The RCC cell line Caki-1 is purchased from the American Type Culture Collection (ATCC) and is stored in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% FBS and 1% antibiotic-antimycotic solution. The cells are cultured at 37 ° C containing 5% CO₂ in a humid atmosphere. Caki-1 cells against PTX (Caki-1 ^PTX) are established by stepwise exposure to PTX at a gradient concentration as previously described (18).

MTT assay is used to detect cell viability. The cells are seeded in 96-well plates with the final concentration of 5 × 10³ cells / well. After the corresponding treatment, the cells are incubated with the MTT solution (1 mg / ml) for 4 hours. Formazan crystals are dissolved in DMSO and measured by absorbance at 570 nm using a microplate reader (SpectraMax M5, Molecular Devices). Cell viability is expressed as a percentage of the vehicle control.

Caki-1 ^PTX cells are seeded in 96-well plates with the final concentration of 2 × 10⁵cells / well. After treated with different doses of PTX (0, 2, 4, 8, 16 nM) for 24 hours, the cells are incubated with 1 μg/ml Hoechst 33258 reagent at 37 ° C for 30 minutes in the dark. (Beyotime Institute of Biotechnology) Remove the Hoechst 33258 reagent and wash the cells with PBS three times (5 min × 3 times). The morphological changes of apoptotic cells are observed under an inverted fluorescence microscope and the images are captured.

Caki-1 ^PTX cells are seeded in 96-well plates with the final concentration of 2 × 10⁵cells / well. After treated with different doses of PTX (0, 2, 4, 8, 16 nM) for 24 hours, the cells are stained with PI / AV-fluorescein isothiocyanate (BD Biosciences) and determined by flow cytometry FACSCalibur instrument (BD Biosciences) according to the manufacturer’s recommendations.

The cells are harvested and lysed with lysis buffer. The protein is extracted from cells, separated by 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Boston, MA, USA). Then, the membrane is blocked with 5% skim milk and incubated overnight at 4 °C with the primary antibodies: survivin (No. 2808, 1:1000, Cell Signaling Technology, USA), PI3K (No. 4249, 1:1000, CST, USA), p-PI3K (No. 4228, 1:1000, CST, USA) AKT (No. 2920, 1:1000, CST, USA) and p-AKT (No. 4060, 1:1000, CST, USA). After that, the membrane is incubated with the corresponding HRP-conjugated secondary antibody for 1 hour at room temperature. Signals are detected using an Enhanced Chemiluminescence (ECL) commercial kit (Amersham Biosciences) according to the manufacturer’s instructions.

For overexpression of Survivin, the full-length Survivin sequence obtained from GenePharma is cloned into the pcDNA3.1-GFP vector. Caki-1 ^PTX cells are seeded into 96-well plates to achieve 60% confluence for transfection. Transfection is performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. Transfection efficiency is determined by western blotting.

All determinations are repeated in triplicate. All data are represented as the mean ± standard deviation (SD). Statistical software SPSS19.0 is used for the assessment. The Student’s t test is used to compare means of two groups and One-Way ANOVA is used for comparing means of multiple samples. Values of P< 0.05 are considered statistically significant.

First, Caki-1 cells and PTX-resistant Caki-1 ^PTX cells are treated with different doses of PTX (0, 2, 4, 8, 16 nM). The results show that PTX inhibits cell viability in Caki-1 and Caki-1 ^PTXcells in a dose-dependent manner, especially in Caki-1 cells. Besides, when the dose of PTX reaches 4 nM, the cell viability of Caki-1 ^PTX cells is significantly reduced, while no significant effect is observed at 2 nM. (Figure 1A, ^*P < 0.05, ^**P < 0.01, ^*** P< 0.001) Flow cytometry and Hoechst 33258 staining show that when the dose of PTX reaches 4 nM, the apoptosis rate of Caki-1 ^PTX cells is significantly increased, while no significant effect is observed at 2 nM. Taken together, these data indicate that PTX inhibits cell viability and inducts apoptosis in RCC cells. Caki-1 ^PTX cells in this study are not sensitive to 2 nM PTX. (Figure 1B-1D, ^*P < 0.05, ^**P < 0.01) Therefore, a 2 nM PTX dose is used for subsequent experiments.

Figure 1.

PTX inhibits cell viability and induces apoptosis in RCC cells. (A) Caki-1 cells and Caki ^PTX cells are treated with different doses of PTX (0, 2, 4, 8, 16 nM) for 24 h. Cell viability is measured by MTT assay. (B-D) Caki^PTX cells are treated with different doses of PTX (0, 2, 4, 8, 16 nM) for 24 h. Cell apoptosis are measured by Hoechst 33258 staining and flow cytometry analysis. (B) Hoechst 33258 staining. (C) Flow cytometry. (D) Cell apoptotic rate in each group. The bars showed means ± SD of three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with control group.

After exposure to 2 nM PTX for 24 hours, Caki-1 ^PTX cells are then treated with RES (0, 10, 30, 50 μM) for another 24 hours. Flow cytometry shows that RES (30, 50 μM) significantly reduce cell viability in a dose-dependent manner, while increasing apoptotic rate compared to the control group (Figure 2A-2B, ^*P < 0.05, ^**P < 0.01). These results indicate that RES enhances the sensitivity of Caki-1 ^PTX cells to PTX.

Figure 2.

RES enhances the sensitivity of PTX resistant cells to PTX. Caki ^PTX cells are exposed to 2 nM PTX for 24 h, and then treated with different doses of RES (0, 10, 30, 50 μM) for another 24 h. (A) Cell viability is measured by MTT assay. (B) Cell apoptosis is measured by flow cytometry assay. The bars showed means ± SD of three independent experiments. ^*P < 0.05, ^**P < 0.01 compared with control group.

To investigate the role of survivin in Caki-1 ^PTX cells, western blot, MTT assay and flow cytometry are used. After exposure to 2 nM PTX for 24 hours, Caki-1 ^PTX cells are then treated with RES (0, 10, 30, 50 μM) for another 24 hours. As shown in Figure 3A, RES treatment (30, 50 μM) downregulate survivin level in a dose-dependent manner. (Figure 3A-3B, *P < 0.05, **P < 0.01) Besides, Caki-1^PTX cells are transfected with pcDNA3.1-GFP or pcDNA3.1-GFP-Survivin after exposure to 2 nM PTX to determine transfection efficiency. As shown in Figure 3C-3D, transfection with pcDNA3.1-GFP-Survivin significantly increases survivin level, whereas there is no significant change after transfection with pcDNA3.1-GFP compared to the control. (* P <0.05, **P <0.01, ## P <0.01) In addition, western blotting also shows that treatment with RES (50 μM) significantly reduces survivin level, whereas overexpression of survivin has opposite effects. Furthermore, elevated survivin in Caki-1^PTX cells significantly attenuates RES (50 μM)-induced cell viability inhibition and apoptosis promotion. (Figure 3E-3F, ## P <0.01, $P <0.05) Our results demonstrate that survivin is involved in the regulation of RES in RCC cells.

Figure 3.

Survivin participates in the regulation of RES in RCC cells. (A-B) Caki ^PTX cells are exposed to 2 nM PTX for 24 h, and then treated with different doses of RES (0, 10, 30, 50 μM) for another 24 h. Protein level of survivin is measured by western blot. P < 0.05, ^**P < 0.01 compared with control group (C-D) Caki ^PTXcells are exposed to 2 nM PTX for 24 h, and then transfected with pcDNA3.1-GFP or pcDNA3.1-GFP-Survivin or RES 50 μM or the combination of RES 50 μM and pcDNA3.1-GFP-Survivin. Protein level of survivin is detected by western blot. ^*P < 0.05, ^**P < 0.01 compared with control group, ^##P < 0.01 compared with RES 50 μM group (E) Cell viability is measured by MTT assay. (F) Cell apoptosis is measured by flow cytometry analysis. The bars showed means ± SD of three independent experiments. ^##P < 0.01 compared with PTX 2 nM group, ^$P < 0.05 compared with PTX 2 nM + RES 50 μM group.

Western blotting analysis is performed to investigate the molecular mechanism of RES on RCC cells. Caki-1^PTX cells are treated with RES (0, 10, 30, 50 μM) for another 24 hours after exposure to 2 nM PTX. As shown in Figure 4A-4B, RES treatment (10, 30, 50 μM) inhibit phosphorylation of PI3K and ATK in a dose dependent manner. (* P <0.05, ** P <0.01) These results indicate that the PI3K/AKT pathway is involved in the regulation of RES in RCC cells.

Figure 4.

PI3K/AKT pathway is involved in the regulation of RES in RCC cells. Caki ^PTXcells are exposed to 2 nM PTX for 24 h, and then treated with different doses of RES (0, 10, 30, 50 μM) for another 24 h. (A) Phosphorylation level of PI3K is monitored by western blot. (B) Phosphorylation level of AKT is monitored by western blotting. The bars showed means ± SD of three independent experiments. ^*P < 0.05, ^**P < 0.01 compared with control group.

To further confirm the role of RES in the PI3K / AKT pathway, the pathway activator IGF-1 is used. Western blotting shows that RES (50 μM) treatment significantly inhibits the phosphorylation levels of PI3K and AKT. However, the opposite result is observed by adding IGF-1 (50 ng / ml). (Figure 5A, ^*P < 0.05, ^**P < 0.01, ^#P < 0.05) Besides, the decrease in p-PI3K and p-AKT further reduces survivin level, while IGF-1 has the opposite effects. (Figure 5B, ^* P< 0.05, ^**P < 0.01, ^#P < 0.05) Furthermore, down-regulation of survivin ultimately inhibits cell viability and promotes apoptosis compared to Caki-1 ^PTX cells treated with 2 nM PTX. (Figure 5C-5D, ^##P < 0.01, ^$P < 0.05) All these results indicate that RES inhibits cell growth and induces apoptosis by inhibiting the PI3K / AKT pathway to downregulate survivin level in PTX resistant cells.

Figure 5.

RES inhibits cell growth and induced apoptosis by inhibition PI3K/AKT pathway to downregulate Survivin level in PTX resistant cells. Caki ^PTXcells are exposed to 2 nM PTX for 24 h, and then treated with RES (50 μM) or/and IGF-1 (50 ng/ml). (A) Phosphorylation level of PI3K is monitored by western blotting. (B) Survivin is detected by western blotting. ^*P < 0.05,^**P < 0.01 compared with control group, ^#P < 0.05 compared with RES 50 μM group (C) Cell viability is measured by MTT assay. (D) Cell apoptosis is measured by flow cytometry analysis. The bars showed means ± SD of three independent experiments. ^##P < 0.01 compared with PTX 2 nM group, ^$P < 0.05 compared with PTX 2 nM + RES 50 μM group.