염주괴불주머니 분획물의 MMP-2, MMP-9 발현 억제 효과

MMP-2 and MMP-9 Inhibitory Effects of Different Solvent Fractions from Corydalis heterocarpa

Abstract

염주괴불주머니(Corydalis heterocarpa)는 두해살이 풀로 노란 꽃이 피고 염주처럼 잘록한 열매를 맺는 것이 특징이다. 또한 우리나라 갯벌에서 서식하는 염생식물로 내건성, 내염성과 같은 독특한 생리적 기전을 가지고 있으며 민간에서 진통 경련 완화 치료제로 사용하고 있다. 본 연구에서는 항산화, 항염증, 항노화 등의 효과가 있는 것으로 보고되어 있는 염주괴불주머니(Corydalis heterocarpa)을 이용하여 용매분획물(H₂O, n-BuOH, 85% aq. MeOH, n-hexane)을 제조하였으며 이를 이용하여 PMA로 유도된 인간 섬유육종세포 HT-1080 세포에서 MMP-2, MMP-9의 발현 조절에 미치는 영향을 확인하였다. 염주괴불주머니 분획물 처리시 TIMP-1 및 TIMP-2를 증가시키면서 MMP-2 및 MMP-9의 mRNA 및 단백질 발현을 감소시키는 것으로 나타났다. 또한 염주괴불주머니 용매분획물 처리시 MAPKs 신호 전달 경로인 p38, JNK, ERK의 인산화를 억제하여 MMPs 발현을 감소시키는 것을 확인할 수 있었다. 특히 염주괴불주머니의 85% aq. MeOH와 n-hexane 용매분획물에서 MMP-2 및 MMP-9 발현이 효과적으로 억제되어 MMP 억제활성이 높은 물질이 들어있을 것으로 사료되며, 이를 통해 염주괴불주머니의 암 전이 억제 소재 개발을 위한 기초자료로 활용될 수 있을 것으로 기대된다.

Natural products have always been an attractive source in terms of novel anti-metastatic compounds which can hinder MMP expression and activity. Corydalis heterocarpa is a salt marsh plant found in the seashores throughout Korea. Its yellow flowers and spikes have been an ingredient in folk medicine to treat spasm and contractions. The present study assessed the potential of different solvent-based fractions from the crude extract of Corydalis heterocarpa (CHE), a halophyte with reported bioactivities, to suppress the PMA-induced MMP expression in human fibrosarcoma HT-1080 cells. The solvent fractions which were named after the solvent used for fractionation (n-hexane, 85% aqueous (aq.) methanol (MeOH), n-butanol (BuOH), and H₂O were shown to inhibit the both elevated mRNA and protein expression levels of MMP-2 and MMP-9 and simultaneously relieved the suppression on the expression of the endogenous MMP inhibitors TIMP-1 and TIMP-2. Results indicated that the CHE fractions might intervene with the PMA-induced activation of the MAPK signaling which is the upstream activator of MMP overexpression. Among tested samples, 85% aq. MeOH and n-hexane fractions of CHE was determined to be the most active and future studies to isolate the bioactive substances responsible for the regulation of the MMP expression are, therefore, urged. In conclusion, C. heterocarpa was shown to be a potential source of anti-metastatic compounds and n-Hexane and MeOH fractions might yield lead molecules to develop novel MMP inhibitors.

Introduction

Matrix metalloproteinases (MMPs) are endopeptidases and break down the extracellular proteins: collagen, elastin, and fibronectin. Owing to their important role on extracellular matrix (ECM) remodeling, they are needed for several biological processes that needs cellular expansion or rebuilding of ECM such as wound healing and angiogenesis [8]. Normally, the expression, activation, and activity of MMPs are tightly regulated, partly via endogenous tissue inhibitors of MMPs (TIMPs). However, under certain pathological situations, especially during the tumor growth and metastasis, MMPs play crucial roles for degradation of the basement membrane and tumor cell migration [3]. Different type of MMPs act on different proteins with different specificity. However, studies showed the overexpression of the MMP-2 and MMP-9 gelatinases are needed for the the tumor cell growth and migration [3]. These two enzymes degrade the type IV collagen of ECM basement membrane. In some types of cancerous tissues such as ovarian, bladder, oral, colon and lung, the expression of MMP-2 and MMP-9 were observed to be upregulated [2, 9, 14].

Up to date, several MMP inhibitors were discovered that inhibit the enzymatic activity of MMPs in order to hinder metastasis [7]. On the other hand, some studies showed that increasing the TIMP levels and related decrease in MMP expression resulted in shrinking in tumors and decrease in migration and invasion [10]. Up to date, several studies focused on developing anti-MMP compounds from both synthetic and natural lead molecules [5]. Phytochemicals allocate a big portion of the MMP inhibitor research and numerous studies reported that extracts of halophytes such as Salicornia herba- cea, Limonium tetragonum [1] and Corydalis saxicola [6] contained MMP-2 and MMP-9 inhibitors. In the present study, solvent fractionated extracts of another halophyte, Corydalis heterocarpa, was assessed for the potential to inhibit up regulated MMP expression in PMA-stimulated HT-1080 cells.

Materials and Methods

Plant material and extraction

The C. heterocarpa var. japonica flowers, leaves and stems were hand-picked from Hae-un-ri (Hyeongyeong-myeon, Muan-gun, Jeollanam-do) in 2003 and sun-dried. Dried plant material was then ground to powder and immersed in 3 l methanol (MeOH) for 2 days and after the MeOH was collected, remaining powder material was kept in 3 l CH₂Cl₂ for 2 more days. The crude extract of C. heterocarpa (CHE; 41.1 g) were obtained from the concentration of combined MeOH and CH₂Cl₂ solvents from previous step. The CHE was later separated between different solvents to obtain solvent fractions of CHE namely n-hexane (7.3 g), 85% aqueous (aq.) MeOH (12.0 g), n-butanol (BuOH) (4.3 g) and H₂O (20.0 g). The CHE and its solvent fractions were dissolved in dimethyl sulfoxide (DMSO). All chemicals used in the extraction and fractionation process were purchased from Samchun Chemical (Seoul, Korea).

Cell culture and cytotoxicity of extracts and solvent fractions

Human HT-1080 fibrosarcoma cells (Korean Cell Line Bank, Seoul, Korea) were fed with Dulbecco’s modified Eagle medium (DMEM, Gibco-BRL, Gaithersburg, MD, USA). Culture medium contained 10% fetal bovine serum (FBS, Gibco-BRL) and cells were grown in in 37℃ temperature in the incubators with 5% CO₂ atmosphere. The HT-1080 cells were stimulated by addition of 10 ng/ml phorbol 12- myristate 13-acetate (PMA) along with sample treatment in order to induce overexpression of MMPs. The non-toxic concentrations of CHE and its solvent fractions were defined according to common MTT assay results.

Gelatin zymography

The effect of CHE fractions on the levels of active MMP-2 and MMP-9 present in the PMA-stimulated HT-1080 culture medium was evaluated by gelatin zymography. The detailed gelatin zymography protocol was previously reported by Bae et al. [1]. The HT-1080 cells were stimulated with 10 ng/ml PMA to induce MMP expression. The cells were then treated with CHE fractions and culture media was harvested after 24 hr incubation for the gelatin zymography. Same amount of cell culture medium samples was loaded onto gels, and MMP-2 and MMP-9 digestion of gelation was stimulated by storing gels in digestion buffer (10 mM CaCl₂, 50 mM Tris–HCl and 150 mM NaCl) for 48 hr. Digested sections of the gels were detected with CAS-400SM Davinch-Chemi imager^TM (Davinch-K, Seoul, Korea) after Coomassie blue staining.

Reverse transcription-polymerase chain reaction analysis

The effect of CHE fractions on the expression of MMP-2 and MMP-9 was analyzed by known RT-PCR analysis method in PMA-stimulated HT-1080 cells after 24 hr treatment with CHE fractions. The mRNA levels were quantified normalized against β-actin levels and given as the relative percentage to that of PMA-stimulated non-treated group. Specific primer sequences for the tested genes and the detailed RT-PCR protocol for the PCR steps and gel staining were reported previously by Bae et al. [1].

Western blotting

The expression levels of MMP-2, MMP-9, TIMP-1, TIMP-2 and phosphorylated MAPK proteins were analyzed by Western blotting in PMA-stimulated HT-1080 cells after 24 hr treatment with CHE fractions. The method reported by Bae et al. [1] was followed to obtain the protein loaded membranes. Primary antibodies against MMP-1 (sc-6837; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP-9 (# 393857; Cell Signaling Technology, Beverly, MA, USA), TIMP-1 (sc-21734; Santa Cruz Biotechnology), TIMP-2 (sc- 21735; Santa Cruz Biotechnology) p-p38 (#4511; Cell Signaling Technology), p-JNK (sc-293136; Santa Cruz Biotechnology), p-ERK (#4370; Cell Signaling Technology), and β-actin (sc-47778; Santa Cruz Biotechnology) were used to detect the protein levels. Visualization of the specific protein bands was carried out with chemiluminescence ECL assay kit (Amersham GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions. Pictures of the stained protein bands were taken with CAS-400SM Davinch-Chemi imager^™ (Davinch-K).

Statistical analysis

All numerical results were given as the mean of three different quantification of the same treatment group ± standard deviation. Any statistical importance was defined at p<0.05 level according to the analysis of variance (ANOVA) and post-hoc Duncan’s multiple range test. The SAS v9.1 software (SAS Institute, Cary, NC, USA) was used for statistical analysis.

Results and Discussion

Effect of CHE fractions on active MMP-2 and MMP- 9 levels

Overly stimulated MMP expression results in disrupted ECM balance and structure. Numerous pathological processes such as tumor metastasis, inflammation and asthma are known to exert this effect in MMP expression profile [3]. Therefore, relieving the stimulatory effect on MMP expression is a target for prevention and treatment of tumor metastasis.

The effect of CHE fractions on the MMP-2 and MMP-9 secretion levels was tested by gelatin zymography. Induced expression of MMPs was expected to lead to increased secretion and activation of MMP-2 and MMP-9 in the extracellular space. Therefore, culture medium of the PMA-stimulated HT-1080 cells were analyzed for the active MMP levels via their degradation of collagen in the loaded gels: visible as hollow spaces after staining. As shown in Fig. 1, PMA can significantly induce active levels of MMP-2 and MMP-9. Treatment with 85% aq. MeOH and n-hexane fractions significantly decreased the MMP-9-dependent degradation at the concentration of 100 μg/ml. Also, H₂O and n-BuOH fractions were not able to decrease the MMP-9 activity significantly even at the highest dose (100 μg/ml). On the other hand, 85% aq. MeOH fraction was the most active fraction among tested samples, almost completely preventing the degradation of collagen by MMP-9. In addition, unlike other CHE fractions, 100 μg/ml treatment of 85% aq. MeOH was also able to significantly decrease MMP-2 activity. Results showed that MeOH fraction of CHE had potential substances that might decrease the secretion and/or activation of MMP-9 levels.

Fig. 1. Effect of Corydalis heterocarpa crude extract (CHE) solvent fractions on the active MMP-2 and MMP-9 levels in PMA-stimulated HT-1080 cell culture conditioned media. HT-1080 cells were stimulated with PMA and treated with indicated concentrations of fractions for 24 hr. Active MMP-2 and MMP-9 levels were evaluated by electrophoresis of enzymes on gelatin containing polyacrylamide gel.

Effect of CHE fractions on MMP-2 and MMP-9 expression

The expression of MMP-2, MMP-9 at the mRNA level was analyzed by RT-PCR. Upon treatment with CHE fractions, PMA-induced MMP-2 and MMP-9 expression was decreased dose-dependently (Fig. 2). Among all tested solvent fractions, the order of overall inhibitory effect of CHE fractions on both MMP-2 and MMP-9 mRNA expression was n-hexane, 85% aq. MeOH, H₂O and n-BuOH. At the highest concentration treated (100 μg/ml), n-hexane fraction decreased the MMP-2 mRNA level to 7.5% and MMP-9 mRNA level to 26.5% of untreated PMA-stimulated group. These values were 39.7% for MMP-2 and 11.2% for MMP-9 in terms of 100 μg/ml 85% aq. MeOH treatment.

Fig. 2. Effect of CHE fractions on the mRNA expression of MMP-2 and MMP-9. The mRNA expression was analyzed by RT-PCR and quantified via the density of the bands and normalized against β-actin. The mRNA expression levels were given as the relative percentage of the PMA-stimulated untreated group. Values are mean ± SD. ^a-dMeans with different letters are significantly different at p<0.05 level.

In addition to mRNA expression levels, the protein levels of MMP-2 and MMP-9 were evaluated by Western blotting. Similar to previous results, PMA-stimulation resulted in significant increase in the MMP-2 and MMP-9 protein levels (Fig. 3A). Treatment with CHE fractions decreased the MMP- 2 and MMP-9 levels in a dose-dependent manner. Among the treated CHE fractions (50 μg/ml), H₂O CHE was the most active for decreasing MMP-9 levels, while n-hexane decreased the MMP-2 levels the most. The mRNA and protein expression levels were not in accordance with the degradation ability of MMP-2 and MMP-9 presented in Fig. 1. Accord- ingly, the effects of the fractions were not in same manner, as well. This difference between gelatin zymography and expression analysis might indicate that although PMA was able to induce expression of MMPs, activation of MMP-2 was not stimulated. Following expression, the activation of MMP-9 occurs in extracellular space while MMP-2 is activated via TIMP-2 dependent membrane type-1 MMP [8]. Therefore, this contrast between the results was suggested to be due to different activation mechanisms for MMPs which might response differently to PMA-stimulation and sample treatment.

Fig. 3. Effect of CHE fractions on the protein levels of (A) MMP-2 and MMP-9 (B) TIMP-1 and TIMP-2 and (C) phosphorylated (p-) p38, ERK and JNK MAPKs. The protein expression levels of PMA-stimulated HT-1080 cells were analyzed by Western blotting after 24 hr treatment with CHE fractions at indicated concentrations. Protein bands were quantified via the density of the bands and normalized against β-actin. The protein expression levels were given as the relative percentage of the PMA-stimulated untreated group. Values are mean±SD. ^a-dMeans with different letters are significantly different at p<0.05 level.

The effect of CHE fractions was also tested on the protein levels TIMP-1 and TIMP-2, the tissue inhibitors of MMP expression. PMA-stimulation of HT-1080 cells resulted in suppressed levels of TIMP-1 and TIMP-2 protein (Fig. 3B). TIMPs are responsible for the regulation of MMP expression as well as the activation of MMPs from their inactive forms. During tumor metastasis, overexpression of MMPs were accompanied with suppressed TIMP levels which enables the upregulated MMP activity to be constant [12]. Therefore, relieving the suppression on TIMP levels were expected to regulate MMP expression back to normal levels [11]. The cells exhibited expected significant decrease in TIMP-1 and TIMP- 2 levels following PMA-stimulation in accordance with MMP overexpression observed in previous results. Treatment with 50 μg/ml CHE fractions was able to revert the effect of PMA-stimulation on TIMP-1 and TIMP-2 levels. While 85% aq. MeOH and n-hexane fractions were brought back TIMP-1 levels similar to that of non-stimulated non-treated group, H₂O and n-BuOH showed similar effects in terms of TIMP-2. The effect of fractions on the TIMP levels were suggested to be the result of suppressed MAPK activation and MMP expression.

Differences between the effects of CHE fractions on different types of MMPs and TIMPs indicated that the potential bioactive substances present in these fractions had different action mechanisms and/or specificities against different steps of the regulatory signaling pathways during MMP expression. Nevertheless, results showed that CHE fractions were able to decrease the PMA-induced MMP expression and increase the TIMP levels.

Effect of CHE fractions on the phosphorylation of MAPKs

Like several other intracellular processes that involve transcriptional activity, expressional regulation of MMPs is partially carried out by the transcriptional activities of AP-1 protein [4]. The activation and subsequent nuclear translocation of AP-1 protein occurs via phosphorylated p38, ERK and JNK MAPK proteins. Nuclear translocation and transcriptional activity of AP-starts MMP-2 and MMP-9 expression [13]. Therefore, effect of CHE fractions on the phosphorylated p38, ERK and JNK MAPKs were analyzed by Western blotting to elucidate the possible mechanism of action that suppressed the MMPs expression. The PMA-stimulation increased the phosphorylated MAPK levels (Fig. 3C). This was expected and suggested the involvement of MAPK activation in MMP-2 and MMP-9 expression. Treatment with CHE fractions decreased the phosphorylated levels of p38, ERK and JNK MAPKs. Among treated CHE fractions (50 μg/ml), n-hexane and 85% aq. MeOH treatment were the most active fractions to suppress elevated phosphorylation of MAPKs.

In conclusion, 85% aq. MeOH and n-hexane fractions of the halophyte C. heterocarpa were shown to act against the PMA-induced overexpression of MMP-2 and MMP-9 enzymes while increasing TIMP levels. It was suggested that CHE fractions might act on MMP expression via suppressing the activation of MAPK signaling in PMA-stimulated HT- 1080 cells. Current results exhibited that C. heterocarpa might contain effective MMP inhibitors which are both able to suppress overexpression of MMPs and increase TIMP expression in cancer cells. A mechanism for this effect was suggested to be through inhibition of MAPK-dependent MMP expression. Overall, C. heterocarpa is suggested as a potential source for anti-metastatic compounds and future studies focusing on isolation of bioactive substances from C. heterocarpa fractions are urged.

The Conflict of Interest Statement

The authors declare that they have no conflicts of interest with the contents of this article.