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-α (TNF-α)-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-κB (NF-κB) subunits, p65 and p50, and IκB 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-κB 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 μg/mL in human breast cancer MDA-MB-231 cells regardless of the presence of TNF-α. MDA-MB-231 cells that were stimulated with TNF-α showed a marked increase of invasion compared to the untreated control, whereas pretreatment with MECF downregulated the TNF-α-induced invasion of MDA-MB-231 cells. Additionally, zymography, western blot analysis, and RT-PCR confirmed that MECF decreased TNF-α-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-α-induced DNA-binding activity of NF-κB, 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-α in the cytosolic compartment. The luciferase assay demonstrated that MECF attenuated TNF-α-induced NF-κB luciferase activity.


CONCLUSIONS
MECF exhibited its anti-invasive capability by downregulating TNF-α-induced MMP-9 expression, resulting from the suppression of NF-κB activity in the human breast cancer cell line MDA-MB-231.
Department of Marine Life Sciences, Jeju National University, Jeju-si 63243, Republic of Korea.
Matharage Gayani
MG
Dilshara
Department of Marine Life Sciences, Jeju National University, Jeju-si 63243, Republic of Korea.
Rajapaksha Gedara Prasad Tharanga
RG
Jayasooriya
Department of Marine Life Sciences, Jeju National University, Jeju-si 63243, Republic of Korea; Nakdonggang National Institute of Biological Resource, Sangju-si, Gyeongsangbuk-do 37242, Republic of Korea.
Chang-Hee
CH
Kang
Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan 47340, Republic of Korea.
Yung-Hyun
YH
Choi
Department of Marine Life Sciences, Jeju National University, Jeju-si 63243, Republic of Korea. Electronic address: immunkim@jejunu.ac.kr.
Gi-Young
GY
Kim
Codium fragile; Invasion; Matrix metalloproteinase-9; Nuclear factor-κB; Tumor necrosis factor-α