PKCδ Sensitizes Neuroblastoma Cells to L-Buthionine-Sulfoximine and Etoposide Inducing Reactive Oxygen Species Overproduction and DNA Damage

Abstract

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches.

p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment

“…In accordance with other studies, 20 we have recently reported that etoposide causes DNA damage and an overproduction of reactive oxygen species (ROS), 21 which have been demonstrated to mediate both cell damage and biological functions. 22 In this regard, herein we show that etoposide induces a dose-dependent increase in the levels of the proapoptotic PKCδ 23 and a parallel decrease of PKC α , the antiapoptotic isoform.…”

“…However, given that PKCs are upstream molecules in the ROS signaling pathway leading to DNA damage and apoptosis, 21, 25, 26 it is important to identify the downstream mediators of the NB response to etoposide and we show that etoposide induces the activation of Akt and MAPKs (i.e. JNK, and p38).…”

Interplay Between Protein Kinase C Isoforms Alpha and Epsilon, Neurofibromin, and the Ras/MAPK Pathway in Neuroblastoma Differentiation

“…Unlike , is commonly expressed (Zeidman et al, 1999b), yet again, transfection studies with full-length, catalytically inactive, or active PKC variants have revealed a pro-apoptotic role (Schultz et al, 2003;Schultz & Larsson, 2004). A major role of PKC in the sensitization of NB cells (SH-SY5Y and SK-N-BE(2C)) to etoposide (Marengo et al, 2011) was just recently described, validating similar observations in other cell types (Griner and Katanietz, 2007). The function of aPKCs in NB differentiation is not clear at present, except recent indications that inhibition of PKC has pro-apoptotic, and antiproliferative effects (Pilai et al, 2011).…”

Role of Glutathione in Cancer Progression and Chemoresistance

“…However, molecular signaling of BSO-induced apoptosis is poorly understood, and, recently, it has been demonstrated that in different leukaemia and lymphoma cells, the death receptor-mediated apoptotic pathway, induced by arsenic trioxide plus BSO, is triggered via JNK activation [58]. Moreover, in neuroblastoma cells susceptible to BSO treatment, DNA damage and apoptosis was triggered via PKC- δ activation and ROS production [59, 60]. …”