Cathepsin L mediates resveratrol-induced autophagy and apoptotic cell death in cervical cancer cells

Abstract

Cathepsins have long been considered as housekeeping molecules. However, specific functions have also been attributed to each of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wortmannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an antiapoptotic role through anti-cat L activity.

Human papillomavirus infection and expression of ATPase family AAA domain containing 3A, a novel anti-autophagy factor, in uterine cervical cancer

“…The loss of mitochondrial membrane integrity would then lead to a failure to keep programmed cell death-related proteins, e.g., apoptosis-inducing factor (AIF) and cytochrome c, in check within the mitochondria. This concept was strengthened by the observations that resveratrol inhibited ATAD3A expression and induced autophagy as well as apoptosis, which corresponded well with results reported by Hsu et al (38).…”

The autophagic paradox in cancer therapy

“…In fact, lysosomal membrane permeabilization (LMP) may underlie the therapeutic actions of some pro-autophagic drugs, such as temozolomide and brevinin-2R (Mathieu et al, 2007;Ghavami et al, 2008). The causational relationship between autophagy and LMP is exemplified by the finding that inhibition of resveratrol-induced autophagy by wortmannin or asparagine results in reduced LMP and resveratrol-associated cytotoxicity (Hsu et al, 2009). The induction of Atg5 and Beclin 1 together with formation of LC3-positive autophagosomes have also been shown to precede lysosomal dysfunction, cathepsin activation and cell death in cells treated with OSU-03012, a celecoxib derivative (Park et al, 2008).…”

“…It is believed that unrestrained autophagy or unwarranted activation of autophagy in the midst of nutritional abundance may result in premature exhaustion of internal feeding materials, thereby making the cell vulnerable in face of real nutritional challenges. Excessive autophagy may also compromise the integrity of lysosomal membrane, resulting in the release of cathepsins into the cytosol (Park et al, 2008;Hsu et al, 2009;Bhoopathi et al, 2010). In fact, lysosomal membrane permeabilization (LMP) may underlie the therapeutic actions of some pro-autophagic drugs, such as temozolomide and brevinin-2R (Mathieu et al, 2007;Ghavami et al, 2008).…”

Cathepsins are involved in virus-induced cell death in ICP4 and Us3 deletion mutant herpes simplex virus type 1-infected monocytic cells

“…Since cathepsin L is known to mediate caspase 3-dependent apoptosis (Hsu et al, 2009), we studied caspase 3 activity in monocytic cells. The HSV-1 protein kinase Us3 blocks caspase 3-mediated apoptosis (Benetti & Roizman, 2007;Hagglund et al, 2002).…”

“…Moreover, the substrate used for the cathepsin L assay was also the substrate for cathepsin B; even if cathepsin B activity was blocked with the cathepsin B inhibitor, the detected cathepsin activity could be the sum of the activities of cathepsins L and B. However, since cathepsin L-inhibitor treatment decreased the amount of apoptosis in d120-infected cells, it appears that cathepsin L is active in d120-infected cells.Since cathepsin L is known to mediate caspase 3-dependent apoptosis (Hsu et al, 2009), we studied caspase 3 activity in monocytic cells. The HSV-1 protein kinase Us3 blocks caspase 3-mediated apoptosis (Benetti & Roizman, 2007;Hagglund et al, 2002).…”