Figure 6.
The signaling pathway enabled by the TSP-1 mimetic peptides in CLL cells activates an immunogenic mode of cell death. (A) Schematic representation of the signaling pathway regulating CD47 peptide targeting in CLL. The PCD pathway enabled by the TSP-1 mimetic peptides in the malignant CLL cells (eg, PKT16) initiates by a CD47 triggering that leads to the activation of the heterotrimeric Gαi proteins and the subsequent decrease of the cAMP levels and PKA activity before phosphorylation of PLCγ1 at Y873. PLCγ1-Y873 cleaves phosphatidylinositol 4,5 bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3), which binds IP3 (IP3R) and ryanodine receptors (RyR) in the endoplasmic reticulum (ER) to provoke a deregulation in the intracellular/extracellular Ca2+ trafficking that induces mitochondrial damage and, finally, CLL cell death. (B) Induction of calreticulin (CALR) exposure by PKT16. CLL cells were treated for 6 hours with PKT16 (100 µM) and subjected to cytofluorometric assessment to detect CALR. (Left) Flow cytometry panels of CALR staining performed on CLL cells untreated (Control) or treated with PKT16 (100 µM, 6 hours). Numbers indicate the percentages of CALR-positive cells. Immunoglobulin G labeling was used as a negative control. (Right) Percentages of CALR-positive cells were recorded, graphed, and represented as a ratio relative to untreated CLL cells. Data are expressed as a mean ± SD (n = 10). Box plots represent the mean of cell death with minimum to maximum values. (C) Release of ATP from CLL cells exposed to PKT16. CLL cells were treated for 6 hours with PKT16 (100 µM), and extracellular ATP was measured in the culture supernatant, plotted, and represented as a ratio relative to untreated CLL cells. Data are expressed as a mean ± SD (n = 8). Box plots represent the mean of cell death with minimum to maximum values. (D) Exodus of nuclear HMGB1 from CLL cells exposed to PKT16. CLL cells were treated for 6 hours with PKT16 (100 µM), and the amounts of HMGB1 were measured in the culture supernatant, plotted, and represented as a ratio relative to untreated CLL cells. Data are expressed as a mean ± SD (n = 7). Box plots represent the mean of cell death with minimum to maximum values. The statistical significance in the figure was calculated by the Student t test.

The signaling pathway enabled by the TSP-1 mimetic peptides in CLL cells activates an immunogenic mode of cell death. (A) Schematic representation of the signaling pathway regulating CD47 peptide targeting in CLL. The PCD pathway enabled by the TSP-1 mimetic peptides in the malignant CLL cells (eg, PKT16) initiates by a CD47 triggering that leads to the activation of the heterotrimeric Gαi proteins and the subsequent decrease of the cAMP levels and PKA activity before phosphorylation of PLCγ1 at Y873. PLCγ1-Y873 cleaves phosphatidylinositol 4,5 bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3), which binds IP3 (IP3R) and ryanodine receptors (RyR) in the endoplasmic reticulum (ER) to provoke a deregulation in the intracellular/extracellular Ca2+ trafficking that induces mitochondrial damage and, finally, CLL cell death. (B) Induction of calreticulin (CALR) exposure by PKT16. CLL cells were treated for 6 hours with PKT16 (100 µM) and subjected to cytofluorometric assessment to detect CALR. (Left) Flow cytometry panels of CALR staining performed on CLL cells untreated (Control) or treated with PKT16 (100 µM, 6 hours). Numbers indicate the percentages of CALR-positive cells. Immunoglobulin G labeling was used as a negative control. (Right) Percentages of CALR-positive cells were recorded, graphed, and represented as a ratio relative to untreated CLL cells. Data are expressed as a mean ± SD (n = 10). Box plots represent the mean of cell death with minimum to maximum values. (C) Release of ATP from CLL cells exposed to PKT16. CLL cells were treated for 6 hours with PKT16 (100 µM), and extracellular ATP was measured in the culture supernatant, plotted, and represented as a ratio relative to untreated CLL cells. Data are expressed as a mean ± SD (n = 8). Box plots represent the mean of cell death with minimum to maximum values. (D) Exodus of nuclear HMGB1 from CLL cells exposed to PKT16. CLL cells were treated for 6 hours with PKT16 (100 µM), and the amounts of HMGB1 were measured in the culture supernatant, plotted, and represented as a ratio relative to untreated CLL cells. Data are expressed as a mean ± SD (n = 7). Box plots represent the mean of cell death with minimum to maximum values. The statistical significance in the figure was calculated by the Student t test.

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