Phytogalactolipid dLGG Inhibits Mouse Melanoma Brain Metastasis through Regulating Oxylipin Activity and Re-Programming Macrophage Polarity in the Tumor Microenvironment.

Current conventional cancer therapies for melanoma brain metastasis (MBM) remain ineffective. In this study, we demonstrated the bioefficacy of a phyto-glyceroglycolipid, 1,2-di-O-α-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (dLGG) alone, or in combination with liposomal doxorubicin (Lip-DOX) or Avastin against MBM in a syngeneic B16BM4COX-2/Luc brain-seeking melanoma mouse model. Treatment with dLGG-10, dLGG-25, dLGG-10 + Avastin-5, Lipo-DOX-2, dLGG-10 + Lipo-DOX-2 or Lipo-DOX-2 + Avastin-5 suppressed, respectively, 17.9%, 59.1%, 55.7%, 16.2%, 44.5% and 72.4% of MBM in mice relative to the untreated tumor control. Metastatic PD-L1+ melanoma cells, infiltration of M2-like macrophages and CD31+ endothelial cells, and high expression levels of 15-LOX/CYP450 4A enzymes in the brain tumor microenvironment of the tumor control mice were significantly attenuated in dLGG-treated mice; conversely, M1-like resident microglia and cytotoxic T cells were increased. A lipidomics study showed that dLGG promoted B16BM4 cells to secrete oxylipins 9,10-/12,13-EpOMEs into the culture medium. Furthermore, the conditioned medium of B16BM4 cells pretreated with dLGG or 9,10-EpOMEs + 12,13-EpOMEs drove M2-like macrophages to polarize into M1-like macrophages in vitro. An ex vivo 3D-culture assay further demonstrated that dLGG, 9,10-EpOME or 9,10-EpOME + 12,13-EpOME pretreatment attenuated B16BM4 cells invading brain tissue, and prevented microglia/macrophages infiltrating into the interface of melanoma plug and brain organ/tissue. In summary, this report provides a novel therapeutic strategy and mechanistic insights into phytogalactolipid dLGG for combating MBM.

Sesquiterpene Lactone Deoxyelephantopin Isolated from Elephantopus scaber and Its Derivative DETD-35 Suppress BRAFV600E Mutant Melanoma Lung Metastasis in Mice.

Melanoma is a highly metastatic disease with an increasing rate of incidence worldwide. It is treatment refractory and has poor clinical prognosis; therefore, the development of new therapeutic agents for metastatic melanoma are urgently required. In this study, we created a lung-seeking A375LM5IF4g/Luc BRAFV600E mutant melanoma cell clone and investigated the bioefficacy of a plant sesquiterpene lactone deoxyelephantopin (DET) and its novel semi-synthetic derivative, DETD-35, in suppressing metastatic A375LM5IF4g/Luc melanoma growth in vitro and in a xenograft mouse model. DET and DETD-35 treatment inhibited A375LM5IF4g/Luc cell proliferation, and induced G2/M cell-cycle arrest and apoptosis. Furthermore, A375LM5IF4g/Luc exhibited clonogenic, metastatic and invasive abilities, and several A375LM5IF4g/Luc metastasis markers, N-cadherin, MMP2, vimentin and integrin α4 were significantly suppressed by treatment with either compound. Interestingly, DET- and DETD-35-induced Reactive Oxygen Species (ROS) generation and glutathione (GSH) depletion were found to be upstream events important for the in vitro activities, because exogenous GSH supplementation blunted DET and DETD-35 effects on A375LM5IF4g/Luc cells. DET and DETD-35 also induced mitochondrial DNA mutation, superoxide production, mitochondrial bioenergetics dysfunction, and mitochondrial protein deregulation. Most importantly, DET and DETD-35 inhibited lung metastasis of A375LM5IF4g/Luc in NOD/SCID mice through inhibiting pulmonary vascular permeability and melanoma cell (Mel-A+) proliferation, angiogenesis (VEGF+, CD31+) and EMT (N-cadherin) in the tumor microenvironment in the lungs. These findings indicate that DET and DETD-35 may be useful in the intervention of lung metastatic BRAFV600E mutant melanoma.

Plant galactolipid dLGG suppresses lung metastasis of melanoma through deregulating TNF-α-mediated pulmonary vascular permeability and circulating oxylipin dynamics in mice.

This study demonstrates the bioefficacy and gives mechanistic insights into a plant galactolipid 1,2-di-O-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (dLGG) against metastatic melanoma using a syngeneic mouse model implanted with B16COX-2/Luc melanoma. dLGG-20 (p.o. dLGG 20 mg/kg) and anti-cancer drug CP-2 (i.p. cisplatin 2 mg/kg) treatment significantly inhibited lung metastasis of melanoma in mice 91 and 57%, respectively, as determined by bioluminescence intensity. Moreover, dLGG-20 and CP-2 treatment prolonged mouse mean survival time. dLGG-20 treatment significantly inhibited the expression levels of several molecular markers, that is, PCNA, MMP2, COX-2, VEGF, vimentin, snail, TGF-ß, ß-catenin, TNF-α, PD-1 and PD-L1 in mouse lung tissues compared to tumor control mice. Significant inhibition of macrophage and neutrophil infiltration and promotion of CD8 + Tc cell recruitment in the lung microenvironment was observed in dLGG-20-treated mice. A LC/MS-based comparative oxylipin metabolomics study showed that dLGG-20 treatment significantly induced (5.0- to 12.8-fold) the 12/15-LOX catalyzed oxylipin products in mouse serum including 17-HDHA from DHA, 15-HEPE from EPA, 8- and 12-HETEs from AA, and CYP450-derived 20-HETE from AA. CP-2 treatment increased 12/15-LOX derived 8-, 11- and 12-HETEs from AA, and CYP450 derived 11,12-EET from AA ad 9,10-DHOME from LA by 5.3- to 8.1-fold. Of note, dLGG and 17-HDHA were more effective than CP in preventing B16 melanoma cell-induced pulmonary vascular permeability in mice through inhibition of TNF-α production, up-regulation of tight junction proteins claudin1 and ZO-2 and deregulation of Src activation. In conclusion, this study shows the novel therapeutic effect of phytoagent dLGG and suggests its potential as a therapeutic agent for metastatic melanoma.

Simvastatin and a Plant Galactolipid Protect Animals from Septic Shock by Regulating Oxylipin Mediator Dynamics through the MAPK-cPLA2 Signaling Pathway.

Sepsis remains a major medical issue despite decades of research. Identification of important inflammatory cascades and key molecular mediators are crucial for developing intervention and prevention strategies. In this study, we conducted a comparative oxylipin metabolomics study to gain a comprehensive picture of lipid mediator dynamics during the initial hyperinflammatory phase of sepsis, and demonstrated, in parallel, the efficacy of simvastatin and plant galactolipid, 1,2-di-O-α-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (dLGG) in the homeostatic regulation of the oxylipin metabolome using a lipopolysaccharide (LPS)-induced sepsis C57BL/6J mouse model. LPS increased the systemic and organ levels of proinflammatory metabolites of linoleic acid including leukotoxin diols (9-,10-DHOME, 12-,13-DHOME) and octadecadienoic acids (9-HODE and 13-HODE) and arachidonic acid-derived prostanoid, PGE2, and hydroxyeicosatetraenoic acids (8-, 12- and 15-HETE). Treatment with either compound decreased the levels of proinflammatory metabolites and elevated proresolution lipoxin A4, 5(6)-EET, 11(12)-EET and 15-deoxy-PGJ2. dLGG and simvastatin ameliorated the effects of LPS-induced mitogen-activated protein kinase (MAPK)-dependent activation of cPLA2, cyclooxygenase-2, lipoxygenase, cytochrome P450 and/or epoxide hydrolase lowered systemic TNF-α and IL-6 levels and aminotransferase activities and decreased organ-specific infiltration of inflammatory leukocytes and macrophages, and septic shock-induced multiple organ damage. Furthermore, both dLGG and simvastatin increased the survival rates in the cecal ligation and puncture (CLP) sepsis model. This study provides new insights into the role of oxylipins in sepsis pathogenesis and highlights the potential of simvastatin and dLGG in sepsis therapy and prevention.