Methanol extract of Codium fragile inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 and invasiveness of MDA-MB-231 cells by suppressing nuclear factor-κB activation

Objective: To evaluate whether the methanol extract of Codium fragile (MECF) regulates tumor necrosis factor-alpha (TNF-alpha)-induced invasion of human breast cancer MDA-MB-231 cells by suppressing matrix metalloproteinase-9 (MMP-9). Methods: Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were performed to analyze the expression of MMP-9 and nuclear factor-kappa B (NF-kappa B) subunits, p65 and p50, and I kappa B in MDA-MB-231 cells. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used for cell viability. MMP-9 activity and invasion were measured by gelatin zymography and a matrigel invasion assay, respectively. NF-kappa B activity was measured by an electrophoretic mobility shift assay and luciferase activity. Results: MECF had no effect on cell viability up to a concentration of 100 mg/mL in human breast cancer MDA-MB-231 cells regardless of the presence of TNF-alpha. MDA-MB-231 cells that were stimulated with TNF-alpha showed a marked increase of invasion compared to the untreated control, whereas pretreatment with MECF downregulated the TNF-alpha-induced invasion of MDA-MB-231 cells. Additionally, zymography, western blot analysis, and RT-PCR confirmed that MECF decreased TNF-alpha-induced MMP-9 expression and activity which is a key regulator for cancer invasion. According to an electrophoretic morbidity shift assay, pretreatment with MECF in MDA-MB-231 cells significantly decreased the TNF-alpha-induced DNA-binding activity of NF-kappa B, which is an important transcription factor for regulating cancer invasion-related genes such as MMP-9. Furthermore, treatment with MECF sustained the expression of p65 and p50 in response to TNF-alpha in the cytosolic compartment. The luciferase assay demonstrated that MECF attenuated TNF-alpha-induced NF-kappa B luciferase activity. Conclusion: MECF exhibited its anti-invasive capability by downregulating TNF-alpha-induced MMP-9 expression, resulting from the suppression of NF-kappa B activity in the human breast cancer cell line MDA-MB-231.