Reelin is a secreted, signaling protein associated with neuronal cell positioning and migration. Recently, reelin was found to be epigenetically silenced in gastric and pancreatic cancers in which down-regulation was associated with increased migratory ability and reduced survival.
SummaryAs antigenic stimulation of the B cell antigen receptor (BCR) is key to chronic lymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinases involved in BCR signalling is an attractive therapeutic approach. We studied the effects of the Src/c-Abl tyrosine kinase inhibitor dasatinib on BCR signal transduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinib induced apoptosis by an average reduction in viability of 33AE7% at 48 h, with dasatinib sensitivity correlating with inhibition of Syk Y348 phosphorylation.Dasatinib inhibited calcium flux, phosphatidylinositol-3-kinase and mitogenactivated protein kinase activation following BCR crosslinking, and blocked the Mcl-1-dependent increase in CLL cell survival on prolonged BCR stimulation. However, the pro-apoptotic effect of dasatinib was abrogated by stromal cell contact alone or in the presence of CD154 and interleukin (IL)-4 (CD154L/IL-4 system). Whilst dasatinib retained the ability to sensitize CLL cells in stromal co-culture to both fludarabine and chlorambucil, the addition of CD154 and IL-4 rendered cells resistant to these drug combinations. We demonstrate that the HSP90 inhibitor 17-DMAG exhibited synergy with dasatinib in vitro, and moreover, induced apoptosis of CLL cells in the CD154L/IL-4 system. Our data provide evidence that dasatinib would be most clinically effective in combination with agents able to target antigenindependent microenvironmental signals.
Monoclonal antibodies (MAbs) form a central part of chronic lymphocytic leukaemia (CLL) treatment. 32 We therefore evaluated whether complement defects in CLL patients reduced the induction of 33 complement-dependent cytoxicity (CDC), using anti-CD20 MAbs rituximab (RTX) and ofatumumab 34 (OFA). OFA elicited higher CDC levels than RTX in all CLL samples examined, particularly the poor 35 prognosis cohorts (11q-and 17p-). Serum sample analyses revealed 38.1% of patients were deficient 36 in one or more complement components, correlating with reduced CDC responses. While a proportion 37 of patients with deficient complement levels initially induced high levels of CDC, on secondary 38 challenge CDC activity in sera was significantly reduced, compared with normal human serum (NHS; 39 p<0.01; n=52). Additionally, high CLL cell number contributed to rapid complement exhaustion. 40 Supplementing CLL serum with NHS or individual complement components, particularly C2, restored 41 CDC on secondary challenge to NHS levels (p<0.0001; n=9). In vivo studies revealed that 42 complement components were exhausted in CLL patient sera post-RTX treatment, correlating with an 43 inability to elicit CDC. Supplementing MAb treatment with fresh frozen plasma may therefore maintain 44 CDC levels in CLL patients with a complement deficiency or high white blood cell count. This study 45 has important implications for CLL patients receiving anti-CD20 MAb therapy.
The combination of topotecan and [(131)I]MIBG compared with either treatment alone gave rise to greater than additive DNA damage, clonogenic cell kill, and tumor growth delay. These effects were dependent on the scheduling of the two agents.
Purpose: Chronic lymphocytic leukemia (CLL) is currently incurable with standard chemotherapeutic agents, highlighting the need for novel therapies. Overcoming proliferative and cytoprotective signals generated within the microenvironment of lymphoid organs is essential for limiting CLL progression and ultimately developing a cure.Experimental Design: We assessed the potency of cyclin-dependent kinase (CDK) inhibitor CR8, a roscovitine analog, to induce apoptosis in primary CLL from distinct prognostic subsets using flow cytometry-based assays. CLL cells were cultured in in vitro prosurvival and proproliferative conditions to mimic microenvironmental signals in the lymphoid organs, to elucidate the mechanism of action of CR8 in quiescent and proliferating CLL cells using flow cytometry, Western blotting, and quantitative real-time PCR.Results: CR8 was 100-fold more potent at inducing apoptosis in primary CLL cells than roscovitine, both in isolated culture and stromal-coculture conditions. Importantly, CR8 induced apoptosis in CD40-ligated CLL cells and preferentially targeted actively proliferating cells within these cultures. CR8 treatment induced downregulation of the antiapoptotic proteins Mcl-1 and XIAP, through inhibition of RNA polymerase II, and inhibition of NF-kB signaling at the transcriptional level and through inhibition of the inhibitor of IkB kinase (IKK) complex, resulting in stabilization of IkBa expression.Conclusions: CR8 is a potent CDK inhibitor that subverts pivotal prosurvival and proproliferative signals present in the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of selective CDK inhibitors as novel therapies for CLL.
Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor-(VEGF-) and fibroblast growth factor-2-(FGF-2)-induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor-induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy-induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF-and FGF-2-specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT-PCR analysis of VEGF and FGF-2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48 -72 hr of treatment with HPR, followed by a progressive accumulation of cells in G 1 at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF-2 receptor-2 was reduced after implantation of HPR-loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors.
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