Prognosis for patients with glioblastoma multiforme (GBM) is poor. Inhibitors of histone deacetylases (HDACi) like trichostatin A (TSA) are promising alternatives to conventional treatment. Deficient tumor suppressor functions, such as TP53 mutations and p14(ARF)/p16(INK4a) deletions, are characteristic for GBM and can cause resistance to DNA damaging agents such as cisplatin and to HDACi like TSA. The type II tumor suppressor Inhibitor of growth 1 (ING1) is involved in DNA damage response and histone modification. We have previously shown that ING1 is downregulated in GBM and involved in glioma-induced angiogenesis and in cisplatin-induced apoptosis in malignant glioma cells. Hence, the goal of our present study was to investigate whether TSA affects ING1 protein expression and also whether modulating ING1 levels affects TSA-induced apoptosis in malignant glioma cells that contain deficient p53 function and inactive pl4(ARF)/p16(INK4a) signaling. If so, we asked, which apoptotic pathway might be the major mediator beyond this interaction. To test whether ING1 proteins function in TSA-induced apoptosis in GBM, we analyzed TSA effects in LN229 GBM cells, which harbor TP53 mutations and INK4a deletion, following ING1 knockdown by siRNA. Expression of ING1, acetylated core histones H3 and H4, and the proapoptotic proteins caspase 3 and Fas-associated death domain (FADD) was determined by Western blotting. Percentages of apoptotic cells were obtained by flow cytometry. TSA induced the major ING1 isoform p33(ING1b) and increased levels of both histone acetylation and apoptosis in LN229 cells. ING1 knockdown cells revealed marked resistance to TSA-induced apoptosis, impairment of caspase 3 activation, and suppression of FADD. The data suggest that ING1 contributes to TSA-induced apoptosis in GBM cells with deficient p53 and p14(ARF)/p16(INK4a) functions, possibly by regulating FADD/caspase 3 signaling.

The inhibitor of growth 1 (ING1) homologue ING4 has previously been implicated as a negative regulator of angiogenesis in a murine glioma and a multiple myeloma model. An association between ING1 and angiogenesis has not been reported yet. Our previous studies using tumor samples from patients have shown that ING1 levels are downregulated in glioblastoma multiforme (GBM), one of the most highly vascularized malignancies. Based on this background, the goal of this study was to test the effects of the major ING1 splicing isoforms, p47ING1a and p33ING1b, on pathological angiogenesis induced by human GBM cells. We used a chorioallantoic membrane (CAM) assay to examine whether LN229 human GBM cells can induce angiogenesis and whether alterations in ING1 expression, such as ING1 knockdown by siRNA or ectopic ING1 overexpression using ING1a and ING1b expression constructs, can affect this process. Increased ING1 protein expression significantly suppressed LN229 cell-induced angiogenesis in the CAM assay. While no effects on the proangiogenic factors VEGF or IL-8 were noted, the expression of angiopoietins (Ang) 1 and 4 were increased by the p47ING1a, but not by the p33ING1b isoform. Levels of Ang-2 were not sensitive to altered ING1 levels. Our data are the first to suggest that ING1 proteins suppress neoangiogenesis in GBM. Moreover, our results may support the idea that ING1 proteins regulate the expression of proteins that are critical for angiogenesis in GBM such as the angiopoietins.