HT-1080 세포주에서 좀보리사초 추출물의 MMP-2와MMP-9 활성 억제효과
Inhibitory Effects of Carex pumila Extracts on MMP-2 and MMP-9 Activities in HT-1080 Cells
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Matrix metalloproteinases (MMPs) are associated with the invasion and metastasis of malignant tumors composed of cancer cells in an increased state of expression. This study evaluates the inhibitory effect of Carex pumila on MMP-2 and MMP-9 activity in phorbol-12-myristate-13-acetate (PMA)- stimulated HT-1080 human fibrosarcoma cells using gelatin zymography, MMPs enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay. C. pumila was extracted twice with dichloromethane (CH2Cl2) and methanol (MeOH). Treatment with CH2Cl2 extract and MeOH extract in PMA-stimulated HT-1080 cells effectively reduced the production of MMP-2 and 9. Also, the combined crude extracts (CH2Cl2 and MeOH) significantly inhibited the enzymatic activities and the expression of MMP-2 and MMP-9 in mRNA and protein levels. The combined crude extracts were partitioned between CH2Cl2 and water. The organic layer was further fractionated with n-hexane, 85% aqueous methanol (85% aq.MeOH) and the aqueous layer was separated into n-butanol and water, successively. Of the fractions, 85% aq.MeOH fraction showed the highest inhibitory activity of MMP-2 and MMP-9 in gelatin zymography and MMP ELISA kit. Furthermore, 85% aq.MeOH fraction most significantly suppressed cell migration. In RT-PCR and Western blot assay, nbutanol and 85% aq.MeOH fractions exerted the greatest inhibition on mRNA and protein expression of MMP-2 and MMP-9, respectively. As a result, C. pumila can be used as a good anti-invasive agent source.
Abstract
Matrix metalloproteinases (MMPs) are associated with the invasion and metastasis of malignant tumors composed of cancer cells in an increased state of expression. This study evaluates the inhibitory effect of Carex pumila on MMP-2 and MMP-9 activity in phorbol-12-myristate-13-acetate (PMA)- stimulated HT-1080 human fibrosarcoma cells using gelatin zymography, MMPs enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay. C. pumila was extracted twice with dichloromethane (CH2Cl2) and methanol (MeOH). Treatment with CH2Cl2 extract and MeOH extract in PMA-stimulated HT-1080 cells effectively reduced the production of MMP-2 and 9. Also, the combined crude extracts (CH2Cl2 and MeOH) significantly inhibited the enzymatic activities and the expression of MMP-2 and MMP-9 in mRNA and protein levels. The combined crude extracts were partitioned between CH2Cl2 and water. The organic layer was further fractionated with n-hexane, 85% aqueous methanol (85% aq.MeOH) and the aqueous layer was separated into n-butanol and water, successively. Of the fractions, 85% aq.MeOH fraction showed the highest inhibitory activity of MMP-2 and MMP-9 in gelatin zymography and MMP ELISA kit. Furthermore, 85% aq.MeOH fraction most significantly suppressed cell migration. In RT-PCR and Western blot assay, nbutanol and 85% aq.MeOH fractions exerted the greatest inhibition on mRNA and protein expression of MMP-2 and MMP-9, respectively. As a result, C. pumila can be used as a good anti-invasive agent source.
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