Synthesis and Cytotoxicity against K562 Cells of 3-O-Angeloyl-20-O-acetyl Ingenol, a Derivative of Ingenol Mebutate

Abstract

Ingenol mebutate possesses significant cytotoxicity and is clinically used to treat actinic keratosis. However, ingenol mebutate undergoes acyl migration which affects its bioactivity. Compound 3-O-angeloyl-20-O-acetyl ingenol (AAI, also known as 20-O-acetyl-ingenol-3-angelate or PEP008) is a synthetic derivative of ingenol mebutate. In this work, we report the AAI synthesis details and demonstrate AAI has higher cytotoxicity than ingenol mebutate in a chronic myeloid leukemia K562 cell line. Our data indicate that the increased activity of AAI originates from the improved intracellular stability of AAI rather than the increased binding affinity between AAI and the target protein protein kinase Cδ (PKCδ). AAI inhibits cell proliferation, induces G2/M phase arrest, disrupts the mitochondrial membrane potential, and stimulates apoptosis, as well as necrosis in K562 cells. Similar to ingenol mebutate, AAI activates PKCδ and extracellular signal regulated kinase (ERK), and inactivates protein kinase B (AKT). Furthermore, AAI also inhibits JAK/STAT3 pathway. Altogether, our studies show that ingenol derivative AAI is cytotoxic to K562 cells and modulates PKCδ/ERK, JAK/STAT3, and AKT signaling pathways. Our work suggests that AAI may be a new candidate of chemotherapeutic agent.

Keywords: 20-O-acetyl-ingenol-3-angelate; 3-O-angeloyl-20-O-acetyl ingenol; PEP008; apoptosis; chronic myeloid leukemia; ingenol mebutat.

Figure 1

Chemical structures of ingenol mebutate (A) and 3-O-angeloyl-20-O-acetyl ingenol (B).

Scheme 1

Synthesis of 3-O-angeloyl-20-O-acetyl ingenol. (a) PTSA·H₂O, acetone; (b) angelic anhydride, lithium hexamethyldisilazide (LHMDS), tetrahydrofuran (THF); (c) 1% HCl, MeOH; and (d) Ac₂O, pyridine.

Figure 2

(A) Cytotoxicity of AAI assayed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) methods. Tested cells in 96-well plates were treated with (0–25 μM) AAI for 72 h, and then the viability was evaluated by MTT assay, as described in the Materials and Methods section; (B) AAI decreases the population of K562 cells in a comparable potency of ingenol mebutate. Cells in 96-well plates were treated with 1 μM AAI or ingenol mebutate for 72 h, and then the cell density was photographed (×200); (C) growth inhibition of AAI against K562 cells. Cells were treated with indicated concentrations of AAI for 36, 48, and 72 h, and then the viability was evaluated by MTT assay; and (D) AAI shows stronger growth inhibition than ingenol mebutate at low concentrations. Cells were treated with indicated concentrations of AAI or ingenol mebutate for 72 h, and then cell viability was evaluated by MTT assay.

Figure 3

AAI induces G2/M phase arrest, apoptosis, and necrosis in K562 cells. (A) AAI time-dependently arrests K562 cells at G2/M phase. K562 cells were treated with 250 nM AAI for 0, 2, 4 and 12 h. Then, cells were collected, fixed, digestion with RNase A, and stained by PI. The DNA contents of the cells were determined with the Aria FACS flow cytometry system; red: G0/G1 or G2/M phase, blue: S phase; (B) K562 cells were treated with AAI (0–25 nM) for 24 h, and then the cell cycle distribution was analyzed; red: G0/G1 or G2/M phase, blue: S phase; (C) histograms show the percentage of cells distributed in G2/M phase after treated with 250 nM AAI for 0, 2, 4 and 12 h; * p < 0.05, ** p < 0.01 versus control; (D) histograms show the percentage of cells distributed in G2/M phase after treated with AAI (0–25 nM) for 24 h; * p < 0.05, ** p < 0.01 versus control; (E) AAI induces both apoptosis and necrosis in K562 cells. K562 cells were untreated or treated with 31.25, 62.5, 125, 250 and 500 nM AAI for 18 h, and then the cells were double-stained with Annexin V-FITC/PI and analyzed by flow cytometry. The percentage of Annexin V-FITC positive cells and/or PI positive cells is indicated; and (F) histogram shows the percentage of necrosis and apoptosis in K562 cells induced by AAI.

Figure 3

AAI induces G2/M phase arrest, apoptosis, and necrosis in K562 cells. (A) AAI time-dependently arrests K562 cells at G2/M phase. K562 cells were treated with 250 nM AAI for 0, 2, 4 and 12 h. Then, cells were collected, fixed, digestion with RNase A, and stained by PI. The DNA contents of the cells were determined with the Aria FACS flow cytometry system; red: G0/G1 or G2/M phase, blue: S phase; (B) K562 cells were treated with AAI (0–25 nM) for 24 h, and then the cell cycle distribution was analyzed; red: G0/G1 or G2/M phase, blue: S phase; (C) histograms show the percentage of cells distributed in G2/M phase after treated with 250 nM AAI for 0, 2, 4 and 12 h; * p < 0.05, ** p < 0.01 versus control; (D) histograms show the percentage of cells distributed in G2/M phase after treated with AAI (0–25 nM) for 24 h; * p < 0.05, ** p < 0.01 versus control; (E) AAI induces both apoptosis and necrosis in K562 cells. K562 cells were untreated or treated with 31.25, 62.5, 125, 250 and 500 nM AAI for 18 h, and then the cells were double-stained with Annexin V-FITC/PI and analyzed by flow cytometry. The percentage of Annexin V-FITC positive cells and/or PI positive cells is indicated; and (F) histogram shows the percentage of necrosis and apoptosis in K562 cells induced by AAI.

Figure 4

AAI induces the loss of MMP in K562 cells. (A) Treatment with AAI (0–500 nM) for 18 h disrupts the MMP in K562 cells. The scatter plot of the flow cytometry analysis shows the distribution of JC-1 aggregates and JC-1 monomers; and (B) AAI at 250 nM time-dependently induces the loss of MMP in K562 cells.

Figure 5

AAI modulates multiple signaling pathways in K562 cells. (A) AAI induces time-dependent activation of PKC and ERK, inactivation of AKT, and inhibition on phosphorylation of JAK and STAT3, and decreases the expression level of surviving; and (B) different concentrations (0–12.5 μM) of AAI treated for 24 h activate PKCδ and ERK, inactivate AKT, inhibit JAK/STAT3 pathway, and decrease the expression level of survivin.

Figure 6

Binding modes of AAI (orange, A) and ingenol mebutate (green, C) to PKCδ Cys2 domain. The binding site of the receptor is shown using transparent surface area, and the H-bond is represented using dashed line; the blue spot is used to show the solvent exposure of the atoms for AAI (B) and ingenol mebutate (D), light green circles: hydrophobic amino acids; magenta circles: hydrophilic amino acids; blue circles: ligand exposure; and the darker color at AAI suggests that these atoms are more solvent exposed; and (E) Energetic contribution of residues at the binding site of PKCδ Cys2 domain to the enthalpy change of AAI (blue) and ingenol mebutate (red).

Figure 6

Binding modes of AAI (orange, A) and ingenol mebutate (green, C) to PKCδ Cys2 domain. The binding site of the receptor is shown using transparent surface area, and the H-bond is represented using dashed line; the blue spot is used to show the solvent exposure of the atoms for AAI (B) and ingenol mebutate (D), light green circles: hydrophobic amino acids; magenta circles: hydrophilic amino acids; blue circles: ligand exposure; and the darker color at AAI suggests that these atoms are more solvent exposed; and (E) Energetic contribution of residues at the binding site of PKCδ Cys2 domain to the enthalpy change of AAI (blue) and ingenol mebutate (red).