Scutellaria barbata D. Don inhibits migration and invasion of colorectal cancer cells via suppression of PI3K/AKT and TGF-β/Smad signaling pathways

Abstract

Metastasis is one of the most aberrant behaviors of cancer cells. Patients with cancers, including colorectal cancer (CRC), have a higher risk of tumor recurrence and cancer-related mortality once metastasis is diagnosed. Existing treatment strategies fail to cure cancer mostly due to the onset of metastasis. Therefore, metastasis remains a challenge in cancer treatment. Some complementary and alternative medical therapies using traditional Chinese medicine have been demonstrated to be clinically effective in cancer treatment. Scutellaria barbata D. Don (SB) is a promising medicinal herb. It was previously reported that the ethanol extract of SB (EESB) is able to promote apoptosis, and inhibit cell proliferation and angiogenesis in human colon cancer cells. However, the anticancer effect of SB and the underlying mechanism require further investigation, particularly its role against metastasis. To further elucidate the antimetastatic effect of SB, MTT and Transwell assays were used in the present study to evaluate the effect of EESB on the proliferation, migration and invasion of the CRC cell line HCT-8. In addition, western blot analysis was performed to detect the expression of matrix metalloproteinases (MMPs), cadherins and other metastasis-associated proteins. EESB significantly reduced HCT-8 cell viability and attenuated the migration and invasion ability of HCT-8 cells in a dose-dependent manner. In addition, EESB decreased the expression of MMP-1, MMP-2, MMP-3/10, MMP-9 and MMP-13, and proteins in the phosphoinositide 3-kinase (PI3K)/AKT and transforming growth factor (TGF)-β/Smad pathways, but not the epithelial-mesenchymal transition (EMT)-related factors E-cadherin and N-cadherin. In conclusion, the results suggested that SB inhibits CRC cell metastasis via the suppression of PI3K/AKT and TGF-β/Smad signaling pathways, which may represent a mechanism by which SB exerts an anticancer effect.

Keywords: PI3K/AKT pathway; Scutellaria barbata D. Don; TGF-β/Smad pathway; colorectal cancer; invasion; migration.

Figure 1.

Effect of EESB on HCT-8 cell viability. Cells were treated with EESB at different concentrations for 24 and 48 h. Cell viability was measured via MTT assay. Data were normalized to the viability of control cells (100%). Data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 vs. the control cells. EESB, ethanol extract of Scutellaria barbata D. Don.

Figure 2.

Effect of EESB on HCT-8 cell density. HCT-8 cells were treated with EESB at various concentrations for 24 h. Density changes were observed using phase-contrast microscopy. Photographic images were captured at a magnification of ×200. Images are representative of three independent experiments. EESB, ethanol extract of Scutellaria barbata D. Don.

Figure 3.

Effect of EESB on HCT-8 cell migration and invasion. HCT-8 cells were treated with EESB at the indicated concentrations for 24 h. The (A and B) migration and (C and D) invasion of HCT-8 cells were determined using Transwell cell culture chambers and Transwell cell culture chambers with Matrigel matrix-coated membranes, respectively. (A) Migrated and (C) invaded cells were stained using crystal violet, and images were captured at a magnification of ×200. The average number of (B) migrated cells and (D) invaded cells were counted in three randomly selected fields. Data were normalized to the migration and invasion of control cells (100%). Data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 vs. the control cells. EESB, ethanol extract of Scutellaria barbata D. Don.

Figure 4.

Effect of EESB on MMP and E-/N-cadherin expression. Cells were treated with EESB at different concentrations for 24 h. (A) MMP, E-cadherin and N-cadherin protein expression levels in HCT-8 cells were determined using western blotting. β-actin was used as the internal control. Images are representatives of three independent experiments. (B) Densitometric analysis. Data are expressed as the mean ± standard deviation and were normalized to the mean protein expression of untreated control (100%). *P<0.05. vs. the control cells. EESB, ethanol extract of Scutellaria barbata D. Don; MMP, matrix metalloproteinase; E-cad, E-cadherin; N-cad, N-cadherin.

Figure 5.

Effect of EESB on the activation of PI3K/AKT and TGF-β/Smad signaling pathways. Cells were treated with EESB at different concentrations for 24 h. (A) PI3K/AKT and TGF-β/Smad protein expression levels were determined by western blotting. β-actin was used as the internal control. Images are representatives of three independent experiments. Densitometric analysis for (B) PTEN, p-PI3K, PI3K, p-AKT and AKT and (C) TGF-β, Smad2/3 and Smad4. Data are expressed as the mean ± standard deviation and were normalized to the mean protein expression of the untreated control (100%). *P<0.05. vs. the control cells. EESB, ethanol extract of Scutellaria barbata D. Don; PI3K, phosphoinositide 3-kinase; TGF-β, transforming growth factor-β; PTEN, phosphatase and tensin homolog; p, phospho.