Anthocyanins from the Fruit of Vitis Coignetiae Pulliat Inhibit TNF-Augmented Cancer Proliferation, Migration, and Invasion in A549 Cells

Abstract

Objective: Anthocyanins belong to a class of flavonoids, exhibiting antioxidant and anti-inflammatory actions have been reported to have anti-cancer effects. Here, we investigated whether anthocyanins can inhibit cancer cell proliferation, invasion, and angiogenesis in human lung cancer A549 cells, which are critically involved in cancer metastasis. Methods: We used anthocyanins from fruits of Vitis coignetiae Pulliat (AIMs) which has been used in Korean folk medicine for the treatment of inflammatory diseases and cancers. We have performed cell proliferation assays, cell invasion assay, gelatin zymography, wound healing assay and western blotting to examine whether anthocyanins can inhibit cancer cell proliferation, invasion, and angiogenesis in A549 cells. Result: AIMs did not inhibit cancer cell proliferation on A549 cells. Also, AIMs suppressed cancer migration, and invasion by supressing MMP-2 and MMP-9 expression. The Immuno-blotting results also revealed that AIMs suppressed the proteins involved in cancer proliferation (COX- 2, C-myc, cyclin D1), migration and invasion (MMP-2, MMP-9), anti-apoptosis (XIAP, and c-IAP2), adhesion and angiogenesis (ICAM-1, VEGF). Conclusion: This study demonstrates that the anthocyanins isolated from fruits of Vitis coignetiae Pulliat inhibit cancer proliferation, cancer migration, and invasion that is involve in cancer-metastasis. This study provides evidence that AIMs might have anti-cancer effects on human lung cancer.

Keywords: Anthocyanins; Vitis coignetiae Pulliat; invasion; anti-cancer effects; lung cancer.

Figure 1

The Inhibitory Effects of Aims on Cancer Cell Proliferation of A549 Lung Cancer Cell. (A) Inhibitory effects of AIMs on cell proliferation were assessed by MTT assay. Cells were treated with indicated concentrations of AIMs for 24h. (B) AIMs greatly influenced the TNF-stimulated cell invasion of A549 cells. (C) A549 cells were treated with TNF. Conditioning medium was used for the measurement of secreted MMP-2 and MMP-9 protein levels by gelatin zymography. The enzyme activities of MMP-2 and MMP-9 in treated cells are expressed as a percentage of their activities in untreated cells. Data are mean ± SD values of three independent experiments. *P< 0.05 versus control.

Figure 2

Inhibitory Effects of Aims on Cancer Cell Migration of A549 Lung Cancer Cells. (A) Cells were grown to 100% confluency on 30-mm cell culture dishes coated with rat tail collagen and then treated with or without AIMs (400 μg/ml). For the group treated with AIMs and TNF, cells were pre-treated with AIMs for 1 hour and then treated with TNF (10 ng/mL). A scratch was made through the cell layer using a pipette tip. After washing with PBS, serum-free media with or without AIMs was added. Photographs of the wounded area were taken at the interval of 0 h, 12 h, 24 h, and 48 h after the scratch to evaluate cell movement into the wounded area.

Figure 3

Inhibitory Effects of Aims on the Metastasis-Related Protein Expression of TNF-Stimulated A549 Cells. (A) AIMs greatly influenced the TNF-stimulated cell invasion of A549 cells. (B) A549 cells were treated with TNF. Whole-cell extracts were prepared (30 mg) and subjected to western blot analysis. (C) Conditioning medium was used for the measurement of secreted MMP-2 and MMP-9 protein levels by gelatin zymography. The enzyme activities of MMP-2 and MMP-9 in treated cells are expressed as a percentage of their activities in untreated cells. Data are mean ± SD values of three independent experiments. *P < 0.05 versus control.

Figure 4

Inhibitory Effects of Aims on the Proteins Involved in Cancer Proliferation, Anti-Apoptosis, Migration, Invasion, and Angiogenesis. Cells (5×10⁴ cells), either left untreated or pre-treated with AIMs for 1 h, were exposed to TNF (10 nM) for indicated times. Whole-cell extracts were prepared, and 30 μg of the whole-cell lysate was analysed by Western blot using antibodies against various TNF-stimulated proteins involved in (A) cancer cell proliferation, (B) anti-apoptosis, and (C) invasion & angiogenesis

Figure 5

Schematic Representation of the Anti-Cancer Effects of Aims on A549 Lung Cancer Cells. Following treatment of TNF, AIMs inhibited the TNF-stimulated proteins involved in cancer cell proliferation, anti-apoptosis, invasion, adhesion, and angiogenesis. Taken together, these data suggested that AIMs may exert anti-metastatic effects on human lung cancer