Normal tissue radioprotection by amifostine via Warburg-type effects.

The mechanism of Amifostine (WR-2721) mediated radioprotection is poorly understood. The effects of amifostine on human basal metabolism, mouse liver metabolism and on normal and tumor hepatic cells were studied. Indirect calorimetric canopy tests showed significant reductions in oxygen consumption and of carbon dioxide emission in cancer patients receiving amifostine. Glucose levels significantly decreased and lactate levels increased in patient venous blood. Although amifostine in vitro did not inhibit the activity of the prolyl-hydroxylase PHD2, experiments with mouse liver showed that on a short timescale WR-1065 induced expression of the Hypoxia Inducible Factor HIF1α, lactate dehydrogenase LDH5, glucose transporter GLUT2, phosphorylated pyruvate dehydrogenase pPDH and PDH-kinase. This effect was confirmed on normal mouse NCTC hepatocytes, but not on hepatoma cells. A sharp reduction of acetyl-CoA and ATP levels in NCTC cells indicated reduced mitochondrial usage of pyruvate. Transient changes of mitochondrial membrane potential and reactive oxygen species ROS production were evident. Amifostine selectively protects NCTC cells against radiation, whilst HepG2 neoplastic cells are sensitized. The radiation protection was correlates with HIF levels. These findings shed new light on the mechanism of amifostine cytoprotection and encourage clinical research with this agent for the treatment of primary and metastatic liver cancer.

Amifostine enhances recovery and expansion of peripheral FAS/CD95+ T- and NK-cell subpopulations during radiotherapy of patients with head-neck cancer.

PURPOSE: Experimental studies suggest that the FAS/APO-1/CD95 (cytokine receptor protein TNF-receptor superfamily, member 6) cell surface molecule is involved in the apoptotic effect of radiotherapy. In this study we investigated the role of amifostine in protecting the CD95+ (CD: cluster of differentiation) lymphocytic subpopulation in patients with head and neck cancer undergoing radiotherapy. MATERIALS AND METHODS: Using flow-cytometry we examined the expression of FAS/CD95 antigen on CD4+ (helper/inducer T cells), CD8+ (suppressor/cytotoxic T cells) and CD56+ (NK, natural killer) T-lymphocytes of 28 patients with head and neck cancer undergoing radiotherapy (with and without amifostine). RESULTS: The numbers of peripheral blood lymphocytes were significantly reduced after treatment from (mean value +/- STD error) 1477 +/- 129 to 1015 +/- 77 for T lymphocytes, 700 +/- 70 to 454 +/- 38 for CD4, 449 +/- 46 to 296 +/- 34 for CD8 and, 140 +/- 18 to 118 +/- 13 for NK, before and after treatment, respectively. CD95 expressing lymphocytes showed a faster recovery rate in patients receiving amifostine. CD95 expressing CD56 lymphocytes increased during radiotherapy in patients receiving daily cytoprotection with amifostine to values higher than the pre-treatment levels (p = 0.004). CONCLUSION: It is suggested that amifostine enhances recovery of T- and NK-lymphocyte subpopulations expressing the CD95 antigen in head-neck cancer patients undergoing RT and may enhance the efficacy of the later by interfering FAS-related immunological pathways.

Early antivascular effects of bevacizumab anti-VEGF monoclonal antibody on colorectal carcinomas assessed with functional CT imaging.

BACKGROUND: The complex effect on anti-VEGF (vascular endothelial growth factor) monoclonal antibodies on the tumor vasculature urges studies to identify the optimal time frames for the administration of such agents with chemotherapy and radiotherapy. PATIENTS AND METHODS: Using CT scan functional imaging, we examined the perfusion changes of contrast medium induced 7 days following administration of bevacizumab (5 mg/kg iv) in 12 patients with colorectal cancer. RESULTS: CT imaging 7 days after the administration of bevacizumab confirmed tumor shrinkage in 3 of 12 cases. The mean Haunsfield units after the injection of contrast medium were significantly lower 7 days following the administration of bevacizumab (P = 0.002). The "perfusion score" significantly decreased after the administration of bevacizumab (P = 0.01). In 5 of 12 cases the perfusion score was by 45% to 75% decreased, in 3 of 12 minor drop of the perfusion score was noted, in 1 of 12 a slight increase was noted, and in 3 of 12 remained unchanged. CONCLUSION: It is concluded that the antivascular effects of bevacizumab are evident within 7 days from administration in 40% of colorectal carcinomas. The mechanisms by which this early antivascular effect may favor the antitumor efficacy of radiotherapy and chemotherapy require further investigation.

Metastatic cancer cells from c-erbB-2 negative primary breast cancer maintain the original c-erbB-2/HIF1alpha phenotype.

The expression of c-erbB-2 and HIF1alpha proteins is linked with an aggressive tumor phenotype and poor survival in breast cancer. In the present study we investigated whether this ominous effect is a result of c-erbB-2/HIF1alpha expressing clone appearance within the primary tumor, by examining the c-erbB-2/HIF1alpha expression status in the primary tumor, node metastasis and cancer cells invading into the lymphovascular spaces. The metastasizing cancer cell clones, whether migrating to the lymph nodes or entering into the systemic circulation maintained the original c-erbB-2/HIF1alphaphenotype of the primary tumor. Migrating c-erbB-2/HIF1alpha negative tumors do so through activation of alternative biologic pathways and not through positive clone appearance. These results strongly support the concept that targeted therapies against c-erbB-2 or against HIF1alpha can be guided with precision by the primary tumor c-erbB-2/HIF1alpha status without demanding the detection of the metastatic tumor phenotype.

Oxygen and glucose consumption in gastrointestinal adenocarcinomas: correlation with markers of hypoxia, acidity and anaerobic glycolysis.

This study gives an insight into tumor metabolic activity by investigating oxygen and glucose content, together with their metabolic products carbon dioxide and acids-pH, in the arterial and venous blood of a tumor. Nineteen patients with gastrointestinal adenocarcinomas undergoing surgery were studied. Biochemical analysis showed that in a large subgroup of tumors, oxygen consumption was reduced while that of glucose was increased in malignant, as compared to normal tissues; these features were more evident in tumors overexpressing lactate dehydrogenase (LDH-5) and hypoxia inducible factors (HIF1alpha/2alpha). An increase in carbon dioxide production in the tumor environment was linked with overexpression of carbonic anhydrase 9 (CA9). The simultaneous overexpression of CA9 and LDH-5 was related to very low pH levels in the veins draining the tumor, suggesting an intense acidification of the tumor microenvironment in such cases. These in vivo data confirm the importance of HIFs and their downstream regulated genes in tumor metabolism, particularly in glycolysis and carbon dioxide buffering.

Endogenous markers of hypoxia/anaerobic metabolism and anemia in primary colorectal cancer.

Anemia has been implicated in the decreased oxygen tension noted within the tumor environment. In a series of 79 colorectal adenocarcinomas we investigated the role of anemia in activating molecular pathways regulated by hypoxia. Preoperative Hb levels were correlated with the immunohistochemical expression of HIF1alpha and HIF2alpha, LDH5, GLUT1, VEGF, DEC1 and BNIP3, and with angiogenesis and the cancer cell proliferation index. Upregulation of HIF1alpha and HIF2alpha proteins, found in 43% and 44.3% of cases, respectively, was not related to anemia (Hb < 10 g%). This is in agreement with other studies suggesting that HIF activation occurs for various reasons, such as poor or irregular vascularity, or oncogene activation. Nevertheless, low Hb levels (<10 g%) were linked to activated anaerobic metabolism (LDH5 overexpression) in a subset of tumors not expressing HIF1alpha (P < 0.01). Overexpression of HIFs, whether linked to anemia or not, was associated with a number of factors related to tumor aggressiveness (assessed as local invasion and nodal metastasis), anaerobic metabolism and intratumoral acidosis (LDH5, GLUT1; increased glucose metabolism to lactate), activation of genes related to necrosis (BNIP3) and angiogenesis (VEGF). Expression of BNIP3 emerged as the strongest independent factor related to transmural invasion and metastasis to lymph nodes. Identification of specific patterns of the hypoxia molecular cascade activated in cancer cells might help in developing specific therapeutic policies.

Hypofractionated accelerated radiotherapy with cytoprotection combined with trastuzumab, liposomal doxorubicine, and docetaxel in c-erbB-2-positive breast cancer.

OBJECTIVES: Trastuzumab, an antic-erbB-2 monoclonal antibody, has become a standard component of chemotherapy for c-erbB-2-positive advanced breast carcinoma. Despite the experimental evidence of its radiosensitizing properties, trastuzumab has never been used in combination with radiotherapy for the treatment of patients with locally advanced disease. PATIENTS AND METHODS: Twenty-two patients with c-erbB-2-positive locally advanced chemoresistant (7 patients) or with high-risk breast cancer (15 patients) were recruited in a treatment protocol combining hypofractionated/accelerated radiotherapy (hypoARC) supported with high-dose amifostine (1000 mg subcutaneous), concurrently with trastuzumab (4 mg/kg every 2 weeks). Thirteen of these patients (including all 7 inoperable cases) received concurrently chemotherapy with liposomal doxorubicin and docetaxel (25 mg/m2 and 40 mg/m2 every 2 weeks, respectively). RESULTS: Administration of trastuzumab together with highly accelerated amifostine-supported radiotherapy was feasible without an increase in early and late radiation toxicity. This was obtained despite the concurrent administration of aggressive chemotherapy. Complete responses were noted in 5 of 7 patients with locally, often far advanced, chemoresistant disease. None of the complete responders or the 15 high-risk breast cancer patients relapsed within the 3- to 26-month follow-up period. CONCLUSION: Inclusion of trastuzumab in the radiochemotherapy protocols for breast cancer does not increase radiation or systemic toxicity. The concurrent administration of aggressive radiotherapy with docetaxel and liposomal doxorubicin is feasible when supported with amifostine. The value of such regimens in the treatment of locally advanced or high risk c-erbB-2 positive breast cancer patients deserves further evaluation.

Lactate dehydrogenase 5 (LDH5) relates to up-regulated hypoxia inducible factor pathway and metastasis in colorectal cancer.

Lactate dehydrogenase 5 (LDH5) is one of the five LDH isoenzymes and, apparently, the most important for promoting anaerobic glycolysis. LDH5 is transcriptionally regulated by the hypoxia inducible factors (HIF) 1alpha and 2alpha. In this study, the possible aggressive advantages that colorectal tumours may gain from a high LDH5 content was investigated. To this end, 75 colorectal adenocarcinomas were studied immunohistochemically for the expression of LDH5, and the results were related to tumor differentiation, lymph node and distant metastases, the expression of HIF1alpha and HIF2alpha, vascular density (VD) and vascular endothelial growth factor (VEGF). A high LDH5 content was noted in 51 of 75 (68%) colorectal adenocarcinomas. The reactivity was nuclear and/or cytoplasmic. Nuclear LDH5 reactivity was correlated with lymph node involvement and distant metastases. There was a direct association between LDH5 up-regulation and HIF1alpha and HIF2alpha accumulation. HIF1alpha was linked with VEGF, VD and also with extramural invasion, nodal and distant metastases. It is concluded that a high LDH5 content in tumor cells is directly related to an up-regulated HIF pathway and is linked with an aggressive phenotype in colorectal adenocarcinomas.

c-erbB-2 related aggressiveness in breast cancer is hypoxia inducible factor-1alpha dependent.

c-erbB-2-positive breast carcinomas are highly aggressive tumors. In vitro data on breast cell lines showed that c-erbB-2 enhanced translational efficiency of hypoxia inducible factor-1alpha (HIF1alpha) production (Laughner et al., Mol Cell Biol 2001;21:3995-4005). We investigated the clinical correlate of this observation to assess whether c-erbB-2 expression was related to HIF1alpha expression, angiogenesis, and prognosis. A series of 180 breast carcinomas of known c-erbB-2 status (90 c-erbB-2-positive and 90 c-erbB-2-negative carcinomas) were stained immunohistochemically for HIF1alpha and CD31 endothelial cell antigen. c-erbB-2 positivity was clearly related to HIF1alpha protein expression and high angiogenesis. However, prognosis was decreased only in cases with simultaneous c-erbB-2 and HIF1alpha expression. If activation of c-erbB-2 in humans results in overexpression of HIF1alpha independently of conditions of hypoxia, as occur in experimental studies, this interaction may represent a main pathway conferring clinical aggressiveness to c-erbB-2-positive breast tumors.

Amifostine induces anaerobic metabolism and hypoxia-inducible factor 1 alpha.

PURPOSE: The cytoprotective mechanism of amifostine (WR-2721) implies free radical scavenging and DNA repair activities. We investigated additional cytoprotective pathways involving intracellular hypoxia and the activation of the hypoxia-inducible factor (HIF) pathway, a key transcription factor regulating glycolysis, angiogenesis and apoptosis, which is also linked with radioresistance. MATERIALS AND METHODS: The glucose and oxygen levels in the peripheral blood of patients receiving 1000 mg amifostine were determined at various time-points in order to investigate the metabolic changes induced by amifostine. MDA468 breast tumor cell lines were incubated with a high amifostine concentration (10 m M) to overcome the natural resistance of cancer cells to influx of the non-hydrolyzed WR-2721, and the HIF1 alpha protein levels were determined by Western blot analysis. In vivo experiments with Wistar rats were performed in order to assess immunohistochemically changes in the intracellular accumulation of HIF1 alpha induced by amifostine (200 mg/kg). RESULTS: By 30 min following amifostine administration, the hemoglobin oxygen saturation and pO(2) levels had increased in the peripheral blood while glucose levels had reduced, providing evidence that normal tissue metabolism switches to glycolytic pathways. Incubation of cell lines with amifostine resulted in HIF1 alpha induction. In Wistar rats administration of amifostine resulted in increased HIF1 alpha accumulation in normal tissues. CONCLUSIONS: Since it is doubtful whether dephosphorylation of amifostine to the active metabolite WR-1065 occurs within tumoral tissues (an acidic environment that lacks vascular alkaline phosphatase activity), intracellular hypoxia and upregulation of HIF1 alpha represents an additional, normal tissue-specific, amifostine cytoprotective pathway.

Amifostine before chemotherapy: improved tolerance profile of the subcutaneous over the intravenous route.

INTRODUCTION: The i.v. administration of the cytoprotective agent amifostine is associated with reversible clinical hypotension, protracted emesis, and malaise in a various percentage of patients. We evaluated, prospectively, whether the s.c. route is a better tolerated alternative to the i.v. route in patients receiving chemotherapy. PATIENTS AND METHODS: Fifty-nine patients treated with "once every 2 weeks" regimens received 1000 mg of amifostine i.v. before chemotherapy. Patients who developed protracted vomiting and malaise and/or clinical hypotension for two consecutive i.v. administrations received the same dose of amifostine s.c. for the subsequent cycles (i.v./s.c. study). In an additional cohort of 12 patients (s.c. study), 1000 mg of amifostine were given s.c. since the first chemotherapy cycle. RESULTS: In the i.v./s.c. study, 8 (13.5%) patients showed protracted emesis/malaise and/or clinical hypotension during the first two cycles. An additional 4 (6.6%) patients developed similar side effects during the subsequent cycles. Switching to the s.c. route, an improved tolerance was noted. In the s.c. study, a total of 76 injections was administered. Protracted vomiting or clinical hypotension was absent, and this tolerance profile was significantly better than the i.v. one (P = 0.001). There were no other systemic side effects related to the s.c. administration. CONCLUSIONS: Amifostine, at a dose of 1000 mg, is better tolerated when administered s.c. Switching to the s.c. route in patients with poor tolerance using the i.v. administration allows the continuation of cytoprotection with minor side effects. Although preliminary, 1000 mg of amifostine effectively protected against the lower, still more frequently administered doses of chemotherapy given once every 2 weeks.