Protection of glioblastoma cells from cisplatin cytotoxicity via protein kinase Cι-mediated attenuation of p38 MAP kinase signaling

Abstract

Glioblastoma multiforme is an aggressive form of brain cancer that responds poorly to chemotherapy and is generally incurable. The basis for the poor response of this cancer to chemotherapy is not well understood. The atypical protein kinases C (PKCi and PKCf) have previously been implicated in leukaemia cell chemoresistance. To assess the role of atypical PKC in glioblastoma cell chemoresistance, RNA interference was used to deplete human glioblastoma cells of PKCi. Transfection of cells with either of two different RNA duplexes specific for PKCi caused a partial sensitisation to cell death induced by the chemotherapy agent cisplatin. To screen for possible mechanisms for PKCi-mediated chemoresistance, microarray analysis of gene expression was performed on RNA from glioblastoma cells that were either untreated or depleted of PKCi. This identified sets of genes that were regulated either positively or negatively by PKCi. Within the set of genes that were negatively regulated by PKCi, the function of the gene coding for GMFb, an enhancer of p38 mitogen-activated protein kinase (MAP kinase) signaling, was investigated further, as the p38 MAP kinase pathway has been previously identified as a key mediator of cisplatin cytotoxicity. The expression of both GMFb mRNA and protein increased upon PKCi depletion, and this was accompanied by an increase in cisplatin-activated p38 MAP kinase signaling. Transient overexpression of GMFb increased cisplatinactivated p38 MAP kinase signaling and also sensitised cells to cisplatin cytotoxicity. The increase in cisplatin cytotoxicity seen with PKCi depletion was blocked by the p38 MAP kinase inhibitor SKF86002. These data show that PKCi can confer partial resistance to cisplatin in glioblastoma cells by suppressing GMFb-mediated enhancement of p38 MAP kinase signaling.

Regulation of glioblastoma cell invasion by PKCι and RhoB

“…The repression of RhoB expression by PKCi also provides a mechanism for (1) the previously reported downregulation of RhoB in human glioblastoma, and (2) the PKCi-mediated loss of actin stress fibers that has been observed in a number of different cell types. These results and our previous study (Baldwin et al, 2006) suggest that PKCi should be evaluated further as a potential drug target for glioblastoma therapy. monoclonal were from BD Biosciences (Mississauga, ON, Canada).…”

“…To assess the role of PKCi in glioblastoma cell motility, we used 'scratch-wound' assays together with RNAi to reduce PKCi protein levels. We have previously shown that RNAi effectively reduces PKCi mRNA and protein levels by 75-90% without affecting mRNA levels of other PKC family members (Baldwin et al, 2006). These assays showed that U87MG cells depleted of PKCi with two different RNA duplexes did not fill in a scratch as rapidly as cells treated with a control RNA duplex, suggesting decreased cell motility ( Figure 1a and Supplementary Figure S1A).…”

“…Microarray expression analysis was previously performed on U87MG cells that were depleted of PKCi by RNAi (Baldwin et al, 2006). Supplementary Table S1 shows microarray analysis data for RhoB mRNA expression.…”

“…Depletion of PKCi using RNAi was carried out as described previously (Baldwin et al, 2006). The control RNA duplex had the sense RNA sequence UAGCGACUAAACACAUCAA (siControl, Dharmacon RNA Technologies Inc. (Lafayette, CO, USA)).…”

“…To assess the role of PKCi in glioblastoma, we previously performed a microarray analysis of gene expression in human glioblastoma cells depleted of PKCi by RNA interference (RNAi) (Baldwin et al, 2006). This identified a number of candidate genes that are potentially regulated by PKCi.…”