Glycosylation of glycolipids in cancer: basis for development of novel therapeutic approaches

Altered networks of gene regulation underlie many pathologies, including cancer. There are several proteins in cancer cells that are turned either on or off, which dramatically alters the metabolism and the overall activity of the cell, with the complex machinery of enzymes involved in the metabolism of glycolipids not being an exception. The aberrant glycosylation of glycolipids on the surface of the majority of cancer cells, associated with increasing evidence about the functional role of these molecules in a number of cellular physiological pathways, has received considerable attention as a convenient immunotherapeutic target for cancer treatment. This has resulted in the development of a substantial number of passive and active immunotherapies, which have shown promising results in clinical trials. More recently, antibodies to glycolipids have also emerged as an attractive tool for the targeted delivery of cytotoxic agents, thereby providing a rationale for future therapeutic interventions in cancer. This review first summarizes the cellular and molecular bases involved in the metabolic pathway and expression of glycolipids, both in normal and tumor cells, paying particular attention to sialosylated glycolipids (gangliosides). The current strategies in the battle against cancer in which glycolipids are key players are then described.Fil: Daniotti, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Vilcaes, Aldo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Torres Demichelis, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Ruggiero, Fernando Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Rodríguez Walker, Macarena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Ganglioside synthesis by plasma membrane-associated sialyltransferase in macrophages

Gangliosides are constituents of the mammalian cell membranes and participate in the inflammatory response. However, little is known about the presence and enzymatic activity of ganglioside sialyltransferases at the cell surface of macrophages, one of the most important immune cells involved in the innate inflammatory process. In the present study, using biochemical and fluorescent microscopy approaches, we found that endogenous ST8Sia-I is present at the plasma membrane (ecto-ST8Sia-I) of murine macrophage RAW264.7 cells. Moreover, ecto-ST8Sia-I can synthetize GD3 ganglioside at the cell surface in lipopolysaccharide (LPS)-stimulated macrophages even when LPS-stimulated macrophages reduced the total ST8Sia-I expression levels. Besides, cotreatment of LPS with an inhibitor of nitric oxide (NO) synthase recovered the ecto-ST8Sia-I expression, suggesting that NO production is involved in the reduction of ST8Sia-I expression. The diminution of ST8Sia-I expression in LPS-stimulated macrophages correlated with a reduction of GD3 and GM1 gangliosides and with an increment of GD1a. Taken together, the data supports the presence and activity of sialyltransferases at the plasma membrane of RAW264.7 cells. The variations of ecto-ST8Sia-I and ganglioside levels in stimulated macrophages constitutes a promissory pathway to further explore the physiological role of this and others ganglioside metabolism-related enzymes at the cell surface during the immune response.Fil: Vilcaes, Aldo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; ArgentinaFil: Garbarino Pico, Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; ArgentinaFil: Torres Demichelis, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; ArgentinaFil: Daniotti, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentin

Selective cytotoxicity of R24-targeted immunotoxin on mouse B16^GD3+ melanoma cells. A

<p>) Wild-type B16 cells (B16^wt) and B16 cells genetically modified to express GD3 (B16^GD3+) grown on coverslips were incubated at 4°C to inhibit intracellular transport, then with R24 antibody for 45 min at 4°C, washed and fixed. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa Fluor⁴⁸⁸ (45 min, 4°C; left panels). Cells were incubated with R24 for 45 min at 4°C and after washing temperature was shifted to 37°C for 30 min to allow the endocytosis of the complex GD3-R24. Then, cells were fixed and R24 antibody detection was carried out as indicated above (30 min, 37°C; right panels). <b>B</b>) B16^GD3+ cells transiently expressing Rab5-GFP or Lamp1-GFP were incubated with R24 for 45 min at 4°C. After washing, temperature was shifted to 37°C for 30 min to allow the endocytosis of the complex GD3-R24. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa Fluor⁵⁴³. Expression of Rab5 and Lamp1 was detected by the intrinsic fluorescence of GFP. In another set of experiments, uptake of Alexa Fluor⁶⁴⁷-transferrin (Tf) was monitored simultaneously with R24 endocytosis. In this case, R24 antibody was detected by using anti-mouse IgG conjugated with Alexa Fluor⁴⁸⁸. Insets in merge panels (right column) show details at higher magnifications. In all experimental conditions, single confocal sections were taken every 0.8 µm parallel to the coverslip. The fluorescence micrographs shown are representative of three independent experiments. Scale bar: 10 µm. <b>C</b>) B16^wt and B16^GD3+ cells were cultured at 37°C for 72 h in 96-well plates at the indicated concentration of monoclonal antibody to GD3 (R24 antibody) in combination with goat antibody to mouse IgG (squares, black lines) or saporin conjugated goat antibody anti mouse IgG secondary antibody (circles, grey lines). The concentration of the secondary antibodies was 0.95 nM. As negative control (100% viability), B16^GD3+ cells were incubated only with culture medium. Cell viability was determined using the colorimetric MTT metabolic activity assay. Absorbance was measured at 595 nm using a multiplate reader. Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. Note that R24-targeted saporin selectively kills B16^GD3+ melanoma cells (** p<0.001, respect to control condition).</p

Targeted delivery of immunotoxin by R24 antibody inhibit the clonogenic growth of CHO-K1^GD3+ cells. A

<p>) A schematical representation of the experimental procedure used in (<b>B</b> and <b>C</b>). <b>B</b>) CHO-K1^GD3+ cells (50–80 cells) were grown in 24-well plates previously coated with 0.5% agar in DMEM supplemented with 20% FBS. Cells were maintained at 37°C in a hummed atmosphere until cell colonies appeared (7 days, upper row. Colonies indicated with arrows). Then, cells were treated for 3 days (7+3 days, lower row) with 0.95 nM Saporin-Ab (Saporin-Ab, middle panel) or 30 nM R24/0.95 nM Saporin-Ab (R24/Saporin-Ab, right panel). CHO-K1^GD3+ cells maintained only with medium were used as negative control (control, left panel). The micrographs are representative of three independent experiments. <b>C</b>) Quantification of the colony area at 7 and 7+3 days at the different conditions indicated in <b>B</b>. Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. Note that the clonogenic growth of CHO-K1^GD3+ cells was severely affected only in presence of R24/Saporin-Ab (*** p<0.0001, respect to control condition at 7+3 days).</p

Immunotoxin inhibits CHO-K1^GD3+ and SK-Mel-28 cells colony formation. A

<p>) A schematical representation of the experimental procedure used in <b>B</b> and <b>C</b>. CHO-K1^GD3+(<b>B</b>) and SK-Mel-28 (<b>C</b>) cells (50–80 cells) were seed in 24-well plates previously coated with 0.5% agar in DMEM supplemented with 20% FBS. Cultures were supplemented with 0.95 nM Saporin-Ab or 30 nM R24/0.95 nM Saporin-Ab and maintained at 37°C in a hummed atmosphere. Quantification of the colony area was performed every day, but only indicated at 7 and 10 days. Cells maintained only with medium were used as negative control (control). Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. Note the drastic inhibition of colony formation only in presence of R24/Saporin-Ab.</p

R24 antibody is specifically internalized in GD3-expressing cells. A

<p>) CHO-K1^GD3+, CHO-K1^WT (GD3-) and SK-Mel-28 cells grown on coverslips were incubated at 4°C to inhibit intracellular transport, then with R24 antibody for 45 min at 4°C, washed and fixed. R24 antibody was detected by using goat anti-mouse IgG conjugated with Alexa Fluor⁴⁸⁸ (45 min, 4°C; left panels). <b>B</b>) Cells were incubated with R24 for 45 min at 4°C and after washing temperature was shifted to 37°C for 30 min to allow the endocytosis of the complex GD3-R24. Then, cells were fixed and R24 antibody detection was carried out as indicated in <b>A</b> (30 min, 37°C; right panel). Single confocal sections were taken every 0.8 µm parallel to the coverslip. The fluorescence micrographs shown are representative of three independent experiments. Scale bar: 10 µm.</p

Selective delivery of saporin via R24 antibody drastically reduces the clonogenic growth of human SK-Mel-28 melanoma cells. A)

<p>SK-Mel-28 cells (50–80 cells) were grown in 24-well plates previously coated with 0.5% agar in DMEM supplemented with 20% FBS. Cells were maintained at 37°C in a hummed atmosphere until cell colonies appeared (7 days). Then, cells were exposed for 3 days (7+3 days) to 0.95 nM Saporin-Ab or 30 nM R24/0.95 nM Saporin-Ab. Colonies are indicated with arrows. <b>B)</b> Quantification of the colony area was performed at 7 and 7+3 days. SK-Mel-28 cells maintained only with medium were used as negative control (control). Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. Note that the clonogenic growth of SK-Mel-28 cells was severely affected only in presence of R24/Saporin-Ab (***p<0.0001, respect to control condition at 7+3 days).</p

CREB3L1-mediated functional and structural adaptation of the secretory pathway in hormone-stimulated thyroid cells

Many secretory cells increase the synthesis and secretion of cargo proteins in response to specific stimuli. How cells couple increased cargo load with a coordinate rise in secretory capacity to ensure efficient transport is not well understood. We used thyroid cells stimulated with thyrotropin (TSH) to demonstrate a coordinate increase in the production of thyroid-specific cargo proteins and ER-Golgi transport factors, and a parallel expansion of the Golgi complex. TSH also increased expression of the CREB3L1 transcription factor, which alone caused amplified transport factor levels and Golgi enlargement. Furthermore, CREB3L1 potentiated the TSH-induced increase in Golgi volume. A dominant-negative CREB3L1 construct hampered the ability of TSH to induce Golgi expansion, implying that this transcription factor contributes to Golgi expansion. Our findings support a model in which CREB3L1 acts as a downstream effector of TSH to regulate the expression of cargo proteins, and simultaneously increases the synthesis of transport factors and the expansion of the Golgi to synchronize the rise in cargo load with the amplified capacity of the secretory pathway.Fil: García, Iris Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Departamento de Bioquímica; ArgentinaFil: Torres Demichelis, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Departamento de Bioquímica; ArgentinaFil: Viale, Diego Luis. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Di Giusto, Pablo. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Departamento de Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ezhova, Yulia. Telethon Institute of Genetics and Medicine; ItaliaFil: Polishchuk, Roman S.. Telethon Institute of Genetics and Medicine; ItaliaFil: Sampieri, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Martinez, Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Sztul, Elizabeth. University of Alabama at Birmingham; Estados UnidosFil: Alvarez, Cecilia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Bioquímica Clínica; Argentin

Targeted delivery of immunotoxin by antibody to ganglioside GD3: a novel drug delivery route for tumor cells.

Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28) and mouse (B16) melanoma cells and Chinese hamster ovary (CHO)-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1(GD3+) cells cultured in attachment-free conditions. A drastic growth inhibition (>80-90%) of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer