Pursuant to our in vitro data
Pursuant to our in vitro data that neutrophilic elastase activated the elastase-dependent oncolytic virus, we initially hypothesized that tumor-infiltrating neutrophils could be the source for elastase in tumor tissue. However, we did not detect neutrophils in the PANC-1 tumor tissue where we observed a beneficial therapeutic effect of the elastase activated virus. Alternative sources of elastase could be macrophages25, 26 or tumor R 568 hydrochloride themselves. It was suggested that tumor cells have a “protease cloud” surrounding their surface. This “protease cloud” was shown to activate reovirus. However, as tumor cells in culture do not activate the NS116-GFP/AE virus in the absence of exogenous protease, it appears to be clear that the source of elastase within such a “protease cloud” is provided or stimulated by cells of the tumor microenvironment. Thus, activation of oncolytic viruses by proteases appears to be a more general and valuable approach to ensure virus targeting to tumors. We have now adapted this approach for influenza virus.
Our data also support previous findings that pancreatic cancer might be an attractive target for influenza virus oncolytic therapy. This virolysis of pancreatic cells may be supported by (1) the inherent susceptibility of pancreatic tissue to influenza virus and (2) by the fact that PANC-1 as well as 90% of pancreatic cancers have a KRAS gene mutation, leading to a suppression of the antiviral PKR pathway. Pancreatic cancer tissue specifically might be a target for elastase-dependent influenza virus given that the pancreatic cancer microenvironment often expresses proteases including elastase.
Modulation of virus entry proteins to target viruses to cancer has previously been conducted with virus families other than orthomyxoviruses.29, 30, 31 In this study, we applied this principle to IAV. A disadvantage of this approach could be that virus-entry-protein-modified viruses may impair viral growth, making production difficult and cumbersome. However, we demonstrated that the elastase-dependent virus grew as efficiently as the trypsin-activated virus in Vero cells, which is the cell line licensed for use for virus production. This indicates that the production of such viruses appears less of a problem.
Materials and Methods
Conflicts of Interest
Acknowledgments This work was supported by internal funds of the Medical University of Vienna (to M.B.) and overhead funds related to EU grants (FP6-518281 and FP6-044512 to M.B.). We thank Julia Bespalova for help in neutrophil isolation. We thank Dr. Lindsay Brammen and Leah Hunter for proofreading the manuscript.
Introduction Melanin is the primary determinant of the skin color. It protects the skin from absorbing ultraviolet (UV) radiation by 50% to 75% and scavenges reactive oxygen species (ROS) (Parvez et al., 2007, Tada et al., 2010). Tyrosinase, a copper-containing monooxy-genase, is a key regulatory enzyme that catalyzes melanin synthesis within melanocytes. The major actions of this enzyme are the hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) and consequent oxidation of L-DOPA to dopaquinone. Dopaquinone is a highly reactive compound that can polymerize spontaneously to form melanin (Prota, 1988). However, overproduction of melanin in the skin may cause hyperpigmentation, melanoma, and could be genotoxic (Brozyna et al., 2007). Therefore, tyrosinase inhibitors have become increasingly important in cosmetics and pharmaceuticals as whitening agents and for the treatment of pigmentory disorders such as melasma, lentigines, and freckles. Elastin is a highly elastic protein found in connective tissues including skin, lung, and arteries and helps in maintaining tissue configuration after stretch or recoil (Daamen et al., 2007, Debelle and Alix, 1999). Overexposure to UV irradiation and oxidative damage up regulate the expression of elastase, a serine protease which hydrolyzes the dermal elastin fiber network (Braverman and Fonferko, 1982, Kim et al., 2006). This may to lead to reduced skin elasticity and the linearity of dermal elastic fibers, inducing wrinkling and sagging (Kambayashi et al., 2001). There are several research studies that suggest both skin-aging and anti-wrinkle effects are directly associated with degradation of elastin (Akazaki et al., 2002). Recently, a number of studies have investigated the interactions between elastase and its inhibitors such as catechin, epigallocatechin gallate, boswellic acids, and purpurin (Kim et al., 2004, Melzig et al., 2001, Thring et al., 2009, Biswas et al., 2015, Bravo et al., 2016). It has been reported that the topical application of specific inhibitors to the surface of human skin may have beneficial effects on UV-irritated and dry skin.