Lipidomic analysis reveals a radiosensitizing role of gamma-linolenic acid in glioma cells

Previous studies have demonstrated that gamma-linolenic acid (GLA) is effective against glioma cells under both in vitro and in vivo conditions. In the present study we determined how GLA alone or in combination with irradiation alters the fatty acid (FA) and lipid profiles, the lipid droplet (LD) content, the lipid biosynthetic gene expression and the apoptosis of glioma cells. In GLA-treated cells direct correlations were found between the levels of various FAs and the expression of the corresponding FA biosynthetic genes. The total levels of saturated and monosaturated FAs decreased in concert with the down-regulation of FASN and SCD1 gene expression. Similarly, decreased FADS1 gene expression was paralleled by lowered arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) contents, while the down-regulation of FADS2 expression was accompanied by a diminished docosahexaenoic acid (22:6 n-3) content. Detailed mass spectrometric analyses revealed that individual treatments gave rise to distinct lipidomic fingerprints. Following uptake, GLA was subjected to elongation, resulting in dihomo-gamma-linolenic acid (20:3 n-6, DGLA), which was used for the synthesis of the LD constituent triacylglycerols and cholesteryl esters. Accordingly, an increased number of LDs were observed in response to GLA administration after irradiation. GLA increased the radioresponsiveness of U87 MG cells, as demonstrated by an increase in the number of apoptotic cells determined by FACS analysis. In conclusion, treatment with GLA increased the apoptosis of irradiated glioma cells, and GLA might therefore increase the therapeutic efficacy of irradiation in the treatment of gliomas

Enantioselective Synthesis of 8-Hydroxyquinoline Derivative, Q134 as a Hypoxic Adaptation Inducing Agent

Hypoxia is a common feature of neurodegenerative diseases, including Alzheimer’s disease that may be responsible for disease pathogenesis and progression. Therefore, the hypoxia-inducible factor (HIF)1 system, responsible for hypoxic adaptation, is a potential therapeutic target to combat these diseases by activators of cytoprotective protein induction. We have selected a candidate molecule from our cytoprotective hydroxyquinoline library and developed a novel enantioselective synthesis for the production of its enantiomers. The use of quinidine or quinine as a catalyst enabled the preparation of enantiomer-pure products. We have utilized in vitro assays to evaluate cytoprotective activity, a fluorescence-activated cell sorting (FACS) based assay measuring mitochondrial membrane potential changes, and gene and protein expression analysis. Our data showed that the enantiomers of Q134 showed potent and similar activity in all tested assays. We have concluded that the enantiomers exert their cytoprotective activity via the HIF1 system through HIF1A protein stabilization

Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

The treatment of metastatic breast cancer remained a challenge despite the recent breakthrough in the immunotherapy regimens. Here, we addressed the multidimensional immunophenotyping of 4T1 metastatic breast cancer by the state-of-the-art single cell mass cytometry (CyTOF). We determined the dose and time dependent cytotoxicity of cisplatin on 4T1 cells by the xCelligence real-time electronic sensing assay. Cisplatin treatment reduced tumor growth, number of lung metastasis, and the splenomegaly of 4T1 tumor bearing mice. We showed that cisplatin inhibited the tumor stroma formation, the polarization of carcinoma-associated fibroblasts by the diminished proteolytic activity of fibroblast activating protein. The CyTOF analysis revealed the emergence of CD11b+/Gr-1+/CD44+ or CD11b+/Gr-1+/IL-17A+ myeloid-derived suppressor cells (MDSCs) and the absence of B220+ or CD62L+ B-cells, the CD62L+/CD4+ and CD62L+/CD8+ T-cells in the spleen of advanced cancer. We could show the immunomodulatory effect of cisplatin via the suppression of splenic MDSCs and via the promotion of peripheral IFN-γ+ myeloid cells. Our data could support the use of low dose chemotherapy with cisplatin as an immunomodulatory agent for metastatic triple negative breast cancer

SMALL MOLECULES DU192, DU283 AND DU325 INDUCE DIFFERENTIATION AND APOPTOSIS OF HUMAN ACUTE PROMYELOCYTIC LEUKEMIA CELLS

Acute myelogenous leukemia (AML) originates from myeloid stem cells or myeloid blasts halted in an immature state during haematopoiesis. AML represents a group of heterogeneous forms of myeloid malignancies with diverse genetic abnormalities and different stages of myeloid differentiation. The human cell line, HL-60 used in this study belongs to a sub-type of AML, namely acute promyelocytic leukemia (APL). Another pathologic condition of myeloid expansion is the ”emergency” granulo-monocytopoiesis in most of the solid malignancies in which, an army of immature myeloid cells leave the bone marrow, called monocytic and granulocytic myeloid-derived suppressor cells (MDSCs). In contrast to AML, MDSCs are not malignant cells, but promote angiogensesis and immunosuppression leading to the progression of cancer. Both in AML and in solid malignancies the differentiation of immature myeloid cells is an already established therapeutic concept. Since the differentiation of AML cells is frequently followed by apoptosis or increases the sensitivity to chemotherapy, we have screened a library of small molecules to mature the human prototype cells, HL-60. In the resazurin assay small molecules DU192, DU283 and DU325 confounded viability of HL-60 cells, half-inhibitory concentration (IC50) values were as follows: 940 nM, 210 nM and 20 nM, respectively. IC50 could not be determined for human primary fibroblasts in the applied concentration range (1.6 nM - 5 µM). Using flow cytometry we obtained ERK phosphorylation as an early response to DU325 stimulation followed by the increase of the percentage of the Bcl-xl and pAkt bright cells. The expression of the members of the AP-1 TF complex, a driver of cellular differentiation, c-Fos, JunB, c-Jun and JunD were elevated on a concentration and time dependent manner detected by qRT-PCR. As a proof of cellular differentiation the expression of haematopoietic stem cell markers CD33 and CD34 decreased. Due to maturation the size and granularity of HL-60 cells increased upon treatment. Matured myeloid cell marker CD11b elevated on the cell surface detected by flow cytometry. We confirmed that differentiation of HL-60 cells was accompanied by apoptosis. We could detect AnnexinV+/PI- early and AnnexinV+/PI+ late apoptotic populations after 24h of treatment. Caspase-3 activated gradually by time detected by the percentage of active caspase-3 positive cells by flow cytometry and the digestion of zDEVD – amino-Luciferin. Finally, as a proof of massive cell death, we have shown the appearance of the hypo-diploid apoptotic cells in the sub-G1 population and the leakage of the lactate-dehydrogenase into the supernatant. We conclude that DU molecules differentiated immature HL-60 cells, which was followed by apoptosis. We propose to further investigate the effects of DU325 on additional human AML cells obtained from clinical samples. On the other hand we plan to systematically investigate the effect of DU325 on the expansion of immature MDSCs in solid malignancies. Funding: GINOP-2.3.2-15-2016-00030 (LGP); János Bolyai Research Scholarship (GJSz, BO/00139/17/8

Single Cell Mass Cytometry Revealed the Immunomodulatory Effect of Cisplatin Via Downregulation of Splenic CD44+, IL-17A+ MDSCs and Promotion of Circulating IFN-γ+ Myeloid Cells in the 4T1 Metastatic Breast Cancer Model

The treatment of metastatic breast cancer remained a challenge despite the recent breakthrough in the immunotherapy regimens. Here, we addressed the multidimensional immunophenotyping of 4T1 metastatic breast cancer by the state-of-the-art single cell mass cytometry (CyTOF). We determined the dose and time dependent cytotoxicity of cisplatin on 4T1 cells by the xCelligence real-time electronic sensing assay. Cisplatin treatment reduced tumor growth, number of lung metastasis, and the splenomegaly of 4T1 tumor bearing mice. We showed that cisplatin inhibited the tumor stroma formation, the polarization of carcinoma-associated fibroblasts by the diminished proteolytic activity of fibroblast activating protein. The CyTOF analysis revealed the emergence of CD11b+/Gr-1+/CD44+ or CD11b+/Gr-1+/IL-17A+ myeloid-derived suppressor cells (MDSCs) and the absence of B220+ or CD62L+ B-cells, the CD62L+/CD4+ and CD62L+/CD8+ T-cells in the spleen of advanced cancer. We could show the immunomodulatory effect of cisplatin via the suppression of splenic MDSCs and via the promotion of peripheral IFN-γ+ myeloid cells. Our data could support the use of low dose chemotherapy with cisplatin as an immunomodulatory agent for metastatic triple negative breast cancer

Synthesis and Cytoprotective Characterization of 8- Hydroxyquinoline Betti Products

The 8-hydroxyquinoline pharmacophore scaffold has been shown to possess a range of activities as metal chelation, enzyme inhibition, cytotoxicity, and cytoprotection. Based on our previous findings we set out to optimize the scaffold for cytoprotective activity for its potential application in central nervous system related diseases. A 48-membered Betti-library was constructed by the utilization of formic acid mediated industrial-compatible coupling with sets of aromatic primary amines such as anilines, oxazoles, pyridines, and pyrimidines, with (hetero)aromatic aldehydes and 8-hydroxiquinoline derivatives. After column chromatography and re-crystallization, the corresponding analogues were obtained in yields of 13–90%. The synthesized analogs were optimized with the utilization of a cytoprotection assay with chemically induced oxidative stress, and the most active compounds were further tested in orthogonal assays, a real time cell viability method, a fluorescence-activated cell sorting (FACS)-based assay measuring mitochondrial membrane potential changes, and gene expression analysis. The best candidates showed potent, nanomolar activity in all test systems and support the need for future studies in animal models of central nervous system (CNS) disorders

Imidazo[1,2-b]pyrazole-7-carboxamides Induce Apoptosis in Human Leukemia Cells at Nanomolar Concentrations

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Lipid droplet binding thalidomide analogs activate endoplasmic reticulum stress and suppress hepatocellular carcinoma in a chemically induced transgenic mouse model

BACKGROUND: Hepatocellular carcinoma (HCC) is the most frequent and aggressive primary tumor of the liver and it has limited treatment options. RESULTS: In this study, we report the in vitro and in vivo effects of two novel amino-trifluoro-phtalimide analogs, Ac-915 and Ac-2010. Both compounds bind lipid droplets and endoplasmic reticulum membrane, and interact with several proteins with chaperone functions (HSP60, HSP70, HSP90, and protein disulfide isomerase) as determined by affinity chromatography and resonant waveguide optical biosensor technology. Both compounds inhibited protein disulfide isomerase activity and induced cell death of different HCC cells at sub or low micromolar ranges detected by classical biochemical end-point assay as well as with real-time label-free measurements. Besides cell proliferation inhibiton, analogs also inhibited cell migration even at 250 nM. Relative biodistribution of the analogs was analysed in native tissue sections of different organs after administration of drugs, and by using fluorescent confocal microscopy based on the inherent blue fluorescence of the compounds. The analogs mainly accumulated in the liver. The effects of Ac-915 and Ac-2010 were also demonstrated on the advanced stages of hepatocarcinogenesis in a transgenic mouse model of N-nitrosodiethylamine (DEN)-induced HCC. Significantly less tumor development was found in the livers of the Ac-915- or Ac-2010-treated groups compared with control mice, characterized by less liver tumor incidence, fewer tumors and smaller tumor size. CONCLUSION: These results imply that these amino-trifluoro-phthalimide analogs could serve potent clinical candidates against HCC alone or in combination with dietary polyunsaturated fatty acids