Protein Kinase C-Mediated in vitro Invasion of Human Glioma Cells through Extracellular-Signal-Regulated Kinase and Ornithine Decarboxylase

Abstract

We investigated the involvement of protein kinase C (PKC) in the in vitro invasiveness of the A-172, U-87 and U-373 human glioma cell lines, as well as the role of ornithine decarboxylase (ODC) and/or extracellular-signal-regulated kinase (ERK) in the actions of PKC. Thus, cells were treated under serum-free conditions with the PKC activator phorbol 12-myristate 13-acetate (PMA), or with the PKC inhibitors bisindolylmaleimide I (GF 109203X) or calphostin C in the absence or presence of the ODC inhibitor D,L-α-difluoromethylornithine (DFMO), and/or the mitogen-activated protein kinase/extracellular-signal-regulated kinase inhibitor 2′-amino-3′-methoxyflavone (PD 098059). Subsequently, cells were assessed for membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA contents, 72-kD latent, and 59/62-kD activated matrix metalloproteinase 2 (MMP-2) in conditioned media, as well as invasiveness. For these purposes, we used Northern blot analysis, gelatine zymography, and an in vitro filter invasion assay, respectively. Data were related to those found with untreated cells. PKC activity was 2- to 3-fold stimulated by PMA (100 nM for 30 min), and about 2-fold inhibited by calphostin C (40 nM for 2 h) or GF 109203X (5 µM for 20 min). This was accompanied by a similar increase or decrease, respectively, in MT1-MMP mRNA expression, 59/62-kD MMP-2 activity, and in vitro invasion. Inhibition of ODC activity (about 2-fold by 24 h DFMO 5 mM), ERK activation (almost completely by 20 min PD 098059 50 µM), or both these enzymes simultaneously led to a reduction by about half in levels of MT1-MMP mRNA, 59/62-kD MMP-2 activity, and invasion in untreated as well as PMA-stimulated cells. The use of these compounds did not significantly alter the inhibitory effects of GF 109203X or calphostin C. Modulation of PKC and/or ERK activity resulted in corresponding changes in ERK and/or ODC activities, but interference with ODC affected neither ERK nor PKC. Our data suggest a regulatory role for PKC, in co-operation with ERK and ODC, in glioma cell invasion, by modulation of MT1-MMP mRNA expression and MMP-2 activation.

Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility

“…Our PKC data lent credence to the hypothesis that claudin-1 might be involved in melanoma malignancy, as PKC is known to be important for melanoma metastasis, and our laboratory as well as others, have shown that mechanisms which upregulate PKC can result in increases in melanoma cell motility (Weeraratna et al, 2002). In addition, PKC activation can increase levels of MMPs, via the p38MAPK pathway (da Rocha et al, 2000;Park et al, 2003). Further data from our laboratory support this observation, showing that the activation of conventional PKCs using phorbol ester results not only in the increase of claudin-1, but also in the increase of MMP-2 secretion (data not shown).…”

Targeting Protein Kinase C: New Therapeutic Opportunities Against High-Grade Malignant Gliomas?

“…Among these is the gene for ornithine decarboxylase (ODC) [68], the rate-limiting enzyme in the biosynthesis of spermine, spermidine, and putrescine [69]. These polyamines have been implicated in cell transformation [70,71], the accelerated G 1 -to-S phase passage of cancer cells [72], as well as processes related to excessive extracellular matrix degradation [68,73,74].…”

“…This could account for the increased ODC mRNA expression and enzyme activity, as well as the progression to malignancy, unchecked S phase transition, growth factor independence, protection against apoptosis, and invasive behavior seen in both non-glioma-and glioma-derived cell lines containing amplified EGF receptor [73][74][75], constitutively active Ras [72], hyperactivated ERK [74,75], and/or overexpressed PKC [69,76,77]. These observations are in accordance with a significant contribution of PKC in well-established signaling pathways for glioma cell proliferation and invasion.…”

“…For instance, exposure of normal or transformed astrocytes or glioma cells to PKC-activating agents such as PMA, led to a more pronounced invasive phenotype, including an increased production of MT1-MMP and MMP-2, enhanced expression of MT1-MMP mRNA, as well as a greater capacity to penetrate through an artificial basement membrane [74,157,167,168]. Inhibition of PKC activity, for instance, by staurosporine, CGP 41251, calphostin C, or GF 109203X elicited the opposite effects [74,167,168].…”

Antitumor effect of antisense ODC adenovirus on human prostate cancer cells

“…The number of invasive cells in rAd-ODC/Ex3as-treated group was 19.478.79, which was far less than that of other groups. As LNCap cell line in essence has little invasion, the Matrigel assay was only carried out in PC-3 32 Similarly, the work of Manni et al 33 supported a role of polyamines in promoting breast cancer aggressiveness, particularly with regard to the development of distant metastasis.…”