HIF and MIF--a nifty way to delay senescence?
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Aging↗
Biomedical subjects
Amit Maity
Publications and source records attributed to Amit Maity.
The chemokine receptor CXCR4: a homing device for hypoxic cancer cells?
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Cell Hypoxia↗
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↗
2-Deoxyglucose and radiosensitization: teaching an old DOG new tricks?
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Deoxyglucose↗
EGFR tyrosine kinase inhibitors decrease VEGF expression by both hypoxia-inducible factor (HIF)-1-independent and HIF-1-dependent mechanisms.
Epidermal growth factor receptor (EGFR) inhibitors can decrease vascular endothelial growth factor (VEGF) expression and tumor angiogenesis. In the current study, we investigate the molecular pathways by which this occurs using two drugs that have been used in the clinic, gefitinib (Iressa) and erlotinib (Tarceva). The decrease in VEGF expression by gefitinib in SQ20B squamous cell carcinoma cells was opposed by adenoviral expression of Akt in these cells. The hypoxia-inducible factor-1 (HIF-1) binding site located at approximately -1 kbp in the VEGF promoter was not required for down-regulation of promoter activity by gefitinib under normoxia. Furthermore, the drug decreased activity of a reporter containing the -88/+54 region. In a gel shift assay, gefitinib led to decreased retardation of a labeled DNA oligonucleotide probe corresponding to the -88/-66 region of the VEGF promoter, which contains Sp1 binding sites. These effects of gefitinib on VEGF promoter activity and DNA binding were both reversed by Akt expression. Phosphorylation of Sp1 was decreased in the presence of gefitinib. Gefitinib also decreases VEGF expression by decreasing HIF-1alpha expression. This occurs due to decreased protein translation without any change in the level of HIF-1alpha mRNA. Together, these results suggest that gefitinib decreases VEGF expression both by decreasing Sp1 binding to the proximal core VEGF promoter and by down-regulating HIF-1alpha expression. Similar results were obtained with erlotinib in SQ20B and gefitinib in HSC3 squamous carcinoma cells. These results indicate that there are at least two separate mechanisms by which EGFR inhibitors decrease VEGF expression.
Binding Sites↗
Pulmonary function abnormalities in children treated with whole lung irradiation.
BACKGROUND: We sought to determine the prevalence of abnormal pulmonary function tests (PFTs) in a cohort of children who had received whole lung irradiation (WLI) for treatment of metastatic disease. PROCEDURE: This was a retrospective (1988-2003) chart review that included all children treated at our institution with WLI who had undergone PFT. Data abstracted included oncologic diagnosis, radiation dose and fractionation, spirometry (FVC, FEV1, FEV1/FVC, FEF25%-75%), plethysmography (TLC, FRC, RV, RV/TLC), diffusing capacity (DLCO), and respiratory muscle strength (MIP, MEP). PFTs were normalized according to standard deviation (Z) scores. RESULTS: Thirty patients were identified who had one or more PFT. The incidence of mild, moderate, or severe reductions in FEV1 was 30%, 10%, and 10%, respectively, with 50% having normal FEV1. Seventeen percent of patients had mild reduction in total lung capacity (TLC), while 13% and 30% had moderate or severe reductions. Thirty-eight percent had mild reductions in diffusing capacity, while 29% and 14% had moderate or severe reductions. CONCLUSIONS: Pulmonary function abnormalities were common in this cohort of children treated with WLI, and may be progressive in nature. Further studies are warranted to identify patients at highest risk.
Adolescent↗
Akt1 activation can augment hypoxia-inducible factor-1alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway.
The phosphoinositide 3-kinase (PI3K)/Akt pathway is commonly activated in cancer; therefore, we investigated its role in hypoxia-inducible factor-1alpha (HIF-1alpha) regulation. Inhibition of PI3K in U87MG glioblastoma cells, which have activated PI3K/Akt activity secondary to phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation, with LY294002 blunted the induction of HIF-1alpha protein and its targets vascular endothelial growth factor and glut1 mRNA in response to hypoxia. Introduction of wild-type PTEN into these cells also blunted HIF-1alpha induction in response to hypoxia and decreased HIF-1alpha accumulation in the presence of the proteasomal inhibitor MG132. Akt small interfering RNA (siRNA) also decreased HIF-1alpha induction under hypoxia and its accumulation in normoxia in the presence of dimethyloxallyl glycine, a prolyl hydroxylase inhibitor that prevents HIF-1alpha degradation. Metabolic labeling studies showed that Akt siRNA decreased HIF-1alpha translation in normoxia in the presence of dimethyloxallyl glycine and in hypoxia. Inhibition of mammalian target of rapamycin (mTOR) with rapamycin (10-100 nmol/L) had no significant effect on HIF-1alpha induction in a variety of cell lines, a finding that was confirmed using mTOR siRNA. Furthermore, neither mTOR siRNA nor rapamycin decreased HIF-1alpha translation as determined by metabolic labeling studies. Therefore, our results indicate that Akt can augment HIF-1alpha expression by increasing its translation under both normoxic and hypoxic conditions; however, the pathway we are investigating seems to be rapamycin insensitive and mTOR independent. These observations, which were made on cells grown in standard tissue culture medium (10% serum), were confirmed in PC3 prostate carcinoma cells. We did find that rapamycin could decrease HIF-1alpha expression when cells were cultured in low serum, but this seems to represent a different pathway.
Cell Line, Tumor↗
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↗
Regulation of histone deacetylase 4 expression by the SP family of transcription factors.
Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4.
Animals↗
Effects of hyperbaric oxygen exposure on experimental head and neck tumor growth, oxygenation, and vasculature.
BACKGROUND: Hyperbaric oxygen (HBO2) is used to promote healing in irradiated tissues, but concern persists about the possibility that it may promote residual tumor growth. METHODS: The tumor growth of SQ20B and Detroit 562 head and neck squamous cell carcinoma xenografts were studied after single-dose irradiation and 5x/week HBO2 treatment at 2.4 atm absolute for 90 minutes. The effect of HBO2 treatment on tumor hypoxia and vasculature was also examined by immunohistochemical analysis. RESULTS: HBO2 treatment increased tumor oxygenation during the treatment interval but did not promote the growth of either irradiated or unirradiated tumors. No increase in tumor vascular endothelial growth factor expression or vascularization was detected. CONCLUSIONS: This study found no evidence for persistent changes in tumor microenvironment or tumor growth promotion caused by hyperbaric oxygen exposure.
Animals↗
Ionizing radiation inhibits tumor neovascularization by inducing ineffective angiogenesis.
The vascular effects of ionizing radiation were examined in K1735 murine melanoma tumors. Single-fraction and fractionated radiation virtually arrested growth of these tumors for about a week, after which they resumed more rapid growth. Tumor microvessel density (MVD) and blood perfusion was unchanged seven days after radiation but decreased at later time points after irradiation, when they had grown 10-fold or more. Together with the finding of severe tumor hypoxia and VEGF induction in the latter tumors, the evidence pointed to vascular insufficiency and inhibited neovascularization in tumors that had grown substantially after radiation. Endothelial cell (EC) death detected by TUNEL staining only transiently increased the day following radiation, whereas EC proliferation detected by Ki-67 staining was increased in irradiated tumors that had grown substantially. The fact that increased EC proliferative activity produced fewer vessels suggests that angiogenesis is defective or ineffective after radiation. These results complement recent genetic evidence that EC damage from radiation plays a major role in tissue damage and antitumor efficacy to highlight the importance of EC and vasculature in radiation response. Our studies further show that radiation impact on tumor vasculature extends beyond near-term induction of EC death to more prolonged effects on their ability to support angiogenesis.
Animals↗
Sp1 is involved in Akt-mediated induction of VEGF expression through an HIF-1-independent mechanism.
Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the -88/-66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate.
Adenoviridae↗
Near complete surgical resection predicts a favorable outcome in pediatric patients with nonbrainstem, malignant gliomas: results from a single center in the magnetic resonance imaging era.
BACKGROUND: Because few reports on outcome in patients with pediatric malignant gliomas during the magnetic resonance imaging era were available, the authors studied the outcomes of children with these tumors at their institution. METHODS: The medical records of 39 patients with nonbrainstem, malignant gliomas who were treated at the Hospital of the University of Pennsylvania/Children's Hospital of Philadelphia between February 1, 1989 and December 31, 2000 were reviewed retrospectively. Magnetic resonance imaging was used to assess tumors at presentation and at follow-up. Progression-free survival (PFS) and overall survival (OS) were determined using the Kaplan-Meier method. Univariate and multivariate analyses were performed using a Cox proportional hazards model. RESULTS: The median follow-up for the 14 surviving patients was 47.6 months. The median PFS for all patients was 12.2 months, and the median OS for all patients was 21.3 months. The extent of surgery was the strongest prognostic factor for predicting outcomes in these patients, with a median survival of 122.2 months in patients who underwent macroscopic total resection compared with 14.1 months in patients who had significant residual disease after surgery. In univariate analyses, other than the extent of surgery, only the absence of visual symptoms at diagnosis significantly predicted improved OS. Local control was improved for patients who underwent better resection and had smaller tumors. In multivariate analyses, although the extent of surgery continued to predict outcomes significantly, histologic grade, which was not significant in the univariate analysis, also was significant. CONCLUSIONS: Children with malignant gliomas appeared to fare better than their adult counterparts. Because the extent of resection was one of the strongest predictors of outcome, the authors concluded that the optimal therapy for these patients would include the maximal possible resection.
Adolescent↗
Craniospinal radiation in the treatment of biopsy-proven intracranial germinomas: twenty-five years' experience in a single center.
PURPOSE: The optimal treatment for intracranial germinomas remains controversial. We report on our 25-year experience using craniospinal irradiation (CSI) for this disease. METHODS AND MATERIALS: Between September 1976 and May 2001, 39 patients with biopsy-proven intracranial germinomas seen at the Children's Hospital of Philadelphia/Hospital of the University of Pennsylvania received CSI. Thirteen of 36 patients (36%) had evidence of spinal dissemination. Median doses to the whole brain, primary site, and spine were 36 Gy (range, 18-44.2 Gy), 50.4 Gy (range, 44-55.8 Gy), and 30.6 Gy (range, 18-40 Gy), respectively. RESULTS: With a median follow-up of 7.1 years (range: 1.5-20.2 years), there have been no documented relapses. This includes 5 patients without spinal dissemination who received 18-19.8 Gy to the craniospinal axis; for these patients, the median length of follow-up was 5.5 years (range, 1.3-6.8 years). One patient, who had no evidence of disease 12.9 years after CSI, died of unknown causes 4 months later. CONCLUSIONS: Our treatment of intracranial germinomas with CSI has yielded outstanding results with no known relapses during a long follow-up period. These results must be considered when evaluating other approaches, such as chemotherapy only or local field irradiation.
Adolescent↗
Surgery with or without radiation therapy in the management of craniopharyngiomas in children and young adults.
PURPOSE: The optimal management of craniopharyngiomas remains controversial, especially in children and young adults. This study reports a single institution's experience with such patients. METHODS AND MATERIALS: Between 1974 and 2001, 76 patients were treated for craniopharyngioma at the Children's Hospital of Philadelphia and the Hospital of University of Pennsylvania (HUP). Of these, 75 patients (97%) were evaluable with long-term follow-up. Although all patients underwent attempted gross total resection, 27 had documentation of less than total resection with 18 of these patients receiving immediate postoperative radiotherapy (RT). An additional 22 patients received RT at HUP after failing surgery alone. RESULTS: Median follow-up for all patients was 7.6 years. The 10-year actuarial overall survival, relapse-free survival, and local control (LC) rates for all patients were 85%, 48%, and 53%, respectively. When comparing the 57 patients treated with surgery alone to the 18 treated with subtotal resection (STR) followed by RT, a significant difference in LC rates at 10 years (42% vs. 84%, respectively; p = 0.004) was noted. However, no statistically significant difference in overall survival was found between the two groups, because RT was highly effective as salvage therapy. Twenty-two patients at HUP treated with RT after relapse had a 10-year ultimate LC rate comparable to that of patients who received RT immediately after STR. CONCLUSION: RT given either immediately after STR or at relapse is effective in controlling craniopharyngiomas.
Adolescent↗
Testicular seminoma 16 years after treatment for CNS germinoma.
Most patients with intracranial germinomas will be cured and become long-term survivors. Physicians caring for these patients should recognize that these patients may be at risk for disease-related and/or treatment-related late sequelae. We report the case of a 27-year-old man who developed testicular seminoma 16 years after treatment for intracranial germinoma. Like their testicular cancer counterparts, long-term survivors of intracranial germinomas may have a susceptibility to develop a subsequent germ cell tumor. These patients require lifelong medical follow-up and should be encouraged to perform testicular self-examination at the appropriate age.
Adult↗
Volumetric considerations in radiotherapy for pediatric parameningeal rhabdomyosarcomas.
PURPOSE: To assess the influence of radiation volume on outcome in pediatric parameningeal rhabdomyosarcomas (PM-RMSs). METHODS AND MATERIALS: Thirty patients ranging in age from 2 to 18 years (median 6) with PM-RMS were treated at the Hospital of the University of Pennsylvania and the Children's Hospital of Philadelphia between August 1988 and December 1999. The histologic subtypes included embryonal (n = 26), alveolar (n = 3), and undifferentiated (n = 1). Twenty-seven patients had Group III and three had Group IV disease. Twenty-seven patients underwent biopsy only and three subtotal resection. All patients were treated with multiple-agent chemotherapy and external beam radiotherapy. In 8 patients, all of whom had intracranial tumor extension, the tumor and whole brain were treated as the initial volume. In the remaining patients, most of those treated before 1992 were treated to the prechemotherapy (CMT) tumor volume for the entire treatment up to the total dose, which ranged from 45 to 59.4 Gy (median dose 57.9). In contrast, patients treated after 1992 generally received radiation initially to the pre-CMT volume (30.6-40 Gy; median dose 36) followed by a conedown to the post-CMT volume to a final dose of 41.4-55.2 Gy (median 50.4). RESULTS: With a median follow-up of 6.2 years (range 2.1-13.3), the actuarial 5-year overall survival, progression-free survival, and local control rate for the entire group was 82%, 76%, and 76%, respectively. Seven failures and five deaths have been documented. In univariate analysis, histologic type, tumor size, and age at diagnosis were found to be predictive of overall survival and local control. No statistically significant difference in overall survival or local control was seen between patients who received a conedown to the post-CMT volume (n = 13) and patients in whom the pre-CMT volume was included for the entire treatment (n = 9). CONCLUSION: Radiotherapy delivered to children with PM-RMS using a shrinking field technique with a post-CMT volume boost was effective and appears to give results comparable to those of patients in whom the pre-CMT volume was treated for the entire course. The use of such tailored treatment fields is likely to lead to fewer late effects and warrants further investigation.
Adolescent↗
PTEN mutation and epidermal growth factor receptor activation regulate vascular endothelial growth factor (VEGF) mRNA expression in human glioblastoma cells by transactivating the proximal VEGF promoter.
Our previous work showed that, compared with parental U87MG human glioblastoma cells, vascular endothelial growth factor (VEGF) mRNA levels are decreased in U87/T691, a derivative line in which epidermal growth factor receptor (EGFR) signaling is inhibited by introduction of a truncated p185(Neu) protein (A. Maity et al., Cancer Res., 60: 5879-5886, 2000). The effect of EGFR activation on VEGF was mediated at the level of transcription via a phosphatidylinositol 3'-kinase (PI3K)-dependent pathway. In the current study we investigated the effect of PTEN, a negative regulator of PI3K signaling commonly mutated in glioblastoma cells, on VEGF expression. Several glioblastoma cell lines containing mutant PTEN, including U87MG, U87/T691, and U251MG, were infected with adenovirus expressing wild-type PTEN. This led to a decrease in the levels of both VEGF mRNA and phosphorylated Akt, a marker for PI3K activation. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing wild-type PTEN decreased VEGF promoter activity using reporters containing either 1.5 kb of the promoter or a fragment extending from -88 to +54 bp. Activity of the -88/+54 VEGF promoter was down-regulated by dominant negative Akt and up-regulated by constitutively active myristoylated Akt. Introduction of wild-type PTEN and pharmacological inhibition of EGFR decreased VEGF mRNA expression and VEGF promoter activity in U87MG cells to a greater extent that did either manipulation by itself. Therefore, in human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally up-regulating the proximal VEGF promoter via the PI3K/Akt pathway.
Endothelial Growth Factors↗