Ginsenoside Rg3 (Rg3) was purchased from Yatai Pharmaceuticals Co., Ltd (Jilin, China) with 98% purity, assayed using high-performance liquid chromatography (HPLC; Fig. 1A). The powder was dissolved in dimethyl sulfoxide (DMSO) in a stock concentration of 100 mg/ml. The final concentration of DMSO in the culture medium was ≤0.1%. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and phosphate-buffered saline (PBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Antibodies obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) were as follows, all used at 1:500 dilution: Mouse anti-cyclin D1 monoclonal antibody (cat. no. sc-8396), mouse anti-p27 monoclonal antibody (cat. no. sc-393380), mouse anti-phosphorylated (phospho)-extracellular signal regulated kinase (Erk)1/2 monoclonal antibody (cat. no. sc-7383), rabbit anti-Erk1/2 polyclonal antibody (cat. no. sc-292838), mouse anti-phospho-c-Jun N-terminal kinase (JNK) monoclonal antibody (cat. no. sc-6254), mouse anti-JNK monoclonal antibody (cat. no. sc-7345), rabbit anti-phophos-p38 polyclonal antibody (cat. no. sc-17852-R), mouse anti-p38 monoclonal antibody (cat. no. sc-81621) and rabbit anti-IGF-1 polyclonal antibody (cat. no. sc-9013). Antibodies obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA) were as follows, all used at 1:1,000 dilution: Mouse anti-B cell lymphoma-2 (Bcl-2) monoclonal antibody (cat. no. 15071), rabbit anti-Bcl-2-associated X protein (Bax) polyclonal antibody (cat. no. 5023), mouse anti-cytochrome C monoclonal antibody (cat. no. 12963), rabbit anti-cytochrome c oxidase (Cox) IV polyclonal antibody (cat. no. 4850), mouse anti-caspase-9 monoclonal antibody (cat. no. 9508), mouse anti-caspase-8 monoclonal antibody (cat. no. 9746), rabbit anti-caspase-3 polyclonal antibody (cat. no. 9665), rabbit anti-phospho-AKT polyclonal antibody (cat. no. 5012), mouse anti-AKT monoclonal antibody (cat. no. 2920), rabbit anti-phospho-mTOR polyclonal antibody (cat. no. 2976) and mouse anti-mTOR monoclonal antibody (cat. no. 2983). IGF-1, rabbit polyclonal anti-retinoblastoma (Rb; cat. no. SAB4502589), mouse monoclonal anti-phospho-Rb (cat. no. R6878) and rabbit polyclonal anti-GAPDH (cat. no. G8795) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used at 1:1,000 dilution. The Cell Counting kit-8 assay (CCK-8), radioimmunoprecipitation assay (RIPA) lysis buffer, bicinchoninic acid (BCA) kit, enhanced chemiluminescence (ECL) system and Fluorescein isothiocyanate (FITC)-Annexin V Apoptosis Detection kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China).

The U266, RPMI8226 and SKO-007 human multiple myeloma cell lines, were obtained from America Type Culture Collection (Rockville, MD, USA) and maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a 5% CO₂ atmosphere.

Multiple myeloma cells were seeded in 96-well plates at a density of 5×10³ cells/well overnight and then treated with Rg3 at different concentrations (0, 20, 40, 60, 80 and 100 µg/ml), and for different durations (6, 12, 24 and 48 h), as indicated. Subsequently, fresh medium containing 10 µl CCK-8 reagent was added, followed by incubation at 37°C for 2 h. The absorbance of each well was measured at 450 nm on a plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).

The cells were washed with cold PBS and lysed with lysis buffer. The protein concentration was determined using the BCA kit. Equal quantities (40 µg) of protein were separated on 12% SDS-PAGE gels (GenScript, Piscataway, NJ, USA) and then transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h, and then probed with appropriate primary antibodies overnight at 4°C, followed by blotting with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (cat. no. A0216) or HRP-labeled goat anti-rabbit IgG (cat. no. A0208) secondary antibodies (1:1,000; Beyotime Institute of Biotechnology) at room temperature for 1 h. The target bands were detected using the ECL system, and the band intensity was determined using ImageJ software (version 1.41; NIH, Bethesda, MD, USA).

The cells were harvested by centrifugation and processed for cell cycle analysis, as described previously (3). Briefly, the cells were digested with 0.25 g/l trypsin and harvested by centrifugation at 1,000 × g for 10 min at room temperature. Following incubation with 70% ethanol at −20°C for 15 min, the cells were stained with 20 mg/ml propidium iodide (PI) and incubated for 30 min at room temperature. The cells were analyzed for DNA content using FACScalibur flow cytometry (BD Biosciences, San Jose, CA, USA). The percentages of cells containing different DNA contents were quantified using CellQuest software (version 5.1; BD Biosciences).

The cellular apoptotic ratios were detected with the FITC-Annexin V Apoptosis Detection kit using flow cytometry. Briefly, the cells were trypsinized and harvested by centrifugation. The cell pellets were re-suspended in a binding buffer of Annexin V-FITC and PI at room temperature in the dark for 15 min. The apoptotic cells were counted using flow cytometry (BD Biosciences), with the percentage of apoptotic cells expressed as the FITC/PI ratio.

The isolation of mitochondrial and cytoplasmic proteins was performed using a Mitochondria Isolation kit (Thermo Fisher Scientific Inc.), according to the manufacturer's protocol. The cytosolic and mitochondrial fractions were analyzed using western blot analysis. Cox IV was used as an internal control for the mitochondrial fraction.

The concentrations of IGF-1 were determined using a Human IGF-I Quantikine ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's protocol. The absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Tek Instruments, Inc.).

All data are presented as the mean ± standard error of the mean, and the n value indicates the number of independent experiments. Data were statistically analyzed using Student's t-test on GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The uncontrolled cell proliferation of cancer cells is vital in the progression of cancer, therefore, the present study evaluated the effects of Rg3 on the viability of multiple myeloma cells. As shown in Fig. 1B, treatment with Rg3 inhibited the viability of the U266 cells in a time- and dose-dependent manner. The inhibition of cell viability in the U266 cells following treatment with Rg3 for 48 h reached the maximal level at 80 µg/ml, and the half maximal inhibitory concentration (IC50) value was 47.5 mg/l. In addition to U266, two other multiple myeloma cell lines, RPMI8226 and SKO-007, were also included in the present study to examine these effects. Rg3 had a more potent effect in inhibiting the viability of the RPMI8226 (IC50=36.8 µg/ml) and SKO-007 (IC50=31.5 µg/ml) cells, compared with the U266 cells (Fig. 1C and D).

Flow cytometric cell cycle analysis was performed using PI staining to determine the effect of Rg3 on cell cycle progression. Treatment of the U266 cells with 20, 40 and 80 µg/ml Rg3 increased the percentage of the cell population in the G₁ phase and decreased the percentage in the S phase. However, Rg3 had no effect on the G2/M phase (Fig. 2A and B). The effect of Rg3 on cell cycle progression was also examined in the SKO-007 cells (Fig. 2C and D). Similarly, the cell cycle of the SKO-007 cells was arrested in the G₁ phase by Rg3, indicating that Rg3 may inhibit multiple myeloma cell proliferation through suppressing the cell cycle transition from the G₁ to the S phase. The cell cycle transition between the G₁ and S phase is strictly regulated by the balance of cyclins and cyclin-dependent kinase inhibitors (18,19). Therefore, to further investigate the mechanisms underlying how Rg3 arrested G₁/S transition, the expression levels of proteins regulating cell cycle progression in U266 cells, including cyclin D1, p27 and phospho-Rb, were determined. The results of the western blot analysis showed that Rg3 decreased the protein expression of cyclin D1 and phosphorylation of Rb, and increased the expression of p27 in a dose-dependent manner (Fig. 2E and F).

To examine the effect of Rg3 on cell survival, its effects on cell apoptosis were determined. Annexin V-FITC/PI staining followed by flow cytometric analysis revealed that treatment of the U266 cells with 20, 40 and 80 µg/ml Rg3 for 48 h significantly increased the percentage of apoptotic cells in the in U266 cell population (Fig. 3A and B). Similar results were observed in the RPMI8226 and SKO-007 cells (data not shown). Bcl-2 and Bax are anti-apoptotic and pro-apoptotic proteins, and the ratio of Bcl-2/Bax appears to be a determinant of cell survival and death. To understand the mechanism by which Rg3 induces cell apoptosis, the expression levels of Bcl-2 and Bax, and the ratio of Bcl-2 to Bax were measured. The results of the western blot analysis showed that Rg3 treatment markedly decreased the protein expression of Bcl-2 and increased the protein expression of Bax in the U266 cells, resulting in a further decrease in the Bcl-2/Bax ratio (Fig. 3C and D). The mitochondria-dependent signaling pathway is critical for cell apoptosis. The release of cytochrome C from the mitochondria into the cytoplasm triggers the downstream apoptotic signal, consequently resulting in cell apoptosis (20). Therefore, the present study examined the release of cytochrome C. As expected, Rg3 treatment caused a significant decrease in the protein expression of cytochrome C in the mitochondria, and an increase in the cytoplasm (Fig. 3E and F). Cytochrome C release sequentially activates downstream apoptosis-associated proteins, including caspases. Western blot analysis demonstrated that Rg3 increased the protein expression levels of cleaved caspase-9, caspase-8 and caspase-3 (Fig. 3G and H). Collectively, these data suggested that mitochondrial dysfunction may underlie, at least partially, the enhanced effect of Rg3 on myeloma cell apoptosis.

The phosphorylation of AKT and downstream mTOR has been reported to be involved in the progression of several types of malignancy, including multiple myeloma (3,21). To clarify the signal transduction pathways by which Rg3 exerts its antitumor effects, the present study first examined the activation of the AKT/mTOR pathway. As shown in Fig. 4A and B, the phosphorylation of AKT and its downstream protein, mTOR, was attenuated by Rg3 treatment in a dose-dependent manner. MAP kinase signaling is also a regulator of cell cycle progression and tumorigenesis (22,23). However, Rg3 treatment did not alter the phosphorylation of Erk1/2, JNK or p38 (Fig. 4C and D). These data excluded the possibility that MAP kinase signaling was involved in the effects of Rg3 on multiple myeloma cell proliferation and survival.

IGF-1 is an important pathway of AKT/mTOR (24). Therefore, the present study investigated whether Rg3 affects this pathway. The results of the western blot analysis showed that Rg3 had no effect on the protein expression of IGF-1 (Fig. 5A). However, the secretion of IGF-1 was markedly decreased following Rg3 treatment (Fig. 5B). In addition, treatment with IGF-1 reversed the Rg3-induced inactivation of the AKT/mTOR pathway, as evidenced by the significant restoration in AKT and mTOR phosphorylation (Fig. 5C and D). The present study further examined whether IGF-1 was involved in the effects of Rg3 on cell proliferation and survival. The results of the CCK-8 assay revealed that the reduction in cell viability induced following Rg3 treatment was gradually inhibited by IGF-1 in a dose-dependent manner (Fig. 5E). As expected, Rg3-induced cell apoptosis was almost eliminated by the addition of IGF-1 (Fig. 5F).