Inhibition of the STAT3 signaling pathway is involved in the antitumor activity of cepharanthine in SaOS2 cells

Abstract

Aim: To investigate the molecular mechanisms underlying the antitumor activity of cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata.

Methods: Human osteosarcoma cell line SaOS2 was used. MTT assay, Hoechst 33342 nuclear staining, flow cytometry, Western blotting and nude mouse xenografts of SaOS2 cells were applied to examine the antitumor activity of CEP in vitro and in vivo. The expression levels of STAT3 and its downstream signaling molecules were measured with Western blotting and immunochemistry analysis. The activity of STAT3 was detected based on the phosphorylation level of STAT3, luciferase gene reporter assay and translocation of STAT3 to the nucleus.

Results: Treatment of SaOS2 cells with CEP (2.5-20 μmol/L) inhibited the cell growth in a concentration- and time-dependent manner. CEP (10 μmol/L) caused cell cycle arrest at G(1) phase and induced apoptosis of SaOS2 cells. CEP (10 and 15 μmol/L) significantly decreased the expression of STAT3 in SaOS2 cells. Furthermore, CEP (5 and 10 μmol/L) significantly inhibited the expression of target genes of STAT3, including the anti-apoptotic gene Bcl-xL and the cell cycle regulators c-Myc and cyclin D1. In nude mouse xenografts of SaOS2 cells, CEP (20 mg·kg(-1)·d(-1), ip for 19 d) significantly reduced the volume and weight of the tumor.

Conclusion: Our findings suggest that inhibition of STAT3 signaling pathway is involved in the anti-tumor activity of CEP.

Figure 1

Growth inhibitive effects of CEP on SaOS2 cell line determined by MTT assay. SaOS2 cells were seeded in 96-well plates and treated with different concentrations of CEP for 12, 24, and 48 h, respectively. Relative cell viability was indicated as the percentage compared to control group (cells treated without CEP).

Figure 2

Cell cycle arrest and apoptosis of SaOS2 cells induced by CEP. (A) Nucleus staining by Hoechst 33342. SaOS2 cells were stained with Hoechst 33342 after incubation with CEP (10 μmol/L) for 0, 24, and 48 h. The red arrow indicates cells in mitotic phase. (B) Cell cycle arrest of SaOS2 cells induced by CEP. Cells were incubated with CEP at 10 μmol/L for 0, 24, and 48 h, and then treated with PI (50 μg/mL). Cell cycle was analyzed by flow cytometry. (C) Apoptosis-inducing effect of CEP on SaOS2 cells. After treatment with CEP (15 μmol/L) for 0, 24, and 48 h, the apoptotic rate of SaOS2 cells was determined by Annexin V-FITC and PI double staining using flow cytometry. (D) Activation of caspase 3 induced by CEP treatment. SaOS2 cells were treated with 0, 5, 10, and 15 μmol/L CEP for 48 h, and for 0, 12, 24, and 48 h at the concentration of 15 μmol/L. The level of caspase 3 was detected by Western blotting. The house-keeping gene β-actin was used as the internal control.

Figure 3

Western blotting for total STAT3 protein. (A) SaOS2 cells were incubated with CEP at the concentrations of 0, 5, 10, and 15 μmol/L for 24 h and protein was collected and analyzed with Western blotting. (B) SaOS2 cells were treated by CEP (10 μmol/L) for 0, 12, 24, and 48 h. Then the STAT3 expression level was determined by Western blotting using β-actin as the internal control. The graphs shown represent mean±SEM of three separate experiments. Results are expressed as percentage of the control (^aP>0.05, ^bP<0.05, ^cP<0.01 vs control).

Figure 4

Inhibition of STAT3 signaling pathway by CEP. (A) Western blotting for p-STAT3, c-Myc, cyclin D1, and Bcl-xL. SaOS2 cells were treated with different concentrations of CEP from 0 to 15 μmol/L for 24 h. For the time course, cells were incubated with CEP (10 μmol/L) for 0, 12, 24, and 48 h. β-actin was used as the internal control. (B) Luciferase assay of APRE plasmids in SaOS2 cells under CEP treatment in different concentrations and at different time points. Cell extracts were prepared and luciferase activities were assayed according to the manufacturer's instruction. Each measured activity of firefly luciferase was normalized by the activity of Renilla luciferase in the same well. The data presents mean relative luciferase activity of three individual experiments (^bP<0.05, ^cP<0.01 vs control).

Figure 5

In vivo experiments. Eighteen nude mice were randomly grouped as a control group (CON) and a CEP-treated group (CEP). When the xenograft model of SaOS2 cells was established, mice in CON group were injected with DMSO while those in CEP group with CEP (20 mg·kg⁻¹·d⁻¹, ip). Body weight, long axon (L) and vertical axon (R) of each mouse were measured every two days. (A) Tumor volume growth curve of SaOS2 grafts in mice. The volumes of tumors were estimated using the formula: V=0.5×L×R². ^cP<0.01. (B) The tumor weights of the two groups. On the twentieth day of CEP treatment, mice were sacrificed and the tumors were weighed after being sected. ^cP<0.01 vs control. (C) Photograph of the tumors. (D) Record of body weights of mice.

Figure 6

Immunohistochemical analyses of STAT3. After treatment of CEP for 20 d, the tumor samples were taken from the sacrificed mice and immunostained with antibodies against total and phosphorylated STAT3. (A) Immunohistochemical staining of total STAT3. STAT3 nuclear localization in control group was indicated with the red arrows. (B) Immunohistochemical staining of phospho-STAT3. Original magnification: ×400.