Saikosaponin-d Enhances the Anticancer Potency of TNF-α via Overcoming Its Undesirable Response of Activating NF-Kappa B Signalling in Cancer Cells

Abstract

Tumor necrosis factor-alpha (TNF- α ) was reported as anticancer therapy due to its cytotoxic effect against an array of tumor cells. However, its undesirable responses of TNF- α on activating NF- κ B signaling and pro-metastatic property limit its clinical application in treating cancers. Therefore, sensitizing agents capable of overcoming this undesirable effect must be valuable for facilitating the usage of TNF- α -mediated apoptosis therapy for cancer patients. Previously, saikosaponin-d (Ssd), a triterpene saponin derived from the medicinal plant, Bupleurum falcatum L. (Umbelliferae), showed to exhibit a variety of pharmacological activities such as antiinflammation, antibacteria, antivirus and anticancer. Recently, we found that Ssd could inhibit the activated T lymphocytes via suppression of NF- κ B, NF-AT and AP-1 signaling. Here, we showed that Ssd significantly potentiated TNF- α -mediated cell death in HeLa and HepG2 cancer cells via suppression of TNF- α -induced NF- κ B activation and its target genes expression involving cancer cell proliferation, invasion, angiogenesis and survival. Also, Ssd revealed a significant potency of abolishing TNF- α -induced cancer cell invasion and angiogenesis in HUVECs while inducing apoptosis via enhancing the loss of mitochondrial membrane potential in HeLa cells. Collectively, these findings indicate that Ssd has a significant potential to be developed as a combined adjuvant remedy with TNF- α for cancer patients.

Figure 1

Inducing effect of Ssd on the TNF-α-mediated cell death through potentiating the loss of mitochondrial membrane potential (ΔΨm). (a) Ssd increases the percentage of cancer cell death in the presence of TNF-α. HeLa and HepG2 cancer cells were treated with 10 μM of Ssd with or without 20 ng/mL TNF-α for 24 h. The LIVE/DEAD Cell Imaging Kit (Life Technologies) reagents were mixed and added to the cells for 15 min. The cells were subjected to fluorescence imaging using Olympus IX71-Applied Precision DeltaVision restoration microscope. Cells with green fluorescence signal represent live cells, while cells with red fluorescence signal represent dead cells. The percentage of dead cells was quantified for HeLa and HepG2 and indicated in bar chart. (b) Ssd enhances the (ΔΨm) loss in the presence of TNF-α. HeLa cells were treated with 10 μM of Ssd with or without 20 ng/mL TNF-α for 4 h, the cells were then stained with 2 μM of JC-1 for 30 min. Fluorescence images were captured using Olympus IX71-Applied Precision DeltaVision restoration microscope and the percentage of cells with (ΔΨm) loss was quantified and indicated in bar chart. Orange-fluorescent signal indicates the cells with hyperpolarized membrane potentials, while green-fluorescent signal indicates the cells with depolarized membrane potentials. **P < 0.01 and ***P < 0.001. Three independent experiments were performed, and more than 5000 cells were scored from 5 different views of captured images.

Figure 2

Suppressive effect of Ssd on the TNF-α-induced NF-κB signaling. (a) Effect of Ssd on TNF-α-induced degradation of IκBα. Upper panel, TNF-α-induced the degradation of IκBα; lower panel, Ssd inhibited TNF-α induced degradation of IκBα. HepG2 cells were pretreated with indicated concentrations of Ssd for 60 min and then incubated with 20 ng/mL TNF-α at 37°C for 15 min. The IκBα in cytosolic extracts was detected by Western blotting. (b) Effect of Ssd on TNF-α-induced phosphorylation and nuclear translocation of NF-κB p65. Upper panel, Ssd inhibited TNF-α-induced nuclear translocation of NF-κB p65; lower panel, Ssd inhibited TNF-α induced phosphorylation of NF-κB p65. HepG2 cells were preincubated with indicated concentrations of Ssd for 60 min, and then the cells were treated with 20 ng/mL TNF-α for 15 min. The cytosolic and nuclear extracts were harvested for detection of p65, while the total cell extracts were harvested for the detection of phosphorylated form of p65. (c) Immunocytochemical analysis of NF-κB p65 nuclear translocation. HeLa cells were pretreated with 10 μM Ssd for 60 min, and then cells were treated with TNF-α for 30 min. Cells were fixed in 4% paraformaldehyde and costained with anti-p65 antibodies (green) and DAPI (blue). Arrows indicate the cells with p65 being translocated into nucleus under magnification 400x. (d) Cytotoxicity of Ssd with or without TNF-α in constitutively active IKK-β transfected HepG2 cells. HepG2 cells were transfected with or without c.IKK-β plasmid being subjected to Ssd treatment in the presence or absence of TNF-α (20 ng/mL) for 72 h. The cell viability was determined by MTT assay. Results are mean ± SD from three independent experiments.

Figure 3

Down-regulatory effect of Ssd on the TNF-α-induced NF-κB-dependent genes expression. (a) Ssd downregulates TNF-α-induced NF-κB dependent cell proliferative genes expression. (b) Ssd downregulates TNF-α-induced NF-κB-dependent invasive genes expression. (c) Ssd downregulates TNF-α-induced NF-κB-dependent angiogenic genes expression. HepG2 and HeLa cells were pretreated with 20 ng/mL TNF-α for 60 min and then subjected to 10 μM of Ssd treatment as indicated in time intervals, and the whole cell extracts were prepared and analyzed by Western blotting. Gel images are representative of three independent experiments.

Figure 4

Diminishing effect of Ssd on the TNF-α-inducted antiapoptotic genes expression. (a) Ssd abates TNF-α-mediated NF-κB-dependent anti-apoptotic genes expression in cancer cells. HepG2 and HeLa cells were pretreated with 20 ng/mL TNF-α for 60 min and then subjected to 10 μM of Ssd treatment for indicated time intervals, and the whole cell extracts were prepared and analyzed by Western blotting. (b) Ssd induces the cleavage of procaspases in cancer cells. HepG2 and HeLa cells were treated with indicated concentrations of Ssd (7.5, 10, 12.5 or 15 μM) for 24 h. The whole cell extracts were then prepared analyzed by Western blot using antibodies against procaspases 3, 7, and 9; while actin was used as loading control. Gel images are representative of three independent experiments.

Figure 5

Inhibitory effect of Ssd on the TNF-α-induced cancer cell invasion. (a) Ssd suppresses the TNF-α-mediated cell invasion in H1299 cells. Images of invasive cells found in the lower layer of ECMatrix chamber were captured by digital camera under microscope with 100X magnification. (b) The number of invasive cells was counted in the lower layer of ECMatrix chamber membrane. ***P < 0.001 for comparison between medium control and TNF-α treatment alone and for Ssd treatment compared to TNF-α treatment alone. (c) The absorbance reading at OD 560 nm of the stained invasive cells solute. **P < 0.01 and ***P < 0.001 compared to medium control and TNF-α treatment alone, respectively. (d) Cytotoxicity of Ssd on H1299 cells. Results are mean ± SD from three independent experiments.

Figure 6

Suppressive effect of Ssd on the HUVEC's tube formation and cell migration. (a) Cytotoxicity assay of Ssd in HUVECs. (b) Cytotoxicity assay of Ssd in CCD19Lu. (c) Ssd inhibits the formation of endothelial cells (EC) tube networks in HUVECs. The HUVECs were incubated with different concentrations of Ssd for 30 min at room temperature before seeding. DMSO or Ssd-treated HUVECs were seeded on polymerized Matrigel in the presence of VEGF; the images of EC tube networks were captured after incubation for 8 h at 37°C. (d) The numbers of the tube formed were quantified under light microscope. (e) Ssd inhibits the cell migration in HUVECs. The HUVECs were incubated onto gelatin-coated plates for 24 h. An artificial wound was created and the denuded area in each well was captured using Motic Image Plus microscope. HUVECs were then incubated with 5 or 10 μM of Ssd for further 16 or 24 h and the denuded areas were captured and analyzed using Java's Image J software. (f) The migration of cells towards the wounds was expressed as percentage of wound closure for 16 and 24 h. Results are presented from three independent experiments.