PP242 was purchased from Active Biochemicals. Co. (Hong Kong); rapamycin was from Sigma Chemical Co. (St. Louis, MO, USA). PP242 and rapamycin were diluted in dimethyl sulfoxide (DMSO). The following antibodies were used: phospho-S6 (S235/236), raptor, cleaved poly(ADP-ribose) polymerase (PARP) and caspase 7 from Cell Signaling Technology, Inc. (Beverly, MA, USA); S6, actin, P-Akt (S473), mTOR, rictor and Akt were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Osteosarcoma cell lines, MG63, U2-OS and Saos-2, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MG63 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), and U2-OS and Saos-2 cells were grown in McCoy’s 5A medium with 10% FBS. All media and FBS were from Gibco-BRL (Rockville, MD, USA). Cells were cultured at 37°C in a humidified atmosphere consisting of 5% CO₂ and 95% air.

Western blotting was performed as previously described (18,19). In brief, cells were lysed in a buffer containing 1% SDS. Equal amounts of whole protein extract were resolved on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Italy), probed overnight at 4°C with antibodies and then revealed using the ECL western blot analysis system.

Drug combination analysis was performed as previously described (19). In briefly, the method describe by Chou and Talalay (20), and multiple drug dose-effect calculations and the combination index plots were generated using CalcuSyn 2.1 software (Biosoft, Cambridge, UK). We assessed the effects of drug interactions by combination index (CI) values. CI<1 indicated synergism, whereas CI=1 and CI>1 indicated additive effect and antagonism, respectively. The pro-apoptosis studies were performed by using 31, 62, 125, 250 and 500 nM of PP242 and rapamycin; both studies were combined with 31, 62, 125, 250 and 500 nM cisplatin treatment.

Human mTOR-, raptor- and rictor-specific siRNAs were chemically synthesized by GenePharma Co., Ltd. (Shanghai, China). Target sequences of these siRNAs were mTOR (50-GAGCCUUGUUGAUCCUUAA-30), raptor (50-CGAGAUUGGACGACCAAAU-30) and rictor (50-GACUAUCCAUAAUCCUUA-30). Cells were transfected with the siRNAs at 60% confluency using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions.

Migration ability was assessed by a wound healing assay. Cells were seeded in 12-well plates and treated with PP242 (200 nM) or rapamycin (200 nM) or transfected with the negative control (NC), mTOR, raptor or rictor siRNA, and grew until reaching 100% confluency. Cells were then treated with 2 μg/ml of mitomycin C for 24 h in a minimum serum medium (containing 0.5% FBS). After using a pipette tip to create a scratch in the confluent cells, the wound distance was visualized at regular intervals of time (24/48 h) at ×100 magnifications using an inverted microscope (Nikon Diaphot). The cell motility assay was performed on a Transwell plate. Cells (1.0×10⁵) were seeded in a Matrigel-coated chamber (BD Biosciences). Cells were seeded in serum-free media and translocated toward complete growth media. After 12 h of incubation with PP242 (200 nM) and rapamycin (200 nM) at 37°C, invaded cell were fixed and stained in dye solution containing 20% methanol and 0.1% crystal violet. The cells which had migrated or invaded were imaged using a BH-2 inverted microscope (Olympus).

Cells were treated with PP242 (500 nM) and rapamycin (500 nM) or transfected with siRNA in medium without serum for 36 h. Apoptotic cells were assessed using a PI assay kit (MultiSciences Biotech Co. Ltd., Beijing, China) according to the manufacturer’s instructions. Briefly, the treated cells were carefully washed three times with PBS, and then staining was performed with a mixture containing 10 mg/ml PI and PBS in the wells and stained for 15 min at 37°C in the dark. Photomicrographs of the stained cells were then recorded under a fluorescence microscope with digital equipment, and the apoptotic cells and viable cells were counted visually. The apoptosis ratio = apoptotic cells/total cells × 100%.

Data analysis was performed with SPSS 13.0. Statistical analysis was performed by applying one-way analysis of variance (ANOVA). Data are presented as means ± standard deviation (SD), and p<0.05 was considered to indicate statistical significance.

We first examined the distinct effects of the targeting of mTORC1 by rapamycin and the targeting of mTORC1/2 by mTOR kinase inhibitor PP242 on their downstream signals in osteosarcoma MG63, U2OS and Saos-2 cell lines. Although these two drugs were able to effectively suppress phosphorylation of S6 (S235/236) in all tested cell lines, only PP242 dose dependently (50–1000 nM) inhibited phosphorylation of Akt (S473), an mTORC2 phosphorylation site. Rapamycin treatment did not cause any significant changes in the phosphorylation level of Akt (S473) (Fig. 1A–C). This indicated that mTOR kinase inhibitors profoundly diminished both mTORC1 and mTORC2 signaling, whereas rapamycin only suppressed mTORC1 in the osteosarcoma cell lines.

Metastasis is the major cause of mortality and morbidity of osteosarcoma patients. Invasion of cancer cells into surrounding tissue and vasculature is an initial step in tumor metastasis. This requires migration of cancer cells (21). The effects of targeted inhibition of mTORC1 and/or mTORC2 on cell migration were examined by wound healing assay. PP242-treated Saos-2 cells exhibited a slower migration speed than the control and rapamycin-treated Saos-2 cells (Fig. 2A and B). In addition, the Transwell assay was performed to confirm the inhibitory effect of PP242 on osteosarcoma cell motility. As expected, the number of migrated cells markedly decreased in the PP242-treated but not in the rapamycin-treated MG63 and Saos-2 cells (p<0.05) (Fig. 2C and D). These results indicated that the inhibitor of mTORC1/2 prevented osteosarcoma cell migration. To further identify the roles of mTORC1 and mTORC2 in osteosarcoma cell migration, the effects of raptor, mTOR or rictor knockdown on migration were examined. Raptor, rictor or mTOR siRNA markedly decreased the protein levels of raptor, rictor or mTOR and reduced the phosphorylation of their outputs S6 (S235/236) and Akt (S473), respectively in Saos-2 cells (Fig. 3A). Knockdown of rictor and mTOR, but not raptor significantly repressed cell migration (p<0.01) (Fig. 3B and C). These results further confirmed the critical role of mTORC2 in osteosarcoma cell migration.

Akt represents an important intracellular survival signaling under a variety of conditions (22). Rapamycin did not inhibit mTORC2/Akt, and accordingly it did not promote apoptosis in osteosarcoma MG63, U2OS and Saos-2 cells (Fig. 4A–C). Consistent with the inhibitory activities of PP242 on mTORC2/Akt (S473) phosphorylation (Fig. 1), the drug significantly enhanced cleavage of PARP, caspase 7 and the number of apoptotic cells in the serum-starved MG63, U2OS and Saos-2 cells (Fig. 4A–C).

To further identify the different role of mTORC1 and mTORC2 in osteosarcoma cell apoptosis, the raptor, rictor or mTOR siRNA was transfected into Saos-2 cells. The results showed that knockdown of mTOR and rictor but not raptor were able to increase the number of apoptotic cells and the level of cleaved-PARP in the Saos-2 cells (Fig. 5). Hence, we demonstrated that targeting of mTORC2 but not mTORC1 promoted apoptosis in the osteosarcoma cells.

Cisplatin is a common chemotherapeutic agent that induces apoptosis in a variety of cancer cell lines including osteosarcoma cells (23). Drug CI analysis is a generalized method for analyzing the effects of multiple drugs and for determining summation, synergism and antagonism.

We used the drug CI analysis to examine the interaction of mTORC1 inhibitor rapamycin or mTORC1/2 inhibitor PP242 with cisplatin. We performed the dose-response studies in osteosarcoma cell lines to obtain the apoptotic rate, and we calculated the CI values by the CalcuSyn program. When PP242 was applied at most concentrations in all tested cell lines, the CI values were <1, indicating synergistic pro-apoptotic interactions between PP242 and cisplatin (Fig. 6). In contrast, rapamycin did not show any synergistic interactions with cisplatin, as the CI values were >1 (Fig. 6). Most importantly, rictor or mTOR knockdown, but not raptor knockdown, markedly enhanced cisplatin-induced cleavage of PARP and the number of apoptotic cells in Saos-2 cells (Fig. 7). Taken together, our results indicate that targeted inhibition of mTORC2 but not mTORC1 markedly enhanced cisplatin-induced apoptosis in osteosarcoma cells.