Human osteosarcoma MG63 cells were acquired from Interlab Cell Line Collection (ICLC; Genova, Italy). Cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2.0 mM L-glutamine, and antibiotic antimycotic solution (100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B; Sigma) in a humidified atmosphere containing 5% CO₂. For the experiments, cells were seeded at 60–70% confluence. After overnight incubation, culture medium was replaced with fresh DMEM containing low percentage of FBS (2%) and cells were treated with the cannabinoid WIN55,212-2 (WIN) (Sigma Aldrich S.R.L., Milan, Italy). Control cells were cultured in the presence of vehicle alone (DMSO).

Cell viability was determined by 3-[4,5-dimethylthiazolyl-2]2,5-diphenyl-tetrazolium bromide (MTT; Sigma Aldrich S.R.L.) assay as previously reported (22).

Protein extracts were prepared by washing the cells in phosphate-buffer saline (PBS) and incubating for 20 min in ice-cold lysis buffer supplemented with protease inhibitor cocktail, as previously reported (23). After sonication three times for 10 sec, proteins were quantified by Bradford method and equal amounts (40 μg) were separated by SDS-PAGE and then electrotransferred to a nitrocellulose membrane for the detection with specific antibodies. The blots were developed using the alkaline phosphatase colorimetric or enhanced chemiluminescence (ECL) labeling systems. Optical densities of the bands were analyzed with Quantity One Imaging software from Bio-Rad Laboratories. The correct protein loading was verified by means of both red Ponceau staining and immunoblotting for actin. The results shown in the figures are representative of four independent experiments with similar results.

RNA was isolated using RNeasy mini kit (Qiagen, Milan, Italy). cDNA was amplified from 1 μg of RNA by using QuantiTect reverse transcription kit (Qiagen) followed by polymerase chain reaction (PCR). The reactions omitting reverse transcriptase enzyme served as negative control. GAPDH was used as a housekeeping gene to demonstrate equal loading of RNA.

The amplified products were resolved by agarose gel electrophoresis (1% agarose, 0.5 μg/ml ethidium bromide; Sigma-Aldrich), and the bands were visualized and photographed with ChemiDoc XRS (Bio-Rad Laboratories Srl, Milan, Italy). The primer sequences (Proligo USA, Milan, Italy) are as follows: SPARC, forward 5′-TGATGATGGTGC AGAGGAAA-3′ and reverse 5′-GGGGGATGTATTTGCAA GG-3′; GAPDH, forward 5′-TGACATCAAGAAGGTGA-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. For PCR analysis, the following protocol was performed: initial denaturation at 94°C for 2 min; denaturation at 94°C for 15 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec for 35 cycles and a final extension at 72°C for 10 min.

Small interfering RNAs (siRNAs) against SPARC (5′-AACAAGACCUU CGACUCUU CC-3′) (siSPARC), CHOP (D-004819-01-0005 and D-004819-02-0005) (siCHOP), and scrambled siRNA (siScr), as a negative non-silencing control, were purchased from Dharmacon RNA Technologies (Chicago, IL, USA) and GRP78 (siGRP78) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cells (10⁵) were seeded in 6-well plates and cultured in antibiotic-free DMEM supplemented with 2.0 mM L-glutamine, until ~50% confluence. Then, cells were transfected with 30 nM siSPARC in the presence of 5 μl Metafectene Pro (Biontex Laboratories GmbH, Martinsried/Planegg, Germany), with 100 nM siCHOP in the presence of 6.2 μl of Metafectene Pro or with 50 nM siGRP78 in the presence of 4 μl Lipofectamine 2000 (Invitrogen Life Technologies, Monza, Italy) in a final volume of 1 ml serum-free medium. The reaction was stopped after 6 h replacing the culture medium with fresh DMEM + 10% FBS. After 24 h from transfection, silenced cells were treated with WIN for another 24 h. Only for SPARC silencing, cells were treated after 48 h from transfection.

The caspase-8/SPARC complex was detected in MG63 cells by immunoprecipitation either with the anti-caspase-8 or the anti-SPARC antibodies. Cell lysates were subjected to centrifugation at 14,000 rpm at 4°C for 15 min. Proteins concentration was evaluated by using Bradford assay and then was adjusted to 500 μg of protein in a final volume of 500 μl of RIPA buffer. After pre-clearing phase by 1 h of incubation at room temperature with 20 μl of protein A/G Plus-agarose beads (Santa Cruz Biotechnology), lysates were spun at 14,000 rpm for 10 min.

Anti-SPARC or anti-caspase-8 antibodies or the appropriate antibodies used as negative controls were added to the supernatant and incubated overnight at 4°C, followed by immunoprecipitation with protein A/G Plus-agarose beads for 2 h of incubation at room temperature. The beads were washed three times with PBS and the bound proteins were detached by boiling the beads in the presence of sample buffer for 5 min. The supernatants were subjected to electrophoresis on SDS-polyacrylamide gels and then electroblotted on nitrocellulose filter for the detection of the immunoprecipitate complex.

Cell viability data were expressed as the mean ± SE and evaluated by the Student's t-test. Differences were considered to indicate a statistically significant result when the P-value was <0.01.

In a previous study we demonstrated that WIN, a synthetic ligand of cannabinoid receptors, is able to sensitize osteosarcoma MG63 and Saos-2 cells to TRAIL action (21). In the present study we first analysed the effect of WIN on modulating the level of SPARC, a factor which plays different roles on cell proliferation and migration. As shown in Fig. 1A, 5 μM WIN, a concentration which is responsible for the morphological and TRAIL-sensitizing effects, induced a time-dependent increase in the level of SPARC in MG63 cells. The effect was already evident after 8 h of treatment and reached the maximum at 24–36 h. Similar results were obtained using semi-quantitative RT-PCR, thus, demonstrating that the increase in SPARC protein was associated with transcriptional activation.

We were interested to clarify whether SPARC can be a critical mediator of the cytotoxic action induced by WIN/TRAIL combined treatment. After confirming that the level of SPARC remained high also in the presence of WIN/TRAIL combined treatment (Fig. 2A), we analysed the effect of the downregulation of SPARC expression by a specific siRNA which knocked down the endogenous protein level by ~70% (Fig. 2B). In SPARC silenced cells, WIN/TRAIL combined treatment exerted smaller effects than that observed in non-silenced cells. After 24 h of treatment with WIN or WIN/TRAIL combination, siSPARC-transfected cells showed a morphology, which was very similar to that observed in control cells (Fig. 2C). Moreover, MTT assay evidenced that in siSPARC-transfected cells the reduction in MG63 cell viability induced by WIN/TRAIL treatment was approximately 40 vs. 80% of that observed in siSicr-transfected cells exposed to the same treatment (Fig. 2D).

Caspase-8 is the main initiating protease activity in extrinsic apoptotic pathway and we previously demonstrated that its activation is a very probable event responsible for the WIN/TRAIL-dependent cytotoxic effect (21). Moreover, as recently reported, SPARC can potentiate the apoptotic pathway by enhancing the signalling cascade in a caspase-8-dependent manner (24), therefore we investigated the involvement of SPARC in caspase-8 activation in our experimental model. First, we analysed the activation of this protease after SPARC silencing. As shown in Fig. 3A, the bands corresponding to the active cleaved forms of caspase-8 observed in WIN/TRAIL-treated cells were almost invisible in silenced cells.

Then, we assessed the possible interaction between SPARC and caspase-8 in MG63 cells by co-immunoprecipitation studies using a caspase-8 antibody (against an epitope to C-terminal region) that recognizes both the full length and the cleaved forms of the protein. As shown in Fig. 3B, caspase-8 and SPARC co-immunoprecipitated in a reciprocal fashion after WIN treatment. Instead, this interaction disappeared when the cells were treated with WIN/TRAIL combination, demonstrating that the cleavage of caspase-8 induced by the combined treatment prevents the SPARC immunoprecipitation.

Based on the previously demonstrated role of WIN-induced ER stress and autophagic process on MG63 cell viability (21), we hypothesized a possible relationship between ER stress induction and the SPARC level. Recently, it has been reported that the overexpression of SPARC induces apoptosis in neuroblastoma cells mediated by the induction of ER stress (25). To establish whether a similar relationship occurs in our experimental system, we analysed the level of CHOP, a marker of ER stress, in WIN-treated SPARC silenced cells. As shown in Fig. 4A, the increase in the level of CHOP, observed after WIN-treatment, was not modified by the reduced SPARC expression in silenced cells. Notably, we demonstrated that SPARC increase was a consequence of the activation of the transcriptional factor CHOP induced by WIN. In fact, CHOP silencing which almost completely suppressed the basal levels of CHOP, markedly reduced WIN-induced SPARC increase (Fig. 4B) while the downregulation of GRP78/Bip, another ER stress marker, whose levels are positively regulated by the cannabinoid, did not modify SPARC upregulation induced by WIN treatment (Fig. 4C).