Improving anticancer effect of aPD-L1 through lowering neutrophil infiltration by PLAG in tumor implanted with MB49 mouse urothelial carcinoma.

BACKGROUND: The PD-L1 antibody is an immune checkpoint inhibitor (ICI) attracting attention. The third-generation anticancer drug has been proven to be very effective due to fewer side effects and higher tumor-specific reactions than conventional anticancer drugs. However, as tumors produce additional resistance in the host immune system, the effectiveness of ICI is gradually weakening. Therefore, it is very important to develop a combination therapy that increases the anticancer effect of ICI by removing anticancer resistance factors present around the tumor. 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 urothelial cancer cells were implanted into the C57BL/6 mice subcutaneously. PLAG at different dosages (50/100 mpk) was 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 once a week. RESULTS: The aPD-L1 monotherapy group inhibited tumor growth of 56% compared to the positive group, while the PLAG and aPD-L1 co-treatment inhibited by 89%. PLAG treatment effectively reduced neutrophils infiltrating localized in tumor and converted to a tumor microenvironment with anti-tumor effective T-cells. PLAG increased tumor infiltration of CD8 positive cytotoxic T-cell populations while effectively inhibiting the infiltration of neoplastic T-cells such as CD4/FoxP3. Eventually, neutrophil-induced tumor ICI resistance was resolved by restoring the neutrophil-to-lymphocyte ratio to the normal range. In addition, regulation of cytokine and chemokine factors that inhibit neutrophil infiltration and increase the killing activity of cytotoxic T cells was observed in the tumors of mice treated with PLAG + aPD-L1. CONCLUSIONS: PLAG effectively turned the tumor-promoting microenvironment into a tumor-suppressing microenvironment. As a molecule that increases the anti-tumor effectiveness of aPD-L1, PLAG has the potential to be an essential and effective ICI co-therapeutic agent.

PLAG co-treatment increases the anticancer effect of Adriamycin and cyclophosphamide in a triple-negative breast cancer xenograft mouse model.

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.

Suppression of tumor progression by thioredoxin-interacting protein-dependent adenosine 2B receptor degradation in a PLAG-treated Lewis lung carcinoma-1 model of non-small cell lung cancer.

Extracellular adenosine in the tumor microenvironment plays a vital role in cancer development. Specifically, activation of adenosine receptors affects tumor cell growth and adenosine release. We examined the anti-tumor efficacy of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) in animal models, revealing the role of PLAG in inhibiting tumor progression by promoting the degradation of adenosine 2B receptors (A2BRs) in tumors. PLAG induced the expression of thioredoxin-interacting protein (TXNIP), a type of α-arrestin that accelerates A2BR internalization by interacting with A2BR complexes containing ß-arrestin. Engulfed receptors bound to TXNIP were rapidly degraded after E3 ligase recruitment and ubiquitination, resulting in early termination of intracellular signals that promote tumor overgrowth. However, in control cancer cells, A2BRs bound to protein phosphatase 2A and were returned to the cell membrane instead of being degraded, resulting in continuous receptor-mediated signaling by pathways including the Raf-Erk axis, which promotes tumor proliferation. A TXNIP-silenced cell-implanted mouse model and TXNIP knockout (KO) mice were used to verify that PLAG-mediated suppression of tumor progression is dependent on TXNIP expression. Increased tumor growth was observed in TXNIP-silenced cell-implanted mice, and the anti-tumor effects of PLAG, including delayed tumor overgrowth, were greatly reduced. However, the anti-tumor effects of PLAG were observed in cancer cell-implanted TXNIP-KO mice, which indicates that PLAG produces anti-tumor effects by enhancing TXNIP expression in tumor cells. These essential functions of PLAG, including delaying tumor growth via A2BR degradation, suggest innovative directions for anticancer drug development.

PLAG alleviates cisplatin-induced cachexia in lung cancer implanted mice.

Chemotherapy-induced cachexia has been a significant challenge to the successful treatment of cancer patients. Chemotherapy leads to loss of muscle, loss of appetite, and excessive weight loss, which makes these necessary treatments intolerable for most patients. Therefore, it is necessary to alleviate cachexia to successfully treat cancer patients. In this study, tumor-implanted mouse models administered cisplatin showed rapid weight loss and reduced feeding rate by the second week of treatment, and 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) effectively alleviated cisplatin-induced cachexia. In mice treated with cisplatin on a sacrificial day after 6 weeks, the weight of the two major leg muscles (quadriceps femoris and gastrocnemius) were reduced by up to 70%, but this muscle reduction was successfully prevented in the PLAG co-treatment group. The distribution and size of muscle fibers that appear in small units in cisplatin-treated mice were restored to normal levels by PLAG co-treatment. Furthermore, myostatin expression levels were upregulated by cisplatin, whereas myostatin decreased to normal levels with muscle recovery in the PLAG co-treated group. Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), which are commonly expressed in cachexia, were significantly increased in cisplatin-treated mice but were reduced to normal levels in PLAG co-treated mice. Glucose absorption, an indicator of muscle tissue activity, decreased with cisplatin treatment and recovered to normal levels with PLAG co-treatment. Overall, PLAG effectively alleviated cisplatin-induced cachexia symptoms and reduced tumor growth in tumor-implanted mice. These findings suggest PLAG may be a promising drug to alleviate cachexia in cancer patients receiving chemotherapy.

1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol ameliorates chemoradiation-induced oral mucositis.

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.

Mitigating Effects of 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on Hematopoietic Acute Radiation Syndrome after Total-Body Ionizing Irradiation in Mice.

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.

HX-1171, a Novel Nrf2 Activator, Induces NQO1 and HMOX1 Expression.

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.

PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) augments the therapeutic effect of pegfilgrastim on gemcitabine-induced neutropenia.

Granulocyte colony-stimulating factor (G-CSF) is widely used for preventing neutropenia during chemotherapy. Polyethylene glycol-conjugated granulocyte colony-stimulating factor (PEG-G-CSF, pegfilgrastim) serves the same purpose but has a longer half-life and greater stability than G-CSF. In this study, we investigated whether 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol, acetylated diglyceride (PLAG), augments the therapeutic effect of pegfilgrastim on chemotherapy-induced neutropenia. We compared neutrophil counts in four groups of mice: control mice, gemcitabine-treated mice, gemcitabine/pegfilgrastim-treated mice, and gemcitabine/pegfilgrastim/PLAG-treated mice. PLAG (50 mg/kg) was orally administered every day during the treatment course. CBC analysis showed that the group treated with PLAG experienced a dramatically increased neutrophil counts on the third day following pegfilgrastim treatment. PLAG had no effect on blood cell apoptosis and neutrophil release from bone marrow. Additionally, pegfilgrastim-induced CXCR2 expression in neutrophils was markedly decreased in PLAG-treated animals. These results suggest that PLAG plays a role in inhibiting neutrophil extravasation, giving rise to an increased number of circulating neutrophils when used with pegfilgrastim during gemcitabine treatment. These data support the potential for PLAG to be used with pegfilgrastim to treat or prevent chemotherapy-induced neutropenia by modulating neutrophil transmigration.

PRDM1, a Tumor-Suppressor Gene, is Induced by Genkwadaphnin in Human Colon Cancer SW620 Cells.

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.