A Phase II trial of epothilone B analogue BMS-247550 (NSC #710428) ixabepilone, in patients with advanced pancreas cancer: a Southwest Oncology Group study.

PURPOSE: The purpose of this Phase II multi-institutional study was to define the efficacy and toxicity of ixabepilone in patients with advance pancreatic adenocarcinoma. PATIENTS AND METHODS: Patients were required to have pancreatic adenocarcinoma and metastatic or recurrent disease that was not amenable to curative resection. Performance status was 0-1, and patients could not have had prior chemotherapy, or chemoradiation therapy for their advanced disease although prior local palliative radiation was allowed. Ixabepilone was administered iv as a 3 hour infusion every 21 days. Initially, the dose was 50 mg/m(2) but this was lowered to 40 mg/m(2) shortly after the trial opened because of concerns about neurotoxicity. RESULTS: Sixty-two patients were registered however 2 were ineligible because they did not have recurrent or metastatic disease. For the 60 eligible patients, 22 had performance status of 0 and 38 performance status of 1. The estimated 6-month survival was 60% (95% CI 48%-72%) with a median survival of 7.2 months and an estimated time to treatment failure of 2.3 months. Out of 56 patients with measurable disease there were 5 confirmed partial responses for a confirmed response probability of 9% (95% CI 3%-20%) and 7 unconfirmed partial responses for an overall response probability of 21% (95% CI 12%-34%). Common toxicities were neutropenia/granulocytopenia, nausea and vomiting and neuropathy. There was one death, cause not determined but judged "possibly" related to treatment. CONCLUSION: Ixabepilone shows encouraging activity in patients with advanced pancreatic cancer and should be investigated further in this disease.

Recombinant human erythropoietin as an adjunct to radiation therapy and cisplatin for stage IIB-IVA carcinoma of the cervix: a Southwest Oncology Group study.

OBJECTIVE: The survival of cervix cancer patients is associated with their hemoglobin (Hgb) level during radiotherapy. The Southwest Oncology Group (SWOG) conducted a phase II trial to determine whether recombinant human erythropoietin (rHuEPO) safely corrects anemia during chemoradiotherapy for cervix cancer. METHODS: Patients had stage IIB-IVA cervix cancer and a Hgb between 8.0 and 12.5 g/dl. All patients received rHuEPO thrice weekly and oral iron starting 10-15 days before their 5-week course of whole pelvic irradiation and weekly cisplatin followed by intracavitary brachytherapy. RESULTS: Fifty-three patients from 26 institutions received the protocol treatment. The mean Hgb was 10.4 +/- 1.3 g/dl on the first day of rHuEPO administration (baseline), 11.0 +/- 1.6 g/dl on the first day of chemoradiotherapy, 11.6 +/- 1.9 g/dl at the midpoint of chemoradiotherapy, and 11.8 +/- 2.2 g/dl at the end of chemoradiotherapy. The target Hgb level of 12.5 g/dl was achieved in 40% of patients (95% CI 26-56%) by the midpoint of Chemoradiotheraphy. Change in Hgb was associated with baseline serum iron (P = 0.008) and transferrin saturation (P = 0.05) levels, but not with baseline Hgb or serum ferritin, or patient age. Seven patients developed deep vein thrombosis. Two-year progression-free survival (PFS) was 43% and overall survival (OS) was 51%. Survival was significantly associated with Hgb level at the end of chemoradiotherapy, but not with the baseline Hgb level. CONCLUSIONS: rHuEPO and iron gradually increased Hgb levels in anemic women with local advanced cervix cancer during chemoradiotherapy. There was a higher than expected incidence of deep vein thrombosis. The progression-free and overall survival rates were lower than reported for women with normal Hgb levels.

Localization of the iron transport proteins Mobilferrin and DMT-1 in the duodenum: the surprising role of mucin.

There are two pathways for inorganic iron uptake in the intestine, the ferric pathway, mediated by the key protein mobilferrin, and the ferrous pathway, mediated by DMT-1. Previous studies reported that the amount of DMT-1 increased in the intestinal mucosa in iron deficiency and the increase was seen in the apical portion of the villus of the duodenal mucosa. Mobilferrin did not quantitatively increase but became localized at the cell membrane. However, studies on fresh tissue have not previously been performed and the localization to the microvillae has not been demonstrated. In order to more definitively localize these proteins immunofluorescent and electron microscopic studies were undertaken. Samples were also subjected to biochemical analysis and Western analysis. In iron-deficient animals both DMT-1 and Mobilferrin were concentrated in the apical surface of the villae. Electron microscopy revealed that the majority of this increase in the amount of these proteins near the luminal surface was due to increased binding of the proteins to mucin in vesicles near the surface. A significant portion of the iron transport proteins was localized in the goblet cells and outside the cell in the luminal mucin, as demonstrated by immunofluorescence, electron microscopy, and isolation of the mucin by cesium chloride gradient centrifugation and Western analysis. A new model for the transport of metal ions was suggested. The metal transport proteins travel from vesicles inside the cell out to the lumen mucin. This increases the surface area and allows a greater portion of the lumen contents to be exposed to the binding proteins. Once the metal is bound to the externalized protein it is internalized into the cell. This explains many of the unique properties of the iron-binding proteins and suggests that it may be a more general model for the absorption of other nutrients.

DMT1: a mammalian transporter for multiple metals.

DMT1 has four names, transports as many as eight metals, may have four or more isoforms and carries out its transport for multiple purposes. This review is a start at sorting out these multiplicities. A G185R mutation results in diminished gastrointestinal iron uptake and decreased endosomal iron exit in microcytic mice and Belgrade rats. Comparison of mutant to normal rodents is one analytical tool. Ectopic expression is another. Antibodies that distinguish the isoforms are also useful. Two mRNA isoforms differ in the 3' UTR: +IRE DMT1 has an IRE (Iron Responsive Element) but -IRE DMT1 lacks this feature. The +/-IRE proteins differ in the distal 18 or 25 amino acid residues after shared identity for the proximal 543 residues. A major function is serving as the apical iron transporter in the lumen of the gut. The +IRE isoform appears to have that role. Another role is endosomal exit of iron. Some evidence indicts the -IRE isoform for this function. In our ectopic expression assay for metal uptake, four metals--Fe2+, Mn2+, Ni2+ and Co2+--respond to the normal DMT1 cDNA but not the G185R mutant. Two metals did not--Cd2+ and Zn2+--and two--Cu2+ and Pb2+--remain to be tested. In competition experiments in the same assay, Cd2+, Cu2+ and Pb2+ inhibit Mn2+ uptake but Zn2+ did not. In rodent mutants, Fe and Mn appear more dependent on DMT1 than Cu and Zn. Experiments based on ectopic expression, specific antibodies that inhibit metal uptake and labeling data indicate that Fe3+ uptake depends on a different pathway in multiple cells. Two isoforms localize differently in a number of cell types. Unexpectedly, the -IRE isoform is in the nuclei of cells with neuronal properties. While the function of -IRE DMT1 in the nucleus is speculative, one may safely infer that this localization identifies new role(s) for this multifunctional transporter. Management of toxic challenges is another function related to metal homeostasis. Airways represent a gateway tissue for metal entry. Preliminary evidence using specific PCR primers and antibodies specific to the two isoforms indicates that -IRE mRNA and protein increase in response to exposure to metal in lungs and in a cell culture model; the +IRE form is unresponsive. Thus the -IRE form could be part of a detoxification system in which +IRE DMT1 does not participate. How does iron status affect other metals' toxicity? In the case of Mn, iron deficiency may enhance cellular responses.

Cellular location of proteins related to iron absorption and transport.

K562 erythroleukemia cells and IEC6 rat cells were examined using confocal microscopy and antibodies raised against DMT-1 (Nramp-2, DCT-1), transferrin receptor (CD71), beta(3) integrin (CD61), mobilferrin (calreticulin), and Hephaestin. The cellular location of each of these proteins was identified by immunofluorescence in both saponin-permeabilized and non-permeabilized cells. Fluorescent reactivity was observed on or near the cell surface of each of these proteins, suggesting that they might participate in surface membrane transport of iron. Fluorescence was observed in the region of the cytoplasm with each antibody to include beta(3) integrin and transferrin receptor. It was pronounced in cells incubated with mobilferrin, Hephaestin, and DMT-1 antibodies. Speckled nuclear fluorescence was observed in cells incubated with anti-DMT-1. While these observations are descriptive, they demonstrate that there are significant concentrations of DMT-1, mobilferrin, and Hephaestin in the cytoplasmic region of cells. This suggests that there may be intracellular roles for these proteins in addition to their serving to transit iron across the cell surface membrane.

The ferrireductase paraferritin contains divalent metal transporter as well as mobilferrin.

Inorganic iron can be transported into cells in the absence of transferrin. Ferric iron enters cells utilizing an integrin-mobilferrin-paraferritin pathway, whereas ferrous iron uptake is facilitated by divalent metal transporter-1 (DMT-1). Immunoprecipitation studies using antimobilferrin antibody precipitated the previously described large-molecular-weight protein complex named paraferritin. It was previously shown that paraferritin functions as an intracellular ferrireductase, reducing ferric iron to ferrous iron utilizing NADPH as the energy source. It functions in the pathway for the cellular uptake of ferric iron. This multipeptide protein contains a number of active peptides, including the ferric iron binding protein mobilferrin and a flavin monooxygenase. The immunoprecipitates and purified preparations of paraferritin also contained DMT-1. This identifies DMT-1 as one of the peptides constituting the paraferritin complex. Since paraferritin functions to reduce newly transported ferric iron to ferrous iron and DMT-1 can transport ferrous iron, these findings suggest a role for DMT-1 in conveyance of iron from paraferritin to ferrochelatase, the enzyme utilizing ferrous iron for the synthesis of heme in the mitochondrion.

Pathways of iron absorption.

Iron is vital for all living organisms but excess iron can be lethal because it facilitates free radical formation. Thus iron absorption is carefully regulated to maintain an equilibrium between absorption and body loss of iron. In countries where meat is a significant part of the diet, most body iron is derived from dietary heme because heme binds few of the dietary chelators that bind inorganic iron. Uptake of heme into enterocytes occurs as a metalloporphyrin in an endosomal process. Intracellular iron is released from heme by heme oxygenase to enter plasma as inorganic iron. Ferric iron is absorbed via a beta(3) integrin and mobilferrin pathway (IMP) which is unshared with other nutritional metals. Ferrous iron uptake is facilitated by a DMT-1 pathway which is shared with manganese. In the iron deficient gut, large quantities of both mobilferrin and DMT-1 are found in goblet cells and intraluminal mucins suggesting that they are secreted with mucin into the intestinal lumen to bind iron to facilitate uptake by the cells. In the cytoplasm, IMP and DMT associate in a large protein complex called paraferritin which serves as a ferrireductase. Paraferritin solublizes iron binding proteins and reduces iron to make iron available for production of iron containing proteins such as heme. Iron uptake by intestinal absorptive cells is regulated by the iron concentration within the cell. Except in hemochromatosis it remains in equilibrium with total body stores via transferrin receptors on the basolateral membrane of absorptive cells. Increased intracellular iron either up-regulates or satiates iron binding proteins on regulatory proteins to alter their location in the intestinal mucosa.