Differentiation therapy: sesamin as an effective agent in targeting cancer stem-like side population cells of human gallbladder carcinoma

Abstract

Background: Recent studies have demonstrated that side population (SP) cells isolated from various cancer cell lines and primary tumors possess stem cell-like properties. Sesamin, a food-derived agent, possesses anti-cancer activities both in vitro and in vivo. The present study was designed to determine whether sesamin also have effects on cancer stem-like SP cells from gallbladder cancer (GBC).

Methods: In this study, we sorted SP cells by flow cytometry. SP cells were cultured and treated with sesamin. Tumor-sphere formation, colony formation, Matrigel invasion and tumorigenic potential were determined. Expression of nuclear NF-κB, IL-6, p-Stat3, Twist, E-cadherin and Vimentin was measured by Western blot, immunofluorescence staining or RT-PCR analysis. Nuclear NF-κB activity and IL-6 protein level were assessed with ELISA. Xenograft tumors were generated in nude mice.

Results: After treated with sesamin, SP cells differentiated into cells expressing the epithelial marker (E-cadherin). Sesamin effectively affected SP cells stem cell-like characteristics (i.e., tumor-sphere formation, colony-formation, Matrigel invasion), weakened the drug-resistance of SP cells and inhibited tumor growth both in vitro and in vivo. Treatment with sesamin significantly reduced the expression of nuclear NF-κB, IL-6, p-Stat3, Twist and Vimentin (a mesenchymal marker) in SP cells. Nuclear NF-κB activity and IL-6 level were also decreased after treatment with sesamin.

Conclusion: Food-derived sesamin directs the epithelial differentiation of cancer stem-like SP cells from GBC, which is associated with attenuation of NF-κB-IL-6-Stat3-Twist signal pathway.

Figure 1

SP cells analysis. The proportion of SP cells in SGC-996 and GBC-SD was analyzed by flow cytomertry. As a control, verapamil (50 μM) was added to inhibit the dye efflux activity.

Figure 2

Biological characteristics of cancer stem-like SP cells. SP cells had higher tumor-sphere formation ability than the non-SP cells under un-differentiating conditions (DMEM/F12 medium supplemented with 20 ng/mL EGF and 10 ng/mL bFGF) (A). SP cells demonstrated much higher colony formation ability (B) and invasion capacity (C). SP cells were more resistant to cisplatin (Cis) compared with non-SP cells (D and E) SP cells fraction decreased gradually under differentiating conditions (DMEM supplemented with 10% FBS) (F). *p < 0.05 vs. non-SP cells group or control group.

Figure 3

Molecular characteristics of SP cells. The protein expression of E-cadherin, Vimentin, Twist, Stat3, p-Stat3 and nuclear NF-κB was detected by Western blot analysis (A). IL-6 mRNA and protein level were measured by real-time reverse transcriptase PCR and ELISA respectively (B and C). *p < 0.05 vs. non-SP cells group.

Figure 4

Immunofluorescence staining for E-cadherin and Vimentin. The expression of Vimentin and E-cadherin in SGC-996 and GBC-SD cells, SP cells in the absence or presence of the FBS, and SP cells treatment with 100 μM sesamin for 7 days.

Figure 5

Sesamin exerted its effects on SP cells. Sesamin significantly decreased SP cells fraction in a dose-dependent manner (A). Sesamin reduced the tumor-sphere formation ability of SP cells (B). Sesamin reduced the invasion capacity of CSCs (C). Sesamin inhibited colony formation of SP cells from SGC-996 and GBC-SD (D). *p < 0.05 vs. control group. Ses: sesamin.

Figure 6

Sesamin suppressed SP cells proliferation and enhanced the chemotherapeutic effect of cisplatin (Cis). Sesamin effectively inhibited the viability of SP cells in a dose- and time-dependent manner (A and B). Sesamin manifested its chemo-sensitization effects on SP cells from both SGC-996 and GBC-SD (C). *p < 0.05 vs. control group. ^#p < 0.05 vs. Cis + Ses combined treatment group. Ses: sesamin.

Figure 7

Effects of sesamin on the protein expression of E-cadherin, Vimentin, Twist, Stat3 and p-Stat3 in SP cells from SGC-996 by Western blotting. SP cells from SGC-996 were treated sesamin for 7 days. Panels show representative bands (A) and histograms represent optical density values normalized to the corresponding β-actin (B-D) or total Stat3 (E). *p < 0.05 vs. control group. Ses: sesamin.

Figure 8

Effects of sesamin on the protein expression of E-cadherin, Vimentin, Twist, Stat3 and p-Stat3 in SP cells from GBC-SD by Western blotting. SP cells from GBC-SD were treated sesamin for 7 days. Panels show representative bands (A) and histograms represent optical density values normalized to the corresponding β-actin (B-D) or total Stat3 (E). *p < 0.05 vs. control group. Ses: sesamin.

Figure 9

Effects of sesamin on the protein expression and activity of nuclear NF-κB in SP cells. Panels show representative bands (A). Histograms represent optical density values normalized to the corresponding lamin (B), and nuclear NF-κB activity (C). *p < 0.05 vs. control group. Ses: sesamin.

Figure 10

Effects of sesamin on the mRNA expression and protein level of IL-6 in SP cells. Histograms represent IL-6 mRNA expression (A) and protein level (B). *p < 0.05 vs. control group. Ses: sesamin.

Figure 11

Effects of sesamin on SP cells-derived tumor growth in vivo. After pretreated with sesamin (100 μM) for 7 days, 1 × 10⁵ SP cells from both SGC-996 and GBC-SD were injected subcutaneously into 6-week-old nude mice (n = 6 each group). Tumor volumes were measured weekly. *p < 0.05 vs. control group. Ses: sesamin.