Anti-metastatic effect of PLAG via interference of neutrophil elastase/PAR2/EGFR signaling on A549 lung cancer orthotopic implantation model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C037. doi:10.1158/1535-7163.TARG-19-C037

Background: Tumor microenvironments (TME) promote tumor growth and induction of metastasis along with the strong presence of tumor-infiltrating neutrophil (TIN), which further contributes to abnormal tumor growth and metastasis. From this tumor-prone environment, we hypothesize that the effective destruction of TIN may enhance the therapeutic efficacy of chemotherapy for reducing the tumor. In this study, we investigated the synergistic effect of PLAG in the MDA-MB-231 breast cancer xenograft model concomitantly treated with the AC-regimen in modulating the effect of TIN. Methods: MDA-MB-231 breast cancer xenograft model was used for evaluation of the tumor growth in the AC-regimen alone and PLAG co-treated animals. AC-regimen was delivered via intraperitoneal injection twice a week with a dose of 2/20 and 5/50 mpk (Doxorubicin/Cyclophosphamide) and PLAG was daily administered with 100 and 250 mpk. Tumor growth was measured in 3-day intervals. Neutrophil chemotaxis-related chemokines, CXCL1/2/8 and circulating neutrophils were also evaluated in a 2-week interval. Expression of apoptosis-related molecular markers (Bax/Bak) and TIN in the tumor lesion was analyzed by immunohistochemistry (IHC) staining. Results: PLAG has synergistic effects on decreasing the tumor burden in the PLAG and AC-treated xenograft model. In AC-treated groups with 2/20 or 5/50 mpk, retardation of tumor growth was observed from the calculated tumor size and regulation of apoptosis-associated markers was proved by TUNEL assay and IHC. Modulated chemokine expression from tumor burden and subsequent neutrophil recruitment were also detected in proportion to the tumor mass. The measured tumor sizes in the PLAG co-treated group were consistently smaller than those from the AC-regimen alone group till the mice were sacrificed. It was confirmed that the tumor burden of the PLAG co-treated group with 5/50 AC-regimen was significantly decreased in a concentration-dependent manner compared to the AC-regimen alone group (p Citation Format: GUEN TAE KIM, SUN YOUNG YOON, KI-YOUNG SOHN, MYUNG-HWAN KIM, JAE WHA KIM. The synergistic effect of PLAG on the antitumor efficacy of AC-regimen via alleviating neutrophil tumor infiltration on breast tumor xenograft model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B054. doi:10.1158/1535-7163.TARG-19-B054

BACKGROUND Euphorbia kansui, a traditional medical herb, has been shown to have anti-tumour and anti-viral activities. Previously, we have reported that E. kansui increases interferon-gamma (IFN-γ) production in natural killer (NK) cells. However, it is not clear how E. kansui regulates IFN-γ secretion by NK cells. RESULTS In this study, E. kansui was separated into six individual compounds from the same chloroform fraction so that the activity of each compound could be compared. E. kansui compounds induced IFN-γ secretion through the phosphorylation of protein kinase D and IκB kinase pathways. Furthermore, E. kansui compounds activated the translocation of p65, a sub-unit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), to the nucleus and induced NF-κB at the transcriptional level. CONCLUSION These findings suggest that E. kansui enhances IFN-γ secretion through the NF-κB pathway in NK cells. © 2015 Society of Chemical Industry

The plant Carpesium abrotanoides (CA) is used in Asian herbal medicines as an insecticide and to treat bruises. However, the effect of single compounds from CA blooms and the mechanism of its immunosuppressive effect remain poorly understood. The aim of this study was to investigate the mechanism of the immunosuppressive effect in the three kinds of immune cells, and the immunosuppressive effect of CA bloom extract (CAE) in acute inflammation models (LPS and ConA-induced inflammation). Interleukin-6, IL-4, IL-13, IFNγ, and IL-10—but not TNFα—were significantly reduced in a dose-dependent manner by 4α,5α-epoxy-10α,14-dihydro-inuviscolide (INV). Furthermore, INV inhibited NF-κB transcriptional activation and IL-10 promoter activity in the same manner as for Bay11. Meanwhile, treatment with dexamethasone reduced the levels of IFNγ, but not IL-10, and resulted in no change in NF-κB transcriptional activation or the IL-10 promoter. INV did not affect PMA-induced IκB kinase complex phosphorylation, IκB degradation, or MAPK and the nuclear translocation of p65, as with DEX. The in vivo, CAE has an immunosuppressive effect on the LPS-induced inflammation response model by inhibiting the plasma level of IFNγ and IL-6 levels. CAE treatment also tends to attenuate the plasma level of IFNγ, IL-4, and IL-6 in ConA-induced inflammation. These findings indicate that INV causes the reduction of the cytokine profile by blocking the NF-κB transcription factor activation and the molecular mechanism by which INV operates could provide new insights into the unique mechanisms responsible for NF-κB inhibition, in contrast to established immunosuppressants, as a therapeutic agent for immunopathological treatment.

// Jimin Kim 1, 2 , Su-Hyun Shin 1, 2 , Jong-Koo Kang 3 and Jae Wha Kim 1, 2 1 Cell Factory Research Center, Division of Systems Biology and Bioengineering, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 2 Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology, Daejeon, Republic of Korea 3 Department of Laboratory Animal Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea Correspondence to: Jae Wha Kim, email: wjkim@kribb.re.kr Keywords: HX-1171; Nrf2 activator; beta cell; diabetes; hyperglycemia Received: July 17, 2017 Accepted: September 04, 2017 Epub: March 23, 2018 Published: May 11, 2018 ABSTRACT Streptozotocin (STZ) acts specifically on pancreatic beta cells, inducing cell destruction and cell dysfunction, resulting in diabetes. Many studies have reported that nuclear factor-erythroid 2-related factor 2 (Nrf2), a main regulator of antioxidant expression, prevents and improves diabetes-related diseases. In this study, we investigated the antidiabetic effect of the newly discovered Nrf2 activator, HX-1171, in the STZ-induced diabetic mouse model. HX-1171 enhanced insulin secretion by reducing STZ-induced cell apoptosis, and decreased intracellular reactive oxygen species (ROS) generation by upregulating the expression of antioxidant enzymes through Nrf2 activation in INS-1 pancreatic beta cells. In STZ-induced diabetic mice, HX-1171 administration significantly lowered blood glucose levels and restored blood insulin levels. In the STZ-only injected mice, the pancreatic islets showed morphological changes and loss of function, whereas the HX-1171-treated group was similar to that of the control group. These results suggest that HX-1171 may be developed as a promising therapeutic agent for diabetes-related diseases.

Previously, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol, acetylated diglyceride) was reported to have an effect on the proliferation of hematopoietic stem cells (HSCs) or to contribute to the prevention of chemotherapy-induced neutropenia. In this study, we examined the role of PLAG in the differentiation of bone marrow cells from HSCs into progenitor cells in mice. After 15days, the lineage-negative cells, especially megakaryocyte/erythrocyte progenitors (MEP), were significantly increased in mice that received daily PLAG administration compared to those in the untreated mice. Furthermore, we explored the possibility that the PLAG-induced increase in MEP will contribute to reduction of chemotherapy-induced thrombocytopenia (CIT) in a thrombocytopenia mouse model. Mice were administrated 5-fluorouracil (5-FU) and PLAG. After 7days, bone marrow cells were analyzed. Treatment with 5-FU powerfully decreased myeloid precursor populations and treatment with 5-FU/PLAG resulted in reduction of decreased myeloid progenitor cell numbers. In addition, numbers of circulating platelets were also increased by PLAG treatment. Taken together, PLAG plays a role in differentiating HSCs toward MEP and alleviating chemotherapy-induced bone marrow cell reduction. Thus PLAG shows its potential to augment the therapeutic effect of anti-cancer drugs-induced thrombocytopenia.

HX-1171 (1-O-hexyl-2,3,5-trimethylhydroquinone) is a novel synthesized vitamin E derivative, which reportedly has positive effects on various diseases and conditions, such as liver fibrosis, hepatic cirrhosis, and cancer. In this study, we analyzed the transcriptional activity induced by HX-1171. Results from reverse transcription polymerase chain reaction and promoter assays reveal that HX-1171 increased the expression of NQO1 and HMOX1, encoding antioxidant-related enzymes, in A549 human lung epithelial cells. The activity of nuclear factor-E2-related factor (Nrf2), a key transcriptional factor for antioxidative enzymes, was examined in HX-1171-treated cells. Confocal microscopy and Western blotting showed that HX-1171 effectively induced the nuclear translocation and transcriptional activity of Nrf2. We conclude that HX-1171, a novel Nrf2 activator, may be a promising therapeutic agent for oxidative stress-induced diseases. J. Cell. Biochem. 118: 3372-3380, 2017. © 2017 Wiley Periodicals, Inc.

EC-18 is a synthetic monoacetyldiaglyceride that is a major constituent in antlers of Sika deer (Cervus nippon Temmenick). In this study, we evaluated the protective effects of EC-18 on Th2-type cytokines, eosinophil infiltration, and other factors in an aluminum hydroxide/ovalbumin (OVA)-induced murine asthma model. Mice were sensitized on days 0 and 14 by intraperitoneal injection of OVA with aluminum hydroxide. On days 21, 22 and 23 after the initial sensitization, the mice received an airway challenge with OVA for 1h using an ultrasonic nebulizer. EC-18 was administered to mice by oral gavage at doses of 30mg/kg and 60mg/kg once daily from day 18 to 23. Methacholine responsiveness was measured 24h after the final OVA challenge, and the bronchoalveolar lavage fluid (BALF) was collected 48h after the final OVA challenge. EC-18 significantly reduced methacholine responsiveness, T helper type 2 (Th2) cytokines, eotaxin-1, immunoglobulin (Ig) E, IgG, and the number of inflammatory cells. In addition, EC-18-treated mice exhibited the reduction in the expression of inducible nitric oxide synthase (iNOS) in lung tissue. In the histological analysis using hematoxylin-eosin stain and periodic acid-Schiff stain, EC-18 attenuated the infiltration of inflammatory cells into the airway and reduced the level of mucus production. Our results showed that EC-18 effectively suppressed the asthmatic response induced by OVA challenge. These effects were considered to be associated with iNOS suppression. In conclusion, this study suggests that EC-18 may be a therapeutic agent for allergic asthma.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), a chemically synthesized monoacetyldiaglyceride, is one of the constituents in Sika deer antlers and has been known traditionally as having immunomodulatory effects. However, the mechanism by which PLAG controls neutrophil migration, which evokes liver injury in the hepatitis animal model, remains largely unknown. This study was designed to evaluate the immunomodulatory effects of PLAG on cytokine secretion and neutrophil migration in vivo and in vitro. The injection of mice with Concanavalin A (Con A) induced leukocyte infiltration in the liver and increased plasma cytokine levels. Pretreatment with PLAG reduced the levels of interleukin (IL)-4, IL-6, IL-10, and CXCL2, but maintained interferon (IFN)-γ levels and modulated neutrophil recruitment towards the liver. Furthermore, the mRNA and protein levels of IL-4 and CXCL2 in liver tissue were also decreased in the Con A-treated mice. Liver histology analyses showed that PLAG reduced Con A-induced hepatic necrosis, which was accompanied by leukocyte infiltration. The in vitro studies revealed that PLAG reduced IL-4 secretion in Con A stimulated T cell and blocked signal transducer and activator of transcription 6 (STAT6) Con A induced hepatocyte. PLAG attenuated IL-4 induced activation of atypical protein kinase C (PKC)/ STAT6 in hepatocytes and inhibited neutrophil migration towards the liver tissue through suppression of IL-8/vascular cell adhesion molecule (VCAM) expression. These results suggest that PLAG could mitigate excess neutrophil migration into liver tissue and potentially have a therapeutic effect on immune-mediated liver injury. This article is protected by copyright. All rights reserved

Acute radiation syndrome (ARS) occurs as a result of partial- or whole-body, high-dose exposure to radiation in a very short period of time. Survival is dependent on the severity of the hematopoietic sub-syndrome of ARS. In this study, we investigated the mitigating effects of a lipid molecule, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), on the kinetics of hematopoietic cells, including absolute neutrophil count (ANC), red blood cells (RBCs) and platelet counts, in mice after gamma-ray total-body irradiation (TBI). Male and female BALB/c mice (11 weeks old) received a LD70/30 dose of TBI. PLAG significantly and dose-dependently attenuated radiation-induced mortality (P = 0.0041 for PLAG 50 mg/kg; P < 0.0001 for PLAG 250 mg/kg) and body weight loss (P < 0.0001 for PLAG 50 and 250 mg/kg) in mice. Single-fraction TBI sharply reduced ANC within 3 days postirradiation and maintained the neutropenic state (ANC < 500 cells/µl) by approximately 26.8 ± 0.8 days. However, administration of PLAG attenuated radiation-induced severe neutropenia (ANC < 100 cells/µl) by effectively delaying the mean day of its onset and decreasing its duration. PLAG also significantly mitigated radiation-induced thrombocytopenia (P < 0.0001 for PLAG 250 mg/kg) and anemia (P = 0.0023 for PLAG 250 mg/kg) by increasing mean platelet and RBC counts, as well as hemoglobin levels, in peripheral blood. Moreover, delayed administration of PLAG, even at 48 and 72 h after gamma-ray irradiation, significantly attenuated radiation-induced mortality in a time-dependent manner. When compared to olive oil and palmitic linoleic hydroxyl (PLH), only PLAG effectively attenuated radiation-induced mortality, indicating that it has a distinctive mechanism of action. Based on these preclinical observations, we concluded that PLAG has high potential as a radiation countermeasure for the improvement of survivability and the treatment of hematopoietic injury in gamma-ray-induced ARS.

Neutrophil activity is prerequisite during chemotherapy. The DAMP (Damage Associated Molecular Pattern) molecules generated by chemotherapy could be effectively trapped by activated neutrophil called ‘NETosis’. Efferocytosis of macrophages should remove most activated neutrophils including NETosis A timely removal of activated neutrophils is essential for the prevention of abnormal activation of immune response and metastatic activity of cancer cells induced by tumor microenvironment (TME). Particularly, appropriate clearance of the activated neutrophils by efferocytosis should be carried out because activated neutrophils have a detrimental effect on TME. In this research, we investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on efferocytosis and its underlying molecular mechanisms. In a co-culture of activated neutrophils with macrophages, PLAG increased the activity of efferocytosis for elimination of activated neutrophils. PLAG accelerated translocation of P2Y2 from lipid rafts to non-lipid-raft plasma membrane domains in macrophages. This repositioning of P2Y2 enables the polarization of the cytoskeleton by association of the receptor with cytoskeletal proteins such as α-tubulin and actin to improve the mobility of macrophages. Through these protein assemble, PLAG encouraged macrophage mobility toward the activated neutrophils. Formation of micelle including PLAG, chylomicron-like structures, was a prerequisite for induction of this macrophage activity. PLAG effect on this activity was not observed in the absence of GPIHBP1, micelle receptor. Taken together, these data showed that PLAG triggered a prompt clearance of activated neutrophils through enhancement of efferocytosis activity. Subsequently, PLAG could have effects on modulation of TME. PLAG could be utilized as an effective lipid-based TME modulator via the prevention of abnormal activation induced by uncontrolled immune response during chemotherapy. Citation Format: Guen Tae Kim, Do Young Lee, Ki-young Sohn, Sun Young Yoon, Jae Wha Kim. PLAG enhances macrophage mobility for efferocytosis of active neutrophils via membrane redistribution of P2Y2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4586.

Chemotherapy-induced neutropenia (CIN) is a complication that arises during cancer treatment and necessitates dose reduction. Preventing CIN and maintaining absolute neutrophil counts (ANC) is critical for successful chemotherapy because a rapid decline of neutrophils increases susceptibility to infection. Here, we investigated whether administration of EC-18 has therapeutic effects on the treatment of CIN in 5-fluorouracil (5-FU)-induced neutropenia mouse model. A single injection of 5-FU 100mg/kg reduced the ANC in the control, EC-18 125 and EC-18 250mg/kg-treated cohort from pre-injection values to 1000 cells/μL. EC-18 125 or 250mg/kg significantly reduced the duration of neutropenia from 7.4±1.14 days to 2.6±0.55 or 3.0±0.71 days, respectively. Moreover, the ANC of all individuals in the control cohort fell to severely neutropenic range (ANC Citation Format: Yong-Jae Kim, Jinseon Jeong, Ki-Young Sohn, Do Young Lee, Sun Young Yoon, Jae Wha Kim. Therapeutic potential of EC-18 as a chemotherapy adjuvant for 5-fluorouracil-induced neutropenia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 360.