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

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.

Keywords: Cannabinoids; ER stress; GRP78/PAR-4 complex.; TRAIL; autophagy; osteosarcoma cells.

Figure 1

Cytotoxic and morphological effects induced by cannabinoids in human osteosarcoma MG63 cells. (A) Effects of anandamide (ANA), meth-anandamide (MethANA) and WIN on MG63 cell viability. Cells were incubated for 48 h with different doses (5, 10 and 20 μM) of cannabinoids. (B) Time and dose-dependent effects of WIN on MG63 cell viability and effect of 100 μM z-VAD in cells treated with 5 μM WIN for 36 h. In A and B, cell survival was estimated by MTT assay, as reported in Materials and Methods, and expressed as the percentage of control cells. Data are the means ± S.E. of four independent experiments involving triplicate assays. ^**p<0.01 versus untreated cells. (C) Fluorescent microscopic evaluation of MG63 cells after acridine orange/ethidium bromide staining (AO/EB). Cells were treated with 5 μM ANA, MethANA and WIN for 36 h. Then, after AO/EB staining, viable cells appear with green nuclei, while dead cells show red nuclei. (Original magnification 200X). (D) Morphological effects induced by cannabinoids after 36 h of treatment evaluated by phase contrast microscopy. (Original magnification 200X). The inset shows a magnification of the cell indicated by the arrow. In C and D, the images are representative of three independent experiments.

Figure 2

WIN induces endoplasmic reticulum stress in osteosarcoma MG63 cells. (A) Time-dependent effect of 5 μM WIN and 5 ng/ml TRAIL on the level of CHOP, TRB3 and GRP78. The results were obtained by immunoblotting employing specific antibodies as reported in Materials and Methods. (B) Morphological effects exerted by the addition of BAPTA-AM to non-silenced or CHOP-silenced MG63 cells. Silencing of CHOP expression was carried out as reported in Materials and Methods. Cytoplasmic vacuolization was analysed by phase contrast microscopy after treatment for 24 h with 5 μM WIN and/or 5 μM BAPTA-AM. (Original magnification 200X). The images are representative of three independent experiments. (C) Effects of BAPTA-AM on the levels of CHOP and GRP78 proteins. Western blotting analysis was performed after treatment for 36 h with the compounds. In A and C, actin blots were included as a loading control.

Figure 3

WIN causes the appearance of autophagic hallmarks in MG63 cells. (A) Time-dependent effect of 5 μM WIN employed alone or in combination with 5 ng/ml TRAIL on the level of non-lipidated (LC3-I) and lipidated (LC3-II) forms of LC3, Beclin1 and p62. The levels of autophagic specific markers were analysed after 8, 16, 36 h of treatment. Cell lysates were analysed by immunoblotting employing specific antibodies as reported in Materials and Methods. Actin blots were included as a loading control. (B-C) Evaluation of autophagic vacuoles induced by WIN treatment, and effect of the autophagic inhibitor, 3-methyladenine (3-MA). MG63 cells were treated for 16 h with 5 μM WIN in the presence or absence of 5 mM 3-MA. After treatment, cells were incubated with MDC (0.05 mM) (B) or AO (2 μg/ml) (C) for 15 min at 37 °C and analyzed using a fluorescent microscope as described in Materials and Methods. (Original magnification 400X). The images are representative of three independent experiments. (D) Flow cytometric examination of red fluorescence emission (FL3) in WIN (red line) and WIN/3MA (green line) -treated cells with respect to control untreated cells (blue line). After incubation with the compounds, cells were treated with 2 μg/ml AO for 15 min at 37 °C. The plot is representative of three independent experiments.

Figure 4

WIN/TRAIL combined treatments induce apoptotic cell death. (A) Effects of cannabinoids and TRAIL on MG63 cell viability. Cells were treated with ANA, MethANA, WIN employed alone or in combination with TRAIL for 36 h at the indicated concentrations. (B) Morphological changes induced by WIN/TRAIL combined treatment in MG63 cells. Cells were treated for 36 h with 5 μM WIN and/or 5 ng/ml TRAIL and analysed by phase contrast microscopy. (Original magnification 200X). The images are representative of three independent experiments. (C) Effects of WIN/TRAIL combined treatment on MG63 cell cycle distribution. Flow cytometric analysis was carried out on propidium iodide stained cells treated for 36 h with WIN and/or TRAIL. The percentage of cells in each phase of the cell cycle was calculated using Expo32 software. (D) Effects of the addition of 5 mM 3-MA or 100 μM z-VAD to WIN or WIN/TRAIL treated cells for 36 h. In A and D, 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 untreated cells. (E) Western blotting analysis of caspase-8, pro-caspase-3 and PARP levels in MG63 cells. After treatment for 36 h with 5 μM WIN and/or TRAIL, cell lysates were analysed via immunoblotting using specific antibodies as reported in Materials and Methods. Actin blots were included as a loading control.

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.

Figure 6

Effects of WIN and WIN/TRAIL treatment on Saos-2 cells. (A) Effects of anandamide (ANA), Meth-anandamide (MethANA) and WIN on Saos-2 cell viability. Cells were incubated for 48 h with different doses (5, 10 and 20 μM) of cannabinoids. (B) Time and dose-dependent effects of WIN on Saos-2 cell viability. In A and B, cell survival was estimated by MTT assay, as reported in Materials and Methods, and expressed as the percentage of control cells. Data are the means ± S.E. of three independent experiments involving triplicate assays. **, p<0.01 versus untreated cells. (C) Effect of WIN and/or WIN/TRAIL combined treatment on the level of CHOP and GRP78 proteins. (D) Time-dependent effect of WIN on the level of Beclin-1 and LC3 protein. (E) Effects of WIN or WIN/TRAIL combined treatment on Saos-2 cell viability. Cells were treated with WIN (5 μM) employed alone or in combination with TRAIL at the indicated concentrations. The effect of caspase inhibitor z-VAD (100 μM) at 36 h of treatment is also shown. Cell viability was estimated by MTT assay as reported in Materials and Methods and expressed as the percentage of control value. Data are the means ± S.E. of three independent experiments involving triplicate assays. **, p<0.01 versus control untreated cells. (F) Effect of WIN and/or WIN/TRAIL combined treatment on the level of PAR-4 protein. For western blotting analysis, after treatment with the compounds, cell lysates were analysed using specific antibodies as reported in Materials and Methods. Actin blots were included as a loading control.

Figure 7

Suggested model of the mechanism of WIN/TRAIL-induced apoptosis on osteosarcoma MG63 cells.