A Natural Bacterial-Derived Product, the Metalloprotease Arazyme, Inhibits Metastatic Murine Melanoma by Inducing MMP-8 Cross-Reactive Antibodies

Abstract

The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent.

TLR4-mediated immunomodulatory properties of the bacterial metalloprotease arazyme in preclinical tumor models

“…As showed previously for active arazyme, 14 treatment of mice with heat-inactivated protease induced high titers of arazyme-specific IgGs (Fig. S1).…”

“…These antibodies were cytotoxic in vitro to B16F10-Nex2 cells, and passive transfer partially inhibited lung metastatic nodule formation. 14 In the present study, we show that the anti-melanoma effect of arazyme also involves a strong immune system activation by a mechanism independent of its proteolytic activity.…”

“…12 Arazyme, a 51.5 kDa metalloprotease secreted by Serratia proteamaculans, a symbiotic bacterium from the Nephila clavata spider, 13 had a strong antitumor effect in a murine metastatic melanoma B16F10-Nex2 model, mediated by the cleavage of tumor cell surface CD44 and the induction of arazyme-specific antibodies that cross-react with tumor matrix metalloprotease 8 (MMP-8). 14 In this study, we show that, in addition to its proteolyticdependent activity, arazyme has a secondary, non-proteolytic antitumor effect dependent on an intact immune system. The anti-melanoma immune response induced by heat-inactivated arazyme was dependent on IFNg, and CD8 C T lymphocytes were identified as the main effector cells for tumor rejection.…”

“…In vitro arazyme-specific antibodies were cytotoxic to tumor cells, an effect increased by complement, and passive transfer to tumor-bearing mice partially inhibited melanoma lung metastasis. 14 In order to verify whether the proteolytic activity of the enzyme was responsible for the antitumor effect, arazyme was heat-inactivated at 56 C, 13 and used in the same treatment protocol. Melanoma-bearing C57Bl/6 male mice inoculated intraperitoneally with active or inactive arazyme on alternate days for 2 weeks showed a significant reduction in the number of metastatic lung nodules compared to PBS-treated mice (Figs.…”

Regulating effect of MMP-9 and TIMP-1 in pi­tuitary adenoma invasion

“…This effect damages the physical barrier around tumor cells, increasing the tumor biological activity by degrading the matrix components, which allows the tumor to escape from the immune system. The changes of the ECM lead to an increase of tumor angiogenesis, leading to further pituitary adenoma proliferation and invasive growth (Ramanujum et al, 2013;Pereira et al, 2014).…”