Resistance to induced apoptosis in the human neuroblastoma cell line SK‐N‐SH in relation to neuronal differentiation

Abstract

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4b-phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by g-enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin-induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro-(Bcl-2, Bcl-x L ) and anti-apoptotic (Bax, Bak) protein expression of the Bcl-2 family.Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl-2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl-x L expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl-2 protein family and the resistance of differentiated SK-N-SH to apoptosis.

Bcl-2 is a key regulator for the retinoic acid-induced apoptotic cell death in neuroblastoma

“…Antiapoptotic Bcl-2 is one of the most important members that inhibits the mitochondria-dependent apoptotic pathway triggered by diverse cytotoxic agents through blocking mitochondrial permeability transition. Indeed, the upregulation of Bcl-2 was associated with the drug-resistant phenotype of certain human tumors (Dole et al, 1994;Lombet et al, 2001). Most intriguingly, our expression studies revealed that antiapoptotic Bcl-2 was constitutively overexpressed in LA-N-5 and RTBM1 cells, whereas its expression levels were extremely low in CHP134 and NB-39-nu cells.…”

Expression of the tumour suppressor gene CADM1 is associated with favourable outcome and inhibits cell survival in neuroblastoma

“…In line with these results, a substantial reduction of cell viability was found in an LDH release assay, suggesting that over-expression of CADM1 in human neuroblastoma cell lines does not only reduce proliferation, but might also function as a proapoptotic or pronecrotic regulator. CADM1 over-expression is known to induce apoptosis in A549 non-small cell lung cancer cells (Mao et al, 2004), but many human neuroblastoma cell lines show considerable resistance to apoptosis when exposed to common apoptosis inducing triggers (Teitz et al, 2000;Lombet et al, 2001). Therefore, further experiments are necessary to more precisely delineate the mechanisms by which CADM1 triggers changes in growth properties and loss of cell viability.…”

Low levels of mycophenolic acid induce differentiation of human neuroblastoma cell lines

“…17,18 Bax expression is not significantly modified. 15 Therefore, although both MPA and retinoic acid induce similar morphologic differentiation in neuroblastoma cells, the molecular mechanisms involved appear to be different. In conclusion, MPA, at concentrations corresponding to those of the active free drug readily attainable therapeutically using mycophenolate mofetil, induces neuroblastoma cells to differentiate toward the neuronal phenotype, confirming a potential role of CellCept in the management of neuroblastoma patients.…”