Cathepsin B fraction active at physiological pH of 7.5 is of prognostic significance in squamous cell carcinoma of human lung

Abstract

Summary In this study we examined both the pH dependence of cathepsin B (cath B) activity and its stability at physiological pH of 7.5 in lung tumours and normal lung tissue by means of fluorogenic assays with Z-Arg-Arg-AMC as specific substrate. Specificity was verified with the cath B blocking inhibitors E-64 and CA-074. With respect to pH dependence of activity, we found a deviation from a normal-shaped pHactivity curve. Besides the typical activity peak at pH 6.0, there were shoulders at pH 4.5-5.5 and at pH 7.0-7.5. This heterogeneity was found in both tumour and normal tissue. To test the stability of cath B at physiological pH of 7.5, homogenates were kept at pH 7.5 for 60 min. Altogether, 82-100% of residual cath B activity was found at pH 5.0-5.5, whereas activity in the range between 5.5 and 7.4 dropped drastically to 26-42%. At pH 7.5, there was still 20-34% residual cath B activity detectable. To test the hypothesis whether the cath B fraction active at pH 7.5 is more abundant in tumour tissues compared with the normal counterparts, we determined this fraction in 91 pairs of lung tumour and normal lung tissue. We found a 2.3-fold increase of median cath B fraction active at pH 7.5 in tumour tissue, although this fraction represented only a small part (about 16%) of the native, acidic (pH 6.0) cath B activity. However, in contrast to native cath B at 6.0, the cath B fraction active at pH 7.5 was related to post-operative probability of survival in curatively operated patients, since activity values higher than 292 (,uEU mg-' protein) were significantly associated with poor prognosis in patients with squamous cell carcinomas (n = 33, P = 0.04). It is concluded that in lung tumour and in normal lung tissue, cath B activity can be divided into at least three fractions with stability optima at different pH values, indicating various forms of cath B. The cath B fraction active at pH 7.5 provides prognostic information in patients with squamous cell carcinoma.

Immunochemical analysis of cathepsin B in lung tumours: an independent prognostic factor for squamous cell carcinoma patients

“…In contrast, cath B activities (cath B AT , cath B A7.5 ) and concentration (cath B C ), measured in tumour homogenates, had no prognostic relevance in the total population of NSCLC patients in a 5-year observation period. This is in contrast to the study by Ebert et al (1994) and Knoch et al (1994), but similar to the recent study (Werle et al, 1997a). The discrepancy with earlier studies is most likely due to longer observation period of 5 years in the present study.…”

“…Previous studies on cath B in lung tumours, using either homogenates (Krepela et al, 1990;Sedo et al, 1991;Ebert et al, 1994;Ledakis et al, 1996;Werle et al, 1997aWerle et al, , 1997b or IHC analysis of cath B (Higashiyama et al, 1991(Higashiyama et al, , 1993Ozeki et al, 1993;Inoue et al, 1994;Sukoh et al, 1994aSukoh et al, , 1994b, showed significantly enhanced expression of the enzyme at activity and protein levels in patients suffering from NSCLC. In this study, we clearly confirmed our earlier results as we found 9.1-fold increased cath B protein concentration as well as 5.6-fold increased total cath B activity and 3.2-fold increase in the fraction of cath B activity, stable at pH 7.5 (Werle et al, 1997b) in tumour compared with normal lung tissue homogenates.…”

“…Both assays were performed exactly as described in detail earlier (Werle et al, 1997a(Werle et al, , 1997b. Total cath B AT activity was measured with the substrate Z-Arg-Arg-AMC at pH 6.0.…”

“…metastasis (reviewed by Berquin and Sloane, 1995;Elliot and Sloane, 1996;Lah and Kos, 1998;Yan et al, 1998). There are a number of evidences, supporting this hypothesis: the levels of cath B in tumours were elevated above the levels in adjacent normal tissue as demonstrated for example in lung carcinoma (Trefz et al, 1989;Krepela et al, 1990;Sukoh et al, 1994aSukoh et al, , 1994bEbert et al, 1994;Ledakis et al, 1996;Werle et al, 1997aWerle et al, , 1997b, breast carcinoma (Lah et al, 1992(Lah et al, , 1995a, colon carcinoma (Shuja et al, 1991), head and neck carcinoma (Budihna et al, 1996), oesophageal carcinoma (Hughes et al, 1998) and glioma (Rempel et al, 1994;Mikkelsen et al, 1995). Elevated levels of the enzyme were also found in sera of tumour patients (Leto et al, 1997;Kos et al, 1997Kos et al, , 1998.…”

Cathepsin B in infiltrated lymph nodes is of prognostic significance for patients with nonsmall cell lung carcinoma

“…The homogenization was performed according to our published protocol. 15 Aliquots of the tissue homogenates were made and kept frozen at Ϫ80°C.…”