XIAP as a Target of New Small Organic Natural Molecules Inducing Human Cancer Cell Death

Abstract

X-linked inhibitor of apoptosis protein (XIAP) is an emerging crucial therapeutic target in cancer. We report on the discovery and characterisation of small organic molecules from Piper genus plants exhibiting XIAP antagonism, namely erioquinol, a quinol substituted in the 4-position with an alkenyl group and the alkenylphenols eriopodols A-C. Another isolated compound was originally identified as gibbilimbol B. Erioquinol was the most potent inhibitor of human cancer cell viability when compared with gibbilimbol B and eriopodol A was listed as intermediate. Gibbilimbol B and eriopodol A induced apoptosis through mitochondrial permeabilisation and caspase activation while erioquinol acted on cell fate via caspase-independent/non-apoptotic mechanisms, likely involving mitochondrial dysfunctions and aberrant generation of reactive oxygen species. In silico modelling and molecular approaches suggested that all molecules inhibit XIAP by binding to XIAP-baculoviral IAP repeat domain. This demonstrates a novel aspect of XIAP as a key determinant of tumour control, at the molecular crossroad of caspase-dependent/independent cell death pathway and indicates molecular aspects to develop tumour-effective XIAP antagonists.

Keywords: Piper eriopodon, alkenylphenols; XIAP antagonists; XIAP-BIR3 domain; apoptosis; caspase-independent cell death; cell death; human cancer cells; phytochemicals; small organic agents.

Figure 1

Identification of new Piper genus-derived compounds. (A) Structures of compounds 1–5. (B) Key correlated spectroscopy (COSY) (bold) and heteronuclear multiple bond correlation (HMBC) (H→C) for compounds 2–5.

Figure 2

Piper genus-derived compounds exhibit cytotoxic effects in human cancer cells. U373 and MCF7 cells were treated with increasing concentrations of gibbilimbol B, eriopodol A, eriopodol B, eriopodol C, and erioquinol for 24 h before 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are expressed by setting the absorbance of the reduced MTT in the respective control (vehicle-treated) samples, i.e., absence of compounds, as 100%. The data points are representative of four independent experiments.

Figure 3

Piper genus-derived compounds exhibit cytotoxic effects in cancer and non-transformed human cells. (A) PC-3/A549 cells were treated with increasing concentrations of eriopodol A and erioquinol while (B) human umbilical vein endothelial cells (HUVEC)/MCF10 cells were treated with increasing concentrations of gibbilimbol B, eriopodol A, and erioquinol, for 24 h before 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are expressed by setting the absorbance of the reduced MTT in the respective control (vehicle-treated) samples, i.e., absence of compounds, as 100%. The data points are representative of four independent experiments.

Figure 4

Time-response of Piper genus-derived compounds on cell viability. MCF7 cells were treated with increasing concentrations of gibbilimbol B, eriopodol A, and erioquinol for 6, 12, and 24 h before 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are expressed by setting the absorbance of the reduced MTT in the respective control (vehicle-treated) samples, i.e., absence of compounds, as 100%. The data points are representative of four independent experiments.

Figure 5

Piper genus-derived compounds induce cell death. (A) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of MCF7 cells treated for 12 h in the absence (CTRL, control) and in the presence of gibbilimbol B/eriopodol A (30 µg/mL) or erioquinol (10 µg/mL). 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) was used for nuclei detection. Scale bar = 50 µm. (B) Bright field microscopy of MCF7 cells treated for 6 h in the absence (CTRL) and in the presence of gibbilimbol B, eriopodol A, or erioquinol at increasing concentrations. Scale bar = 100 µm. (C) 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) staining of MCF7 cells treated for 6 h in the absence (CTRL) and in the presence of gibbilimbol B/eriopodol A (30 µg/mL) or erioquinol (10 µg/mL). Scale bar = 10 µm. Lower panels represent enlarged image details. (D) Evaluation by flow cytometry of Annexin V-fluorescein isothiocyanate(FITC)/propidium iodide (PI) staining in MCF7 cells treated in the absence (CTRL) and in the presence of 10 µg/mL erioquinol, for 3 and 6 h. Quadrants are drawn, and relative proportion of labelled cells is indicated. The events shown in the lower left-hand quadrant are unlabeled cells. Images and data are representative of four independent experiments.

Figure 6

Piper genus-derived compounds induce cell death. Western blot analysis of cleaved-caspase 9 and 7 in MCF7 cells treated for increasing times in the absence (CTRL, control) and in the presence of (A) gibbilimbol B or (B) eriopodol A. Vinculin was used as internal standard. Right panels: densitometric analysis expressed as fold change of CTRL. Images and data are representative of three-five independent experiments. * p < 0.01, ** p < 0.001, and *** p < 0.0001 relative to CTRL. (C) Western blot analysis of cleaved-caspase 7 in MCF7 cells treated for increasing times in the absence (CTRL) and in the presence of 10 µg/mL erioquinol. The stain-free gel was used as loading control. Images are representative of three independent experiments. PC: positive control. (D) Immunofluorescence imaging of cleaved-caspase 7 (punctate red pattern) in MCF7 cells treated for 3 and 6 h in the absence (CTRL) and in the presence of gibbilimbol B/eriopodol A (30 µg/mL) or erioquinol (10 µg/mL). 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) (blue) and phalloidin (green) were used for nuclei and cytoskeleton detection, respectively. Images are representative of four independent experiments. Scale bar = 25 µm.

Figure 7

Piper genus-derived compounds induce cell death and mitochondrial dysfunction. (A) Immunofluorescence (confocal) imaging of cleaved-caspase 7 (punctate red pattern) in U373 cells treated for 6 h in the absence (CTRL, control) and in the presence of gibbilimbol B/eriopodol A (30 µg/mL) or erioquinol (10 µg/mL). 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) (blue) and phalloidin (green) were used for nuclei and cytoskeleton detection, respectively. Scale bar = 25 µm. (B) Bright field microscopy (upper panels) and DAPI staining (lower panels) of U373 cells treated for 6 h in the absence (CTRL) and in the presence of of gibbilimbol B/eriopodol A (30 µg/mL) or erioquinol (10 µg/mL). Scale bars = 50 µm (bright field) and 10 µm (DAPI). Lower panels represent enlarged image details. (C) Quantitative analysis of tetramethylrhodamine methyl ester (TMRM) fluorescence changes over time in MCF7 (left panel) and U373 (righ panel) cells in the absence (CTRL) and in the presence of gibbilimbol B/eriopodol A (30 µg/mL) or erioquinol (10 µg/mL). Results are expressed by setting TMRM fluorescence in the respective control (vehicle-treated) samples, i.e., absence of compounds, as 1. *** p < 0.0001 relative to CTRL. Images and data are representative of four independent experiments.

Figure 8

Confocal microscopy for co-localisation of cytochrome c with mitochondria. (A) MCF7 and (B) U373 cells were treated for 3 h in the absence (CTRL, control) and in the presence of gibbilimbol B/eriopodol A (30 µg/mL) or erioquinol (10 µg/mL). Cells were then stained for cytochrome c (green) and the mitochondrial marker COX IV). 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) (blue) was used for nuclei detection. The images are representative of three independent experiments. Scale bars: 10 µm (MCF7) and 25 µm (U373). Panels on the right represent enlarged image details.

Figure 9

Confocal microscopy for reactive oxygen species (ROS) detection. (A) MCF7 and (B) U373 cells were treated for 6 h in the absence (CTRL, control) and in the presence of gibbilimbol B/eriopodol A (30 µg/mL) or erioquinol (10 µg/mL). Cells were then stained for ROS (2’-7’dichlorofluorescin diacetate - DCFH-DA, green). 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) (blue) and phalloidin (red) were used for nuclei and cytoskeleton detection, respectively. The images are representative of three independent experiments. Scale bar: 25 µm.

Figure 10

Piper genus-derived compounds induce caspase-dependent and independent loss of cell viability. (A) MCF7 cells were cultured in the absence (CTRL, control) and in the presence of 30 μg/mL gibbilimbol B/eriopodol A or 10 µg/mL erioquinol for 6 h, before 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The pan-caspase inhibitor Z-VAD-(OMe)-FMK (50 μM) or its vehicle were used as well. Results are expressed by setting the absorbance of the reduced MTT in the CTRL, as 100%. Data are representative of four-twelve independent experiments. *** p < 0.0001 relative to the respective compound alone, i.e., + Z-VAD vehicle. (B) MCF7 (left panel) and U373 (right panel) cells were treated with increasing concentrations of erioquinol for 24 h before MTT assay. Erioquinol was administered both in the absence (vehicle) or in the presence of 50 μM necrostatin-1 and 10 μM ferrostatin-1 (2 h pre-treatment), a necroptosis and ferroptosis inhibitor, respectively. Results are expressed by setting the absorbance of the reduced MTT in the control samples (absence of erioquinol) as 100%. The data points are representative of four independent experiments.

Figure 11

X-linked inhibitor of apoptosis protein (XIAP) as a molecular target of Piper genus-derived compounds. (A) Molecular docking and (C) dynamics analysis of embelin (orange), erioquinol (green), eriopodol A (purple) and gibbilimbol B (blue) in complex with the baculovirus IAP repeat (BIR)-3 domain of XIAP (PDB code 5C83). Interacting residues are displayed in wireframe, hydrogen bonds are displayed in yellow dot lines and π-π stacking interactions are displayed in blue dot lines. (B) Protein-ligand root mean square deviation (RMSD) trajectory of the atomic positions for ligands (red, Lig fit Prot) and the receptor (blu, C⍺ positions) BIR-3 domain of XIAP, for the dynamics trajectory of 50 ns.

Figure 12

X-linked inhibitor of apoptosis protein (XIAP) as a molecular target of Piper genus-derived compounds. (A) Western blot analysis of XIAP in MCF7 cells both untransfected (CTRL, control) or transfected for 24 h with a XIAP-specific and scrambled targeting (scr) siRNA (50 nM). Lactate dehydrogenase (LDH) was used as internal standard. Low panel: densitometric analysis expressed as fold change of scr siRNA. Images and data are representative of three independent experiments. **P < 0.001 relative to scr siRNA. (B) MCF7 cells were transfected for 24 h with a XIAP-specific or scr siRNA (50 nM) and then cultured in the absence (CTRL) and in the presence of 30 μg/mL gibbilimbol B/eriopodol A or 10 µg/mL erioquinol for 6 h, before 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are expressed by setting the absorbance of the reduced MTT in the scr siRNA CTRL, as 100%. Data are representative of three independent experiments. * p < 0.0001 relative to the respective scr siRNA. (C) Western blot analysis of cleaved-caspase 7 in MCF7 cells transfected for 24 h with a XIAP-specific or scr siRNA (50 nM) and then cultured in the presence of 30 μg/mL gibbilimbol B and eriopodol A for 6 h. LDH was used as internal standard. Images are representative of three independent experiments. (D) Real-time PCR and (E) Western blot analysis of XIAP mRNA and protein expression, respectively, in MCF7 cells treated for increasing times in the absence (CTRL) and in the presence of 30 μg/mL gibbilimbol B/eriopodol A or 10 µg/mL erioquinol. β-actin (PCR) and LDH (Western blot) were used as internal standards. PCR results are expressed as fold change of respective CTRL, set as 1. Images and data are representative of three independent experiments.

Figure 13

Schematic picture depicting cell death mechanisms of Piper genus-derived compounds. Escape of both intrinsic and extrinsic apoptosis is a common feature of cancer cells. (A) This hallmark is often carried out by overexpressing anti-apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP), which prevents the execution of apoptosis by binding of its baculovirus IAP repeat (BIR) 3 domain to already active initiator caspase 9. In order to counteract this resistance to cell death, several cancer pharmacological therapies have the aim of removing the ‘molecular brakes’ to apoptosis sensitising cancer cell to undergo loss of viability. The approach we described includes the use of three compounds from Piper genus plants which were predicted to bind XIAP-BIR3 domain. (B) Two of the compounds (gibbilimbol B and eriopodol A) were shown to induce a classical pro-apoptotic response, including mitochondrial outer membrane polarisation, release of cytochrome c, and subsequent activation of both initiator and effector caspases. (C) Despites triggering a similar response at the mitochondria level, erioquinol does not act through the apoptotic machinery, and results in a caspase-independent cell death characterised by cytoplasmic reactive oxygen species (ROS) accumulation.