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Involvement of PAR-4 in cannabinoid-dependent sensitization of osteosarcoma cells to TRAIL-induced apoptosis.

Notaro A, Sabella S, Pellerito O, Di Fiore R, De Blasio A, Vento R, Calvaruso G, Giuliano M - Int. J. Biol. Sci. (2014)

Bottom Line: In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes.WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization.However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.

View Article: PubMed Central - PubMed

Affiliation: 1. Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.

ABSTRACT
The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.

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WIN treatment induces PAR-4 upregulation and cell surface GRP78 translocation. (A) Time dependent effect of WIN or WIN/TRAIL combined treatment on the level of PAR-4 protein. After treatment with 5 μM WIN employed alone or in combination with 5 ng/ml TRAIL, cell lysates were analysed by immunoblotting using a specific antibody as reported in Materials and Methods. (B) Effects of WIN treatment on surface GRP78 levels. Upper panel: Determination of surface GRP78 by immunofluorescence. Cells were treated for 24 h with 5 μM WIN, incubated with anti-GRP78 antibody followed by FITC-conjugated secondary antibody and analyzed using an inverted fluorescent microscope as described in Materials and Methods. Nuclei were counterstained with Hoechst 33342 (blue). Lower panel: Cytometric analyses showing cell surface expression of GRP78 in MG63 cells. The open histograms indicate isotype control, filled histograms indicate the expression of GRP78 in untreated and WIN-treated cells. (C) PAR-4 level in CHOP-silenced cells. Silencing of CHOP expression was carried out as reported in Materials and Methods. After WIN treatment cell lysates were analysed by immunoblotting using specific antibody as reported in Materials and Methods. In A and C, actin blots were included as a loading control. (D) Effects of WIN/TRAIL combined treatment in CHOP silenced cells. Silencing of CHOP expression was carried out as reported in Materials and Methods. Cells were treated with 5 μM WIN and/or 5 ng/ml TRAIL for 24 h. MG63 cell viability was estimated by MTT assay as reported under Materials and Methods and expressed as the percentage of control value. Data are the means ± S.E. of four independent experiments involving triplicate assays. **, p<0.01 versus control untreated cells.
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Figure 5: WIN treatment induces PAR-4 upregulation and cell surface GRP78 translocation. (A) Time dependent effect of WIN or WIN/TRAIL combined treatment on the level of PAR-4 protein. After treatment with 5 μM WIN employed alone or in combination with 5 ng/ml TRAIL, cell lysates were analysed by immunoblotting using a specific antibody as reported in Materials and Methods. (B) Effects of WIN treatment on surface GRP78 levels. Upper panel: Determination of surface GRP78 by immunofluorescence. Cells were treated for 24 h with 5 μM WIN, incubated with anti-GRP78 antibody followed by FITC-conjugated secondary antibody and analyzed using an inverted fluorescent microscope as described in Materials and Methods. Nuclei were counterstained with Hoechst 33342 (blue). Lower panel: Cytometric analyses showing cell surface expression of GRP78 in MG63 cells. The open histograms indicate isotype control, filled histograms indicate the expression of GRP78 in untreated and WIN-treated cells. (C) PAR-4 level in CHOP-silenced cells. Silencing of CHOP expression was carried out as reported in Materials and Methods. After WIN treatment cell lysates were analysed by immunoblotting using specific antibody as reported in Materials and Methods. In A and C, actin blots were included as a loading control. (D) Effects of WIN/TRAIL combined treatment in CHOP silenced cells. Silencing of CHOP expression was carried out as reported in Materials and Methods. Cells were treated with 5 μM WIN and/or 5 ng/ml TRAIL for 24 h. MG63 cell viability was estimated by MTT assay as reported under Materials and Methods and expressed as the percentage of control value. Data are the means ± S.E. of four independent experiments involving triplicate assays. **, p<0.01 versus control untreated cells.

Mentions: Finally, we wondered what was the link between the ability of WIN to induce ER stress and the sensitization of MG63 cells to TRAIL action. Some evidence indicates that the tumor suppressor protein PAR-4 (Prostate Apoptosis Response-4, also known as PAWR) is able to activate apoptotic pathway by interacting with GRP78 and inducing its translocation on cell surface 25 where GRP78 behaves as a receptor for soluble ligands to induce apoptosis 26. To explore the possible involvement of this pathway, we firstly analyzed the expression level of PAR-4 in WIN-treated cells. As shown in Fig. 5A, the level of PAR-4 increased after 8 and 16 h of treatment with WIN and further in WIN/TRAIL treated cells, indicating an actual involvement of this factor in the early phases of apoptotic process. In the presence of TRAIL alone, no variation in the level of PAR-4 was observed (not shown). Then, we detected the presence of GRP78 on cell membrane of non-permeabilized WIN-treated MG63 cells by immunofluorescence microscopy. As shown in Fig. 5B (upper panel), a marked increase in green fluorescence, indicative of an enhanced GRP78 expression on cell surface, was observed after WIN treatment for 24 h. The results were quantified by cytofluorimetric analysis that indicated an increase of about 2.5-fold in green fluorescence (lower panel).


Involvement of PAR-4 in cannabinoid-dependent sensitization of osteosarcoma cells to TRAIL-induced apoptosis.

Notaro A, Sabella S, Pellerito O, Di Fiore R, De Blasio A, Vento R, Calvaruso G, Giuliano M - Int. J. Biol. Sci. (2014)

WIN treatment induces PAR-4 upregulation and cell surface GRP78 translocation. (A) Time dependent effect of WIN or WIN/TRAIL combined treatment on the level of PAR-4 protein. After treatment with 5 μM WIN employed alone or in combination with 5 ng/ml TRAIL, cell lysates were analysed by immunoblotting using a specific antibody as reported in Materials and Methods. (B) Effects of WIN treatment on surface GRP78 levels. Upper panel: Determination of surface GRP78 by immunofluorescence. Cells were treated for 24 h with 5 μM WIN, incubated with anti-GRP78 antibody followed by FITC-conjugated secondary antibody and analyzed using an inverted fluorescent microscope as described in Materials and Methods. Nuclei were counterstained with Hoechst 33342 (blue). Lower panel: Cytometric analyses showing cell surface expression of GRP78 in MG63 cells. The open histograms indicate isotype control, filled histograms indicate the expression of GRP78 in untreated and WIN-treated cells. (C) PAR-4 level in CHOP-silenced cells. Silencing of CHOP expression was carried out as reported in Materials and Methods. After WIN treatment cell lysates were analysed by immunoblotting using specific antibody as reported in Materials and Methods. In A and C, actin blots were included as a loading control. (D) Effects of WIN/TRAIL combined treatment in CHOP silenced cells. Silencing of CHOP expression was carried out as reported in Materials and Methods. Cells were treated with 5 μM WIN and/or 5 ng/ml TRAIL for 24 h. MG63 cell viability was estimated by MTT assay as reported under Materials and Methods and expressed as the percentage of control value. Data are the means ± S.E. of four independent experiments involving triplicate assays. **, p<0.01 versus control untreated cells.
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Related In: Results  -  Collection

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Figure 5: WIN treatment induces PAR-4 upregulation and cell surface GRP78 translocation. (A) Time dependent effect of WIN or WIN/TRAIL combined treatment on the level of PAR-4 protein. After treatment with 5 μM WIN employed alone or in combination with 5 ng/ml TRAIL, cell lysates were analysed by immunoblotting using a specific antibody as reported in Materials and Methods. (B) Effects of WIN treatment on surface GRP78 levels. Upper panel: Determination of surface GRP78 by immunofluorescence. Cells were treated for 24 h with 5 μM WIN, incubated with anti-GRP78 antibody followed by FITC-conjugated secondary antibody and analyzed using an inverted fluorescent microscope as described in Materials and Methods. Nuclei were counterstained with Hoechst 33342 (blue). Lower panel: Cytometric analyses showing cell surface expression of GRP78 in MG63 cells. The open histograms indicate isotype control, filled histograms indicate the expression of GRP78 in untreated and WIN-treated cells. (C) PAR-4 level in CHOP-silenced cells. Silencing of CHOP expression was carried out as reported in Materials and Methods. After WIN treatment cell lysates were analysed by immunoblotting using specific antibody as reported in Materials and Methods. In A and C, actin blots were included as a loading control. (D) Effects of WIN/TRAIL combined treatment in CHOP silenced cells. Silencing of CHOP expression was carried out as reported in Materials and Methods. Cells were treated with 5 μM WIN and/or 5 ng/ml TRAIL for 24 h. MG63 cell viability was estimated by MTT assay as reported under Materials and Methods and expressed as the percentage of control value. Data are the means ± S.E. of four independent experiments involving triplicate assays. **, p<0.01 versus control untreated cells.
Mentions: Finally, we wondered what was the link between the ability of WIN to induce ER stress and the sensitization of MG63 cells to TRAIL action. Some evidence indicates that the tumor suppressor protein PAR-4 (Prostate Apoptosis Response-4, also known as PAWR) is able to activate apoptotic pathway by interacting with GRP78 and inducing its translocation on cell surface 25 where GRP78 behaves as a receptor for soluble ligands to induce apoptosis 26. To explore the possible involvement of this pathway, we firstly analyzed the expression level of PAR-4 in WIN-treated cells. As shown in Fig. 5A, the level of PAR-4 increased after 8 and 16 h of treatment with WIN and further in WIN/TRAIL treated cells, indicating an actual involvement of this factor in the early phases of apoptotic process. In the presence of TRAIL alone, no variation in the level of PAR-4 was observed (not shown). Then, we detected the presence of GRP78 on cell membrane of non-permeabilized WIN-treated MG63 cells by immunofluorescence microscopy. As shown in Fig. 5B (upper panel), a marked increase in green fluorescence, indicative of an enhanced GRP78 expression on cell surface, was observed after WIN treatment for 24 h. The results were quantified by cytofluorimetric analysis that indicated an increase of about 2.5-fold in green fluorescence (lower panel).

Bottom Line: In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes.WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization.However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.

View Article: PubMed Central - PubMed

Affiliation: 1. Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Polyclinic, Palermo, Italy.

ABSTRACT
The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.

Show MeSH
Related in: MedlinePlus