The RPMI8226 cell line was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and the ARP-1 cell line was kindly provided by Professor Cai Zhen (Zhejiang University, Hangzhou, China). The two cell lines were cultured in RPMI-1640 (HyClone Laboratories; GE Healthcare, Chicago, IL, USA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories; GE Healthcare) at 37°C in an incubator with 5% CO₂. GSK-470 and MG-132 were purchased from Selleck Chemicals (Houston, TX, USA). IGF-1 was purchased from PeproTech (Rocky Hill, NJ, USA).

The inhibitory effect of GSK-470 and/or MG-132 on the proliferation of MM cell lines was examined by modified microculture tetrazolium (MTT) assay. Cells (2×10⁴ cells/well) were cultured in 96-well plates and treated with GSK-470 and/or MG-132 at the indicated concentrations for 24 h. Following this incubation, 20 µl of MTT solution (5 mg/ml) was added into each well and then the plates were incubated for an additional 4 h at 37°C. Following removal of the supernatant, 200 µl dimethyl sulfoxide (DMSO) was added to each well, and the absorbance at a wavelength of 570 nm was assessed using an enzyme-linked immunosorbent assay plate reader.

After treatment with GSK-470 and/or MG-132 at the indicated concentrations for 24 h, MM cells (2×10⁵ cells) were washed twice in cold phosphate-buffered saline (PBS), then the cell pellets were resuspended in 500 µl of binding buffer and stained with 5 µl of Annexin V-FITC and 10 µl of propidium iodide (PI; Biouniquer, Suzhou, China) for 15 min in the dark. Cells were analyzed by FACScan flow cytometer (Becton-Dickinson, San Diego, CA, USA). Expression of green fluorescent protein (GFP) was also assessed by the FACScan flow cytometer.

For analyses of protein expression, cells treated with GSK-470 and/or MG-132 were collected by centrifuging and washed with cold PBS. Then, the cells were lysed in lysis buffer. The cytoplasm and nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions as previously described (23). The lysate was quantified using the bicinchoninic acid (BCA) assay, and equal amounts of protein was subjected to 6–12% SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with fat-free milk for 2 h, the membranes were incubated with primary antibodies at 4°C overnight, and then incubated with secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) for 2 h. Antibody binding was detected by enhanced chemiluminescence using an ECL detection kit. The primary antibodies used here were as follows and all diluted to 1:1,000 for western blot experiment: Total PTEN (cat. no. 9188), phosphor-PTEN at Ser380/Thr382/383 (cat. no. 7960), total PDK1 (cat. no. 3062), phospho-PDK1 at Ser241 (cat. no. 3061), total mTOR (cat. no. 2972), phosphor-mTOR at Ser2448 (cat. no. 5536), phosphor-mTOR at Ser 2481 (cat. no. 2974), total AKT (cat. no. 9272), phosphor-AKT at Ser473 (cat. no. 4060), phosphor-AKT at Thr308 (cat. no. 13038), PI3Kp110α (cat. no. 4255), poly(adenosine diphosphate-ribose) polymerase (PARP) (cat. no. 9542) and caspase-8 (cat. no. 9746), caspase-9 (cat. no. 9505) and caspase-3 (cat. no. 9662), were purchased from Cell Signaling Technology with the exception of Lamin B1 (cat. no. 12987-1-AP; Proteintech Group Inc., Chicago, IL, USA) and β-actin (cat. no. sc-69879; Santa Cruz Biotechnolgy, Santa Cruz, CA, USA).

Cells were treated with 2 µM GSK-470 and/or 200 nM MG-132 for 24 h, washed and fixed on the 0.1% poly-L-lysine (Wuhan Boshide Bio Inc, Wuhan, China)-treated slides with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 and then blocked with goat serum for 30 min. Subsequently, the slides were incubated overnight with antibody PTEN (cat. no. ab32199; Abcam, Cambridge, UK) diluted 1:100 at 4°C, rinsed three times with PBS, and then incubated with secondary antibody: Alexa Fluor 488 (cat. no. A10468; Invitrogen; Thermo Fisher Scientific, Inc.) diluted 1:500 for 1 h at 37°C. Next, the slides were repeatedly rinsed three times with PBS, and then incubated with 100 nM rhodamine phalloidin (Cytoskeleton, Denver, CO, USA) for 30 min. Before incubation with 0.5 mg/ml DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min, the slides were again rinsed three times with PBS. The slides were then observed under an Olympus confocal microscope (Olympus FV1000; Olympus Corp., Tokyo, Japan).

Experimental results are presented as the mean ± standard deviation (SD). Statistical analysis was performed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference. Synergisms in the combination treatments were analyzed using CalcuSyn software (Biosoft, Cambridge, UK).

We first assessed the cytotoxic effect of GSK-470 on the human MM cell lines. As shown in Fig. 1A, both RPMI8226 and ARP-1 cell lines exhibited a sensitivity to GSK-470 in a dose-dependent manner as determined by an MTT assay. The 50% inhibition (IC₅₀) values of GSK-470 at 48 h were 5.04 µM in the RPMI8226 cells and 2.21 µM in the ARP-1 cells, respectively. Since IL-6 acts as an important mediator of cell survival, migration and drug resistance in MM (11), we next evaluated the effect of GSK-470 on RPMI8226 in the presence of exogenous IL-6. RPMI8226 cells were incubated in FBS-free RPMI-1640 medium containing 1–10 µM GSK-470 and 100 ng/ml IL-6. Stimulation by IL-6 increased cell growth of myeloma cells, but GSK-470 was able to suppress this stimulation (Fig. 1B). Following treatment of ARP-1 cells with GSK-470 at various concentrations (100–2,500 nM) for 24 h, we revealed that GSK-470 triggered a time-dependent cleavage of caspase-8, −9 and −3, followed by PARP cleavage (Fig. 1C). These data indicated that GSK-470 induced cytotoxicity and apoptosis in myeloma cells.

GSK-470 significantly inhibited the phosphorylation of AKT at Thr308 (Fig. 1C). Since inhibition of PTEN phosphorylation is important for PTEN protein stability to proteasome-mediated degradation (24), we next examined the effect of GSK-470 on the phosphorylation of PTEN in ARP-1 cells. GSK-470 induced a notable inhibition of phosphor-PTEN at Ser380 and Thr382/383 (Fig. 1C), which may accelerate the proteasome-mediated degradation of PTEN (24).

Since a previous study reported that the combined use of PDK1 inhibitor BX-912 and bortezomib exerts synergistic effect in MM cells (21), we examined whether the sensitivity of RPMI8226 cells to GSK-470 was enhanced by MG-132. For this purpose, we treated RPMI8226 cells with a series of concentrations of GSK-470 and MG-132 either alone or in combination for 24 h and assessed cell viability using an MTT assay. As revealed in Fig. 2A, co-treatment with GSK-470 and MG-132 led to significantly lower cell viability than either GSK-470 or MG-132 treatment alone. The combination index values were <1, which indicated a synergistic effect between GSK-470 and MG-132 (Fig. 2B). Flow cytometric analysis revealed an enhanced apoptosis of cells following exposure to treatment with GSK-470 plus MG-132 (Fig. 2C). We also evaluated the cytotoxicity triggered by GSK-470 and MG-132 against primary myeloma cells. The myeloma cells from a patient newly diagnosed with MM were treated with GSK-470 (5 µM) and/or MG-132 (400 nM) for the detection of cell viability and apoptosis. Combination of GSK-470 with MG-132 resulted in an enhanced cytotoxicity against primary myeloma cells and produced more apoptotic cells (Fig. 3).

In order to characterize the cytotoxicity of GSK-470 combined with MG-132 against MM cells, we examined the alteration of the apoptotic pathway induced by the co-treatment. Western blot analysis indicated that RPMI8226 cells treated with GSK-470 or MG-132 alone demonstrated a weak activation of both the extrinsic and intrinsic apoptosis pathway, indicated by cleavage of caspase-8, caspase-9, caspase-3 and PARP. In contrast, an enhanced activation of cleaved caspase molecules was observed in the myeloma cells treated with the combination therapy (Fig. 4A).

The PI3K/AKT/mTOR signaling pathway is frequently hyperactivated in MM cells, which is associated with resistance to antimyeloma agents (20,25). Our data indicated that GSK-470 inhibited PI3K p110α protein expression. However, co-treatment with GSK-470 and MG-132 led to a markedly enhanced inhibition of PI3K p110α (Fig. 4B). Western blot analysis also indicated that GSK-470 decreased phosphorylation of AKT at Thr308 and phosphorylation of mTOR on Ser2448, a downstream molecule of AKT, consistent with the notion that Thr308 residues of AKT are the main effectors of PDK1 in cancer cells (20). Additionally, MG-132 exhibited inhibitory effects on the phosphorylation of AKT at Ser473 and phosphorylation of mTOR at Ser2481, a marker for mTORC2 activity. Notably, the combination therapy led to almost complete elimination of phosphorylated AKT (Ser473/Thr308) and activity of mTORC1 as well as mTORC2. Furthermore, significantly upregulated expression of PTEN was observed in the MG-132-treated cells compared to the untreated cells, which was consistent with a recent observation which revealed that bortezomib increased PTEN expression in drug-resistant breast cancer cells (26).

Recent studies demonstrated that in the nucleus PTEN exerts an important tumor-suppressive function (27). In the present study, PTEN expression was slightly elevated in nucleus of cells treated with GSK-470 compared to the untreated control. Whereas, a higher level of PTEN was found in the nuclear protein extracts of RPMI8226 cells treated with MG-132, but not in the cytoplasmic extracts. Furthermore, combination of GSK-470 and MG-132 resulted in a significantly increased level of PTEN protein in the nucleus but decreased PTEN protein expression in the cytoplasm (Fig. 5A). To confirm the subcellular localization of PTEN in MM cells, confocal microscopy studies were performed (Fig. 5B). Intense staining of PTEN was observed at the nuclear periphery and in the cytoplasm of MG-132-treated cells. Notably, PTEN was found mainly in the nucleus when cells were treated with the combination therapy. Collectively, these findings demonstrated that combination of PDK1 inhibitor GSK-470 and proteasome inhibitor MG-132 resulted in the nuclear accumulation of PTEN.