• Biomolecules & Therapeutics
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• 제22권6호
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• pp.540-546
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• 2014

The high mortality rates associated with cancer reflect the metastatic spread of tumor cells from the site of their origin. Metastasis, in fact, is the cause of 90% of cancer deaths. Therefore, considerable effort is being made to inhibit metastasis. In the present study, we screened ketotifen for anti-migratory and anti-invasive activities against MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer cells. Cancer cell migration and invasion were measured using multi-well chambers. Additionally, western blots were used to examine the effects of ketotifen on the expressions of CDC42, Rho, Rac, and matrix metalloproteinase 9 (MMP-9). The results showed that ketotifen dose-dependently suppressed the migration and invasion of MDA-MB-231 and HT-1080 cells. Ketotifen also suppressed the expressions of CDC42, Rac, and Rho, which, significantly, are involved in MDA-MB-231 and HT-1080 cancer cell migration. Moreover, ketotifen suppressed the expression and activity of MMP-9, which is involved in degradation of the extracellular matrix leading to invasion. The overall data suggested that ketotifen suppresses the migration and invasion of MDA-MB-231 and HT-1080 cancer cells via inhibition of CDC42, Rac, Rho, and MMP-9 expression.

• 한국식품과학회지
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• 제43권2호
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• pp.213-219
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• 2011

등골나물은 국화과 여러해살이 식물로 한방에서는 고혈압, 폐렴, 황달, 홍역, 요통 등에 사용한다고 알려져 있다. 본 연구에서는 등골나물의 꽃 부위를 추출하여 등골나물 추출물이 유방암 세포인 MDA-MB-231 세포의 이동, 침윤 및 부착에 미치는 영향을 조사하였다. 그 결과 MDA-MB-231 세포의 이동, 침윤 및 부착은 등골나물 추출물의 농도($0-20{\mu}g/mL$)가 증가할수록 현저하게 감소하였다. 등골나물 추출물은 MMP-9, MMP-2의 활성을 억제하였고, TIMP-1의 발현은 감소시킨 반면 TIMP-2의 발현은 증가시켰다. 또한, 등골나물 추출물은 uPA, VEGF 그리고 ICAM의 mRNA 및 단백질 수준을 현저히 감소시켰다. 특히, 등골나물 헥산 분획물이 유방암세포의 이동을 현저하게 억제하였다. 이상의 결과로부터 등골나물 추출물은 MMP-9, MMP-2, uPA, TIMP-1 및 ICAM의 감소, TIPM-2의 증가를 통해 유방암세포의 전이를 억제하는 것으로 판단된다. 따라서 본 연구는 이러한 효능을 지닌 등골나물 추출물을 암전이에 효과가 있는 암예방제나 항암제로 개발할 수 있는 가능성을 제시한다.

• 생명과학회지
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• 제24권1호
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• pp.8-13
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• 2014

여성에게 특히 높은 발병률을 보이는 유방암은 다양한 인자에 의해 암세포 침투와 전이가 발생한다. 그중에서 MMP-9은 유방암에서 높은 발현율을 나타내며, 유방암 세포의 침투와 전이에 주요하게 작용한다. MMP-9은 다양한 성장인자와 사이토카인, PMA 등에 의해 발현이 유도되며 세포 내 자연적으로 발생된 내생적 억제인자인 TIMP-1과 -2에 의해 그 활성 및 발현이 조절된다. MMP-9의 활성 및 발현을 저해하기 위해 사용한 물질은 모링가 추출물로 모링가의 모든 부위가 다양한 질환을 치료하는 것으로 알려져 있다. 이 연구에서는 대표적으로 모링가씨, 뿌리, 잎, 열매 추출물질을 이용하여 MMP-9의 활성억제 유무를 연구하였으며, MCF-7 세포에서 모링가 뿌리추출물이 MMP-9을 가장 효과적으로 억제하였다. 또한 MDA-MB-231을 이용한 비교실험에서도 세포특이성 없이 MMP-9의 활성 및 발현이 모링가 뿌리 추출물에 의해 억제되었고 단백질과 mRNA 발현에서도 동일한 결과가 도출되었다. 뿐만 아니라 암세포 침투실험에서도 모링가 뿌리 추출물에 의해 암세포 침투가 50% 이상 감소 되었으나, TIMP-1과 -2 mRNA 발현은 모링가 뿌리 추출물에 의해 저해되지 않았으므로 모링가 뿌리 추출물이 MMP-9 활성 및 발현 저해를 통해 암세포 침투를 저해시키는 것으로 예상된다. 그리고 MMP-9 활성 및 발현 조절기전을 확인하기 위해 MAP kinase의 인산화를 확인한 실험에서 모링가 뿌리 추출물이 ERK와 JNK의 인산화를 억제시킴을 확인하였다. 결론적으로 이 실험을 통해 모링가 추출물 중 뿌리 추출물이 MMP-9 활성 및 발현저해에 가장 효과적이며, ERK와 JNK의 인산화 조절을 통해 MMP-9의 활성 및 발현을 직접적으로 조절함을 확인할 수 있었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.402-407
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• 2006

Vascular endothelial cells release proteinases that degrade the extracellular matrix (ECM), thus enabling cell migration during angiogenesis and vasculogenesis. Sildenafil citrate stimulates the nitric oxide-cyclic guanosine monophosphate pathway through inhibition of phosphodiesterase type V (PDE5). In this report, we examined the mechanisms underlying sildenafil citrate-induced cell migration using cultured mouse aortic endothelial cells (MAECs). Sildenafil citrate induced migration and proteinase secretion by murine endothelial cells. Sildenafil citrate induced the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9, which is inhibited by $NF-{\kappa}B$ inhibitors. Sildenafil citrate also induced the secretion of plasmin, which is inhibited by PI 3'-kinase inhibitors. It is suggested that sildenafil citrate-induced migrating activity in endothelial cells may be accomplished by increased secretion of proteinases.

• Biomolecules & Therapeutics
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• 제20권3호
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• pp.286-292
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• 2012

All-trans retinoic acid (ATRA) is currently used in adjuvant differentiation-based treatment of residual or relapsed neuroblastoma (NB). It has been reported that short-term ATRA treatment induces migration and invasion of SH-SY5Y via transglutaminase-2 (Tgase-2). However, the detailed mechanism of Tgase-2's involvement in NB cell invasion remains unclear. Therefore we investigated the role of Tgase-2 in invasion of NB cells using SH-SY5Y cells. ATRA dose-dependently induced the invasion of SH-SY5Y cells. Cystamine (CTM), a well known tgase inhibitor suppressed the ATRA-induced invasion of SH-SY5Y cells in a dose-dependent manner. Matrix metalloproteinase -9 (MMP-9) and MMP-2, well known genes involved in invasion of cancer cells were induced in the ATRA-induced invasion of the SH-SH5Y cells. Treatment of CTM suppressed the MMP-9 and MMP-2 enzyme activities in the ATRA-induced invasion of the SH-SY5Y cells. To confirm the involvement of Tgase-2, gene silencing of Tgase-2 was performed in the ATRA-induced invasion of the SH-SH5Y cells. The siRNA of Tgase-2 suppressed the MMP-9 and MMP-2 activity of the SH-SY5Y cells. MMP-2 and MMP-9 are well known target genes of NF-${\kappa}B$. Therefore the relationship of Tgase-2 and NF-${\kappa}B$ in the ATRA-induced invasion of the SH-SY5Y cells was examined using siRNA and CTM. ATRA induced the activation of NF-${\kappa}B$ in the SH-SY5Y cells and CTM suppressed the activation of NF-${\kappa}B$. Gene silencing of Tgase-2 suppressed the MMP expression by ATRA. These results suggested that Tgase-2 might be a new target for controlling the ATRA-induced invasion of NBs.

• 대한화장품학회:학술대회논문집
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• 대한화장품학회 2003년도 IFSCC Conference Proceeding Book I
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• pp.590-600
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• 2003

The matrix metalloproteinases (MMPs) are a family of enzymes responsible for degrading connective tissue. MMPs catalyze the breakdown of collagen from the extracellular matrix, leading to wrinkle formation and accelerated skin aging. Furthermore, ultraviolet irradiation causes increased expression of certain MMPs. In the extracellular matrix turnover, MMPs are interacting with endogenous regulators named tissue inhibitors of metalloproteinases (TIMPs). Using peptide substrate assays, it has been demonstrated that TIMP-MMP complexes interact highly specifically with $K_{i}$ values of 10$^{-9}$ -10$^{-16}$ M. Therefore applications for TIMP as inhibitor of collagen degradation are suggested for cosmetic anti-aging products to prevent wrinkle formation and loss of elasticity. To date four TIMP proteins (TIMP-1, TIMP-2, TIMP-3 and TIMP-4) have been identified which show a high degree in sequence similarity. The production of human TIMP-2, a 194-residue nonglycosylated protein, was performed by fed-batch culture of Escherichia coli. TIMP-2 accumulated in the bacterial cells in an insoluble form as inclusion bodies. The inclusion bodies were solubilized and the protein refolded to yield the native TIMP-2 in the active form. The integrity of the protein was confirmed by mass analysis, Edman sequencing and gel shift experiments with authentic samples. The inhibitory activity of the refolded and purified TIMP-2 was demonstrated with MMP-1 and MMP-2 assays using synthetic fluorogenic peptide substrates.s.

• 환경생물
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• 제27권1호
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• pp.58-65
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• 2009

Naringenin은 flavonoid구조의 감귤류 과피에 다량 함유되어 있으며 항암 및 항산화 등의 다양한 생리활성을 가지는 것으로 보고되었다. 이에 본 연구에서는 HT-1080 섬유육종세포의 전이에 대한 영향을 조사하였다. 먼저 Naringenin이 암세포의 이동에 미치는 영향을 알아보기 위해 migrationassay를 한 결과, Naringenin이 암세포의 이동을 농도 의존적으로 억제시켰다. 암의 전이에 있어서 매우 중요한 역할을 하는 단백질분해효소인 matrix metalloproteinase-9 (MMP-9)의 활성도를 측정하기 위해 gelatin zymography를 한 결과, Naringenin이 PMA에 의해 증가된 MMP-9의 효소 활성도를 농도 의존적으로 감소시켰다. 또한 MMP-9와 TIMP-1의 유전자 발현에 대한 Naringenin의 영향을 RT-PCR방법으로 조사한 결과, Naringenin이 PMA에 의해 증가된 MMP-9의 mRNA발현을 농도 의존적으로 감소시켰으나 TIMP-1의 mRNA발현에는 변화가 없었다. MMP-9발현 감소에 관여하는 전사조절인자를 확인하기 위해 promoterassay를 한 결과, Naringenin이 PMA에 의해 증가된 MMP-9 및 NF-$\kappa$B, AP-1의 Promoter활성을 농도 의존적으로 감소시 켰다. 또한 MMP-9의 전자조절인자인 NF-$\kappa$B와 AP-1의 결합 활성도를 electrophoretic mobility shift assay로 확인한 결과 Naringenin이 PMA에 의해 증가된 NF-$\kappa$B와 AP-1의 결합 활성도를 농도 의존적으로 감소시켰다. 결론적으로 Naringenin이 전사조절인자인 NF-$\kappa$B와 AP-1의 활성을 억제함으로써 MMP-9의 유전자 발현 및 효소 활성을 억제하고 그 결과 암세포의 이동과 침윤을 억제하는 것을 알 수 있다.

• Biomolecules & Therapeutics
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• 제22권5호
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• pp.414-419
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• 2014

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that regulate cell-matrix composition and are also involved in processing various bioactive molecules such as cell-surface receptors, chemokines, and cytokines. Our group recently reported that MMP-3, -8, and -9 are upregulated during microglial activation and play a role as proinflammatory mediators (Lee et al., 2010, 2014). In particular, we demonstrated that MMP-8 has tumor necrosis factor alpha (TNF-${\alpha}$)-converting enzyme (TACE) activity by cleaving the prodomain of TNF-${\alpha}$ and that inhibition of MMP-8 inhibits TACE activity. The present study was undertaken to compare the effect of MMP-8 inhibitor (M8I) with those of inhibitors of other MMPs, such as MMP-3 (NNGH) or MMP-9 (M9I), in their regulation of TNF-${\alpha}$ activity. We found that the MMP inhibitors suppressed TNF-${\alpha}$ secretion from lipopolysaccharide (LPS)-stimulated BV2 microglial cells in an order of efficacy: M8I>NNGH>M9I. In addition, MMP inhibitors suppressed the activity of recombinant TACE protein in the same efficacy order as that of TNF-${\alpha}$ inhibition (M8I>NNGH>M9I), proving a direct correlation between TACE activity and TNF-${\alpha}$ secretion. A subsequent pro-TNF-${\alpha}$ cleavage assay revealed that both MMP-3 and MMP-9 cleave a prodomain of TNF-${\alpha}$, suggesting that MMP-3 and MMP-9 also have TACE activity. However, the number and position of cleavage sites varied between MMP-3, -8, and -9. Collectively, the concurrent inhibition of MMP and TACE by NNGH, M8I, or M9I may contribute to their strong anti-inflammatory and neuroprotective effects.

• 생명과학회지
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• 제15권2호
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• pp.231-235
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• 2005

기질 금속단백분해효소(MMP)는 기저막이나 간질성 조직 등에 있는 세포외기질 성분을 분해하여 상처 치유, 태아의 발생, 종양세포의 침윤과 전이 등을 포함하여 조직이 재구성되는 과정에서 생리학적 및 병리학적인 과정 양쪽 모두에 매우 중요한 역할을 한다. 본 연구에서는 MMP-9 유전자의 발현을 억제하는 물질을 천연물에서 탐색하였고, 탐색된 시료 중에서 높은 역가를 가진 낙우송과의 Metasequoia glyptostroboides가 선택되었다. 여러 가지 flavonoid 성분을 가지고 있는 Metasequoia glyptostroboides를 이용하여 4개의 biflavonoid와 2개의 monoflavonoid 구조의 물질을 분리하였고, 이 flavonoid들은 sciadopitysin, isoginkgetin, bilobetin, 2, 3-dihydrohino-kiflavone, luteolin, apigenin으로 정제하여 구조를 밝히고 zymography, WST-1을 이용한 세포독성시험, northern blot 등의 실험을 통하여 각 화합물의 구조적인 특성과 함께 MMP-9 유전자 발현을 억제하는 효과가 있는 것을 확인하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권7호
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• pp.2719-2724
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• 2015

Objective: The aim of this study was to investigate the clinical significance of annexin a1 (ANXA1) and provide molecular evidence to support that decreased ANXA1 expression could enhance cancer migration and invasion in pancreatic ductal adenocarcinoma (PDAC). Materials and Methods: Immunohistochemistry of a tissue microarray with 162 surgically resected PDAC specimens was performed to examine the expression of ANXA1. We also investigated the relationship between ANXA1 expression and clinicopathological factors and prognosis of PDAC patients. We further studied the role of ANXA1 in PDAC cell proliferation, migration and invasion by cell proliferation assay, migration assay and matrigel invasion assay with reduced ANXA1 expression by RNAi. Western blotting was used to detect matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression. We also detected MMP-9 enzyme activity by gelatin zymography. Results: Decreased expression of ANXA1 was significantly associated with poor differentiation, lymph node metastasis and advanced TNM stage of PDAC patients (p<0.05). Moreover, decreased expression of ANXA1 was correlated with poor survival (p<0.05). Furthermore, we found that ANXA1 knockdown inhibited cell proliferation, induced G1 phase cell cycle arrest, increased PDAC cell migration and invasion capacity compared with controls. In addition, Western blotting showed that ANXA1 knockdown increased the MMP-9 protein level and decreased TIMP-1 expression. Gelatin zymography showed that MMP-9 enzyme activity was also elevated. Conclusions: Negative ANXA1 expression is a most unfavorable prognostic factor for PDAC patients. ANXA1 knockdown inhibits cell proliferation by inducing G1 phase cell cycle arrest and increases migration and invasion of PDAC cells through up-regulating MMP-9 expression and activity, implying that ANXA1 may serve as a promising prognostic biomarker and therapeutic target for PDAC.