Beneficial effects of cathepsin inhibition to prevent chemotherapy-induced intestinal mucositis

Abstract

One of the main secondary toxic side effects of anti-mitotic agents used to treat cancer patients is intestinal mucositis. Previous data showed that cathepsin D activity, contributing to the proteolytic lysosomal pathway, is up-regulated during intestinal mucositis in rats. At the same time, cathepsin inhibition limits intestinal damage in animal models of inflammatory bowel diseases. The aim of this study was to evaluate the effects of cathepsin inhibition on methotrexate-induced mucositis in rats. Male Sprague-Dawley rats received saline solution subcutaneously as the control group or 2·5 mg/kg of methotrexate for 3 days (D0-D2). From D0 to D3 methotrexate-treated rats also received intraperitoneal injections of pepstatin A, a specific inhibitor of cathepsin D or E64, an inhibitor of cathepsins B, H and L, or vehicle. Rats were euthanized at D4 and jejunal samples were collected. Body weight and food intake were partially preserved in rats receiving E64 compared with rats receiving vehicle or pepstatin A. Cathepsin D activity, used as a marker of lysosomal pathway, was reduced both in E64 and pepstatin-treated rats. However, villus atrophy and intestinal damage observed in methotrexate-treated rats were restored in rats receiving E64 but not in rats receiving pepstatin A. The intramucosal concentration of proinflammatory cytokines, interleukin-1β and cytokine-induced neutrophil chemoattractant (CINC)-2, was markedly increased in methotrexate-treated rats receiving vehicle or pepstatin A but not after E64 treatment. In conclusion, a large broad inhibition of cathepsins could represent a new potential target to limit the severity of chemotherapy-induced mucositis as opposed to the inhibition of cathepsin D alone.

Fig. 1

Food intake (a) and body weight changes (b) in control (closed circles) and methotrexate (MTX)-treated rats receiving vehicle (open circles), pepstatin A (open triangles) or E64 (closed triangles). Values are means ± standard error of the mean from five rats in each group. #P < 0·05 versus all other groups, †P < 0·05 versus MTX-treated rats receiving vehicle or pepstatin A.

Fig. 2

Histology, villus height and damage score in the jejunum. Representative observations (×5) (a), villus height (b) and damage score (c) in jejunal mucosa of control [(1) or open bars] and methotrexate (MTX)-treated rats receiving vehicle [(2) or closed bars], pepstatin A [(3) or dark grey bars] or E64 [(4) or grey bars]. Values are means ± standard error of the mean from five rats in each group. *P < 0·05 versus control rats, †P < 0·05 versus MTX-treated rats receiving vehicle or pepstatin A.

Fig. 3

Intramucosal concentrations and mRNA levels of cytokine-induced neutrophil chemoattractant (CINC)-2 (a,d), interleukin-1β (b,e) and tumour necrosis factor-α (c,f) in the jejunum of control (open bars) and methotrexate (MTX)-treated rats receiving vehicle (closed bars), pepstatin A (dark grey bars) or E64 (grey bars). Values are means ± standard error of the mean from five rats in each group. *P < 0·05 versus control rats, †P < 0·05 versus MTX-treated rats receiving vehicle or pepstatin A.

Fig. 4

Cathepsin D expression (a) and activity (b) in the jejunum of control (open bars) and methotrexate (MTX)-treated rats receiving vehicle (closed bars), pepstatin A (dark grey bars) or E64 (grey bars). Values are means ± standard error of the mean from five rats in each group. *P < 0·05 versus control rats, ‡P < 0·05 versus MTX-treated rats.

Fig. 5

Chymtrypsin-like (a) and peptidase (b) activities of proteasome in the jejunum of control (open bars) and methotrexate (MTX)-treated rats receiving vehicle (closed bars), pepstatin A (dark grey bars) or E64 (grey bars). Values are means ± standard error of the mean from five rats in each group. *P < 0·05 versus control rats.