have various biological effects (Baek et ai., 1995; Kim et ai., 1996; Lee et ai., ..... 47, 3863-3867. Ota, T., Maeda, M., and Odashima, S. (J 99 J) Mechanism of.
Mol. Celis, Vol. 9, No.5, pp. 476-483
Molecules and
Cells © Springer-Verlag 1999
Glucocorticoid Receptor-induced Down-regulation of MMP-9 by Ginseng Components, PD and PT Contributes to Inhibition of the Invasive Capacity of HT1080 Human Fibrosarcoma Cells Moon-Taek Park, Hee-Jae Chat, Joo-Won Jeong, Shin-II Kim\ Hae-Young Chung 2, Nam Deuk Kim 2 , Ok Hee Kim4, and Kyu-Won Kim * Department of Molecular Biology and Pusan Cancer Research Center, Pusan National University, Pusan 609-735, Korea; I Research Institute of Basic Sciences, Pusan National University, Pusan 609-735, Korea; 2 Department of Pharmacy, Pusan National University, Pusan 609-735, Korea; 3 Korea Ginseng and Tobacco Research Institute, Taejon 305-333, Korea; 4 Pathology Department, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul 122-020, Korea. (Received on April 21, 1999)
regulation of MMP-9 by PD and PT contributes to reduce the invasive capacity of HTI080 cells.
We examined the effects of the purified ginseng components, panaxadiol (PD) and panaxatriol (PT), on the expression of matrix metalloproteinase-9 (MMP-9) in highly metastatic HTI080 human fibrosarcoma cell line. A significant down-regulation of MMP-9 by PD and PT was detected by Northern blot analysis. However, the expression of MMP-2 was not changed by treatment with PD and PT. Quantitative gelatin based zymography confirmed a markedly reduced expression of MMP-9, but not MMP-2 in the treatment of PD and PT. To investigate whether the reduced level of MMP9 by PD and PT affects the invasive capacity of HTl080 cells, we conducted an in vitro invasion assay with PD and PT treated cells. The results of the in vitro invasion assay revealed that PD and PT reduced tumor cell invasion through a reconstituted basement membrane in the transwell chamber. Because of the similarity of chemical structure between PD, PT and dexamethasone (Dexa), a synthetic glucocorticoid, we investigated whether the down-regulation of MMP-9 by PD and PT were mediated by the nuclear translocation of glucocorticoid receptor (GR). Increased GR in the nucleus of HTI080 human fibrosarcoma cells treated by PD and PT was detected by immunocytochemistry. Western blot and gel retardation assays confirmed the increase of GR in the nucleus after treatment with PD and PT_ These results suggest that GR-induced down-
Proteo lysis of the extracellul ar matrix and base ment membrane has been implicated in a number of di stinct steps in metastasis (Liotta et ai., 1980). Because the basement membrane is primarily composed of several kinds of collagens, the coll agenases have been the focus of many studies seeki ng to establi sh a role for proteinases in determini ng metastatic spread (Salo et ai., 1982). There are ev idences that collagenases are involved in the metastatic process - metastatic cell lines express proteinases capable of degrading type IV coll agen, and inhibition of expression or activity of these proteinases reduces in vasion and metastasis (Lee et ai., 1992; Mignatti et ai., 1986; Nakajima et ai., 1989; Schultz et ai. , 1988; Wang et ai., 1988). Ginseng, the root of Panax ginseng C. A. Meyer, is a medicinal plant used worldwide and has been reported to have various biol ogical effects (Baek et ai., 1995 ; Kim et ai., 1996; Lee et ai., 1994; 1996; 1998; Nah et ai. , 1995). Gi nse ng ex tracts have been reported to inhibit th e
* To whom correspondence should be addressed. Tel: 82-5 1-510-2277; Fax: 82-5 1-513-9258 E-Mail : k.imkw@ hyowon.cc. pusan.ac.i'"
-*-
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PO (3 days) PO (6 days)
-e--
PT (3 days) --8-- PT (6 days)
20
0 0
10
20
40
30
50
11M
F ig. 2. Effects of PD and PT on the cell viability. The number of viable cells was checked by MIT assay. MIT assay of HTl080 cell s was conducted after treatment with 10- 50 f.lM of PD or PT for 3 or 6 d.
A PO
+
+
3
6
(10 f.lM) days
479
The effects of PD and PT on the repression of the MMP-9 expression were confirmed at the protein level by the gelatin-based zymography. As expected, treatment with PD and PT reduced the ·enzyme activity of MMP-9 . As demonstrated in Fig. 4, treatment with 10 11M of PD for 6 d showed significant decrease of MMP-9 expression . Whereas treatment for 3 d did not show much change. Treatment with 40 11M of PT for 3 d and 20-40 11M for 6 d significantly reduced the activity of MMP-9 , whereas, treatment with 10 to 30 11M for 3 d and 10 11M for 6 d did not change the level of MMP-9 drastically. This result indicated that the PT reduces the protein level of MMP-9 in a dose- and a time-dependent manner. From the observation of gelatin-based zymography, the level of MMP-2 was reduced by neither PD nor PT. The same results are obtained from the Northern blot analysis demonstrated in Fig. 3. However, the action of PD and PT on the down-regulation of MMP-9 by the gelatin-based zymography was less effective than that of MMP-9 by Northern blot analysis. These results may be due to the difference between the amounts of transcribed mRNA and translated protein level of MMP-9 . Anti- invasive effects of PD and PT We examined whether down-regulation of MMP-9 by PD and PT affects the invasive capacity of HTl080 cells by in vitro invasion assay. The invasive capacity of HT1080 cells through a reconstituted basement membrane (Matrigel) to the collagen-coated lower surface of the filters was inhibited by PD and PT (Fig. 5). HTl080 cells treated with 10 11M of PD or 40 11M of PT for 6 d, comparing with a control , inhibited its invasion into Matrigel by about 70 and 80%, respectively. This result suggested that the down-regulation of MMP-9 expression by PD and PT contribute to the inhibition of invasive capacity of the HTl080 cells.
+-- MMP-9
+-- MM P-2
+-- 288 +-- 188
B PT
10
40
10 20
A
PD
MMP-9
+
+
3
6
(10 11M) days
+- MM P-9
+- MMP-2 MM P-2 B
PT 288 188
Fig. 3. Effects of PO (A) and PT (B) on the expression of MMP9 and MMP-2. HTl080 cells were treated with 10 f.lM of PD or 10-40 f.lM of PT and cultured for 3 or 6 d. Express ions of MMP-9 and MMP-2 were assayed by Northern blot an alysis. Filters were hybridi zed with 32P_Iabeled cDNA probes of MMP-9 and MMP-2. Molecular sizes of the transcripts are 3.1 and 2.8 kb for MMP-2 and MMP-9, respectively.
10
20
30
3
40
10
20 6
30
40 11M days
+- MM P-9 +- MMP-2 Fig. 4. Gelatin based zymography of the culture medium of HT I 080 cells treated with PO (A) and PT (B). After treating with 10-40 f.lM of PT or 10 f.lM of PD for 3 or 6 d, the culture media were used in gelatin based electrophoresi s and stained with Coomassie Brilliant Blue. Arrows indicate MMP-9 and MMP-2, respecti vel y.
PO and PT Inhibit HTl080 Invasion via GR Pathway
480 120
ec
Control
.... GR
Days
0
80
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ro
40
>
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6
3
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in the Western blot ana lysis. After treatment with 10 JlM of PO and 40 JlM of Pt for 3 or 6 d, the cell were harvested and nuclear proteins (40 Jlg) were purified and transferred to Hybo nd-ECL. The fi lters were blotted with anti-GR antibody and detected by ECL.
60
""0 ""0
3
Fig. 7. Effects of PO and PT on the nuclear translocation of GR
~ ~
!!!
PT
100
u '0
PD
20
Control PD 10 JlM PT 40 JlM
Fig.s. Anti-invasive activity of PO and PT. The effects of PO and PT on the invasive capacity of HT I 080 cells were meas ured by in vitro invasion assay. The assay was performed after treatment with 10 JlM of PO or 40 JlM of PT for 6 d. HTl080 cells treated with PO and PT were incubated on the transwell chamber for 16 h. The number of invaded cells was counted and mean va lues were determined under X400 light microscopy.
PD and PT-induced nuclear translocation of GR
In
order to determine whether PD and PT induce the translocation of OR from cytosol to nucleus, HT1080 cell s were stained with anti-OR antibody after treatments with 10 11M of PD and 40 11M of PT for 3 or 6 d. Figure 6 shows that the immunoactivity of OR in untreated cells was mainly localized in the cytosol whereas the treatments with 10 11M of PD and 40 11M of PT for 3 or 6 d drastically induced the translocation of OR from cytosol to nucl eus, respectively. To confirm whether PD a nd PT promote the translocation of OR, we carried out Western blot analysis and gel retardation assay using nuclear extracts of HTI 080
cells treated with PD and PT. Figure 7 shows that the treatments with 10 11M of PD and 40 11M of PT for 3 or 6 d increased the amount of nuclear OR, respectively on the protein level. Furthermore, in the treatment with 40 11M of PT for 3 or 6 d, the amo unt of translocated OR was increased more than that for the treatment with 10 11M of PD for 3 or 6 d. These results suggested that the effect of PT on the nuclear translocation of OR is stronger than that of PD. We then performed a gel retardation assay to exami ne the amount of nucl ear OR interaction with its specific DNA recognition seq ue nces, ORE. After inc ubation of the nuclear extracts with a radio labeled double stranded recognition seq ue nces, OR formed a spec ifi c , retarded comp lex with a radiolabeled oli gonucl eotide, ORE (Fig. 8) . Similar to the result of
: Competitor : Fold Molar Excess
N.S. N.S. GR/GRE
Free probe
Control
PD 10 JlM (3 day) PD 10 JlM (6 day)
PT 40 JlM (3 day) PT 40 JlM (6 day)
Fig.6. PO and PT induce the nucl ear translocation of GR in the immunocytochemistry. HT 1080 cel ls treated with 10 JlM of PO and 40 JlM of PT for 3 or 6 d and immunocytochemistry were carried out as described in Materi al and Methods.
Fig. 8. Effects of PO and PT on the binding of GR to its binding site on g lucoco rti co id responsive e lemen t (GRE) in the gel retardation assay. Nuclear extracts ( 10 Jlg) from HTI080 cells treated with 10 /J.M of PO and 40 11M of PT for 3 or 6 d were reacted with a 32P-labelled GRE binding oligon ucleotides and fractionated on the polyacry lamide ge l. In order to determine the specificity of binding between GR and GRE, 25- or 100-fold unlabeled cold GRE was used as a spec ific competitor (N.S. indicates nonspecific) .
Moon-Taek Park et al.
Western blot analysis, the amount of GR/GRE complex showed a higher increase after treatment with 40 ~M of PT than after treatment with 10 ~M of PD. When a 25-fold and 100-fold molar excess of unlabeled GRE was added, the formation of the GR/GRE complex was efficiently prevented by unlabeled GRE. These results demonstrated that the nuclear translocation of GR is induced by PD or PT and that the translocated nuclear GR may affect the repression of MMP-9 gene expression.
Discussion Arresting or retarding molecular events involved in tumor invasion, such as the expression and secretion of MMPs, defines a new category of cytostatic prevention of tumor metastasis (Schultz et ai., 1988). Regulation of the level of MMP may be a key step in the complex steps of tumor invasions. And among these MMPs , correlative evidence for the involvement of MMP-2 and MMP-9 in the invasive phenotype has been reported (Levy et al ., 1991 ; Monteagudo et ai. , 1990; Ura et al., 1989). We recently demonstrated that UA has an inhibitory effect on the invasive activity of the HT1080 cells in a concentration dependent manner and this action of UA can partially be attributed to the down-regulation of the expression of MMP-9 (Cha et al., 1996). Since the chemical structure ofPD and PT resembles that of VA , we assumed that PD and PT might act through the same manner as that of VA. In the present study, we investigated whether PD and PT have an inhibitory effect on the expression ofMMP9 in the HTl080 cells. The results of Northern blot analysis showed that the expression of MMP2 was not changed significantly, whereas the expression of MMP-9 was reduced by PD and PT in a concentrationdependent manner (Fig. 3). To confirm if the decreased mRNA level of MMP-9 in the PD and PT-treated HTl080 cells resu lted in inhibition of matrix degradation, metalloproteinase activity in PD and PT treated HTI080 cells was analyzed by gelatin-based zymography. Two bands of gelatinolysis, MMP-9 and MMP-2, were detected in the conditioned media from HTI080 cells. The gelatinolytic activity of MMP-9 was remarkably downregulated in the PD and PT-treated HT1080 cells, whereas that of MMP-2 was almost the same (Fig. 4). These results are in concert with the results of Northern blot analysis and suggest that the decreased transcription of the MMP-9 gene result in its decreased activity. Down-regulation of MMP-9 by PD and PT suggests the possibility that these ginseng components have a capacity to inhibit tumor cell invasion. Thus, we analyzed whether the invasive capacity of HTl080 cells was inhibited by PD and PT. As shown in Fig. 5, treatment with 10 ~M of PD or 40 ~M of PT for 6 d significantly repressed the invasive capability of HTl080 cells, and PT repressed the invasive capability of HT 1080 cells more than PD.
481
It has been previously reported that the glucocorticoid hormone was a potent inhibitor of collagenases containing AP-I binding motif in their promoter region, and that the repression of these collagenases by the glucocorticoid hormone was mediated by GR interfering AP-l. Dexa activates and translocates GR from the cytosol to nucleus by dissociating it from Hsp90. The translocated GR inhibits the activity of AP-l by directly binding to it (Jonat et ai. , 1990; Liu et ai. , 1995; Nicholson et al., 1990). MMP-9 contains AP-l binding sequences on its promoter region (Sato and Seiki, 1993). The chemical structure of PD and PT is similar to that of Dexa. Thereby, the decreased expression of MMP-9 by PD and PT could be due to the reduced transactivation activity of AP-l through the interaction with nuclear GR translocated by PD and PT. In the treatments with PD and PT for 3 or 6 d, the nuclear translocation of GR was significantly induced (Fig. 6) and it was confirmed by Western blot analysis and Gel retardation assay using nuclear extracts of HT1080 cells treated with PD and PT (Figs. 7 and 8). These results suggest that the nuclear translocation of GR induced by PT is more effective than that by PD. In conclusion , PD and PT inhibit tumor cell invasion in vitro by inhibiting the transcription of the MMP-9 gene required for the degradation of basement membrane. This down-regulation of MMP-9 by PD and PT might be mediated through the translocation of GR from the cytosol to nucleus. Acknowledgments This work was supported by the research fund (1997) from the Korea Ginseng and Tobacco Research Institute and G7 project (1541-211).
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