Biomedical subjects

George J Cerniglia

Publications and source records attributed to George J Cerniglia.

10 recordsLinked to original sources

PubMed2007

Bortezomib sensitizes human head and neck carcinoma cells SQ20B to radiation.

The proteasome inhibitor bortezomib was tested in a cell screen as a single agent with good efficacy in multiple hematologic and solid cancer cell lines. Phase II/III studies have supported the use of bortezomib in hematologic malignancies. In solid tumors, however, the results have been poor. There is data that bortezomib can induce PTEN expression resulting in down-regulation of PI3K-Akt signaling. We and others have shown that down-regulation of Akt results in radiation sensitization. We therefore evaluated the use of bortezomib in the head and neck cancer cell line SQ20B as a radiation sensitizer. SQ20B have a constitutively active mutation in EGFR resulting in a robust Akt response. We found that 10 nM of bortezomib decreased Akt signaling to almost undetectable. This same concentration decreased the surviving fraction after 2 Gy (SF2) from 0.77 to 0.45. Given that radiation is usually given at 2 Gy increments daily for 30 or more treatments, the exponential difference in log kill could be as high as 7 logs. The dose of bortezomib is also 2 logs less as a sensitizer than that required for single agent efficacy. Further studies should be done to explore this model in vivo.

Antineoplastic Agents↗

PubMed2006

Nelfinavir down-regulates hypoxia-inducible factor 1alpha and VEGF expression and increases tumor oxygenation: implications for radiotherapy.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) expression. We examined the effect of nelfinavir, an HIV protease inhibitor that inhibits Akt signaling, on VEGF and HIF-1alpha expression and on angiogenesis, tumor oxygenation, and radiosensitization. Nelfinavir decreases VEGF expression under normoxia via the transcription factor Sp1, which regulates the proximal core VEGF promoter. Nelfinavir decreased Sp1 phosphorylation and decreased Sp1 binding to a probe corresponding to the proximal VEGF promoter in a gel shift assay. Nelfinavir also decreased the hypoxic induction of HIF-1alpha, which also regulates the VEGF promoter, most likely by decreasing its translation. The effect of nelfinavir on VEGF expression had the functional consequence of decreasing angiogenesis in an in vivo Matrigel plug assay. To determine the effect this might have on tumor radiosensitization, we did tumor regrowth assays with xenografts in nude mice. The combination of nelfinavir and radiation increased time to regrowth compared with radiation alone whereas nelfinavir alone had little effect on tumor regrowth. This radiosensitizing effect was greater than suggested by in vitro clonogenic survival assays. One possible explanation for the discordance is that nelfinavir has an effect on tumor oxygenation. Therefore, we examined this with the hypoxia marker EF5 and found that nelfinavir leads to increased oxygenation within tumor xenografts. Our results suggest that nelfinavir decreases HIF-1alpha/VEGF expression and tumor hypoxia, which could play a role in its in vivo radiosensitizing effect. These data support the use of nelfinavir in combination with radiation in future clinical trials.

Animals↗

PubMed2006

HIV protease inhibitors decrease VEGF/HIF-1alpha expression and angiogenesis in glioblastoma cells.

Glioblastomas are malignant brain tumors that are rarely curable, even with aggressive therapy (surgery, chemotherapy, and radiation). Glioblastomas frequently display loss of PTEN and/or epidermal growth factor receptor activation, both of which activate the PI3K pathway. This pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha expression. We examined the effects of two human immunodeficiency virus protease inhibitors, nelfinavir and amprenavir, which inhibit Akt signaling, on VEGF and HIF-1alpha expression and on angiogenesis. Nelfinavir decreased VEGF mRNA expression and VEGF secretion under normoxia. Downregulation of P-Akt decreased VEGF secretion in a manner similar to that of nelfinavir, but the combination of the two had no greater effect, consistent with the idea that nelfinavir decreases VEGF through the PI3K/Akt pathway. Nelfinavir also decreased the hypoxic induction of VEGF and the hypoxic induction of HIF-1alpha, which regulates VEGF promoter. The effect of nelfinavir on HIF-1alpha was most likely mediated by decreased protein translation. Nelfinavir's effect on VEGF expression had the functional consequence of decreasing angiogenesis in in vivo Matrigel plug assays. Similar effects on VEGF and HIF-1alpha expression were seen with a different protease inhibitor, amprenavir. Our results support further research into these protease inhibitors for use in future clinical trials for patients with glioblastoma multiformes.

Brain Neoplasms↗

PubMed2005

HIV protease inhibitors block Akt signaling and radiosensitize tumor cells both in vitro and in vivo.

In tumor cells with mutations in epidermal growth factor receptor (SQ20B), H-Ras (T24), or K-Ras (MIAPACA2 and A549), the inhibition of Akt phosphorylation increases radiation sensitivity in clonogenic assays, suggesting that Akt is a potential molecular target when combined with therapeutic radiation. Insulin resistance and diabetes are recognized side effects of HIV protease inhibitors (HPIs), suggesting that these agents may inhibit Akt signaling. Because activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is common in human cancers, we hypothesized that HPIs can inhibit Akt activity resulting in increased tumor cell sensitivity to ionizing radiation-induced cell death. Five first-generation HPIs were subsequently tested and three of the five (amprenavir, nelfinavir, and saquinavir but not ritonavir or indinavir) inhibited Akt phosphorylation at Ser473 at serum concentrations routinely achieved in HIV patients. In both tumor cell colony formation assays and tumor regrowth delay experiments, combinations of drug and radiation exerted synergistic effects compared with either modality alone. In addition, in vivo, doses of amprenavir or nelfinavir comparable with the therapeutic levels achieved in HIV patients were sufficient to down-regulate phosphorylation of Akt in SQ20B and T24 xenografts. Finally, overexpression of active PI3K in cells without activation of Akt resulted in radiation resistance that could be inhibited with HPIs. Because there is abundant safety data on HPIs accumulated in thousands of HIV patients over the last 5 years, these agents are excellent candidates to be tested as radiation sensitizers in clinical trials.

Animals↗

PubMed2005

Live cell imaging reveals distinct roles in cell cycle regulation for Nek2A and Nek2B.

Two splice variants of Nek2 kinase, a member of the NIMA-related family, have been identified as Nek2A and Nek2B. Nek2A regulates centrosome disjunction, spindle formation checkpoint signaling, and faithful chromosome segregation. A specific role for Nek2B has not yet been identified. Here, we have examined the distinct roles of Nek2A and Nek2B using timelapse video microscopy to follow the fate of cells progressing through the cell cycle in the absence of either Nek2A or Nek2B. We show that the down-regulation of Nek2B leads to a mitotic delay in the majority of cells. Upon exiting mitosis, cells exhibit mitotic defects such as the formation of multinucleated cells. Such phenotypes are not observed in cells that exit mitosis in the absence of Nek2A. These observations suggest that Nek2B may be required for the execution of mitotic exit.

Alternative Splicing↗

PubMed2005

Farnesyltransferase inhibitor effects on prostate tumor micro-environment and radiation survival.

BACKGROUND: Ras activation by mutation, overexpression, or receptor signaling can increase tumor cell survival after irradiation. METHODS: We examined whether inhibiting Ras activity with farnesyltransferase inhibitors (FTI) altered the radiosensitivity and tumor micro-environment in prostate tumors. RESULTS: Treatment with FTIs L-744,832 or FTI-277 reduced clonogenic survival of prostate tumor cells expressing oncogenic H-ras after irradiation. PI3-kinase/Akt and MAPK signaling pathways were downregulated by FTIs in these cells. FTI treatment reduced tumor hypoxia and also reduced MMP-9 expression in tumors with activated mutant H-ras. FTI treatment did not, however, increase apoptosis in irradiated intestine, demonstrating that acute radiation injury of this normal tissue was not enhanced by FTIs. CONCLUSIONS: FTIs can enhance the killing of prostate tumors with activated H-Ras. Together with the absence of increased acute toxicity to normal bowel, these results imply that FTI treatment should be further studied as a possible adjuvant to radiotherapy in the treatment of abdominal cancers with activated Ras signaling.

Alkyl and Aryl Transferases↗

PubMed2004

Inhibition of centrosome separation after DNA damage: a role for Nek2.

DNA damage results in cell cycle arrest in G2. Centrosomes also separate in G2, raising the question of whether separation occurs during the DNA damage-induced G2 arrest. Nek2, the mammalian homologue of NIMA, is a cell cycle-regulated serine/threonine protein kinase that regulates centrosome separation during G2. Here we show that damaged cells fail to activate Nek2. Both Nek2 levels and activity are reduced after DNA damage. Radiation inhibits the premature centrosome splitting induced by overexpression of Nek2, indicating that Nek2 is involved in activation of the G2 checkpoint and is not secondary to cell cycle arrest. We confirm using siRNA that centrosome separation and cell growth are impaired in the absence of Nek2. These studies define a previously unreported DNA damage response of inhibition of centrosome separation mechanistically linked to Nek2.

Base Sequence↗

PubMed2003

Radiation sensitization of human cancer cells in vivo by inhibiting the activity of PI3K using LY294002.

Multiple genetic alterations such as in Ras or EGFR can result in sustained signaling through PI3K. Our previous experiments have shown that resistance to radiation results from PI3K activity in cells in culture. Here we examined whether inhibition of PI3K in vivo would sensitize tumors to radiation. The human bladder cancer cell line T24 has amplified and mutated H-Ras resulting in sustained PI3K activity and phosphorylation of the downstream target of PI3K, Akt. Nude mice bearing T24 tumor cell xenografts were randomly assigned to one of four groups: control, radiation alone, the PI3K inhibitor LY294002 alone, or combined LY294002 and radiation. The LY294002 was delivered intraperitoneally to the mice. Downregulation of Akt was documented by Western blot analysis of tumor lysates. In vivo sensitization was measured using clonogenic assays or regrowth assays.A dose of 100 mg/kg of LY294002, but not 50 mg/kg, consistently eliminated the phosphorylation of Akt. This inhibition was transient, and Akt activity returned after 30 min. This dose resulted in severe respiratory depression and lethargy resolving without lethality. It is not possible to tell whether these side effects of LY294002 were mechanism-based or idiosyncratic. The PI3K inhibitor LY294002 by itself had minimal antitumor effect. The combination of LY294002 and radiation resulted in significant and synergistic reduction in clonogenicity and growth delay. Inhibition of PI3K by LY294002 can synergistically enhance radiation efficacy. This acts as a proof of principle that inhibition of the Ras to PI3K pathway could be useful clinically.

Animals↗

PubMed2002

Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction.

PURPOSE: Locoregional recurrence is the dominant form of treatment failure in head and neck (H&N) cancer. The epidermal growth factor receptor (EGFR) is frequently amplified in this disease (<or=80%) and can lead to activation of phosphatidylinositol-3-kinase (PI3K), both directly and indirectly through Ras. We have shown previously that radioresistance could be conferred via the Ras-PI3K pathway. Here we investigate the contribution of EGFR to this pathway and its impact on treatment outcome. EXPERIMENTAL DESIGN: In a series of 38 H&N cancer patients, overexpression of EGFR by immunohistochemical staining was assessed. PI3K signaling was evaluated by staining for phosphorylated Akt (P-Akt), a downstream target of PI3K. Both EGFR and P-Akt were then related to outcome. Radiation survival was determined in the SQ20B cell line, a radioresistant squamous cell line derived from a recurrent laryngeal cancer, after pharmacological blockade of EGFR with Iressa, of Ras by the FTI L744,832, or of PI3K by LY294002. RESULTS: A significant association was found between P-Akt staining and local control in the patient series. Two-year local control was 100% for patients staining 0-1+ for P-Akt as compared with 70.6% for patients staining 2-3+ (P = 0.04). In our series of 38 H&N cancers, 30 (78.9%) of the specimens were strongly (3+) positive for EGFR, whereas 25 (65.8%) were moderately to strongly (2-3+) positive for P-Akt. Pharmacologically inhibiting EGFR, Ras, and PI3K led to radiosensitization of SQ20B cells. CONCLUSIONS: Evaluation of PI3K activation by Akt phosphorylation might be a prognostic marker for response to therapy, and PI3K could be a useful target for therapy. These results also suggest that signaling from EGFR to PI3K can lead to radioresistance.

Adult↗

PubMedDate not supplied

C-Raf-1 protein kinase is not essential for Ras transformation of mouse embryo fibroblasts.

Transfection of primary cells with mutated oncogenic ras plus a cooperating oncogene such as myc results in the acquisition of the transformed cell phenotype. The pathways downstream of Ras that are required for transformation are an active topic of research. The Raf-MEKK-MAP kinase pathway is triggered by activation of Ras and thought to be important in Ras transformation of rodent fibroblasts. To further explore the involvement of this pathway, fibroblasts from homozygous knock out c-Raf-1 mouse embryos (20 KO) and wild-type c-Raf-1 mouse embryos (16 WT) were transfected with H-ras and myc(v). The resulting cell line derived from the knock out cells grew slower both in tissue culture and had a longer latency period as tumors than the transformed cell line from the wild-type cells. Both cell lines were however able to form tumors in nude mice. These results suggest that c-Raf-1 is not required for Ras transformation in this system.

Animals↗