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Tumor Biology |
Department of Internal Medicine and Molecular Therapeutics, University of Tokushima School of Medicine, Tokushima 770-8503, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MMPs3
are a family of structurally related zinc-ion-dependent endopeptidases that include the collagenases, stromelysins, gelatinases, and MT-MMPs (1)
. The first three types are secreted as proenzymes, are activated by cleavage of the NH2 terminus, and have highly conserved regions, particularly at the catalytic site, which encloses a zinc ion. MMP activity is tightly controlled in several ways: at the transcriptional level, by regulated activation of latent proenzymes, and by the presence of natural inhibitors (such as 
2-macrogloblin) and a specific family of tissue inhibitors of metalloproteinase (2)
. MMPs play crucial roles in tumor invasion and angiogenesis by mediating degradation of extracellular matrix (3)
. In many types of neoplasm, including lung cancer, higher levels of activated MMPs have been demonstrated in more invasive and/or metastatic tumors and may give prognostic information independent of stage (4, 5, 6, 7, 8)
. Gelatinases (MMP-2 and MMP-9) are the major proteases in lung cancer, and they closely correlate with invasive and metastatic potentials (9
, 10)
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As reviewed by Zucker et al. (11) , because host stroma cells, as well as tumor cells, produce a variety of MMPs and promote tumor progression, it is possible that host cell-derived MMPs promote tumor progression. In this study, we used two metastasis models (12 , 13) of human lung cancer cell lines that express various levels of MMPs and a selective MMP inhibitor, ONO-4817 (14) , to determine whether metastasis of lung cancer to multiple organs is dependent solely on the expression of MMPs by tumor cells.
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MATERIALS AND METHODS |
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Chemicals.
ONO-4817 [(2S,4S)-N-hydroxy-5-ethoxymethyloxy-2-methyl-4-(4-phenoxybenzoyl)aminopentanamide] was synthesized by Ono Pharmaceutical Co. (Osaka, Japan) as reported previously (14)
. ONO-4817 is a novel synthetic hydroxamic acid-based nonpeptide compound designed to be administered p.o. It binds reversibly to the zinc-binding region of MMPs such as MMP-2, -8, -9, -12, and -13, but not MMP-1, -3, or -7, and has a selective inhibitory spectrum. ONO-4817 suppressed the release of proteoglycan from the cartilage of the knee joints in an arthritis model of guinea pigs. Pharmacokinetic studies showed that plasma concentrations of ONO-4817 were >10 µmol/l at 1 h and 1 µmol/l at 4 h after p.o. administration at a dose of 100 mg/kg.
Measurement of MMP Activity.
The activities of MMP-1, -2, and -9 were measured by commercially available assay kits (Biotrak; Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom), as reported previously (16)
. To evaluate the effect of ONO-4817 on the inhibition of MMP activity, samples (culture supernatants of PC14PE6 cells in serum-free conditions) were incubated with various concentrations of ONO-4817 for 1 h at 37°C. The resultant solutions were measured for MMP-1, -2, and -9 activity by the respective ELISAs. The detection limits of the ELISAs for MMP-1, -2, and -9 were 0.3, 0.75, and 0.25 ng/ml, respectively.
MT1-, MT2-, and MT3-MMP mRNA Expression.
mRNA expression of MT1-, MT2-, and MT3-MMP was determined by RT-PCR. The total cellular RNA was extracted from the culture of seven lung cancer cell lines by use of ISOGEN (Nippon Gene Co., Toyama, Japan) according to the protocol recommended by the manufacturer. One µg of total RNA was reverse transcribed. The primers for MT1-, MT2-, and MT3-MMP and ß-actin were as follows: MT1-MMP sense (5'-CGC TAC GCC ATC CAG GGT CTC AAA-3') and antisense (5'-CGG TCA TCA TCG GGC AGC ACA AAA-3'); MT2-MMP sense (5'-ACA ACC ACC ATC TGA CCT TTA GCA-3') and antisense (5'-AGC TTG AAG TTG TCA ACG TCC TCC-3'); MT3-MMP sense (5'-TTA CTT CTG GCG GGG CTT GCC TTG CCT CCT AGT AT-3') and antisense (5'-ACA GTA CAG TAT GTG GCG CGG TGT TCC TTT-3'); ß-actin sense (5'-ACG CAT TTG GTC GTA TTG GG-3') and antisense (5'-TAG ATT TTG GAG GGA TCT CGC-3'; Ref. 17
). RT-PCR was performed as reported previously (17)
with some modifications. The reaction mixture containing the cDNA was derived from 1 µg of total RNA, 20 pmol of the sense and antisense primers, 1 unit of Taq DNA polymerase, and 200 pmol of each deoxynucleotide triphosphate, in a final volume of 50 µl. The Taq DNA polymerase, reaction buffer, and deoxynucleotide triphosphates were from the commercial kit (TaKaRa Taq; Takara Shuzo, Kyoto, Japan). Amplification was performed in sequential cycles and included 30 s of denaturation, 1 min of primer annealing at 62°C (MMP-16 at 58°C), and 1 min and 50 s of extension at 72°C. Amplification was carried out for 28 cycles for MT1-, MT2-, and MT3-MMP. Amplification of ß-actin only involved 30 sequential cycles of 1 min of denaturation, 1 min of primer annealing at 58°C, and 2 min of extension at 72°C, with the last cycle including incubation for 4 min at 72°C.
Animals.
Animal studies were performed in accordance with guidelines established by the Tokushima University Committee on Animal Care and Use. Male athymic mice and SCID mice 6–8 weeks of age were obtained from Clea Japan (Osaka, Japan) and were maintained under specific pathogen-free conditions throughout this study.
In Vivo Metastasis Models.
We used two kinds of metastasis models in this study. As the lung metastasis model, 1 x 106 PC14, PC14PE6, or A549 cells, or 5 x 105 H226 cells were injected into nude or SCID mice (13)
. As the multiple-organ metastasis model, SCID mice were pretreated with antimouse interleukin-2 receptor ß-chain antibody to deplete NK cells (12)
. Two days later, the mice were inoculated with 1 x 106 SBC-3/DOX or RERF-LC-AI cells or 5 x 106 H69/VP cells. In both models, the mice were given food mixed with or without 1% ONO-4817 for the indicated periods. Four to eight weeks after tumor cell inoculation, the mice were killed, the major organs were removed, and metastasis formation in the liver, kidney, and lymph nodes was determined macroscopically. The number of lung metastases was determined after staining of the lung with Bouin’s solution. Because some of the mice inoculated with PC14 or PC14PE6 cells developed pleural effusions in addition to lung metastasis (13)
, the effect of ONO-4817 treatment on the formation of pleural effusion was also determined in these mice.
FIZ.
Fresh tissues with or without metastatic lesions were dissected from tumor-bearing mice and embedded in Cryomold O.C.T.4583 Compound (Miles, Inc., Elkhart, IN) and immediately frozen on dry ice. These frozen blocks were then sliced sequentially by use of a TISSUTEK II cryostat microtome (Miles) to prepare serial frozen thin sections (8-µm thick). These thin sections were placed on a polyethylene terephthalate base film coated with cross-linked gelatin (7 µm thickness) with (FIZ-GI) or without (FIZ-GN) 1,10-phenanthroline (inhibitor of MMPs but not trypsin), as described previously (18)
. The films with sections were incubated in a moist chamber (Cosmo Bio Co., Ltd. Tokyo, Japan) at 37°C for 6 h. After the incubation, the specimens were stained for 4 min with Biebrich Scarlet (Wako Pure Chemical Industries, Ltd., Osaka, Japan). These films were then rinsed for 10 min with distilled water. The degraded gelatin was not stained with Biebrich Scarlet, and areas of gelatinolytic activity were visualized as white to weakly red areas on the red background. Gelatinolysis observed on FIZ-GN films, but not FIZ-GI films, indicated MMP activity. Gelatinolysis observed on both FIZ-GN and FIZ-GI films indicated activity of proteases other than MMPs (such as trypsin). One of the serial sections was fixed with 4% paraformaldehyde fixative and rinsed thoroughly with PBS, followed by staining with Gill’s H&E solution (Wako Pure Chemical Industries). For quantitative analysis of the gelatinolytic activity, the photographic image observed under the light microscope was scanned by a Nikon Scantouch 210 scanner (Nikon, Tokyo, Japan).
Statistical Analysis.
The statistical significances of difference in in vivo data were analyzed by the Mann-Whitney t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PC14 and PC14PE6 cells expressed MMP-1, -2, and -9 activity, whereas H226 cells expressed only MMP-2 activity. A549, SBC-3/DOX, H69/VP, and RERF-LC-AI cells expressed very low or undetectable MMP-1, -2, and -9 activity under in vitro condition.
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. H69/VP cells expressed no detectable MT1-MMP mRNA. All seven cell lines expressed MT2-MMP mRNA. Interestingly, none of adenocarcinoma cell lines tested expressed detectable levels of MT3-MMP mRNA, whereas others did.
Production of Metastasis and Expression of MMPs by Human Lung Cancer Cell Lines in Vivo.
In the lung metastasis model, adenocarcinoma (A549, PC14, PC14PE6) or squamous cell carcinoma (H226) cells injected i.v. into SCID or nude mice produced metastasis only in the lungs, although some of the mice inoculated with PC14 or PC14PE6 cells developed malignant pleural effusions (Table 1)
. In the multiple-organ metastasis model, lung squamous cell carcinoma (RERF-LC-AI) or small cell carcinoma (SBC-3/DOX and H69/VP) cells injected i.v. into SCID mice depleted of NK cells produced metastases in the liver, kidneys, and systemic lymph nodes (but not to the lungs; Table 2
). FIZ analysis revealed that although lung parenchyma had no detectable gelatinolytic activity, liver and kidney parenchyma showed high and low MMP activity, respectively, irrespective of the tumor cell injection (Fig. 2)
. In the lung metastasis model, gelatinolytic activity was observed in lung lesions produced by A549, PC14PE6, and H226 cells on FIZ-GN films (Fig. 3)
. The gelatinolytic activity of lung colonies of A549 cells, but not PC14PE6 or H226 cells, was detected on FIZ-GI films, suggesting that the gelatinolytic activity of tumors produced by PC14PE6 and H226 cells was attributable to MMPs but that that the activity of A549 tumors was mainly attributable to other proteases, such as trypsin. In the multiple-organ metastasis model, nontumor parenchyma of the liver and kidney had high and low gelatinolytic activity, respectively, on FIZ-GN film, but tumors produced by SBC-3/DOX cells did not, consistent with the lower potential of this cell line to express MMPs in vitro. The gelatinolytic activity was not detected on FIZ-GI film, suggesting that the liver and kidney have high and low MMP activity, respectively, and that SBC-3/DOX cells do not express MMP activity even under in vivo conditions. These findings indicate the heterogeneity of both tumor cells and organ microenvironments in terms of MMP expression.
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. In contrast, treatment with ONO-4817 showed no effect on MMP-1 activity produced by PC14PE6 cells, consistent with previous findings (14)
. ONO-4817 at 10 µM did not affect proliferation of the seven cell lines used in this in vitro study (data not shown).
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; Fig. 5A
). Treatment of mice bearing PC14 or PC14PE6 cells with ONO-4817 significantly reduced the number of lung metastases. The formation of pleural effusion in the mice was also inhibited by treatment with ONO-4817. H226 cells developed numerous small lesions (<2 mm) on the surface of the lung. Although >150 lesions were observed in the lungs even in the mice treated with ONO-4817, the size of each lesion was clearly smaller in treated mice than in control mice (Fig. 5B)
. Consistent with the size of the lung lesions, the lung weight of ONO-4817-treated mice was significantly lower than that of control mice (Table 1)
. FIZ analysis showed that treatment of tumor-bearing mice with ONO-4817 abrogated the gelatinolytic activity of tumors produced by H226 cells, but not A549 cells on FIZ-GN films (Fig. 6)
. These results suggest that MMPs may be essential for the formation of lung metastases by tumor cells with high MMP expression (such as PC14, PC14PE6, and H226), but not tumor cells without MMP expression (such as A549).
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; Fig. 5C
). Mice inoculated with RERF-LC-AI, SBC-3/DOX, or H69/VP cells did not develop lung metastases, irrespective of treatment with ONO-4817. FIZ analysis revealed that ONO-4817 treatment inhibited the MMP activity of nontumor parenchyma of the liver and kidney but had no effect on MMP activity of tumors produced by SBC-3/DOX cells (Fig. 6)
. These findings suggest that MMP activity expressed by liver parenchyma, but not tumors, may be necessary for the production of liver metastases by SBC-3/DOX cells.
Effect of MMP Inhibition by ONO-4817 Started after Tumor Cell Inoculation.
MMPs have been suggested to promote tumor invasion and angiogenesis (11)
. To address what events in metastasis formation were interrupted by ONO-4817, the mice inoculated with H226 or SBC-3/DOX cells were treated with ONO-4817 starting on the indicated days until the end of the experiments. As shown in Tables 3
and 4
, ONO-4817 treatment started by days 28 and 7 inhibited the formation of lung metastases by H226 cells and liver metastases (but not kidney or lymph node metastases) by SBC-3/DOX cells, respectively, suggesting that ONO-4817 may inhibit early steps of the metastatic process under particular organ microenvironments after tumor cell injection in these animal models. In addition, vascularization in metastatic lesions was evaluated by immunostaining for CD31. There was, however, no significant difference in the microvessel density in the metastatic lesions (lung and liver) between the control group and the ONO-4817-treated group (data not shown).
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DISCUSSION |
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Eccles et al. (20) reported an effect of MMP inhibition by Batimastat in a spontaneous metastasis model of rat mammary carcinoma (HOSP.1P) that expressed both MMP-2 and MMP-9. They found that inhibition of a broad spectrum of MMPs by Batimastat led to inhibition of lung metastasis but had little effect on lymph node metastasis. Consistent with their results, MMP inhibition by ONO-4817 did not significantly inhibit lymph node metastasis in the present model. In addition, ONO-4817 had no significant effect on kidney metastasis, although low levels of gelatinolytic activity were detected in the kidney parenchyma. Although we could not evaluate gelatinolytic activity of normal lymph nodes because of availability, one explanation for the lower efficiency of MMP inhibition in these organs is that distribution of ONO-4817 in these organs may not be adequate for inhibiting metastasis. The second possibility is that the level of MMP activity in the parenchyma of these organs, unlike the liver, may be less than the threshold level for promoting metastasis formation.
MMPs have been suggested to promote tumor invasion and angiogenesis by basement membrane degradation (3) . In our experimental metastasis models, H226 and SBC-3/DOX cells distributed to the lung and liver by 1 h after inoculation (data not shown) and developed micrometastases by 10 days (13 , 21) . MMP inhibition by ONO-4817 started by days 7 and 28 reduced metastasis formation by SBC-3/DOX and H226 cells, respectively, suggesting that MMPs are necessary for early events of the metastatic processes, such as extravasation, of these cell lines. The reason that MMP inhibition by ONO-4817 did not reduce liver metastasis when started on day 14 is unclear at present. One explanation is that large tumors express more MMPs, and hence the level of MMP inhibition by ONO-4817 might not have been adequate to inhibit angiogenesis of large nodules. However, this is probably not the case because we could not detect gelatinolytic activity even in large tumors (5 mm in diameter) produced by SBC-3/DOX cells in our experimental conditions. Therefore, MMPs (MMP-2 and -9) might not be the major factor involved in the invasion or angiogenesis for large nodules, as described previously (11) .
Several preclinical studies have suggested that MMPs produced by tumor cells have critical importance in tumor progression; therefore, inhibition of MMP activity is suggested to be one promising strategy for molecular targeted modalities against cancer metastasis (11) . In fact, many MMP inhibitors have been developed (11 , 19) . The first generation of MMP inhibitors, such as Batimastat, have broad inhibitory activity against almost all MMPs and is not appropriate for p.o. administration. The second generation, such as Marimastat, MMI270 (CGS27023A), and Prinomastat, also show broad activity spectra, but can be administered p.o. The third generation, such as ONO-4817 and BAY12-9566, were designed to have selective spectra of MMP inhibition, and many compounds of this generation do not inhibit MMP-1 (19) . The antitumor potential of many of these compounds has been evaluated in animal models with tumor cells expressing high MMP activity. Recent studies, however, revealed several deficiencies in suppression of cancer progression by MMP inhibitors. Batimastat was reported to inhibit the growth of primary tumors (in the skin, peritoneal cavity, and orthotopic liver) and lung metastasis (20 , 22, 23, 24, 25) , but to promote liver metastasis (26) . MMI-166 was shown to inhibit liver metastasis and orthotopic pancreas tumors (27) , but not orthotopic colon tumors (28) . These results suggest organ heterogeneity in the efficacy of MMP inhibitors. Consistent with these previous findings, we here demonstrated that one of the third-generation MMP inhibitors (ONO-4817) inhibited metastasis of human lung cancer cell lines to the liver and lung (except A549 cells) but not to the kidney or lymph nodes. ELISA and FIZ analyses for evaluating MMP activity in vitro and in vivo, respectively, further suggested that the antimetastatic effect of this drug by MMP inhibition depends on expression of MMPs derived not only from tumor cells but also from host microenvironments. It was previously documented that the antitumor effect of MMP inhibitors was directly correlated with the level of MMP expression by tumors (23) and that the effect of MMP inhibitors was sometimes heterogeneous. as described above. In the present study, we demonstrated that the therapeutic efficacy of MMP inhibitors can be differentially affected by heterogeneity of host-derived MMP expression.
In conclusion, we demonstrated that MMP expression by the host microenvironment varied among organs. In addition, metastasis formation could be facilitated if MMPs were expressed at the sites of tumor growth irrespective of their source (tumors or nontumor parenchyma). Therefore, the antitumor effect of MMP inhibitors may be dependent on MMPs derived not only from tumor cells but also from organ-specific microenvironments.
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FOOTNOTES |
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1 This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, Sports and Culture of Japan, and the Public Trust Haraguchi Memorial Cancer Research Fund. 
2 To whom requests for reprints should be addressed, at Department of Internal Medicine and Molecular Therapeutics, University of Tokushima School of Medicine, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. Phone: 81-88-633-7127; Fax: 81-88-633-2134; E-mail:ssone{at}clin.med.tokushima-u.ac.jp
3 The abbreviations used are: MMP, matrix metalloproteinase; MT-MMP, membrane-type MMP; FBS, fetal bovine serum; RT-PCR, reverse transcription-PCR; NK, natural killer; FIZ, film in situ zymography.
Received 2/19/02. Accepted 8/16/02.
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