Survivin, a member of the inhibitor of apoptosis (IAP) gene family, plays an important role in both the regulation of cell cycle and the inhibition of apoptosis, and is frequently overexpressed in many tumor types. In neuroblastomas, the expression of survivin correlates with a more aggressive and histologically unfavorable disease. Survivin is predominantly a cytoplasmic protein that is expressed in a cell cycle-dependent manner, increasing in the G2/M phase of the cell cycle followed by a rapid decline in the G1 phase. Recently, the role of survivin in resistance to chemotherapy has become an area of intensive investigation. In this study, we demonstrate a phase-specific resistance due to survivin in staurosporine (STS)-induced apoptosis in the human neuroblastoma cell line SK-N-MC. G2/M-arrested cultures show an upregulation of survivin expression and are more resistant, whereas G1-phase cells that show decreased levels of survivin are more sensitive to apoptosis. Localization studies revealed differences in the distribution of survivin in two synchronized populations, with G1 cells having weakly positive staining confined to the nucleus, in contrast to G2/M cells that depicted a more uniform and intense expression of survivin throughout the cell. In our experimental system, STS induced apoptosis through the mitochondrial-caspase 9-mediated pathway. Retention of survivin in G1 cells by inhibition of the ubiquitin-proteosome pathway or inhibition of caspase 9 protected the cells against apoptosis. Our data suggest that survivin exerts its antiapoptotic effect by inhibiting caspase 9 activity, an important event in STS-mediated apoptosis. In context with cell cycle-dependent responses to chemotherapy, the data from this study suggest the possibility of exploiting the survivin pathway for inducing apoptosis in tumor cells.

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Background: The role of TNF-α in affecting the fate of tumors is controversial, while some studies have reported apoptotic or necrotic effects of TNF-α, others provide evidence that endogenous TNF-α promotes growth and development of tumors. Understanding the mechanism(s) of TNF-α mediated growth arrest will be important in unraveling the contribution of tissue associated macrophages in tumor resistance. The aim of this study was to investigate the role of Cyclin Dependent Kinase Inhibitors (CDKI) -p21 cip/waf1 and p27 kip1 in TNF-α mediated responses in context with p53 and activation of NF-κB and Akt pathways. The study was done with human glioma cell lines -LN-18 and LN-229 cells, using monolayer cultures and Multicellular Spheroids (MCS) as in vitro models.

Tumor associated macrophages (TAMs) constitute a substantial mass in gliomas. The activated macrophages secrete various cytokines that affect diverse functions of tumors. The aim of this study was to elucidate the role of Akt and NF-kappaB pathways in resistance to TNF-alpha mediated cell death in human gliomas using monolayers and multicellular spheroids (MCS) as in vitro models. Akt and NF-kappaB are constitutively expressed and intimately involved in progression of gliomas. The activation of these pathways also renders the tumors resistant to conventional treatments including chemotherapy. While PI3K/Akt is shown to regulate the NF-kappaB activation in diverse systems, other studies place NF-kappaB upstream of Akt activation. Using a stable IkappaBalpha mutant LN-18 cell line and pharmacological inhibitors to PI3K/Akt (LY294002) and Akt (Akt2), we provide evidence that Akt and NF-kappaB are activated independently on stimulation with TNF-alpha and both the pathways contribute towards resistance to TNF-alpha mediated cell death. TNF-alpha-induced NF-kappaB activation independent of PI3K/Akt pathway was also confirmed in human glioma cell lines-LN-229 and U373MG. We also show that NF-kappaB and Akt are activated during spheroidogenesis and their expression is further enhanced on stimulation with TNF-alpha implicating their involvement in resistance to cell death. The findings thus underscore the relevance of spheroids as appropriate in vitro models for studying the signaling pathways in drug induced resistance.