Schlüsselwörter
Bronchialkarzinom
Matrix-Metalloproteinase
Gelatin-Zymographie
Casein-Zymographie
Zusammenfassung
Hintergrund: Die Matrix-Metalloproteinasen (MMP)-2, -9 und -7 werden mit Tumorinvasion, -metastasierung und -angiogenese in Verbindung gebracht. Bisher ist jedoch ungeklärt, welche Rolle sie bei der Entwicklung des frühen Bronchialkarzinoms spielen. Wir haben die Aktivität von MMP-2, -7 und -9 in Bronchialkarzinomproben im Frühstadium gemessen. Material und Methoden: Bei 60 Patienten mit nicht-kleinzelligem Bronchialkarzinom im Stadium I wurden sowohl normales Lungengewebe (N) als auch Tumorgewebe (T) entnommen. Die Aktivität von MMP-2 und MMP-9 wurde mittels Gelatin-Zymographie bestimmt, und die Aktivität von MMP-7 mittels Casein-Zymographie. Desweiteren wurde das Mengenverhältnis der aktiven Form von MMP-2 im Tumorgewebe verglichen mit normalem Gewebe bestimmt und das Überleben in den Gruppen mit unterschiedlichem MMP-2-T:N-Verhältnis verglichen. Ergebnisse: Sowohl MMP-2 als auch MMP-9 wurden in allen Tumorgeweben und normalen Geweben detektiert. Interessanterweise war die MMP-9-Aktivität im Tumorgewebe verglichen mit normalem Lungengewebe signifikant reduziert, während die MMP-2-Aktivität signifikant erhöht war. Die Überlebensrate in der Gruppe mit MMP-2-T:N > 2,5 war 57,45%, was im Vergleich zur Gruppe mit T:N ≤ 2,5 (86,78%) signifikant niedriger war. Schlussfolgerung: Unsere Ergebnisse deuten an, dass MMP-2, nicht jedoch MMP-9 und MMP-7, an der frühen Tumorinvasion, Metastasierung und Angiogenese beim nicht-kleinzelligen Bronchialkarzinom beteiligt sein könnten.
Introduction
Matrix metalloproteinases (MMPs), especially MMP-2, -9 and -7, may play crucial roles in invasion and metastasis of malignant tumors [1]. MMPs can degrade extracellular matrix (ECM) and intensify the attack of cancer cells on the normal tissue. In addition, they may play a key role in tumor angiogenesis [2]. Increased expression/activity of MMPs is also found in lung cancer patients [3]. Recently, MMP inhibitors have been developed to prevent and treat invasion, metastasis, and angiogenesis of malignant tumors including lung cancer. However, as far as we are aware, no clinical trial has so far achieved satisfying results. Although chemical components, clinical trial design, and patient selection might be important factors, a better understanding of the role of MMPs in the pathophysiology of malignant tumors may be needed [4]. In this study, we measured the activity of MMP-2, -9 and -7 in tumor tissues of early-stage non-small cell lung cancer (NSCLC).
Material and Methods
Tissue Collection
Tumor tissues and corresponding normal lung tissues from a total of 60 consecutive stage-I NSCLC patients (male: n = 49; female: n = 11) were collected during surgery between August 2009 and December 2010. Tissue samples were frozen with liquid nitrogen immediately after being removed and stored at -80 °C. According to the classification system of the International Union against Cancer (UICC), 36 cases were pT1N₀M₀Ia and 24 cases were pT2N₀M₀Ib. In addition, 25 cases were squamous carcinoma and 35 cases were adenocarcinoma. The age range of the patients was 44-86 years, and the average age was 63.3 ± 6.3 years. The basic clinical feature of the patients are summarized in table 1.
Gelatin Zymography
Gelatin zymography was used to measure the activity of MMP-2 and MMP-9 as described previously [5]. Briefly, tissue extracts were electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel containing 0.1% gelatin and rinsed in dd-H2O followed by incubation with a bulky volume of renaturation buffer (2.7% Triton-X 100 in dd-H2O) at room temperature for 1 h with gentle shaking. Enzyme activity was developed in 50 mM Tris (ph 7.5), 0.2 M NaCl, 5 mM CaCl2 and 0.2% Brij-35 at 37 °C for 18 h and stained with Coomassie Blue. MMP activity was analyzed with NIH image software (edition 1.62). Activities of pro-MMP-2 and MMP-2 were quantified at 66 and 62 kDa, respectively. Activity of MMP-9 was considered a lucid band at 92 KDa (fig. 1). MMP activities were calculated according to the method of Davies et al. [6]. Briefly, the ratio of activated MMP-2 to total MMP-2 activity (62 kDa/66 kDa + 62 kDa) was calculated based on the gelatinolytic activities measured by computer-assisted image analysis. We then determined the T (tumor):N (normal tissue) ratio of the active form of MMP-2 by comparing the percentage of the active form in tumor tissue with that in normal tissue in each case.
Fig. 1
A representative gelatin zymography indicating the activities of MMP-2 and MMP-9 in normal lung (N) and tumor tissues (T) of non-small cell lung cancer. MMP-2 activity in tumor tissue was significantly higher than that in normal lung tissue, whereas MMP-9 activity in tumor tissue was significantly lower than that in normal lung tissue.
Casein Zymography
Casein zymography was used for measuring MMP-7 activity. Tissue extracts were electrophoresed on a 8% polyacrylamide gel containing 2 mg/ml casein. After electrophoresis, gels were washed in 2.5% Triton-X 100 and incubated for 48 h at 37 °C in 50 mmol/l Tris-HCl (pH 7.4), 10 mmol/l CaCl2, 1 mmol/l ZnCl2, and 0.02% NaN3, followed by staining with 0.1% Coomassie Blue. A pregnant woman's amniotic fluid was used (with added sample buffer at a ratio of 1:1) as positive control. Activity of MMP-7 was quantified at 20 kDa.
Follow-Up
Patients were followed at 3-month intervals after operation. Follow-up evaluation included blood examination and chest X-ray every 3 months and chest computed tomography every 6 months. The follow-up period ranged from 4 to 75 months (median 32 months). We compared the survival in the different MMP-2 T:N ratio groups.
Statistical Analysis
Results are expressed as the mean ± standard error (S.E.), and were compared by Student's t test. Differences were considered significant at p ≤ 0.05. Survival was analyzed according to the Kaplan-Meier method, and differences in distribution were evaluated by the log-rank test.
Results
The activities of pro-MMP-2 and MMP-2 were quantified at 66 kDa and 62 kDa, respectively. As shown in figure 1, both pro-MMP-2 and MMP-2 activities were detected in tumor and normal lung tissues. However, activity of MMP-2 (62 kDa/62 kDa + 66 kDa) was significantly higher in tumor tissues (45.5 ± 8.4%) compared to normal lung tissue (25.9 ± 10.5%) (p < 0.001). The T:N ratio ranged from 1.2 to 3.8. No difference was found between the MMP-2 activity in squamous carcinoma and adenocarcinoma. On the other hand, activity of MMP-9 was detected in both normal lung tissue and tumor tissues. Interestingly, activity of MMP-9 was significantly lower in tumor tissues (1,189.3 ± 537 pixel) than in normal lung tissue (1,557.7 ± 422.5 pixel) (p = 0.021) (fig. 1). There was no difference between the MMP-9 activity in squamous carcinoma and adenocarcinoma. As shown in figure 2, although we used different concentrations of tissue extracts and repeated the measurement several times, activity of MMP-7 was only detected in the positive control at 20 kDa, but neither in the tumor tissues nor the normal lung tissues.
Fig. 2
A representative casein zymography indicating the activities of MMP-7 in normal lung (N) and tumor tissues (T) of non-small cell lung cancer. A human amniotic fluid specimen was used as positive control and detected at 20 kDa. MMP-7 activity was not detectable in any tumor and normal lung tissues.
Follow-Up
During the follow-up period, local and distant cancer recurrence was identified in 14 of the 60 (23.3%) stage-I patients. The sites of recurrence include bone in 2, contralateral lung in 5, brain in 3, mediastinal lymph nodes in 3, and in the same lung in 1 patient. Patients were divided into 2 groups according to differences in the MMP-2 T:N ratio. In the group with T:N ratio > 2.5, the survival rate was 57.45% which was significantly reduced compared with the group with T:N ratio ≤ 2.5 (86.78%) (p = 0.015, log-rank test) (fig. 3). The recurrence-free survival rate of the group with T:N ratio > 2.5 was 46.81% which was significantly reduced compared with the group with T:N ratio ≤ 2.5 (81.23%) (p = 0.001, log-rank test) (fig. 4).
Fig. 3
Survival rate of the group with T:N ratio > 2.5 was 57.45% which was significantly reduced compared to the T:N ratio ≤ 2.5 group (86.78%) (p = 0.015, log-rank test).
Fig. 4
Recurrence-free survival rate of the group with T:N ratio > 2.5 was 46.81% which was significantly reduced compared to the T:N ratio ≤ 2.5 group (81.23%) (p = 0.001, log-rank test).
Discussion
MMPs play a key role in cancer progression. MMPs such as interstitial collagenase (MMP-1) and type IV collagenases (MMP-2, MMP-9) are involved in the initial breakdown of collagen and basement membrane components during tumor growth and invasion. Overexpression of MMP-1, MMP-2 and MMP-9, MMP-11, MMP-13, MMP-14-17, MMP-24 and MMP-25, and TIMP-1 and TIMP-2 positively correlated with more advanced-stage disease and highly invasive and meta-static potential of carcinomas [7,8,9].
Previous studies found that protein expression of MMPs was upregulated in lung cancer, and their upregulation appeared to be associated with a poor prognosis. However, protein expression is not always representative of activity. Thus, in the present study, we measured the activities of MMPs. Several studies have shown that MMP-2 activity may be associated with a poor prognosis in lung cancer and other cancers [10]. We found that MMP-2 activity was significantly increased in tumor tissue of NSCLC. This finding is consistent with a previous study; using zymography and immunohistochemistry, Suzuki et al. [10] found that MMP-2 was detected in 5/5 NSCLC, whereas MMP-9 was detected in only 1/5 NSCLC, which suggested that MMP-2 may play a more important role in NSCLC invasion. We previously showed that MMP-2 has a high capacity to decompose tumor extracellular matrix tenascin-C [5]. In this study, we showed that the survival rate of patients with an MMP-2 T:N ratio > 2.5 was 57.45% which was significantly reduced compared to that of patients with a T: N ratio ≤ 2.5 (86.78%). Furthermore, recurrence-free survival in MMP-2 T:N ratio > 2.5 patients was significantly reduced. Thus, MMP-2 may play a more important role in invasion, metastasis, and angiogenesis of early-stage NSCLC.
A number of studies have reported that protein and mRNA expression of MMP-7 was upregulated in certain lung cancer tissues [11]. Interestingly, we did not detect MMP-7 activity in cancer tissues of early-stage NSCLC. Thus, MMP-7 may not play an important role in invasion, metastasis, and angiogenesis of early-stage NSCLC. In addition, although MMP-9 activity was detected in cancer tissues, MMP-9 activity was surprisingly lower in cancer tissues compared to normal lung tissues. Thus, the role of MMP-9 in early-stage NSCLC needs to be further studied.
In summary, a significant increase in MMP-2, but not MMP-9 and MMP-7, was found in tumor tissue of early-stage NSCLC. Development of a selective MMP-2 inhibitor may be beneficial to prevent and treat invasion, metastasis, and angiogenesis of early-stage NSCLC.
Disclosure Statement
We declare that we have no conflict of interest.
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