fenretinide and Neuroblastoma

Neuroblastoma is a rare pediatric cancer characterized by a wide clinical behavior and adverse outcome despite aggressive therapies. New approaches based on targeted drug delivery may improve efficacy and decrease toxicity of cancer therapy. Furthermore, nanotechnology offers additional potential developments for cancer imaging, diagnosis, and treatment. Following these lines, in the past years, innovative therapies based on the use of liposomes loaded with anticancer agents and functionalized with peptides capable of recognizing neuroblastoma cells and/or tumor-associated endothelial cells have been developed. Studies performed in experimental orthotopic models of human neuroblastoma have shown that targeted nanocarriers can be exploited for not only decreasing the systemic toxicity of the encapsulated anticancer drugs, but also increasing their tumor homing properties, enhancing tumor vascular permeability and perfusion (and, consequently, drug penetration), inducing tumor apoptosis, inhibiting angiogenesis, and reducing tumor glucose consumption. Furthermore, peptide-tagged liposomal formulations are proved to be more efficacious in inhibiting tumor growth and metastatic spreading of neuroblastoma than nontargeted liposomes. These findings, herein reviewed, pave the way for the design of novel targeted liposomal nanocarriers useful for multitargeting treatment of neuroblastoma.

Topics: Animals; Antineoplastic Agents; Bortezomib; Doxorubicin; Drug Delivery Systems; Fenretinide; Humans; Liposomes; Neuroblastoma

Fenretinide (4-HPR) is a synthetic retinoid that has cytotoxic activity against cancer cells. Despite substantial in vitro cytotoxicity, response rates in early clinical trials with 4-HPR have been less than anticipated, likely due to the low bioavailability of the initial oral capsule formulation. Several clinical studies have shown that the oral capsule formulation at maximum tolerated dose (MTD) achieved <10 µmol/L concentrations in patients. To improve bioavailability of 4-HPR, new oral powder (LYM-X-SORB®, LXS) and intravenous lipid emulsion (ILE) formulations are being tested in early-phase clinical trials. ILE 4-HPR administered as five-day continuous infusion achieved over 50 µmol/L at MTD with minimal systemic toxicities; multiple complete and partial responses were observed in peripheral T cell lymphomas. The LXS oral powder 4-HPR formulation increased plasma levels approximately two-fold at MTD in children without dose-limiting toxicities and demonstrated multiple complete responses in recurrent neuroblastoma. The clinical activity observed with new 4-HPR formulations is attributed to increased bioavailability. Phase I and II clinical trials of both LXS 4-HPR and ILE 4-HPR are in progress as a single agent or in combination with other drugs. Impact statement One of the critical components in drug development is understanding pharmacology (especially pharmacokinetics) of the drugs being developed. Often the pharmacokinetic properties, such as poor solubility leading to poor bioavailability, of the drug can limit further development of the drug. The development of numerous drugs has often halted at clinical testing stages, and several of them were due to the pharmacological properties of the agents, resulting in increased drug development cost. The current review provides an example of how improved clinical activity can be achieved by changing the formulations of a drug with poor bioavailability. Thus, it emphasizes the importance of understanding pharmacologic characteristics of the drug in drug development.

Topics: Administration, Oral; Antineoplastic Agents; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Fenretinide; Humans; Infusions, Intravenous; Lymphoma, T-Cell; Neuroblastoma; Treatment Outcome

The synthetic retinoid fenretinide [N-(4 hydroxyphenyl)retinamide] induces apoptosis of cancer cells and acts synergistically with chemotherapeutic drugs, thus providing opportunities for novel approaches to cancer therapy. The upstream signaling events induced by fenretinide include an increase in intracellular levels of ceramide, which is subsequently metabolized to GD3. This ganglioside triggers the activation of 12-Lox (12-lipoxygenase) leading to oxidative stress and apoptosis via the induction of the transcription factor Gadd153 and the Bcl-2-family member protein Bak. Increased evidence suggests that the apoptotic pathway activated by fenretinide is p53-independent and this may represent a novel way to treat tumors resistant to DNA-damaging chemotherapeutic agents. Therefore, fenretinide offers increased clinical benefit as a novel agent for cancer therapy, able to complement the action of existing chemotherapeutic treatment regimes. Furthermore, synergy between fenretinide and chemotherapeutic drugs may facilitate the use of chemotherapeutic drugs at lower concentrations, with possible reduction in treatment-associated morbidity.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Caspases; CCAAT-Enhancer-Binding Proteins; Ceramides; Enzyme Activation; Fenretinide; Gangliosides; Genes, p53; Humans; Membrane Proteins; Neuroblastoma; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Sialyltransferases; Transcription Factor CHOP; Transcription Factors

Fenretinide is thought to induce apoptosis via increases in ceramide levels but the mechanisms of ceramide generation and the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear. In SH-SY5Y neuroblastoma cells, evidence suggests that acid sphingomyelinase activity is essential for the induction of ceramide and apoptosis in response to fenretinide. Downstream of ceramide, apoptosis in response to fenretinide is mediated by increased glucosylceramide synthase activity resulting in increased levels of gangliosides GD3 and GD2 via GD3 synthase. GD3 is a key signalling intermediate leading to apoptosis via the activation of 12-Lipoxygenase, and the parallel induction of GD2 suggests that fenretinide might enhance the response of neuroblastoma to therapy with anti-GD2 antibodies.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Ceramides; Fenretinide; Gangliosides; Humans; Neuroblastoma; Signal Transduction

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Bone Morphogenetic Proteins; Cell Differentiation; Chromosomes, Human, Pair 1; Combined Modality Therapy; Diploidy; DNA-Binding Proteins; Fenretinide; Gangliosides; Humans; Infant; Loss of Heterozygosity; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Sympathetic Nervous System; Transcription Factors; Tretinoin

Synthetic retinoids such as fenretinide [N-(4-hydroxyphenyl)retinamide] induce apoptosis of neuroblastoma cells, act synergistically with chemotherapeutic drugs, and may provide opportunities for novel approaches to neuroblastoma therapy. Fenretinide-induced cell death of neuroblastoma cells is caspase dependent and results in the release of cytochrome c from mitochondria independently of changes in permeability transition. This is mediated by a signaling pathway characterized by the generation of reactive oxygen species (ROS) via 12-lipoxygenase (12-LOX), and an oxidative-stress-dependent induction of the transcription factor, GADD153 and the BCL2-related protein BAK. Upstream events of fenretinide-induced signaling involve increased levels of ceramide as a result of increased sphingomyelinase activity, and the subsequent metabolism of ceramide to gangliosides via glucosylceramide synthase and GD3 synthase. These gangliosides may be involved in the regulation of 12-LOX leading to oxidative stress and apoptosis via the induction of GADD153 and BAK. The targeting of sphingomyelinases or downstream effectors such as 12-LOX or GADD153 may present novel approaches for the development of more effective and selective drugs for neuroblastoma therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arachidonate 12-Lipoxygenase; CCAAT-Enhancer-Binding Proteins; Cell Death; Cell Line, Tumor; Cytochromes c; Fenretinide; Humans; Mitochondria; Models, Biological; Neuroblastoma; Oxidative Stress; Permeability; Reactive Oxygen Species; Retinoids; Signal Transduction; Transcription Factor CHOP; Transcription Factors

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). Here, we investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-immunoliposomes (anti-GD2-SIL) showed specific, competitive binding to, and uptake by, various NB cell lines. Moreover, anti-GD2-SIL-HPR presented increased selectivity and efficacy in inhibiting NB cell proliferation through the induction of apoptosis, compared to free drug and SL-HPR. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. However, similar, but significantly less potent antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 mAb alone (P=0.0297 and P=0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that, although anti-GD2 mAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only following treatment with anti-GD2-SIL-HPR (P<0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Division; Disease Models, Animal; Fenretinide; Gangliosides; Humans; Liposomes; Mice; Neuroblastoma; Tumor Cells, Cultured

Unlike 13-cis retinoic acid, the synthetic retinoid fenretinide induces apoptosis of neuroblastoma cells and in vitro acts synergistically with the chemotherapeutic drugs, cisplatin, etoposide and carboplatin. The stress-induced transcription factor GADD153 and the Bcl2-related protein Bak are upregulated in response to fenretinide. Although fenretinide is a partial retinoic acid receptor (RAR)-beta/gamma agonist, RARbeta/gamma antagonists do not block the induction of GADD153 or Bak by fenretinide. Conversely, the induction of GADD153 and Bak is blocked by antioxidants. Neither GADD153 or Bak were induced by chemotherapeutic agents but over expression of GADD153 results in increased sensitivity to fenretinide-induced apoptosis. Therefore, fenretinide induces apoptosis via RAR-dependent and -independent pathways in which the RAR-independent pathway is characterised by the reactive oxygen species-dependent induction of GADD153 and Bak. The targeting of GADD153 and Bak in neuroblastoma cells may be novel pathways for the development of drugs inducing apoptosis of neuroblastoma with improved tumour specificity.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; CCAAT-Enhancer-Binding Proteins; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Neuroblastoma; Receptors, Retinoic Acid; Transcription Factor CHOP; Transcription Factors; Tumor Cells, Cultured

Angiogenesis is a critical event in the progression of human neuroblastoma. This mini-review summarizes our literature and experimental data concerning the use of anti-angiogenic molecules, such as TNP-470 and fenretinide, in neuroblastoma treatment.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Fenretinide; Humans; Neovascularization, Pathologic; Neuroblastoma

In vitro studies that showed RA could cause growth arrest and differentiation of myelogenous leukemia and neuroblastoma led to clinical trials of retinoids in APL and neuroblastoma that increased survival for both of those diseases. In the case of APL, ATRA has been the drug of choice, and preclinical and clinical data support direct combinations of ATRA with cytotoxic chemotherapy. For neuroblastoma, a phase I study defined a dose of 13-cis-RA, which was tolerable in patients after myeloablative therapy, and a phase III trial that showed postconsolidation therapy with 13-cis-RA improved EFS for patients with high-risk neuroblastoma. Preclinical studies in neuroblastoma indicate that ATRA or 13-cis-RA can antagonize cytotoxic chemotherapy and radiation, so use of 13-cis-RA in neuroblastoma is limited to maintenance after completion of cytotoxic chemotherapy and radiation. A limitation on the antitumor benefit of ATRA in APL is the marked decrease in drug levels that occurs during therapy as a result of induction of drug metabolism, resulting in a shorter drug half-life and decreased plasma levels. Although early studies sought to overcome the pharmacologic limitations of ATRA therapy in APL, the demonstration that ATO is active against APL in RA-refractory patients has led to a focus on studies employing ATO. Use of 13-cis-RA in neuroblastoma has avoided the decreased plasma levels seen with ATRA. It is likely that recurrent disease seen during or after 13-cis-RA therapy in neuroblastoma is due to tumor cell resistance to retinoid-mediated differentiation induction. Studies in neuroblastoma cell lines resistant to 13-cis-RA and ATRA have shown that they can be sensitive, and in some cases collaterally hypersensitive, to the cytotoxic retinoid fenretinide. Fenretinide induces tumor cell cytotoxicity rather than differentiation, acts independently from RA receptors, and in initial phase I trials has been well tolerated. Clinical trials of fenretinide, alone and in combination with ceramide modulators, are in development.

Topics: Antineoplastic Agents; Child; Clinical Trials as Topic; Fenretinide; Humans; Isotretinoin; Leukemia, Myeloid, Acute; Neoplasms; Neuroblastoma; Randomized Controlled Trials as Topic; Receptors, Retinoic Acid; Retinoids; Tretinoin

Retinoids are derivatives of vitamin A that include all- trans-retinoic acid (ATRA), 13-cis-retinoic acid, (13-cis-RA), and fenretinide (4-HPR). High levels of either ATRA or 13-cis-RA can cause arrest of cell growth and morphologic differentiation of human neuroblastoma cell lines. Phase I trials have shown that higher and more sustained drug levels were obtained with 13-cis-RA relative to ATRA. A phase III randomized trial showed that high-dose pulse therapy with 13-cis-RA given after completion of intensive chemoradiotherapy (with or without autologous bone marrow transplantation) significantly improves event-free survival in high-risk neuroblastoma. Because 4-HPR achieves multi-log cell kills in neuroblastoma cell lines that are resistant to ATRA and 13-cis-RA, a pediatric phase I trial is in progress to determine the maximum tolerated dose of 4-HPR, with a view toward giving 4-HPR after completion of myeloablative therapy and 13-cis-RA.

Topics: Antineoplastic Agents; Child; Drug Administration Schedule; Fenretinide; Humans; Isotretinoin; Neuroblastoma; Randomized Controlled Trials as Topic; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Patients with relapsed/refractory stage 4 high-risk neuroblastoma were enrolled on a phase I study (NANT2004-03) of intravenous fenretinide emulsion. Pharmacokinetic samples were collected during and after the infusion, and the levels were measured using an HPLC system. A likely case of a fatal drug interaction between fenretinide, ceftriaxone, and acetaminophen is described, including the pharmacokinetics of fenretinide, laboratory data, and post-mortem autopsy in a pediatric neuroblastoma patient treated on this study.. On Day 4 of a scheduled 5-day-infusion of intravenous fenretinide, the patient developed a fever, acetaminophen was started, ceftriaxone initiated for possible bacteremia, and fenretinide level doubled from 56 to 110 μM. Over the next three days, although blood cultures remained negative, the patient's condition deteriorated rapidly. Acute liver failure was diagnosed on Day 7, and the patient expired on Day 20 of fulminant hepatic failure with associated renal, cardiac, and hemorrhagic/coagulation toxicities. Autopsy showed extensive hemorrhagic necrosis of the liver, marked bile duct proliferation, and abundant hemosiderin, consistent with cholestasis and drug toxicity.. After extensive review of patient data, the clinical course, and the literature, we conclude that observed hepatic toxicity was likely due to a drug interaction between fenretinide and concomitant ceftriaxone and acetaminophen. None of the other 16 patients treated on this study experienced significant hepatic toxicity. Although the prevalence of cholestasis with ceftriaxone usage is relatively high, the potential drug interaction with these concomitant medications has not been previously reported. Concomitant use of fenretinide, ceftriaxone, and acetaminophen should be avoided.

Topics: Acetaminophen; Ceftriaxone; Child; Dose-Response Relationship, Drug; Drug Interactions; Fatal Outcome; Fenretinide; Humans; Injections, Intravenous; Male; Neuroblastoma; Radiography

A phase I study was conducted to determine the maximum-tolerated dose, dose-limiting toxicities (DLTs), and pharmacokinetics of fenretinide (4-HPR) delivered in an oral powderized lipid complex (LXS) in patients with relapsed/refractory neuroblastoma.. 4-HPR/LXS powder (352-2,210 mg/m(2) /day) was administered on Days 0-6, in 21-day courses, by standard 3 + 3 design.. Thirty-two patients (median age = 8 years, range 3-27 years) enrolled with 30 evaluable for dose escalation. Prior therapies included stem cell transplantation/support (n = 26), 13-cis-retinoic acid (n = 22), (125/131) I-MIBG (n = 13), and anti-GD2 antibody (n = 6). 170+ courses were delivered. Course 1 DLTs were a Grade 3 (n = 1) alkaline phosphatase at 352 mg/m(2) /day. Other major toxicities were Grade 4 (n = 1) alkaline phosphatases on Courses 5 and 6 at 774 mg/m(2) /day, and Grade 3 (n = 1) ALT/AST elevation on Course 2 at 1,700 mg/m(2) /day. Of 29 response-evaluable patients, six had stable disease (SD) (4-26 courses); four with marrow- or bone disease-only had complete responses (CR) (10-46 courses). 4-HPR plasma levels were several folds higher (P < 0.05) than previously reported using capsular fenretinide. The Day 6 mean peak 4-HPR plasma level at 1,700 mg/m(2) /day was 21 µM. An MTD was not reached.. 4-HPR/LXS oral powder obtained higher plasma levels, with minimal toxicity and evidence of anti-tumor activity, than a previous capsule formulation. A recommended phase II schedule of 4-HPR/LXS powder is 1,500 mg/m(2) /day, TID, on Days 0-6, of a 21-day course.

Topics: Adolescent; Adult; Antineoplastic Agents; Child; Child, Preschool; Female; Fenretinide; Humans; Male; Maximum Tolerated Dose; Neoplasm Recurrence, Local; Neuroblastoma; Young Adult

To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma.. Patients received 7 days of fenretinide: 2,475 mg/m(2)/d divided TID (<18 years) or 1,800 mg/m(2)/d divided BID (≥18 years) every 21 days for a maximum of 30 courses. Patients with stable or responding disease after course 30 could request additional compassionate courses. Best response by course 8 was evaluated in stratum 1 (measurable disease on CT/MRI ± bone marrow and/or MIBG avid sites) and stratum 2 (bone marrow and/or MIBG avid sites only).. Sixty-two eligible patients, median age 5 years (range 0.6-19.9), were treated in stratum 1 (n = 38) and stratum 2 (n = 24). One partial response (PR) was seen in stratum 2 (n = 24 evaluable). No responses were seen in stratum 1 (n = 35 evaluable). Prolonged stable disease (SD) was seen in 7 patients in stratum 1 and 6 patients in stratum 2 for 4 to 45+ (median 15) courses. Median time to progression was 40 days (range 17-506) for stratum 1 and 48 days (range 17-892) for stratum 2. Mean 4-HPR steady-state trough plasma concentrations were 7.25 μmol/L (coefficient of variation 40-56%) at day 7 course 1. Toxicities were mild and reversible.. Although neither stratum met protocol criteria for efficacy, 1 PR + 13 prolonged SD occurred in 14/59 (24%) of evaluable patients. Low bioavailability may have limited fenretinide activity. Novel fenretinide formulations with improved bioavailability are currently in pediatric phase I studies.

Topics: 3-Iodobenzylguanidine; Administration, Oral; Adolescent; Antineoplastic Agents; Capsules; Child; Child, Preschool; Disease-Free Survival; Female; Fenretinide; Humans; Infant; Iodine Radioisotopes; Magnetic Resonance Imaging; Male; Neoplasm Recurrence, Local; Neuroblastoma; Radiopharmaceuticals; Survival Rate; Tomography, X-Ray Computed; Young Adult

Pharmacokinetic data on fenretinide (4-HPR) are scant, thus limiting the rational use of the drug. We investigated the pharmacokinetics of 4-HPR and its active metabolite 4-oxo-fenretinide (4-oxo-4-HPR).. Pharmacokinetics were assessed in 18 children (3 for each dose) with neuroblastoma who received oral 4-HPR once daily for 28 days at the doses of 100, 300, 400, 600, 1,700 and 4,000 mg/m(2)/day. 4-HPR and 4-oxo-4-HPR were determined by HPLC in plasma collected up to 48 h after the first and 28th administration.. After single administration, 4-HPR mean C (max) ranged from 0.9 to 6.6 microM and these concentrations roughly doubled at steady state (range 1.6-14.5 microM). 4-HPR mean t (1/2) was 22 h. 4-HPR pharmacokinetics were linear in the dose range 100-1,700 mg/m(2); less than dose-proportional increase in exposure was found at 4,000 mg/m(2). At steady state, pharmacologically relevant plasma concentrations (range 0.7-10 microM and 0.4-5 microM for 4-HPR and 4-oxo-4-HPR, respectively) were maintained during the 24 h dosing interval in the dose range 300-4,000 mg/m(2).. 4-HPR pharmacokinetics supports once-daily dosing. Steady state concentrations of 4-HPR and 4-oxo-4-HPR in children with neuroblastoma are in line with those found to have in vitro growth inhibitory effects in neuroblastoma cells.

Topics: Administration, Oral; Adolescent; Adult; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Child; Child, Preschool; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Screening Assays, Antitumor; Female; Fenretinide; Half-Life; Humans; Male; Neuroblastoma

To determine the maximal tolerated dosage (MTD) of oral fenretinide given as intact capsules for 7 days, repeated every 21 days, in children with high-risk solid tumors.. Children 21 years of age or younger received daily doses from 350 mg/m2 to 3,300 mg/m2 (divided into two or three doses), with pharmacokinetics during course one. The MTD was defined as zero to one of six patients with dose-limiting toxicity (DLT), with at least two of three or two of six DLT at next higher dose.. Fifty-four patients, age 2 years to 20 years (median, 9 years), were treated: neuroblastoma (n = 39), Ewing sarcoma (n = 5), and other (n = 10). Prior therapy included autologous stem cell transplantation (n = 42), 13-cis-RA (n = 35), and 9-cis-RA (n = 1). One of four patients at 1,050 mg/m2 with prior liver transplant had grade 3 ALT/abdominal pain/nausea/dehydration and grade 4 AST/emesis. At 1,860 mg/m2, one of seven patients had grade 3 hypoalbuminemia/hypophosphatemia. At 2,475 mg/m2, one of eight patients had grade 3 alkaline phosphatase; three of five patients had DLT at 3,300 mg/m2: grade 3 AST/ALT (n = 1), grade 4 bilirubin/grade 3 AST/ALT (n = 1), pseudotumor cerebri (n = 1). Pseudotumor cerebri also occurred at 600 mg/m2 and 800 mg/m2. There was one complete response and 13 patients with stable disease (SD) for 8 or more courses in 30 assessable neuroblastoma patients. SD for 8 or more courses was seen in one of five Ewing sarcoma patients and one melanoma patient. Mean N-4-hydroxyphenyl retinamide plasma level (day 7, steady-state concentration) was 9.9 mumol/L at MTD.. The pediatric MTD of oral capsular fenretinide was 2,475 mg/m2 per day, which achieved levels active against neuroblastoma in vitro with minimal toxicity. Response data support a phase II trial in neuroblastoma.

Topics: Administration, Oral; Adolescent; Adult; Antineoplastic Agents; Child; Child, Preschool; Drug Administration Schedule; Female; Fenretinide; Humans; Male; Neoplasm Recurrence, Local; Neoplasms; Neuroblastoma; Treatment Outcome; Vitamin A

Fenretinide (4HPR), a synthetic retinoid, induces apoptosis in neuroblastoma cells. A Phase I study in children with neuroblastoma was designed to determine maximum tolerated dose, toxicity, and pharmacokinetics.. Fifty-four patients received oral 4HPR, once daily, for 28 days, followed by a 7-day interruption, for up to 6 courses. The starting dose was 100 mg/m(2)/day. At least 3 patients were entered at each escalating 4HPR dose level. Pharmacokinetic sampling was performed on days 1 and 28 of the first course.. Fifty-four patients, of whom 53 were evaluable, received doses between 100 and 4000 mg/m(2)/day for a total of 168 courses. Additional dose escalation was precluded by capsule number intake. A total of 34 of 53 evaluable patients showed manageable, reversible toxicities, which were not dose related. One dose-limiting toxicity (nyctalopia grade 3) occurred after the 1000 mg/m(2)/day dose. Twelve patients showed grade 2 toxicity: skin xerosis (6 cases); nyctalopia (3 cases); hepatic toxicity (1 case); diarrhea (1 case); and headache (1 case). Stable disease was observed in 41 patients for a median period of 23 months (range 2-35+). After first administration, average 4HPR peak plasma levels ranged from 0.6 to 6 micro M (after 100 and 4000 mg/m(2)/day, respectively) and increased 2-fold (to 1.3 and 12.9 micro M, respectively) after the 28-day treatment. 4HPR half-life increased from 17 h after the first administration to 25 h after the 28(th) administration. Incidence of grade 2-3 toxicity was 0 of 12 (0%), 7 of 22 (31%), and 4 of 8 (50%) with peak 4HPR concentrations <3 micro M, 3-10 micro M, and >10 micro M, respectively. After repeated treatment, retinol levels decreased from 20 to 10% of pretreatment levels after all of the doses.. In children, 4HPR administration up to 4000 mg/m(2)/day over 28 days, followed by a 7-day interruption, results in manageable toxicity and in drug plasma concentrations comparable with those that induce apoptosis in neuroblastoma cell lines.

Topics: Adolescent; Adult; Antineoplastic Agents; Child; Child, Preschool; Female; Fenretinide; Humans; Male; Neuroblastoma; Vitamin A

In a previous study, we demonstrated that the combination of fenretinide with lenalidomide, administered by a novel nanomicellar formulation (FLM), provided a strong antitumor effect in a neuroblastoma TrkB-expressing tumor. In this study, we tested the nanomicellar combination in an. FLM was administered to mice bearing human NLF xenografts to evaluate its efficacy in comparison with the nanomicelles containing fenretinide alone (FM). Confocal laser-scanning fluorescence microscopy images of the NLF cells treated with FLM and FM allowed us to estimate the nanomicelle ability to transport the encapsulated drugs inside the tumor cells. Flow cytometric analysis of the cells from treated tumors was performed to assess the effect of treatment on GD2 expression and NK cell infiltration.. FLM and FM decreased the growth of NLF xenografts at comparable extents during the treatment period. Afterwards, FLM induced a progressive tumor regression without regrowth, while FM treatment was followed by regrowth within 15-20 days after the end of treatment. Both FLM and FM were able to penetrate the tumor cells transporting the encapsulated drugs. FLM transported higher amount of fenretinide inside the cells. Also, FLM treatment strongly increased GD2 expression in treated tumors and slightly decreased the NK infiltration compared to FM.. FLM treatment induced a superior antitumor response than FM in NLF xenografts, presumably due to the combined effects of fenretinide cytotoxicity and lenalidomide antiangiogenic activity. The ability of FLM to penetrate tumor cells, transporting the encapsulated drugs, substantially improved the therapeutic efficiency of this system. Moreover, the enhancement of GD2 expression in FLM treated tumors offers the possibility to further increase the antitumor effect by the use of anti-GD2 CAR-T cells and anti-GD2 antibodies in combination with FLM in multimodal therapies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Drug Delivery Systems; Female; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Killer Cells, Natural; Lenalidomide; Mice, Nude; Micelles; Microscopy, Confocal; N-Myc Proto-Oncogene Protein; Nanostructures; Neuroblastoma; Xenograft Model Antitumor Assays

Recurrent high-risk neuroblastoma is a childhood cancer that often fails to respond to therapy. Fenretinide (4-HPR) is a cytotoxic retinoid with clinical activity in recurrent neuroblastoma and venetoclax (ABT-199) is a selective inhibitor of the antiapoptotic protein B-cell lymphoma-2 (BCL-2). We evaluated activity of 4-HPR + ABT-199 in preclinical models of neuroblastoma. Patient-derived cell lines and xenografts from progressive neuroblastoma were tested. Cytotoxicity was evaluated by DIMSCAN, apoptosis by flow cytometry, and gene expression by RNA sequencing, quantitative RT-PCR, and immunoblotting. 4-HPR + ABT-199 was highly synergistic against high BCL-2-expressing neuroblastoma cell lines and significantly improved event-free survival of mice carrying high BCL-2-expressing patient-derived xenografts (PDX). In 10 matched-pair cell lines [established at diagnosis (DX) and progressive disease (PD) from the same patients], BCL-2 expression in the DX and PD lines was comparable, suggesting that BCL-2 expression at diagnosis may provide a biomarker for neuroblastomas likely to respond to 4-HPR + ABT-199. In a pair of DX (COG-N-603x) and PD (COG-N-623x) PDXs established from the same patient, COG-N-623x was less responsive to cyclophosphamide + topotecan than COG-N-603x, but both DX and PD PDXs were responsive to 4-HPR + ABT-199. Synergy of 4-HPR + ABT-199 was mediated by induction of NOXA via 4-HPR stimulation of reactive oxygen species that induced expression of ATF4 and ATF3, transcription factors for NOXA. Thus, fenretinide + venetoclax is a synergistic combination that warrants clinical testing in high BCL-2-expressing neuroblastoma.

Topics: Animals; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Cell Culture Techniques; Cell Line, Tumor; Disease Models, Animal; Female; Fenretinide; Humans; Mice; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Currently >50% of high-risk neuroblastoma (NB) patients, despite intensive therapy and initial partial or complete response, develop recurrent NB due to the persistence of minimal residual disease (MRD) that is resistant to conventional antitumor drugs. Indeed, their low therapeutic index prevents drug-dose escalation and protracted administration schedules, as would be required for MRD treatment. Thus, more effective and less toxic therapies are urgently needed for the management of MRD. To address this aim, we evaluated a new combination of fenretinide and lenalidomide, both endowed with antitumor activity and low-toxicity profiles. New nanomicelles were prepared as carriers for this combination to maximize bioavailability and accumulation at the tumor site because of the enhanced permeability and retention (EPR) effect.. New nanomicelles containing the fenretinide-lenalidomide combination (FLnMs) were prepared by a one-step method, providing high drug encapsulation and micelle dimensions suitable for tumor accumulation. Their administration to mice bearing human NB xenografts allowed us to evaluate their efficacy in comparison with the nanomicelles containing fenretinide alone (FnMs).. Treatment by FLnMs significantly decreased the tumor growth of NB xenografts. FLnMs were more active than FnMs despite comparable fenretinide concentrations in tumors, and lenalidomide alone did not show cytotoxic activity in vitro against NB cells. The tumor mass at the end of treatment with FLnMs was predominantly necrotic, with a decreased Ki-67 proliferation index.. FLnMs provided superior antitumor efficacy in NB xenografts compared to FnMs. The enhanced efficacy of the combination was likely due to the antiangiogenic effect of lenalidomide added to the cytotoxic effect of fenretinide. This new nanomicellar combination is characterized by a low-toxicity profile and offers a novel therapeutic option for the treatment of high-risk tumors where the persistence of MRD requires repeated administrations of therapeutic agents over long periods of time to avoid recurrent disease.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Drug Stability; Female; Fenretinide; Humans; Injections, Subcutaneous; Lenalidomide; Mice; Mice, Nude; Micelles; Nanoparticles; Neuroblastoma; Xenograft Model Antitumor Assays

All-trans-N-(4-hydroxyphenyl)retinamide or fenretinide (4-HPR) acts by reactive oxygen species (ROS) and dihydroceramides (DHCers). In early-phase clinical trials 4-HPR has achieved complete responses in T-cell lymphomas (TCL) and neuroblastoma (NB) and signals of activity in ovarian cancer (OV). We defined the activity of 4-HPR metabolites in N-(4-methoxyphenyl)retinamide (MPR), 4-oxo-N-(4-hydroxyphenyl)retinamide (oxoHPR), and the 4-HPR isomer 13-cis-fenretinide (cis-HPR) in NB, OV, and TCL cell lines cultured in physiological hypoxia.. We compared the effect of 4-HPR, cis-HPR, oxoHPR, and MPR on cytotoxicity, ROS, and DHCers in a panel of TCL, NB, and OV cell lines cultured in bone marrow level physiological hypoxia (5% O2), utilizing a fluorescence-based cytotoxicity assay (DIMSCAN), flow cytometry, and quantitative mass spectrometry.. 4-HPR (10 µmol/l) achieved more than three logs of cell kill in nine of 15 cell lines. Cytotoxicity of 4-HPR and oxoHPR was comparable; in some cell lines, cis-HPR cytotoxicity was lower than 4-HPR, but additive when combined with 4-HPR. MPR was not cytotoxic. ROS and DHCers were equivalently increased by 4-HPR and oxoHPR in all cell lines (P<0.01), to a lesser extent by cis-HPR (P<0.01), and not increased in response to MPR (P>0.05). Mitochondrial membrane depolarization, caspase-3 cleavage, and apoptosis (TUNEL) were all significantly increased by 4-HPR and oxoHPR (P<0.01).. Cytotoxic and pharmacodynamic activity was comparable with 4-HPR and oxoHPR, lower with cis-HPR, and MPR was inactive. Neither MPR or cis-HPR antagonized 4-HPR activity. These data support focusing on achieving high 4-HPR exposures for maximizing antineoplastic activity.

Topics: Antineoplastic Agents; Apoptosis; Cell Proliferation; Drug Synergism; Female; Fenretinide; Humans; Hypoxia; Lymphoma, T-Cell; Neuroblastoma; Ovarian Neoplasms; Reactive Oxygen Species; Tumor Cells, Cultured

Although the administration of retinoids represents an important part of treatment for children suffering from high-risk neuroblastomas, approximately 50% of these patients do not respond to this therapy or develop resistance to retinoids during treatment. Our study focused on the comparative analysis of the expression of five genes and corresponding proteins (DDX39A, HMGA1, HMGA2, HOXC9 and PBX1) that have recently been discussed as possible predictive biomarkers of clinical response to retinoid differentiation therapy. Expression of these five candidate biomarkers was evaluated at both the mRNA and protein level in the same subset of 8 neuroblastoma cell lines after treatment with natural or synthetic retinoids. We found that the cell lines that were HMGA2-positive and/or HOXC9-negative have a reduced sensitivity to retinoids. Furthermore, the experiments revealed that the retinoid-sensitive cell lines showed a uniform pattern of change after treatment with both natural and sensitive retinoids: increased DDX39A and decreased PBX1 protein levels. Our results showed that in NBL cells, these putative protein biomarkers are associated with sensitivity or resistance to retinoids, and their endogenous or induced expression can distinguish between these two phenotypes.

Topics: Adolescent; Antineoplastic Agents; Bexarotene; Biomarkers, Pharmacological; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; DEAD-box RNA Helicases; Drug Resistance, Neoplasm; Female; Fenretinide; HMGA1a Protein; HMGA2 Protein; Homeodomain Proteins; Humans; Infant; Infant, Newborn; Isotretinoin; Male; Nervous System Neoplasms; Neuroblastoma; Paraffin Embedding; Pre-B-Cell Leukemia Transcription Factor 1; Tissue Fixation; Tretinoin; Young Adult

Maintenance therapy with 13-cis-retinoic acid and immunotherapy (given after completion of intensive cytotoxic therapy) improves outcome for high-risk neuroblastoma patients. The synthetic retinoid fenretinide (4-HPR) achieved multiple complete responses in relapse/refractory neuroblastoma in early-phase clinical trials, has low systemic toxicity, and has been considered for maintenance therapy clinical trials. Difluoromethylornithine (DFMO, an irreversible inhibitor of ornithine decarboxylase with minimal single-agent clinical response data) is being used for maintenance therapy of neuroblastoma. We evaluated the cytotoxic activity of DFMO and fenretinide in neuroblastoma cell lines.. We tested 16 neuroblastoma cell lines in bone marrow-level hypoxia (5% O. At clinically achievable levels (100 μM), DFMO significantly decreased (P < 0.05) polyamine putrescine and achieved modest cytotoxicity (<1 log (90% cytotoxicity). Prolonged exposures (7 days) or culture in 2% and 20% O. DFMO as a single agent has minimal cytotoxic activity for neuroblastoma cell lines.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Eflornithine; Fenretinide; Humans; Inhibitory Concentration 50; Neuroblastoma

We previously reported that concurrent ketoconazole, an oral anti-fungal agent and P450 enzyme inhibitor, increased plasma levels of the cytotoxic retinoid, fenretinide (4-HPR) in mice. We have now determined the effects of concurrent ketoconazole on 4-HPR cytotoxic dose-response in four neuroblastoma (NB) cell lines in vitro and on 4-HPR activity against two cell line-derived, subcutaneous NB xenografts (CDX) and three patient-derived NB xenografts (PDX). Cytotoxicity in vitro was assessed by DIMSCAN assay. Xenografted animals were treated with 4-HPR/LXS (240 mg/kg/day) + ketoconazole (38 mg/kg/day) in divided oral doses in cycles of five continuous days a week. In one model, intratumoral levels of 4-HPR and metabolites were assessed by HPLC assay, and in two models intratumoral apoptosis was assessed by TUNEL assay, on Day 5 of the first cycle. Antitumor activity was assessed by Kaplan-Meier event-free survival (EFS). The in vitro cytotoxicity of 4-HPR was not affected by ketoconazole (p ≥ 0.06). Ketoconazole increased intratumoral levels of 4-HPR (p = 0.02), of the active 4-oxo-4-HPR metabolite (p = 0.04), and intratumoral apoptosis (p ≤ 0.0006), compared to 4-HPR/LXS-alone. Concurrent ketoconazole increased EFS in both CDX models compared to 4-HPR/LXS-alone (p ≤ 0.008). 4-HPR + ketoconazole also increased EFS in PDX models compared to controls (p ≤ 0.03). Thus, concurrent ketoconazole decreased 4-HPR metabolism with resultant increases of plasma and intratumoral drug levels and antitumor effects in neuroblastoma murine xenografts. These results support the clinical testing of concurrent ketoconazole and oral fenretinide in neuroblastoma.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cytochrome P-450 CYP3A Inhibitors; Drug Administration Schedule; Drug Synergism; Fenretinide; Humans; Ketoconazole; Mice; Neuroblastoma; Treatment Outcome; Xenograft Model Antitumor Assays

A simple and accurate high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its metabolites, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) and N-(4-methoxyphenyl)retinamide (4-MPR), in human plasma. Plasma samples were prepared using protein precipitation with ethanol. Chromatographic separation of the three analytes and N-(4-ethoxyphenyl)retinamide (4-EPR), an internal standard, was achieved on a Zorbax SB-C18 column (3.5μm, 50×2.1mm) using gradient elution with the mobile phase of 0.1% formic acid in water and acetonitrile (pH* 2.4) at a flow rate of 0.5mL/min. Electrospray ionization (ESI) mass spectrometry was operated in the positive ion mode with multiple reaction monitoring (MRM). The calibration curves obtained were linear over the concentration range of 0.2-50ng/mL with a lower limit of quantification of 0.2ng/mL. The relative standard deviation of intra-day and inter-day precision was below 7.64%, and the accuracy ranged from 94.92 to 105.43%. The extraction recoveries were found to be higher than 90.39% and no matrix effect was observed. The analytes were stable for the durations of the stability studies. The validated method was successfully applied to the analyses of the pharmacokinetic study for patients treated with 4-HPR in a clinical trial.

Topics: Acetonitriles; Algorithms; Chromatography, Liquid; Clinical Trials, Phase I as Topic; Fenretinide; Humans; Hydrogen-Ion Concentration; Ions; Limit of Detection; Mass Spectrometry; Neuroblastoma; Quality Control; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Temperature; Tretinoin; Water

Neuroblastoma is a childhood neural crest tumor. Fenretinide, a retinoic acid analogue, induces accumulation of mitochondrial reactive oxygen species and consequent apoptosis in neuroblastoma cells. The p75 neurotrophin receptor (p75NTR) enhances the antineuroblastoma cell efficacy of fenretinide in vitro. We examined the role of the retinoid binding protein, CRABP1, in p75NTR-mediated potentiation of the efficacy of fenretinide. Knockdown and overexpression, respectively, of either p75NTR or CRABP1 were effected in neuroblastoma cell lines using standard techniques. Expression was determined by qRT-PCR and confirmed at the protein level by Western blot. Metabolic viability was determined by Alamar blue assay. While protein content of CRABP1 correlated roughly with that of p75NTR in the three neuroblastoid or epithelioid human neuroblastoma cell lines studied, manipulation of p75NTR expression resulted in cell line-dependent, variable change in CRABP1 expression. Furthermore, in some cell lines, induced expression of CRABP1 in the absence of p75NTR did not alter cell sensitivity to fenretinide treatment. The effects of manipulation of p75NTR expression on CRABP1 expression and the effects of CRABP1 expression on fenretinide efficacy are therefore neuroblastoma cell line-dependent. Potentiation of the antineuroblastoma cell effects of fenretinide by p75NTR is not mediated solely through CRABP1.

Topics: Antineoplastic Agents; Apoptosis; Biomarkers; Cell Line; Cell Line, Tumor; Cell Survival; DNA, Complementary; Fenretinide; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Nerve Tissue Proteins; Neuroblastoma; Reactive Oxygen Species; Receptors, Nerve Growth Factor; Receptors, Retinoic Acid

Retinoid therapy has contributed to improved outcomes in neuroblastoma. Clinical trials of fenretinide report favorable toxicity and disease stabilization in patients with high risk (HR) neuroblastoma. Skeletal effects have been described with other retinoids, but not with fenretinide to date. Two patients with HR, metastatic, refractory neuroblastoma received protracted courses of oral fenretinide for more than 5 years' duration. Both developed premature long bone physeal closure, causing limb length discrepancies; their neuroblastoma remains in remission. The radiographic and clinical findings reported suggest these skeletal abnormalities may be a consequence of treatment with 13-cis-retinoic acid (13cisRA) followed by prolonged oral fenretinide exposure.

Topics: Antineoplastic Agents; Child; Child, Preschool; Female; Fenretinide; Growth Plate; Humans; Isotretinoin; Male; Neuroblastoma

ABT-751 is a colchicine-binding site microtubule inhibitor. Fenretinide (4-HPR) is a synthetic retinoid. Both agents have shown activity against neuroblastoma in laboratory models and clinical trials. We investigated the antitumor activity of 4-HPR + the microtubule-targeting agents ABT-751, vincristine, paclitaxel, vinorelbine, or colchicine in laboratory models of recurrent neuroblastoma. Drug cytotoxicity was assessed in vitro by a fluorescence-based assay (DIMSCAN) and in subcutaneous xenografts in nu/nu mice. Reactive oxygen species levels (ROS), apoptosis, and mitochondrial depolarization were measured by flow cytometry; cytochrome c release and proapoptotic proteins were measured by immunoblotting. 4-HPR + ABT-751 showed modest additive or synergistic cytotoxicity, mitochondrial membrane depolarization, cytochrome c release, and caspase activation compared with single agents in vitro; synergism was inhibited by antioxidants (ascorbic acid, α-tocopherol). 4-HPR + ABT-751 was highly active against four xenograft models, achieving multiple maintained complete responses. The median event-free survival (days) for xenografts from 4 patients combined was control = 28, 4-HPR = 49, ABT-751 = 77, and 4-HPR + ABT-751 > 150 (P < 0.001). Apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL) was significantly higher in 4-HPR + ABT-751-treated tumors than with single agents (P < 0.01) and was inhibited by ascorbic acid and α-tocopherol (P < 0.01), indicating that ROS from 4-HPR enhanced the activity of ABT-751. 4-HPR also enhanced the activity against neuroblastoma xenografts of vincristine or paclitaxel, but the latter combinations were less active than 4-HPR + ABT-751. Our data support clinical evaluation of 4-HPR combined with ABT-751 in recurrent and refractory neuroblastoma. Mol Cancer Ther; 15(11); 2653-64. ©2016 AACR.

Topics: Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Disease Models, Animal; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fenretinide; Humans; Membrane Potential, Mitochondrial; Mice; Neoplasm Recurrence, Local; Neuroblastoma; Reactive Oxygen Species; Sulfonamides; Tumor Burden; Xenograft Model Antitumor Assays

Retinoids are an important component of neuroblastoma therapy at the stage of minimal residual disease, yet 40-50% of patients treated with 13-cis-retinoic acid (13-cis-RA) still relapse, indicating the need for more effective retinoid therapy. Vorinostat, or Suberoylanilide hydroxamic acid (SAHA), is a potent inhibitor of histone deacetylase (HDAC) classes I & II and has antitumor activity in vitro and in vivo. Fenretinide (4-HPR) is a synthetic retinoid which acts on cancer cells through both nuclear retinoid receptor and non-receptor mechanisms. In this study, we found that the combination of 4-HPR + SAHA exhibited potent cytotoxic effects on neuroblastoma cells, much more effective than 13-cis-RA + SAHA. The 4-HPR + SAHA combination induced caspase-dependent apoptosis through activation of caspase 3, reduced colony formation and cell migration in vitro, and tumorigenicity in vivo. The 4-HPR and SAHA combination significantly increased mRNA expression of thymosin-beta-4 (Tβ4) and decreased mRNA expression of retinoic acid receptor α (RARα). Importantly, the up-regulation of Tβ4 and down-regulation of RARα were both necessary for the 4-HPR + SAHA cytotoxic effect on neuroblastoma cells. Moreover, Tβ4 knockdown in neuroblastoma cells increased cell migration and blocked the effect of 4-HPR + SAHA on cell migration and focal adhesion formation. In primary human neuroblastoma tumor tissues, low expression of Tβ4 was associated with metastatic disease and predicted poor patient prognosis. Our findings demonstrate that Tβ4 is a novel therapeutic target in neuroblastoma, and that 4-HPR + SAHA is a potential therapy for the disease.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Drug Resistance, Neoplasm; Fenretinide; Humans; Hydroxamic Acids; Neuroblastoma; Thymosin; Vorinostat

Neuroblastoma is the most common extracranial solid tumor of childhood. The retinoic acid analogue, fenretinide (4-hydroxyphenyl retinamide; 4-HPR), induces apoptosis in neuroblastoma cells in vitro and is currently in clinical trials for children with refractory neuroblastoma. We have previously shown that expression of the p75 neurotrophin receptor (p75NTR) enhances apoptosis induction and mitochondrial accumulation of reactive oxygen species by 4-HPR in neuroblastoma cells. We now examine the signaling events that underlie this effect.. Systematic examination of pro- and anti-apoptotic signaling effectors was performed by Western blot. Specific inhibitors of JNK phosphorylation and scavengers of mitochondrial reactive oxygen species were used to demonstrate the roles of these phenomena in the enhancement of fenretinide efficacy.. The present studies demonstrate that enhancement of 4-HPR-induced apoptosis by p75NTR is dependent upon p38MAPK phosphorylation, JNK phosphorylation, caspase 3 activation, Akt cleavage, and decreased Akt phosphorylation. In addition, treatment with 4-HPR results in upregulation of MKK4 and MEKK1, and phosphorylation of MKK3/6. Efforts to enhance the efficacy of 4-HPR and to identify those tumors most likely to respond to it might exploit these effectors of 4-HPR-induced apoptosis.. Pharmacological agents that enhance MKK4 or MEKK1 expression or JNK expression or phosphorylation may enhance efficacy of 4-HPR in neuroblastomas that do not express high levels of p75NTR.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Down-Regulation; Fenretinide; Gene Knockdown Techniques; Humans; MAP Kinase Kinase 4; Mitochondria; Nerve Tissue Proteins; Neuroblastoma; Oncogene Protein v-akt; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; Receptors, Nerve Growth Factor; Signal Transduction; Transfection

Neuroblastoma is an embryonal tumor originating from the simpatico-adrenal lineage of the neural crest. It approximately accounts for about 15% of all pediatric oncology deaths. Despite advances in multimodal therapy, metastatic neuroblastoma tumors at diagnosis remain a clinical challenge. Retinoids are a class of compounds known to induce both terminal differentiation and apoptosis/necrosis of neuroblastoma cells. Among them, fenretinide (HPR) has been considered one of the most promising anti-tumor agent but it is partially efficacious due to both poor aqueous solubility and rapid metabolism. Here, we have developed a novel HPR formulation, by which the drug was encapsulated into sterically stabilized nanoliposomes (NL[HPR]) according to the Reverse Phase Evaporation method. This procedure led to a higher structural integrity of liposomes in organic fluids for a longer period of time, in comparison with our previous liposomal formulation developed by the film method. Moreover, NL[HPR] were further coupled with NGR peptides for targeting the tumor endothelial cell marker, aminopeptidase N (NGR-NL[HPR]). Orthotopically xenografted neuroblastoma-bearing mice treated with NGR-NL[HPR] lived statistically longer than mice untreated or treated with free HPR (NGR-NL[HPR] vs both control and HPR: P<0.0001). Also, NL[HPR] resulted in a statistically improved survival (NL[HPR] vs both control and HPR: P<0.001) but to a less extent if compared with that obtained with NGR-NL[HPR] (NGR-NL[HPR] vs NL[HPR]: P<0.01). Staining of tumor sections with antibodies specific for neuroblastoma and for either pericytes or endothelial cells evidenced that HPR reduced neuroblastoma growth through both anti-tumor and anti-angiogenic effects, mainly when delivered by NGR-NL[HPR]. Indeed, in this group of mice a marked reduction of tumor progression, of intra-tumoral vessel counts and VEGF expression, together with a marked down-modulation of matrix metalloproteinases MMP2 and MMP9, was observed. In conclusion, the use of this novel targeted delivery system for the apoptotic and antiangiogenic drug, fenretinide, could be considered as an adjuvant tool in the future treatment of neuroblastoma patients.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Female; Fenretinide; Humans; Liposomes; Mice; Mice, Nude; Neovascularization, Pathologic; Neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Combining passive immunotherapy with an antibody to the disialoganglioside GD2 (ch14.18/SP2/0) and cytokines with 13-cis-retinoic acid for post-myeloablative maintenance therapy increased survival in high-risk NB, but the overall prognosis for these children is still in need of improvement. Fenretinide (4-HPR) is a synthetic retinoid that has shown clinical activity in recurrent NB and is cytotoxic to a variety of cancer cells, in part via the accumulation of dihydroceramides, which are precursors of GD2. We investigated the effect of 4-HPR on CHO-derived, ch14.18-mediated anti-NB effector functions, complement-dependent cytotoxicity (CDC), and antibody-dependent and antibody-independent cellular cytotoxicity (ADCC and AICC, respectively). Here, we demonstrate for the first time that pretreatment of fenretinide-resistant NB cells with 4-HPR significantly enhanced ch14.18/CHO-mediated CDC and ADCC and AICC by both human natural killer cells and peripheral blood mononuclear cells. Treatment with 4-HPR increased GD2 and death receptor (DR) expression in resistant NB cells and induced an enhanced granzyme B and perforin production by effector cells. Blocking of ganglioside synthesis with a glucosylceramide synthase inhibitor abrogated the increased ADCC response but had no effect on the AICC, indicating that GD2 induced by 4-HPR mediates the sensitization of NB cells for ADCC. We also showed that 4-HPR induced increased GD2 and DR expression in a resistant NB xenograft model that was associated with an increased ADCC and AICC response using explanted tumor target cells from 4-HPR-treated mice. In summary, these findings provide an important baseline for the combination of 4-HPR and passive immunotherapy with ch14.18/CHO in future clinical trials for high-risk NB patients.

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Cell Line, Tumor; Coculture Techniques; Complement System Proteins; Drug Resistance, Neoplasm; Female; Fenretinide; Gangliosides; Humans; Killer Cells, Natural; Mice; Neuroblastoma; Receptors, Death Domain; Xenograft Model Antitumor Assays

Neuroblastoma is a common, frequently fatal, neural crest tumor of childhood. Chemotherapy-resistant neuroblastoma cells typically have Schwann cell-like ("S-type") morphology and express the p75 neurotrophin receptor (p75NTR). p75NTR has been previously shown to modulate the redox state of neural crest tumor cells. We, therefore, hypothesized that p75NTR expression level would influence the effects of the redox-active chemotherapeutic drug fenretinide on neuroblastoma cells.. Transfection and lentiviral transduction were used to manipulate p75NTR expression in these cell lines. Sensitivity to fenretinide was determined by concentration- and time-cell survival studies. Apoptosis incidence was determined by morphological assessment and examination of cleavage of poly-ADP ribose polymerase and caspase-3. Generation and subcellular localization of reactive oxygen species were quantified using species- and site-specific stains and by examining the effects of site-selective antioxidants on cell survival after fenretinide treatment. Studies of mitochondrial electron transport employed specific inhibitors of individual proteins in the electron transport chain.. Knockdown of p75NTR attenuates fenretinide-induced accumulation of mitochondrial superoxide and apoptosis. Overexpression of p75NTR has the opposite effects. Pretreatment of cells with 2-thenoyltrifluoroacetone or dehydroascorbic acid uniquely prevents mitochondrial superoxide accumulation and cell death after fenretinide treatment, indicating that mitochondrial complex II is the likely site of fenretinide-induced superoxide generation and p75NTR-induced potentiation of these phenomena.. Modification of expression of p75NTR in a particular neuroblastoma cell line modifies its susceptibility to fenretinide. Enhancers of p75NTR expression or signaling could be potential drugs for use as adjuncts to chemotherapy of neural tumors.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Survival; Electron Transport; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Indicators and Reagents; Mitochondria; Nerve Tissue Proteins; Neuroblastoma; Oxidation-Reduction; Reactive Oxygen Species; Receptors, Nerve Growth Factor; RNA, Small Interfering; Signal Transduction

Despite the successful introduction of 13-cis retinoic acid (13cisRA) therapy for the treatment of neuroblastoma, approximately 50% patients do not respond or experience relapse. A retinoid analogue, fenretinide [N-(4-hydroxyphenyl) retinamide; 4-HPR] can induce apoptosis in neuroblastoma cell lines and could have clinical use after therapy with 13cisRA. However, there are important questions concerning potential retinoid drug interactions which need to be addressed. The aim of this study was to investigate the influence of retinoic acid pre-treatment on fenretinide-induced apoptosis and fenretinide metabolism in neuroblastoma cell lines. Apoptosis was measured by flow cytometry of propidium iodide-stained neuroblastoma cells and a live-cell imaging assay. Intracellular fenretinide metabolism was determined by HPLC analysis. Pre-treatment of neuroblastoma cell lines with retinoic acid (RA) resulted in a significant decrease in the apoptotic response to fenretinide in three of the four lines tested. Comparison between responsive and non-responsive cell lines suggested that RA sensitivity was required to promote fenretinide resistance, and that this was mediated by up-regulation of Bcl-2 and the inhibition of pro-apoptotic fenretinide signalling pathways. Induction of the oxidative metabolism of fenretinide after RA pre-treatment did not significantly impact on intracellular parent drug levels and is unlikely to explain the decreased apoptotic response observed. The interaction between RA and fenretinide could have important implications for the scheduling of fenretinide in therapeutic protocols for neuroblastoma.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Chromatography, High Pressure Liquid; Drug Interactions; Fenretinide; Flow Cytometry; Humans; Neuroblastoma; Tretinoin

This study reports on the preparation and evaluation of amphiphilic macromolecules based on branched polyethylene glycol covalently linked with alkyl hydrocarbon chains. These macromolecules easily dissolved in an aqueous environment, with formation of micellar nanoaggregates endowed with hydrophobic inner cores capable of hosting fenretinide by complexation. The complexes increased fenretinide aqueous solubility, while hindering its release as a free drug in an aqueous environment. Particle size analysis indicated dimensional suitability of the complexes for intravenous administration. Neuroblastoma cell lines (SH-SY5Y and NGP) exhibited increased sensitivity to fenretinide in complex as compared to free drug, associated with higher intracellular concentrations of fenretinide observed after treatment with the complex. Transmission electronic microscopy images revealed endocytosis of the micellar complex. Moreover, fenretinide conversion to its metabolite 4-oxo-fenretinide was delayed in cells treated with the complex, further supporting the hypothesis that fenretinide may be absorbed by micellar transport and exposed to the cytoplasm for conversion to its metabolite only after micelle destabilization.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Crystallization; Drug Design; Fenretinide; Humans; Micelles; Nanocapsules; Neuroblastoma; Polyethylene Glycols

The major limitation to successful chemotherapy of neuroblastoma (NB) is the toxicity and the poor bioavailability of traditional drugs.. We synthesised an amphiphilic dextrin derivative (DX-OL) able to host fenretinide (4-HPR) by complexation. In this study, we have investigated the effects of 4-HPR-loaded amphipilic dextrin (DX-OL/4-HPR) in comparison with 4-HPR alone both in vitro on human NB cells and in vivo in pseudometastatic NB models. The haemolysis assay was used as a measure of the potential damage caused by the pharmaceutical formulation in vivo. Pharmacokinetic experiments were performed to assess drug plasma levels in mice treated with free or complexed 4-HPR.. DX-OL/4-HPR exerted a more potent cytotoxic activity on NB cells. Complexed 4-HPR significantly increased the proportion of sub-G1 cells with respect to free 4-HPR. Dextrin derivatives showed no haemolytic activity, indicating their suitability for parenteral administration. DX-OL/4-HPR increased the lifespan and the long-term survival of treated mice over controls. The analysis of drug plasma levels indicates that the complexed drug has a higher AUC due to a reduced clearance from the blood.. Our data suggest that DX-OL/4-HPR is an injectable formulation that is able to improve drug aqueous solubility and bioavailability.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biological Availability; Cell Division; Cell Line, Tumor; Disease Models, Animal; Drug Delivery Systems; Female; Fenretinide; Humans; Infusions, Intravenous; Mice; Mice, Nude; Neuroblastoma

Malignant neuroblastomas are childhood tumors that remain mostly incurable. We explored efficacy of N-(4-hydroxyphenyl) retinamide (4-HPR) and (-)-epigallocatechin-3-gallate (EGCG) in altering expression of oncogenic microRNAs (OGmiRs) and tumor suppressor miRs (TSmiRs) for controlling growth of human malignant neuroblastoma SK-N-BE2 and IMR-32 cells. Combination of 4-HPR and EGCG most significantly decreased expression of OGmiRs (miR-92, miR-93, and miR-106b) and increased expression of TSmiRs (miR-7-1, miR-34a, and miR-99a) in both cell lines. Overexpression of miR-93 and miR-7-1, respectively, decreased and increased efficacy of treatments. Thus, alterations in expression of specific OGmiRs and TSmiRs by 4-HPR and EGCG inhibited growth of malignant neuroblastomas.

Topics: Antineoplastic Agents; Apoptosis; Catechin; Cell Line, Tumor; Cell Survival; Fenretinide; Humans; MicroRNAs; Neuroblastoma

Lestaurtinib (CEP-701), a multi-kinase inhibitor with potent activity against the Trk family of receptor tyrosine kinases, has undergone early phase clinical evaluation in children with relapsed neuroblastoma. We studied the interaction of CEP-701 with isotretinoin (13cRA) and fenretinide (4HPR), two retinoids that have been studied in children with high-risk neuroblastoma.. In vitro growth inhibition was assessed following a 72-hour drug exposure using the sulforhodamine B (SRB) assay in eight neuroblastoma cell lines with variable TrkB expression. When appropriate, the combination index (CI) of Chou-Talalay was used to characterize the interaction of 13cRA (non-constant ratio) or 4HPR (constant ratio) with CEP-701.. The median (range) IC(50) of single-agent CEP-701 across all cell lines was 0.09 (0.08-0.3) μM. The combination of 13cRA and CEP-701 resulted in additive to synergistic interactions in four of the five cell lines studied. Addition of 1 or 5 μM of 13cRA decreased the median (range) CEP-701 IC(50) 1.5-fold (1.1-2.8-fold) and 1.7-fold (1.5-1.8-fold), respectively. With 10 μM 13cRA, less than 50% of cells survived when combined with various concentrations of CEP-701. The combination of 4HPR and CEP-701 trended toward being antagonistic, with a median (range) CI at the ED(50) of 1.3 (1.1-1.5).. The combination of 13cRA and CEP-701 was additive or synergistic in a spectrum of neuroblastoma cell lines, suggesting that these agents can be potentially studied together in the setting of minimal residual disease following intensive chemoradiotherapy for children with high-risk neuroblastoma.

Topics: Antineoplastic Agents; Brain Neoplasms; Carbazoles; Cell Line, Tumor; Drug Evaluation, Preclinical; Fenretinide; Furans; Humans; Isotretinoin; Neuroblastoma; Receptor, trkB

To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5-10 μM, 24 h) resulted in ROS induction (114-633%) and increased GSH levels (68-120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25-100 μM, 24 h). The 4HPR-BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110-150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR-BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR-BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR-BSO combination might be a promising new strategy in the treatment of neuroblastoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Buthionine Sulfoximine; Cell Line; Drug Synergism; Fenretinide; Glutathione; Growth Inhibitors; Humans; Neuroblastoma; Reactive Oxygen Species; Spheroids, Cellular

Neuroblastoma is the most prevalent extracranial solid tumor mainly in pediatric patients. We explored the efficacy of the combination of 2[(3-[2,3-dichlorophenoxy]propyl)amino]ethanol (2,3-DCPE, a small molecule inhibitor of the anti-apoptotic protein Bcl-x(L)) and N-(4-hydroxyphenyl) retinamide (4-HPR, a synthetic retinoid) in inducing differentiation and apoptosis in human malignant neuroblastoma cells. Immunofluorescence confocal microscopy and flow cytometry showed that the highest level of Bcl-x(L) expression occurred in SK-N-DZ cells followed by SH-SY5Y and IMR-32 cells. Combination of 20 μM 2,3-DCPE and 1 μM 4-HPR acted synergistically in decreasing viability of SK-N-DZ and SH-SY5Y cells. In situ methylene blue staining and protein gel blotting showed the efficacy of this combination of drugs in inducing neuronal differentiation morphologically and also biochemically with upregulation of the neuronal markers such as neurofilament protein (NFP) and neuron specific enolase (NSE) and downregulation of the differentiation inhibiting molecules such as N-Myc and Notch-1 in SK-N-DZ and SH-SY5Y cells. Annexin V-FITC/PI staining showed the synergistic action of this combination therapy in increasing apoptosis in both cell lines. Protein gel blotting manifested that combination therapy increased apoptosis with downregulation of the anti-apoptotic proteins Bcl-x(L), Bcl-2 and Mcl-1 and upregulation of the pro-apoptotic proteins Bax, p53, Puma (p53 upregulated modulator of apoptosis), and Noxa, ultimately causing activation of caspase-3. In conclusion, our results appeared highly encouraging in advocating the use of 2,3-DCPE and 4-HPR as a novel combination therapy for increasing both differentiation and apoptosis in human malignant neuroblastoma cells having Bcl-x(L) overexpression.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Cell Differentiation; Cell Line, Tumor; Cell Survival; Chlorobenzenes; Drug Synergism; Ethanolamines; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma

Fenretinide (4-HPR) is a cytotoxic retinoid with minimal systemic toxicity that has shown clinical activity against recurrent high-risk neuroblastoma. To identify possible synergistic drug combinations for future clinical trials, we determined whether ABT-737, a small-molecule BH3-mimetic that inhibits most proteins of the antiapoptotic Bcl-2 family, could enhance 4-HPR activity in neuroblastoma.. Eleven neuroblastoma cell lines were tested for the cytotoxic activity of 4-HPR and ABT-737 as single agents and in combination using the DIMSCAN fluorescence digital imaging cytotoxicity assay. The effect of these agents alone and in combination on mitochondrial membrane depolarization and apoptosis (by flow cytometry), cytochrome c release, caspases, Bax-α, t-Bid, and Bak activation, and subcutaneous xenografts in nu/nu mice was also determined.. Multilog synergistic cytotoxicity was observed for the drug combination in all of the 11 neuroblastoma cell lines tested, including MDR lines and those insensitive to either drug as single agents. 4-HPR + ABT-737 induced greater mitochondrial membrane depolarization and mitochondrial cytochrome c release, greater activation of caspases, Bax-α, t-Bid, and Bak, and a higher level of apoptosis than either drug alone. In vivo, 4-HPR + ABT-737 increased the event-free survival of the MDR human neuroblastoma line CHLA-119 implanted subcutaneously in nu/nu mice (194.5 days for the combination vs. 68 days for ABT-737 and 99 days for 4-HPR).. Thus, the combination of 4-HPR with a BH3-mimetic drug warrants clinical trials in recurrent neuroblastoma.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Caspases; Cell Line, Tumor; Cytochromes c; Disease-Free Survival; Female; Fenretinide; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Neuroblastoma; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Xenograft Model Antitumor Assays

Fenretinide induces apoptosis in neuroblastoma by induction of reactive oxygen species (ROS). In this study, we investigated the role of mitochondria in fenretinide-induced cytotoxicity and ROS production in six neuroblastoma cell lines. ROS induction by fenretinide was of mitochondrial origin, demonstrated by detection of superoxide with MitoSOX, the scavenging effect of the mitochondrial antioxidant MitoQ and reduced ROS production in cells without a functional mitochondrial respiratory chain (Rho zero cells). In digitonin-permeabilized cells, a fenretinide concentration-dependent decrease in ATP synthesis and substrate oxidation was observed, reflecting inhibition of the mitochondrial respiratory chain. However, inhibition of the mitochondrial respiratory chain was not required for ROS production. Co-incubation of fenretinide with inhibitors of different complexes of the respiratory chain suggested that fenretinide-induced ROS production occurred via complex II. The cytotoxicity of fenretinide was exerted through the generation of mitochondrial ROS and, at higher concentrations, also through inhibition of the mitochondrial respiratory chain.

Topics: Antineoplastic Agents; Antioxidants; Dose-Response Relationship, Drug; Down-Regulation; Drug Evaluation, Preclinical; Electron Transport; Fenretinide; Humans; Mitochondria; Models, Biological; Neuroblastoma; Organophosphorus Compounds; Reactive Oxygen Species; Tumor Cells, Cultured; Ubiquinone

Fenretinide (4-HPR), a synthetic retinoid currently used in clinic for cancer therapy and prevention, markedly lowers plasma retinol levels, an effect associated with nyctalopia. Our aim was to investigate the relationship between 4-HPR pharmacokinetics, plasma retinol reduction and incidence of nyctalopia.. Children with neuroblastoma, participating in a phase I trial, were treated with oral 4-HPR, once a day for 28-day courses followed by a 7-day drug interruption, with escalating dose levels from 100 to 4,000 mg/m(2) per day. Blood samples were collected at baseline and up to 48 h after the 1st (50 patients) and 28th (41 patients) administration, and the plasma concentrations of 4-HPR and retinol were measured by HPLC.. After the first administration, nadir retinol concentrations were reached at 16-20 h post-dosing; the extent of retinol reduction was related to 4-HPR dose and plasma concentrations as well as to pretreatment retinol concentrations. After repeated treatments, nadir retinol concentrations (10-20% of baseline values) were maintained during the 24 h dosing interval and were similar at all doses; the extent of retinol reduction was significantly (r = 0.97, P < 0.0001) related to pretreatment retinol concentrations. After a single dose, the relationship between 4-HPR pharmacokinetics and pharmacodynamics indicated a counterclockwise hysteresis suggesting the presence of an effect compartment. At steady state, the hysteresis collapsed suggesting that the 4-HPR concentrations in plasma and in the effect compartments were in equilibrium. Nyctalopia was not related to the administered dose, but was significantly associated (P = 0.05) with lower nadir retinol concentrations (0.11 +/- 0.012 vs. 0.17 +/- 0.015 microM).. During 4-HPR chronic treatment, plasma retinol reduction is not proportional to the dose. Plasma retinol levels of 0.11 microM could be considered as a safety biomarker in children with neuroblastoma. Finally, since initial retinol levels strongly predict the extent of retinol reduction, retinol decrease could be used to monitor 4-HPR compliance.

Topics: Antineoplastic Agents; Biomarkers, Pharmacological; Child; Chromatography, High Pressure Liquid; Clinical Trials, Phase I as Topic; Dose-Response Relationship, Drug; Female; Fenretinide; Humans; Male; Neuroblastoma; Night Blindness; Vitamin A

The proteasome inhibitor bortezomib inhibited cell growth and angiogenesis in neuroblastoma. Bortezomib has been shown to induce synergistic activity when combined with other antineoplastic agents. Here we have investigated the antitumor activity of bortezomib in combination with fenretinide, a synthetic retinoid, against neuroblastoma cells.. Different neuroblastoma cell lines were tested for sensitivity to bortezomib and fenretinide, given alone or in different dose-dependent and time-dependent combination schedules. Cell proliferation, cell viability, and apoptosis were evaluated by measuring 3H-thymidine incorporation, trypan blue staining, DNA fragmentation, and western blot analysis. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay. An orthotopic neuroblastoma mouse model was used to examine in vivo sensitivity.. Each compound alone was able to induce a dose-dependent inhibition of cell proliferation, with a significant enhanced antiproliferative effect for the drugs used in combination. This inhibition was characterized by marked G2-M and G1 cell cycle arrest with nearly complete depletion of S phase. Bortezomib and fenretinide in association triggered an increased apoptosis through activation of specific genes of the endoplasmic reticulum stress compared with either drug tested alone. Tumor-bearing mice treated with bortezomib plus fenretinide lived statistically significantly longer than mice treated with each drug alone. Histologic evaluation and chorioallantoic membrane analysis of primary tumors showed that the combined therapeutic activity of bortezomib and fenretinide rested upon antitumor and antiangiogenic mechanisms.. These findings provide the rationale for the development of a new therapeutic strategy for neuroblastoma based on this pharmacologic combination.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Cell Line, Tumor; Cell Proliferation; Chick Embryo; Endoplasmic Reticulum; Fenretinide; Humans; Mice; Neuroblastoma; Pyrazines; Xenograft Model Antitumor Assays

Resistance of high-risk metastatic neuroblastoma (HR-NB) to high dose chemotherapy (HD-CT) raises a major therapeutic challenge in pediatric oncology. Patients are treated by maintenance CT. For some patients, an adjuvant retinoid therapy is proposed, such as the synthetic retinoid fenretinide (4-HPR), an apoptotic inducer. Recent studies demonstrated that NB metastasis process is enhanced by the loss of caspase-8 involved in the Integrin-Mediated Death (IMD) process. As the role of caspase-8 appears to be critical in preventing metastasis, we aimed at studying the effect of 4-HPR on caspase-8 expression in metastatic neuroblasts.. We used the human IGR-N-91 MYCN-amplified NB experimental model, able to disseminate in vivo from the primary nude mouse tumor xenograft (PTX) into myocardium (Myoc) and bone marrow (BM) of the animal. NB cell lines, i.e., IGR-N-91 and SH-EP, were treated with various doses of Fenretinide (4-HPR), then cytotoxicity was analyzed by MTS proliferation assay, apoptosis by the propidium staining method, gene or protein expressions by RT-PCR and immunoblotting and caspases activity by colorimetric protease assays.. The IGR-N-91 parental cells do not express detectable caspase-8. However the PTX cells established from the primary tumor in the mouse, are caspase-8 positive. In contrast, metastatic BM and Myoc cells show a clear down-regulation of the caspase-8 expression. In parallel, the caspases -3, -9, -10, Bcl-2, or Bax expressions were unchanged. Our data show that in BM, compared to PTX cells, 4-HPR up-regulates caspase-8 expression that parallels a higher sensitivity to apoptotic cell death. Stable caspase-8-silenced SH-EP cells appear more resistant to 4-HPR-induced cell death compared to control SH-EP cells. Moreover, 4-HPR synergizes with drugs since apoptosis is restored in VP16- or TRAIL-resistant-BM cells. These results demonstrate that 4-HPR in up-regulating caspase-8 expression, restores and induces apoptotic cell death in metastatic neuroblasts through caspase-8 activation.. This study provides basic clues for using fenretinide in clinical treatment of HR-NB patients. Moreover, since 4-HPR induces cell death in caspase-8 negative NB, it also challenges the concept of including 4-HPR in the induction of CT of these patients.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Bone Marrow; Caspase 8; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Enzyme Activation; Etoposide; Fenretinide; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neuroblastoma; Reverse Transcriptase Polymerase Chain Reaction; TNF-Related Apoptosis-Inducing Ligand; Xenograft Model Antitumor Assays

Neuroblastomas, which mostly occur in children, are aggressive metastatic tumors of the sympathetic nervous system. The failure of the previous therapeutic regimens to target multiple components of N-Myc pathway resulted in poor prognosis. The present study investigated the efficacy of the combination of N-(4-hydroxyphenyl) retinamide (4-HPR, 0.5 microM) and genistein (GST, 25 microM) to control the growth of human neuroblastoma cells (SH-SY5Y and SK-N-BE2) harboring divergent molecular attributes. Combination of 4-HPR and GST down regulated N-Myc, Notch-1, and Id2 to induce neuronal differentiation. Transition to neuronal phenotype was accompanied by increase in expression of e-cadherin. Induction of neuronal differentiation was associated with decreased expression of hTERT, PCNA, survivin, and fibronectin. This is the first report that combination of 4-HPR and GST mediated reactivation of multiple tumor suppressors (p53, p21, Rb, and PTEN) for early cell cycle exit (due to G1/S phase arrest) in neuroblastoma cells. Reactivation of tumor suppressor(s) repressed N-Myc driven growth factor mediated angiogenic and invasive pathways (VEGF, b-FGF, MMP-2, and MMP-9) in neuroblastoma. Repression of angiogenic factors led to the blockade of components of mitogenic pathways [phospho-Akt (Thr 308), p65 NF-kappaB, and p42/44 Erk 1/2]. Taken together, the combination of 4-HPR and GST effectively blocked survival, mitogenic, and angiogenic pathways and activated proteases for apoptosis in neuroblastoma cells. These results suggested that combination of 4-HPR and GST could be effective for controlling the growth of heterogeneous human neuroblastoma cell populations.

Topics: Apoptosis; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Fenretinide; Flow Cytometry; Genes, p53; Glutathione Transferase; Humans; Mutation; Neuroblastoma; Proto-Oncogene Proteins c-myc; Signal Transduction

Langerhans cell histiocytosis (LCH) has previously been reported in association with other malignancies. The pathogenesis of LCH and its relationship to other malignancies is poorly understood. We present a novel case of a child who developed an LCH bone lesion while receiving a Phase I protocol therapy with oral fenretinide/Lym-X-Sorb (4-HPR/LXS) powder for neuroblastoma.

Topics: Bone Neoplasms; Child; Fenretinide; Histiocytosis, Langerhans-Cell; Humans; Male; Neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For increasing the therapeutic index of anticancer drugs, while reducing side effects, one of the most promising strategies in modern chemotherapy is based on the development of innovative drug delivery systems, such as liposomes. "Anticancer drug"-loaded liposomes have demonstrated enhanced ability to target to the affected area, as well as increased antitumor efficacy compared to conventional drugs. Liposomes tend to extravasate preferentially and to accumulate into tumor interstitial fluids, due to the defective structure of the new angiogenic vessels within the tumor masses. This inherent tumor selectivity can be increased further by coupling tumor-specific antibodies or other targeting moieties to the surface of the lipid envelope. Here, we describe the methodology used in these studies, as well as the antitumor results obtained by the use of several "anticancer drugs," encapsulated into antibody- and peptide-targeted liposomal formulations, against NB.

Topics: Animals; Antineoplastic Agents; Doxorubicin; Drug Delivery Systems; Fenretinide; Gangliosides; Gold; Liposomes; Mice; Microscopy, Electron, Transmission; Neoplasm Transplantation; Neovascularization, Pathologic; Neuroblastoma

Fenretinide is a synthetic retinoid that interferes with the attachment of retinol to retinol binding protein. It may inhibit accumulation of A2E and lipofuscin, and is proposed as therapy for Stargardt disease. It is currently used for cancer therapy, and mild depression of rod function and dark adaptation is a side effect at standard dosage. We studied two youngsters (aged between 12 and 13) receiving high doses as compassionate treatment for neuroblastoma: 800 mg daily for 1 out of every 3 weeks, for roughly 2 years. Goldmann-Weekers dark adaptometry, ISCEV standard ERG and mfERG were performed, and blood was analyzed for vitamin A. Neither child complained of night blindness or showed retinal fundus abnormalities. On initial exam, dark adaptation thresholds were elevated by 3 log units, and there were no detectable rod ERG responses. However, cone responses and mfERG were normal. Retesting one subject 3 months after stopping the drug revealed normal rod thresholds (slightly delayed) and low normal rod ERG responses. Serum vitamin A levels were normal from both subjects, but there is no record of whether the samples were drawn during cycles on or off drug. Our study demonstrates that high dose Fenretinide can suppress rod function quite completely, although serum vitamin A and rod function apparently return to normal or near normal levels rapidly once the drug is stopped. It is intriguing that cone function and access to vitamin A seems largely independent of Fenretinide effects on retinol availability.

Topics: Adolescent; Antineoplastic Agents; Child; Dark Adaptation; Electroretinography; Female; Fenretinide; Humans; Male; Neuroblastoma; Retinal Diseases; Retinal Rod Photoreceptor Cells; Vitamin A

The efficacy and mechanism of action of fenretinide (4-HPR), a vitamin A analogue, was investigated in a panel of six neuroblastoma cell lines and multicellular tumor spheroids. The latter are three dimensional cell aggregates and as such, a model for micrometastases. In all cell lines, the production of reactive oxygen species (ROS) increased with 163-680% after 1 h of treatment with 4-HPR. In addition, a decrease of the mitochondrial membrane potential of 30-75% was observed after 4 h of incubation with 4-HPR. A 6-12-fold difference was observed between the IC50 values for cell proliferation and viability between the most sensitive (IMR32) and most resistant (NASS) cell line towards 4-HPR. Flow cytometric analysis showed an increased amount of apoptotic bodies and no cell-cycle arrest. The antioxidant Trolox completely inhibited the accumulation of 4HPR-induced ROS and prevented the 4HPR-associated cytotoxicity. In all neuroblastoma spheroids, 4-HPR induced a complete cytostasis at clinical relevant concentrations (3-10 microM). Immunohistochemical analysis of 4-HPR-treated spheroids showed a decreased staining for proliferation marker Ki-67 and an increased staining for cleaved-PARP, a marker of apoptosis. Our results suggest that 4-HPR might be a promising agent for the treatment of micrometastases and high-risk neuroblastoma.

Topics: Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Cell Aggregation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromans; Dose-Response Relationship, Drug; Fenretinide; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Immunohistochemistry; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Neuroblastoma; Reactive Oxygen Species; Spheroids, Cellular; Time Factors

Fenretinide-induced apoptosis of neuroectodermal tumour cells is mediated through generation of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, mitochondrial cytochrome c release and caspase activation. The present study describes the requirement of the BH3-domain only protein Noxa for this process and its regulation by p53. Noxa expression was induced by fenretinide in neuroblastoma and melanoma cells, including those with mutated p53, and this induction was abolished by antioxidants. Knockdown of p53 by RNA interference (RNAi) demonstrated upregulation of Noxa protein levels in response to fenretinide was p53-independent, although evidence suggested that Noxa may be transcriptionally regulated by p53. The ER stress-inducing agent thapsigargin also induced p53-independent Noxa expression. Conversely, Noxa transcription in response to the chemotherapeutic agents cisplatin or temozolomide was inhibited by p53 knockdown. Apoptosis in response to cisplatin or temozolomide was also inhibited by abrogation of p53 expression yet apoptosis in response to fenretinide or thapsigargin was unaffected. RNAi-mediated down-regulation of Noxa inhibited apoptosis in response to fenretinide or thapsigargin, whereas apoptosis induced by cisplatin or temozolomide was unaffected. These data demonstrate the importance of Noxa induction in determining the apoptotic response to fenretinide and emphasise the role of Noxa in p53-independent apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Fenretinide; Gene Expression Regulation; Humans; Melanoma; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Thapsigargin; Tumor Suppressor Protein p53

The role of dihydroceramide desaturase as a key enzyme in the de novo pathway of ceramide generation was investigated in human neuroblastoma cells (SMS-KCNR). A novel assay using water-soluble analogs of dihydroceramide, dihydroceramidoids (D-erythro-dhCCPS analogs), was used to measure desaturase activity in situ. Conversion of D-erythro-2-N-[12'-(1''-pyridinium)-dodecanoyl]-4,5-dihydrosphingosine bromide (C(12)-dhCCPS) to its 4,5-desaturated counterpart, D-erythro-2-N-[12'-(1''-pyridinium)dodecanoyl]sphingosine bromide (C(12)-CCPS), was determined by liquid chromatography/mass spectrometry analysis. The validity of the assay was confirmed using C(8)-cyclopropenylceramide, a competitive inhibitor of dihydroceramide desaturase. A human homolog (DEGS-1) of the Drosophila melanogaster des-1 gene was recently identified and reported to have desaturase activity. Transfection of SMS-KCNR cells with small interfering RNA to DEGS-1 significantly blocked the conversion of C(12)-dhCCPS to C(12)-CCPS. The associated accumulation of endogenous dihydroceramides confirmed DEGS-1 as the main active dihydroceramide desaturase in these cells. The partial loss of DEGS-1 inhibited cell growth, with cell cycle arrest at G(0)/G(1). This was accompanied by a significant decrease in the amount of phosphorylated retinoblastoma protein. This hypophosphorylation was inhibited by tautomycin and not by okadaic acid, suggesting the involvement of protein phosphatase 1. Additionally, we found that treatment of SMS-KCNR cells with fenretinide inhibited desaturase activity in a dose-dependent manner. An increase in dihydroceramides (but not ceramides) paralleled this process as measured by liquid chromatography/mass spectrometry. There were no effects on the mRNA or protein levels of DEGS-1, suggesting that fenretinide acts at the post-translational level as an inhibitor of this enzyme. Tautomycin was also able to block the hypophosphorylation of the retinoblastoma protein observed upon fenretinide treatment. These findings suggest a novel biological function for dihydroceramides.

Topics: Base Sequence; Blotting, Western; Cell Cycle; Cell Division; Cell Line, Tumor; Ceramides; Chromatography, Liquid; DNA Primers; Fenretinide; Humans; Mass Spectrometry; Neuroblastoma; Oxidoreductases; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering

Endoplasmic reticulum (ER) malfunction, leading to ER stress, can be a consequence of genome instability and hypoxic tissue environments. Cancer cells survive by acquiring or enhancing survival mechanisms to counter the effects of ER stress and these homeostatic responses may be new therapeutic targets. Understanding the links between ER stress and apoptosis may be approached using drugs specifically to target ER stress responses in cancer cells. The retinoid analogue fenretinide [N-(4-hydroxyphenyl) retinamide] is a new cancer preventive and chemotherapeutic drug, that induces apoptosis of some cancer cell types via oxidative stress, accompanied by induction of an ER stress-related transcription factor, GADD153. The aim of this study was to test the hypothesis that fenretinide induces ER stress in neuroectodermal tumour cells, and to elucidate the role of ER stress responses in fenretinide-induced apoptosis. The ER stress genes ERdj5, ERp57, GRP78, calreticulin and calnexin were induced in neuroectodermal tumour cells by fenretinide. In contrast to the apoptosis-inducing chemotherapeutic drugs vincristine and temozolomide, fenretinide induced the phosphorylation of eIF2alpha, expression of ATF4 and splicing of XBP-1 mRNA, events that define ER stress. In these respects, fenretinide displayed properties similar to the ER stress inducer thapsigargin. ER stress responses were inhibited by antioxidant treatment. Knockdown of ERp57 or ERdj5 by RNA interference in these cells increased the apoptotic response to fenretinide. These data suggest that downregulating homeostatic ER stress responses may enhance apoptosis induced by oxidative stress-inducing drugs acting through the ER stress pathway. Therefore, ER-resident proteins such as ERdj5 and ERp57 may represent novel chemotherapeutic targets.

Topics: Activating Transcription Factor 4; Alternative Splicing; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; DNA-Binding Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Eukaryotic Initiation Factor-2; Fenretinide; HSP40 Heat-Shock Proteins; Humans; Molecular Chaperones; Neuroblastoma; Neuroectodermal Tumors; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Oxidative Stress; Phosphorylation; Protein Disulfide-Isomerases; Reactive Oxygen Species; Regulatory Factor X Transcription Factors; RNA, Messenger; RNA, Small Interfering; Transcription Factors; Tumor Cells, Cultured; X-Box Binding Protein 1

Cytotoxicity assays in 96-well tissue culture plates allow rapid sample handling for multicondition experiments but have a limited dynamic range. Using DIMSCAN, a fluorescence digital image system for quantifying relative cell numbers in tissue culture plates, we have developed a 96-well cytotoxicity assay with a >4-log dynamic range.. To overcome background fluorescence that limits detection of viable cells with fluorescein diacetate, we used 2'4'5'6'-tetrabromofluorescein (eosin Y) to quench background fluorescence in the medium and in nonviable cells to enhance the reduction of background fluorescence achieved with digital image thresholding. The sensitivity and linearity of the new assay were tested with serial dilutions of neuroblastoma and leukemia cell lines. DIMSCAN was compared with other in vitro cytotoxicity assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and trypan blue dye exclusion.. Without background fluorescence reduction, scans produced a nearly flat curve across various cell concentrations from 100 to 10(6) cells per well. Either digital image thresholding or eosin Y dramatically reduced background fluorescence, and combining them achieved a linear correlation (r > 0.9) of relative fluorescence to viable cell number over >4 logs of dynamic range, even in the presence of 4 x 10(4) nonviable cells per well. Cytotoxicity of deferoxamine for neuroblastoma cell lines measured by the DIMSCAN assay achieved dose-response curves similar to data obtained by manual trypan blue counts or colony formation in soft agar but with a wider dynamic range. Long-term cultures documented the clonogenic ability of viable cells detected by DIMSCAN over the entire dynamic range. The cytotoxicity of two drug combinations (buthionine sulfoximine + melphalan or fenretinide + safingol) was tested using both DIMSCAN and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the wider dynamic range of DIMSCAN facilitated detection of synergistic interactions.. DIMSCAN offers the ability to rapidly and efficiently conduct cytotoxicity assays in 96-well plates with a dynamic range of >4 logs. This assay enables rapid testing of anticancer drug combinations in microplates.

Topics: Antineoplastic Combined Chemotherapy Protocols; Buthionine Sulfoximine; Cell Proliferation; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Inhibitors; Eosine Yellowish-(YS); Fenretinide; Fluorescent Dyes; Humans; Leukemia; Melphalan; Microscopy, Fluorescence; Neuroblastoma; Protein Kinase C; Sphingosine; Tumor Cells, Cultured; Tumor Stem Cell Assay

Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is a cytotoxic retinoid that suffers from a wide interpatient variation in bioavailability when delivered orally in a corn oil capsule. The poor bioavailability of the capsule formulation may have limited responses in clinical trials, and the large capsules are not suitable for young children. To support the hypothesis that a novel organized lipid matrix, LYM-X-SORB, can increase the oral bioavailability of fenretinide, fenretinide in LYM-X-SORB matrix and in a powderized LYM-X-SORB formulation was delivered to mice.. Fenretinide was delivered orally to mice as the contents of the corn oil capsule, in LYM-X-SORB matrix (4-HPR/LYM-X-SORB matrix) or in a LYM-X-SORB matrix powderized with sugar and flour (4-HPR/LYM-X-SORB oral powder). Levels of 4-HPR, and its principal metabolite, N-(4-methoxyphenyl)retinamide, were assayed in plasma and tissues.. In a dose-responsive manner, from 120 to 360 mg/kg/d, delivery to mice of 4-HPR in LYM-X-SORB matrix, or as 4-HPR/LYM-X-SORB oral powder, increased 4-HPR plasma levels up to 4-fold (P<0.01) and increased tissue levels up to 7-fold (P<0.01) compared with similar doses of 4-HPR delivered using capsule contents. Metabolite [N-(4-methoxyphenyl)retinamide] levels mirrored 4-HPR levels. Two human neuroblastoma murine xenograft models showed increased survival (P<0.03), when treated with 4-HPR/LYM-X-SORB oral powder, confirming the bioactivity of the formulation.. 4-HPR/LYM-X-SORB oral powder is a novel, oral drug delivery formulation, suitable for pediatric use, which warrants further development for the delivery of fenretinide in the treatment of cancer. A phase I clinical trial in pediatric neuroblastoma is in progress.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Biological Availability; Cell Line, Tumor; Drug Delivery Systems; Fatty Acids; Fenretinide; Humans; Lysophosphatidylcholines; Mice; Monoglycerides; Neuroblastoma; Peripheral Nervous System Neoplasms; Powders; Tissue Distribution

The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Benzyl Compounds; Cell Line, Tumor; Child; DNA Damage; DNA, Single-Stranded; Enzyme Inhibitors; Fenretinide; Flow Cytometry; Gamma Rays; Humans; Hydrocarbons, Fluorinated; Hydrogen Peroxide; In Situ Nick-End Labeling; Melphalan; Neuroblastoma; Oxidants; Reactive Oxygen Species; Topotecan

N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) has been shown to be active toward many tumors without appreciable side effects. However its in vitro activity does not match a correspondent efficacy in vivo. The main reason is that the drug's hydrophobicity hinders its bioavailability in the body fluids. Even if the drug is previously dissolved in organic solvents, such as ethanol or DMSO, the subsequent dilution in body fluids trigger its precipitation in fine aggregates characterized by very low dissolution efficiency, never reaching amounts suitable for therapeutic response. To date no intravenous formulation of 4-HPR exists on the market. The 4-HPR linkage to a hydrophilic polymer by a covalent bond easily hydrolyzable in aqueous environment is expected to increase the drug's aqueous solubility, providing the free drug after hydrolysis of the covalent bond. This may be a useful tool for the preparation of aqueous intravenous formulations of 4-HPR. For this purpose, we linked 4-HPR to polyvinylalcohol (PVA) by a carbonate bond at different drug/hydroxy vinyl monomer molar ratios. We demonstrated that conjugation increased 4-HPR aqueous solubility and strongly inhibited neuroblastoma cell proliferation. In addition, in an in vivo neuroblastoma metastatic model, we obtained a significant antitumor effect as a consequence of the improved drug bioavailability.

Topics: Animals; Antineoplastic Agents; Biological Availability; Cell Line, Tumor; Cell Proliferation; Dimethyl Sulfoxide; Drug Delivery Systems; Fenretinide; Humans; Infusions, Intravenous; Mice; Mice, Nude; Models, Chemical; Neoplasm Metastasis; Neuroblastoma; Polyvinyl Alcohol; Solubility

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Shape; Dose-Response Relationship, Drug; Fenretinide; Humans; Neuroblastoma; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Transfection; Tretinoin

4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a recently identified metabolite of fenretinide (4-HPR). We explored the effectiveness of 4-oxo-4-HPR in inducing cell growth inhibition in ovarian, breast, and neuroblastoma tumor cell lines; moreover, we investigated the molecular events mediating this effect in two ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/HPR) to 4-HPR. 4-oxo-4-HPR was two to four times more effective than 4-HPR in most cell lines, was effective in both 4-HPR-sensitive and 4-HPR-resistant cells, and, in combination with 4-HPR, caused a synergistic effect. The tumor growth-inhibitory effects of 4-oxo-4-HPR seem to be independent of nuclear retinoid receptors (RAR), as indicated by the failure of RAR antagonists to inhibit its effects and by its poor ability to bind and transactivate RARs. Unlike 4-HPR, which only slightly affected the G(1) phase of the cell cycle, 4-oxo-4-HPR caused a marked accumulation of cells in G(2)-M. This effect was associated with a reduction in the expression of regulatory proteins of G(2)-M (cyclin-dependent kinase 1 and cdc25c) and S (cyclin A) phases, and with an increase in the expression of apoptosis-related proteins, such as p53 and p21. Apoptosis was induced by 4-oxo-4-HPR in both 4-HPR-sensitive and 4-HPR-resistant cells and involved activation of caspase-3 and caspase-9 but not caspase-8. We also showed that 4-oxo-4-HPR, similarly to 4-HPR, increased reactive oxygen species generation and ceramide levels by de novo synthesis. In conclusion, 4-oxo-4-HPR is an effective 4-HPR metabolite that might act as therapeutic agent per se and, when combined with 4-HPR, might improve 4-HPR activity or overcome 4-HPR resistance.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Caspase 8; Caspase 9; Caspases; Cell Cycle Proteins; Cell Division; Cell Growth Processes; Cell Line, Tumor; Ceramides; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Female; Fenretinide; G2 Phase; Humans; Neuroblastoma; Ovarian Neoplasms; Reactive Oxygen Species

Fenretinide induces apoptosis in SH-SY5Y neuroblastoma cells via a signaling pathway involving the production of reactive oxygen species (ROS), 12-lipoxygenase activity and the induction of the GADD153 transcription factor. NF-kappa B is a key element of many cell signaling pathways and adopts a pro- or anti-apoptotic role in different cell types. Studies have suggested that NF-kappa B may play a pro-apoptotic role in SH-SY5Y cells, and in other cell types NF-kappa B activation may be linked to lipoxygenase activity. The aim of this study was to test the hypothesis that NF-kappa B activity mediates fenretinide-induced apoptosis in SH-SY5Y neuroblastoma cells. Using a dominant-negative construct for Ikappa Balpha stably transfected into SH-SY5Y cells, we show that apoptosis, but not the induction of ROS, in response to fenretinide was blocked by abrogation of NF-kappa B activity. In parental SH-SY5Y cells, fenretinide induced NF-kappa B activity and Ikappa Balpha phosphorylation. These results suggest that NF-kappa B activity links fenretinide-induced ROS to the induction of apoptosis in SH-SH5Y cells, and may be a target for the future development of drugs for neuroblastoma therapy.

Topics: Apoptosis; Fenretinide; Flow Cytometry; Humans; I-kappa B Proteins; Neuroblastoma; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Reactive Oxygen Species; Transfection; Tumor Cells, Cultured

Fenretinide (4-HPR) is a synthetic retinoid whose apoptosis-inducing effects have been demonstrated in many tumor types. The precise mechanism of its apoptotic action is not fully understood. To further study the mechanism by which 4-HPR exerts its biological effects in neuroblastoma (NB) and to identify the genes that contribute to the induction of apoptosis, we determined the sensitivity of eight NB cell lines to 4-HPR. Additionally, cDNA microarray analysis was performed on a 4-HPR-sensitive cell line to investigate the temporal changes in gene expression, primarily focusing on the induction of proapoptotic genes. BBC3, a transcriptionally regulated proapoptotic member of the BCL2 family, was the most highly induced proapoptotic gene. Western analysis confirmed the induction of BBC3 protein by 4-HPR. Furthermore, the induction of BBC3 was associated with the sensitivity to this agent in the cell lines tested. Finally we demonstrated that BBC3 alone is sufficient to induce cell death in the 4-HPR-sensitive and resistant NB cell lines, and that siRNA against BBC3 significantly decreases apoptosis induced by 4-HPR. Our results indicate that BBC3 mediates cell death in NB cells in response to 4-HPR.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Blotting, Western; Cell Division; Cell Line, Tumor; Cell Survival; Colorimetry; DNA, Complementary; DNA, Neoplasm; Fenretinide; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Microarray Analysis; Neoplasm Proteins; Neuroblastoma; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Up-Regulation

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arachidonate 12-Lipoxygenase; Cell Line, Tumor; Ceramides; Enzyme Induction; Enzyme Inhibitors; Fenretinide; Gangliosides; Glucosyltransferases; Humans; Neuroblastoma; Oxidoreductases; Reactive Oxygen Species; RNA, Small Interfering; Sphingomyelin Phosphodiesterase

The lipid second messenger ceramide, which is generated by acidic and neutral sphingomyelinases or ceramide synthases, is a common intermediate of many apoptotic pathways. Metabolism of ceramide involves several enzymes, including glucosylceramide synthase and GD3 synthase, and results in the formation of gangliosides (GM3, GD3, and GT3), which in turn promote the generation of reactive oxygen species (ROS) and apoptosis. Fenretinide, a retinoic acid derivative, is thought to induce apoptosis via increases in ceramide levels, but the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear.. SH-SY5Y and HTLA230 neuroblastoma cells were treated with fenretinide in the presence or absence of inhibitors of enzymes important in ceramide metabolism (fumonisin B1, inhibitor of ceramide synthase; desipramine, inhibitor of acidic and neutral sphingomyelinases; and PDMP, inhibitor of glucosylceramide). Small interfering RNAs were used to specifically block acidic sphingomyelinase or GD3 synthase activities. Apoptosis, ROS, and GD3 expression were measured by flow cytometry.. In neuroblastoma cells, ROS generation and apoptosis were associated with fenretinide-induced increased levels of ceramide, glucosylceramide synthase activity, GD3 synthase activity, and GD3. Fenretinide also induced increased levels of GD2, a ganglioside derived from GD3. Inhibition of acidic sphingomyelinase but not of neutral sphingomyelinase or ceramide synthase, blocked fenretinide-induced increases in ceramide, ROS, and apoptosis. Exogenous GD3 induced ROS and apoptosis in SH-SY5Y cells but not in SH-SY5Y cells treated with baicalein, a specific 12-lipoxygenase inhibitor. Exogenous GD2 did not induce apoptosis.. A novel pathway of fenretinide-induced apoptosis is mediated by acidic sphingomyelinase, glucosylceramide synthase, and GD3 synthase, which may represent targets for future drug development. GD3 may be a key signaling intermediate leading to apoptosis via the activation of 12-lipoxygenase.

Topics: Antineoplastic Agents; Apoptosis; Arachidonate 12-Lipoxygenase; Cell Line, Tumor; Ceramides; Desipramine; Enzyme Activation; Enzyme Inhibitors; Fenretinide; Flow Cytometry; Fumonisins; Gangliosides; Gene Expression Regulation, Neoplastic; Glucosyltransferases; Humans; Morpholines; Neuroblastoma; Oxidoreductases; Reactive Oxygen Species; RNA, Small Interfering; Sphingomyelin Phosphodiesterase

Fenretinide, which mediates apoptosis in neuroblastoma cells, is being considered as a novel therapeutic for neuroblastoma. The cytotoxic mechanisms of fenretinide, however, have not been fully elucidated. Sustained-activation of JNK and p38 MAPK signaling has been shown recently to have a pivotal role in stress-induced apoptosis. Whether fenretinide activates the signaling in neuroblastoma cells is not known. In the present study, fenretinide induced sustained-activation of both JNK and p38 MAPK in neuroblastoma cells. Pretreatment with the antioxidant L-ascorbic acid almost completely inhibited the accumulation of fenretinide-induced intracellular reactive oxygen species (ROS), activation of JNK and p38 MAPK and apoptosis. Intracellular ROS production and activation of stress signaling was not altered by fenretinide in resistant neuroblastoma cells. Our study demonstrates that in neuroblastoma cells, fenretinide induces sustained-activation of JNK and p38 MAPK in an ROS-dependent manner and indicates that JNK and p38 MAPK signaling might mediate fenretinide-induced apoptosis. Our results also indicate that suppression of the fenretinide-induced ROS productive system and the downstream JNK and p38 MAPK signaling pathways causes neuroblastoma cells to become resistant to fenretinide.

Topics: Antineoplastic Agents; Apoptosis; Fenretinide; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Neuroblastoma; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid Fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). We investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-SIL showed specific, competitive binding to and uptake by, various NB cell lines. In in vitro cytotoxicity studies, NB cells, incubated with 30 microM HPR entrapped in anti-GD2-immunoliposomes, showed a significant reduction in cellular growth compared to free HPR, HPR entrapped in Ab-free liposomes or anti-GD2 empty liposomes. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. Similar, but significantly less potent, antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 MAb alone (p = 0.0297 and p = 0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that although anti-GD2 MAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only after treatment with anti-GD2-SIL-HPR (p < 0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.

Topics: Animals; Apoptosis; Cell Division; Disease Models, Animal; Fenretinide; Humans; Liposomes; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neuroblastoma; Survival Analysis; Time Factors; Tumor Cells, Cultured

The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving signaling pathways mediated by free radicals or reactive oxygen species (ROS). The aim of this study was to identify mechanisms generating ROS and apoptosis of neuroblastoma cells in response to fenretinide. Fenretinide-induced ROS or apoptosis of SH-SY5Y or HTLA 230 neuroblastoma cells were not blocked by Nitro l-argenine methyl ester (l-NAME), an inhibitor of nitric oxide synthase. Flavoprotein-dependent superoxide-producing enzymes such as NADPH oxidase were also not involved in fenretinide-induced apoptosis or ROS generation. Similarly, ketoconazole, a cytochrome P450 inhibitor, and inhibitors of cyclooxygenase (COX) were also ineffective. In contrast, inhibition of phospholipase A(2) or lipoxygenases (LOX) blocked the induction of ROS and apoptosis in response to fenretinide. Using specific inhibitors of LOX, blocking 12-LOX but not 5- or 15-LOX inhibited both fenretinide-induced ROS and apoptosis. The effects of eicosatriynoic acid, a specific 12-LOX inhibitor, were reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid and 12 (S)-hydroxyeicosatetraenoic acid. The targeting of 12-LOX in neuroblastoma cells may thus be a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cytochrome P-450 Enzyme System; Eicosanoic Acids; Enzyme Activation; Enzyme Inhibitors; Fenretinide; Free Radicals; Humans; Leukotrienes; Lipoxygenase; NADPH Oxidases; Neuroblastoma; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phospholipases A; Prostaglandin-Endoperoxide Synthases; Reactive Oxygen Species; Tumor Cells, Cultured

Unlike 13-cis-retinoic acid, the synthetic retinoid fenretinide [N-(4-hydroxyphenyl)retinamide] induces apoptosis of neuroblastoma cells by mechanisms involving retinoic acid receptors and oxidative stress. After screening a cDNA array for apoptosis-related genes, the Bcl2-related protein Bak was identified as a fenretinide-inducible gene in SH-SY5Y neuroblastoma cells, and this was confirmed by Western blotting and flow cytometry. Although fenretinide acts synergistically in vitro with chemotherapeutic drugs, these drugs did not induce Bak expression. Retinoic acid receptor antagonists did not block the induction of Bak by fenretinide. Conversely, Bak induction was blocked by the antioxidant vitamin C. Overexpression of Bak increased apoptosis in both the presence and absence of fenretinide, whereas expression of antisense Bak inhibited fenretinide-induced apoptosis. Bak expression was also induced in cells overexpressing the stress-induced transcription factor GADD153, but Bak expression was inhibited in cells expressing an antisense GADD153 construct. These results suggest that Bak is a downstream mediator of an oxidative stress pathway leading to apoptosis of SH-SY5Y neuroblastoma cells in response to fenretinide.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Blotting, Western; Cell Line, Tumor; Fenretinide; Flow Cytometry; Humans; Membrane Proteins; Neuroblastoma

Fenretinide induces apoptosis of neuroblastoma cells in vitro and interacts synergistically with the chemotherapeutic drugs cisplatin and etoposide. The stress-inducible transcription factor known as growth and DNA damage (GADD)-inducible transcription factor 153 is induced in response to fenretinide and in other cell types modulates apoptosis via pro- and antiapoptotic members of the BCL2 family. Because BCL2-family proteins are important in apoptosis induced by chemotherapeutic drugs, GADD153 may be a key mediator of synergy between fenretinide and chemotherapeutic drugs. To investigate this, GADD153 cDNA in sense and antisense orientations was stably transfected into SH-SY5Y neuroblastoma cells using a tetracycline-inducible vector. Increased expression of GADD153 raised the background level of apoptosis and increased apoptosis induced by fenretinide or the chemotherapeutic drugs cisplatin and etoposide. However, there was no increase in synergy between fenretinide and chemotherapeutic drugs. Conversely, expression of antisense-GADD153 virtually abolished the induction of apoptosis in response to fenretinide but overall had no significant effect on apoptosis induced by chemotherapeutic drugs. The effect of antisense-GADD153 on synergy between chemotherapeutic drugs and fenretinide varied with the drug used: there was no effect on synergy between fenretinide and cisplatin, but the combination of fenretinide with etoposide became antagonistic. These results suggest that mechanisms mediating synergy between fenretinide and chemotherapeutic drugs lie upstream of GADD153.

Topics: Antineoplastic Agents; Apoptosis; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; DNA Damage; Dose-Response Relationship, Drug; Drug Synergism; Fenretinide; Humans; Neuroblastoma; Transcription Factor CHOP; Transcription Factors

The synthetic retinoid fenretinide induces apoptosis of neuroblastoma cells and in vitro acts synergistically with chemotherapeutic drugs used to treat neuroblastoma. The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving cellular signaling pathways as yet incompletely defined but, in part, involving the generation of reactive oxygen species (ROS). In an attempt to characterize the mechanism of action of fenretinide, cDNA array filters were screened to identify apoptotic genes regulated in response to treatment of SH-SY5Y cells with fenretinide. Expression of the stress-induced transcription factor, GADD153, was up-regulated at both the protein and mRNA levels in response to fenretinide. Overexpression of GADD153 increased apoptosis in the presence and absence of fenretinide, whereas reduced expression of GADD153 by expression of antisense DNA abrogated the response to fenretinide. Although fenretinide is a partial retinoic acid receptor (RAR)-beta/gamma agonist, RARbeta/gamma antagonists did not block the induction of GADD153 by fenretinide; conversely, the induction of GADD153 was blocked by antioxidants. Enzyme inhibitors were used to identify pathways mediating the ROS-dependent effects of fenretinide: inhibitors of phospholipase A(2) and lypoxygenases (LOX), and specific inhibitors of 12-LOX, but not 5-LOX or 15-LOX, inhibited the induction of ROS, apoptosis, and GADD153 in response to fenretinide. The inhibition of ROS and apoptosis was reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid (12-HpETE) and 12 (S)-hydroxyeicosatetraenoic acid (12-HETE). Fenretinide did not increase free arachidonic acid levels, but increased LOX activity without a detectable increase in 12-LOX protein. These results suggest that fenretinide induces apoptosis via RAR-dependent and -independent pathways in which the RAR-independent pathway is characterized by a fenretinide-dependent increase in 12-LOX activity, leading to the induction of GADD153. The targeting of 12-LOX and/or GADD153 in neuroblastoma cells may thus present a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity.

Topics: Antineoplastic Agents; Apoptosis; Arachidonate 12-Lipoxygenase; CCAAT-Enhancer-Binding Proteins; Drug Synergism; Fenretinide; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Reactive Oxygen Species; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Transcription Factor CHOP; Transcription Factors; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR; fenretinide) is cytotoxic to a variety of cancer cell lines, and we previously showed an association between ceramide generation and 4-HPR cytotoxicity for neuroblastoma cell lines (B. J. Maurer et al., J. Natl. Cancer Inst. (Bethesda), 91: 1138-1146, 1999). Here we determine whether the increased ceramide mediated by 4-HPR in the CHLA-90 human neuroblastoma cell line results from de novo ceramide synthesis. Treatment of CHLA-90 with 4-HPR for 2 h, in the presence of [(3)H]palmitic acid, caused sequential formation of [(3)H]sphinganine (220% over control) and [(3)H]ceramide (160% over control), with sphinganine returning to baseline at 4 h, and ceramide continuing to increase (215% over control). 4-HPR treatment did not accelerate cellular decay of sphingomyelin. Preincubation of cells with either L-cycloserine, an inhibitor of serine palmitoyltransferase (SPT), or fumonisin B(1), an inhibitor of ceramide synthase, retarded ceramide formation in response to 4-HPR treatment, although sphinganine was still generated when 4-HPR and FB(1) were present. Data from in vitro enzyme assays using microsomes showed that preexposure of intact cells to 4-HPR resulted in a time (175% over control; 6 h)- and dose-dependent increase (173% over control; 10 microM) in SPT activity as well as a time (265% over control)- and dose-dependent increase (215% above control; 10 microM) in ceramide synthase activity. Our results show that 4-HPR-mediated ceramide generation is derived from the de novo synthetic pathway by coordinate activation of SPT and ceramide synthase. Knowledge of these biochemical events is of utility when downstream modulators of ceramide metabolism are used to heighten the cytotoxic response to chemotherapy.

Topics: Acyltransferases; Antineoplastic Agents; Ceramides; Enzyme Activation; Enzyme Induction; Fenretinide; Humans; Neuroblastoma; Oxidoreductases; Serine C-Palmitoyltransferase; Sphingosine; Tumor Cells, Cultured

Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor- (VEGF-) and fibroblast growth factor-2- (FGF-2)-induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor-induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy-induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF- and FGF-2-specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT-PCR analysis of VEGF and FGF-2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48-72 hr of treatment with HPR, followed by a progressive accumulation of cells in G(1) at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF-2 receptor-2 was reduced after implantation of HPR-loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors.

Topics: Adenocarcinoma; Adrenal Glands; Animals; Anticarcinogenic Agents; Antineoplastic Agents; Cell Cycle; Cell Division; Cell Line; Cell Movement; Chick Embryo; Chorion; Endometrial Neoplasms; Endothelial Growth Factors; Endothelium; Enzyme-Linked Immunosorbent Assay; Female; Fenretinide; Fibroblast Growth Factor 2; Flow Cytometry; Humans; Immunohistochemistry; Kinetics; Lymphokines; Neovascularization, Pathologic; Neuroblastoma; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Cytochrome c Group; Fenretinide; Free Radicals; Humans; Mitochondria; Neuroblastoma; Oligopeptides; Poly(ADP-ribose) Polymerases; Receptors, Retinoic Acid; Tumor Cells, Cultured

Retinoic acid therapy improves the survival of children with neuroblastoma and 13-cis retinoic acid now forms an important component of treatment for residual disease of stage IV neuroblastoma after chemotherapy. However, although 13-cis retinoic acid induces differentiation, other retinoids are effective at inducing apoptosis of neuroblastoma in vitro, including the novel compounds fenretinide and CD437 and these may be alternative retinoids for neuroblastoma therapy. The aim of our study was to evaluate the ability of fenretinide, CD437 (6-¿3-(1-adamantyl)-4-hydroxyphenyl¿ -2-naphthalene carboxylic acid) and different retinoic acid isomers to induce apoptosis of neuroblastoma in conjunction with the chemotherapeutic drugs, cisplatin, etoposide and carboplatin. Neuroblastoma cell lines were treated with retinoids prior to treatment with chemotherapeutic agents and flow cytometry used to measure apoptosis and free radical generation. Pre-treatment of neuroblastoma cell lines with fenretinide or CD437 prior to treatment with cisplatin, etoposide or carboplatin synergistically increased apoptosis, an effect not seen with 13-cis, all-trans or 9-cis retinoic acid. Contrary to retinoic acid isomers or chemotherapeutic drugs, apoptosis of neuroblastoma cells induced by fenretinide or CD437 was accompanied by the generation of intracellular free radicals. Quenching of fenretinide- or CD437-induced free radicals with antioxidants abolished the synergistic response seen with the subsequent addition of chemotherapeutic agents. Therefore, the generation of free radicals by fenretinide or CD437 may be the key property of these retinoids leading to synergistic responses with chemotherapeutic drugs. Clearly, these synthetic retinoids provide new opportunities for novel neuroblastoma therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carboplatin; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Drug Synergism; Etoposide; Fenretinide; Flow Cytometry; Free Radicals; Humans; Neuroblastoma; Retinoids; Time Factors; Tretinoin; Tumor Cells, Cultured

High-dose, pulse-13-cis-retinoic acid (13-cis-RA) given after intensive cytotoxic therapy improves event-free survival for high-risk neuroblastoma (NB), but more than 50% of patients have tumor recurrence.. We conducted multistep selection for resistance to all-trans-retinoic acid (ATRA) in NB cell lines with (SMS-KCNR and LA-N-5) or without (SMS-LHN) MYCN genomic amplification.. After 12 exposures to 10 microM ATRA, the two MYCN-amplified cell lines (KCNR 12X RR and LA-N-5 12X RR) showed partial resistance to the cytostatic/differentiation effects of ATRA; complete resistance was seen in LHN 12X RR. ATRA-selected cells showed general RA resistance (cross-resistance to 13-cis-RA). Transient (KCNR 12 X RR, LA-N-5 12X RR) or sustained (LHN 12X RR) novel overexpression of c-myc was associated with RA resistance. RA-insensitive overexpression of MYCN by transduction in SMS-LHN also conferred RA resistance. Both parental and RA-resistant lines showed 2-4 logs of cell kill in response to N-(4-hydroxyphenyl)retinamide (4- HPR, fenretinide). Compared to parental lines, 4-HPR achieved 1-3 log greater cell kills in RA-resistant LHN 12X RR, LA-N-5 12X RR, KCNR 12X RR, and MYCN-transduced SMS-LHN or SK-N-RA. NB cell lines (n = 26) from 21 different patients showed that 16 of 26 (62%) were sensitive to 4-HPR (LC(90) < 10 microM), including lines established at relapse after myeloablative and/or 13-cis-RA therapy.. Thus, RA-resistant NB cell lines can be sensitive (and in some cases collaterally hypersensitive) to 4-HPR.

Topics: Antineoplastic Agents; Drug Resistance, Neoplasm; Fenretinide; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

The RARbeta/gamma-selective retinoids fenretinide and CD437 induce caspase-dependent apoptosis but generate free radicals independently of caspases. Apoptosis, but not free radical generation, induced by these retinoids is inhibited by RARbeta/gamma-specific antagonists. Both fenretinide and CD437 induce apoptosis synergistically with cisplatin, carboplatin, or etoposide. However, antioxidants inhibit this synergy to the level obtained with chemotherapeutic drugs alone, and this implies that free radical generation is important in the synergistic response. Since apoptosis induced by fenretinide or CD437 is mediated by apoptotic pathways involving RARs and/or mitochondria and differs from mechanisms of chemotherapy-induced apoptosis this may explain the strong synergistic response seen between these synthetic retinoids and chemotherapeutic drugs. These results suggest that fenretinide or CD437 may be useful adjuncts to neuroblastoma therapy.

Topics: Antineoplastic Agents; Apoptosis; Child; Drug Synergism; Fenretinide; Free Radicals; Humans; Neuroblastoma; Retinoids; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Ceramides; Fenretinide; Humans; Neuroblastoma; Reactive Oxygen Species; Tumor Cells, Cultured

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extracellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a "DNA ladder" on gel electrophoresis, by propidium iodide staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to beta 1 integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interactions by means of perturbing antibody against beta 1 subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings indicate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Cell Adhesion; DNA Fragmentation; DNA, Neoplasm; Down-Regulation; Fenretinide; Flow Cytometry; Humans; Integrin beta1; Integrins; Neuroblastoma; Oligonucleotides, Antisense; Polymerase Chain Reaction; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

Retinoids exert various important biological effects in the control of normal growth, differentiation, and fetal development. While retinoic acid (RA) has entered clinical trials as a differentiation-promoting agent, it is only recently that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has been shown to be of potential clinical interest in cancer chemoprevention and treatment. Since thus far no data exist on the effects of HPR on neural crest cell-derived tumors, we have examined its in vitro effects on neuroblastoma (NB) cell lines and found that at relevant pharmacological concentrations it induces a dose-dependent growth inhibition. The antiproliferative effects of HPR were, in six of six cell lines tested, drastically more potent that those induced by an equimolar dose of RA. Time course growth analysis showed that HPR at 3 x 10(-6) M induces a very rapid (24-72 h) fall in thymidine uptake (> 90%), whereas at 3 x 10(-7) M it exhibits cytostatic effects. In contrast to RA, HPR did not show morphological changes typical of NB cell maturation nor did it induce the expression of any cytoskeletal protein associated with neuronal differentiation. DNA flow cytofluorimetric analysis revealed that HPR did not induce an arrest in a specific phase of the cell cycle while triggering apoptosis. This phenomenon was evidenced both by the visualization of "DNA ladders" on gel electrophoresis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. With the latter method, apoptotic cells were detectable as early as 3-6 h after treatment of NB cells with 10(-5) M HPR, while more than 50% of cells were apoptotic by 24-72 h following exposure to 3 x 10(-6) M HPR. In contrast, RA induced a low rate of apoptosis in NB cells only after 3-5 days. Time lapse photomicroscopy showed that NB cells treated with HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses in the nuclear periphery, a typical feature of apoptotic cells. In conclusion, our study demonstrates that contrary to the differentiation-promoting activity of RA, HPR dramaticall

Topics: Apoptosis; Cell Cycle; Cell Differentiation; Cell Division; Cytoskeletal Proteins; DNA Damage; DNA, Neoplasm; Fenretinide; Fluorescent Antibody Technique; Humans; Neuroblastoma; Neurons; Tretinoin

N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid with anti-cancer properties and lower toxicity than all-trans retinoic acid (RA). Neuroblastoma cells treated with HPR and observed by fluorescence microscopy showed clear signs of apoptosis, such as chromatin condensation and margination, nuclear fragmentation and the presence of "apoptotic bodies". Moreover, measurements on a cell-by-cell basis by the flow-cytometric DNA-content in situ-terminal-deoxinucleotidyl-transferase(TDT) assay showed that apoptosis induced by HPR was dose- and time-dependent and that the fraction of apoptotic cells increased from approximately 15% at 1.25 microM at 2 days after treatment up to approximately 90% at 5 microM and 8 days of continuous treatment. Additionally, we found that cells were induced into apoptosis independently from the cell-cycle phase. In contrast, equimolar or higher doses of RA, from 5 microM to 80 microM, were able to inhibit growth by differentiation, but failed to induce apoptosis. We conclude that the functional effects of HPR and RA in LA-N-5 neuroblastoma cells are mediated by apoptosis and differentiation respectively, suggesting a potential clinical use of HPR in the management of neuroblastoma patients.

Topics: Apoptosis; Cell Count; Cell Cycle; DNA, Neoplasm; Dose-Response Relationship, Drug; Fenretinide; Flow Cytometry; Humans; Microscopy, Fluorescence; Neuroblastoma; Tumor Cells, Cultured

Topics: Apoptosis; DNA, Neoplasm; Fenretinide; Humans; Neuroblastoma; Tumor Cells, Cultured