Circulating hormones and breast cancer risk in premenopausal women: a randomized trial of low-dose tamoxifen and fenretinide.

Tamoxifen and fenretinide have been extensively studied and exhibit breast cancer-preventing activity. We aimed to assess their effect on sex hormones, sex hormone binding globulin (SHBG) and retinol, and their association with mammographic density (MD) and breast cancer events. In a double-blind, placebo-controlled trial, premenopausal women at risk for breast cancer were randomized to tamoxifen 5 mg/day, fenretinide, both agents, or placebo for 2 years. We measured MD and circulating concentrations of follicle-stimulating hormone, luteinizing hormone (LH), estradiol, progesterone, testosterone, androstenedione, dehydro-epiandrosteronesulfate, prolactin, SHBG, and retinol at baseline and on yearly intervals. The associations with breast cancer events were evaluated through competing risk and Cox regression survival models. Low-dose tamoxifen markedly and enduringly increased SHBG, whereas the increases in testosterone, estradiol, and prolactin and reduction in LH weakened after 1 year. Fenretinide increased testosterone and androstenedione and decreased retinol. MD correlated directly with SHBG and inversely with retinol. After a median follow-up of 12 years, the 10-year cumulative incidence of breast cancer events was 37 % in women with SHBG ≤ 59.3 nmol/L, 22 % in women with SHBG between 59.3 and 101 nmol/L, and 19 % in women with SHBG > 101 nmol/L (P = 0.018). The difference among SHBG tertiles remained statistically significant at multivariable analysis: HR = 2.26 (95 % CI 1.04, 4.89) for the lowest versus the highest tertile. We conclude that low-dose tamoxifen or fenretinide exhibits favorable hormonal profiles as single agents, further supporting their administration for prevention of breast cancer in premenopause. Notably, SHBG levels were inversely associated with breast neoplastic events.

AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway.

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker ß-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.

Enhanced anti-neuroblastoma activity of a fenretinide complexed form after intravenous administration.

OBJECTIVES: The major limitation to successful chemotherapy of neuroblastoma (NB) is the toxicity and the poor bioavailability of traditional drugs. METHODS: 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. KEY FINDINGS: 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. CONCLUSIONS: Our data suggest that DX-OL/4-HPR is an injectable formulation that is able to improve drug aqueous solubility and bioavailability.

4-oxo-N-(4-hydroxyphenyl)retinamide: two independent ways to kill cancer cells.

BACKGROUND: The retinoid 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a polar metabolite of fenretinide (4-HPR) very effective in killing cancer cells of different histotypes, able to inhibit 4-HPR-resistant cell growth and to act synergistically in combination with the parent drug. Unlike 4-HPR and other retinoids, 4-oxo-4-HPR inhibits tubulin polymerization, leading to multipolar spindle formation and mitotic arrest. Here we investigated whether 4-oxo-4-HPR, like 4-HPR, triggered cell death also via reactive oxygen species (ROS) generation and whether its antimicrotubule activity was related to a ROS-dependent mechanism in ovarian (A2780), breast (T47D), cervical (HeLa) and neuroblastoma (SK-N-BE) cancer cell lines. METHODOLOGY/PRINCIPAL FINDINGS: We provided evidence that 4-oxo-4-HPR, besides acting as an antimicrotubule agent, induced apoptosis through a signaling cascade starting from ROS generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and upregulation of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Through time-course analysis and inhibition of the ROS-related signaling pathway (upstream by vitamin C and downstream by PLAB silencing), we demonstrated that the antimitotic activity of 4-oxo-4-HPR was independent from the oxidative stress induced by the retinoid. In fact, ROS generation occurred earlier than mitotic arrest (within 30 minutes and 2 hours, respectively) and abrogation of the ROS-related signaling pathway did not prevent the 4-oxo-4-HPR-induced mitotic arrest. CONCLUSIONS/SIGNIFICANCE: These data indicate that 4-oxo-4-HPR anticancer activity is due to at least two independent mechanisms and provide an explanation of the ability of 4-oxo-4-HPR to be more potent than the parent drug and to be effective also in 4-HPR-resistant cell lines. In addition, the double mechanism of action could allow 4-oxo-4-HPR to efficiently target tumour and to eventually counteract the development of drug resistance.

Colorectal tumors are effectively eradicated by combined inhibition of {beta}-catenin, KRAS, and the oncogenic transcription factor ITF2.

Colorectal carcinomas (CRC) harbor well-defined genetic abnormalities, including aberrant activation of ß-catenin (ß-cat) and KRAS, but independent targeting of these molecules seems to have limited therapeutic effect. In this study, we report therapeutic effects of combined targeting of different oncogenes in CRC. Inducible short hairpin RNA (shRNA)-mediated silencing of ß-cat, ITF2, or KRAS decreased proliferation by 88%, 72%, and 45%, respectively, with no significant apoptosis in any case. In contrast, combined blockade of ß-cat and ITF2 inhibited proliferation by 99% with massive apoptosis. Similar effects occurred after combined shRNA against ß-cat and KRAS. In vivo, single oncogene blockade inhibited the growth of established tumors by up to 30%, whereas dual ß-cat and ITF2 targeting caused 93% inhibition. Similar tumor growth suppression was achieved by double ß-cat/KRAS shRNA in vivo. Our findings illustrate an effective therapeutic principle in CRC based on a combination targeting strategy that includes the ITF2 oncogene, which represents a novel therapeutic target.

Relationship among pharmacokinetics and pharmacodynamics of fenretinide and plasma retinol reduction in neuroblastoma patients.

PURPOSE: 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. PATIENTS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Human CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand target both tumor cells and tumor vasculature.

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

Antimitotic effect of the retinoid 4-oxo-fenretinide through inhibition of tubulin polymerization: a novel mechanism of retinoid growth-inhibitory activity.

The retinoid 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR), a metabolite of fenretinide (4-HPR) present in plasma of 4-HPR-treated patients, is very effective in inducing growth inhibition and apoptosis in several cancer cell lines. 4-Oxo-4-HPR and 4-HPR have different mechanisms of action because 4-oxo-4-HPR, unlike 4-HPR, causes marked cell accumulation in G2-M phase. Here, we investigated the molecular events involving 4-oxo-4-HPR-induced cell cycle perturbation in ovarian (A2780 and IGROV-1) and breast (T47D, estrogen receptor+ and BT-20, estrogen receptor-) cancer cells. 4-Oxo-4-HPR induced a delay of mitosis (with mitotic index increasing 5- to 6-fold in all cell lines) without progression beyond the anaphase, as shown by cyclin B1 expression. 4-Oxo-4-HPR induced multipolar spindle formation and phosphorylation of BUBR1, resulting in activation of the spindle checkpoint. Multipolar spindles were not due to impairment of pole-focusing process, loss of centrosome integrity, or modulation of the expression levels of molecules associated with spindle aberrations (Kif 1C, Kif 2A, Eg5, Tara, tankyrase-1, centractin, and TOGp). We show here that 4-oxo-4-HPR targets microtubules because, in treated cells, it interfered with the reassembly of cold-depolymerized spindle microtubules and decreased the polymerized tubulin fraction. In cell-free assays, 4-oxo-4-HPR inhibited tubulin polymerization (50% inhibition of microtubule assembly at 5.9 micromol/L), suggesting a direct molecular interaction with tubulin. In conclusion, by showing that 4-oxo-4-HPR causes mitotic arrest through antimicrotubule activities, we delineate a new molecular mechanism for a retinoid.

Randomized double-blind 2 x 2 trial of low-dose tamoxifen and fenretinide for breast cancer prevention in high-risk premenopausal women.

PURPOSE: Tamoxifen and fenretinide are active in reducing premenopausal breast cancer risk and work synergistically in preclinical models. The authors assessed their combination in a two-by-two biomarker trial. PATIENTS AND METHODS: A total of 235 premenopausal women with pT1mic/pT1a breast cancer (n = 21), or intraepithelial neoplasia (IEN, n = 160), or 5-year Gail risk > or = 1.3% (n = 54) were randomly allocated to either tamoxifen 5 mg/d, fenretinide 200 mg/d, their combination, or placebo. We report data for plasma insulin-like growth factor I (IGF-I), mammographic density, uterine effects, and breast neoplastic events after 5.5 years. RESULTS: During the 2-year intervention, tamoxifen significantly lowered IGF-I and mammographic density by 12% and 20%, respectively, fenretinide by 4% and 10% (not significantly), their combination by 20% and 22%, with no evidence for a synergistic interaction. Tamoxifen increased endometrial thickness principally in women becoming postmenopausal, whereas fenretinide decreased endometrial thickness significantly. The annual rate of breast neoplasms (n = 48) was 3.5% +/- 1.0%, 2.1% +/- 0.8%, 4.7% +/- 1.3%, and 5.2% +/- 1.3% in the tamoxifen, fenretinide, combination, and placebo arms, respectively, with hazard ratios (HRs) of 0.70 (95% CI, 0.32 to 1.52), 0.38 (95% CI, 0.15 to 0.90), and 0.96 (95% CI, 0.46 to 1.99) relative to placebo (tamoxifen x fenretinide adverse interaction P = .03). There was no clear association with tumor receptor type. Baseline IGF-I and mammographic density did not predict breast neoplastic events, nor did change in mammographic density. CONCLUSION: Despite favorable effects on plasma IGF-I levels and mammographic density, the combination of low-dose tamoxifen plus fenretinide did not reduce breast neoplastic events compared to placebo, whereas both single agents, particularly fenretinide, showed numerical reduction in annual odds of breast neoplasms. Further follow-up is indicated.

PLAB induction in fenretinide-induced apoptosis of ovarian cancer cells occurs via a ROS-dependent mechanism involving ER stress and JNK activation.

Fenretinide [N-(4-hydroxyphenyl)-retinamide (4HPR)] is a synthetic retinoid with antitumor activity that induces apoptosis in various types of cancer cell. We showed previously that 4HPR upregulates the proapoptotic gene placental bone morphogenetic protein (PLAB), which is a mediator of 4HPR-induced apoptosis in ovarian cancer cells. Here, we investigated the signaling cascade involving PLAB that mediates the apoptotic effect. In 4HPR-sensitive ovarian cancer cells, 4HPR-induced reactive oxygen species (ROS) are involved in PLAB upregulation and apoptosis, both events abrogated by the antioxidants vitamin C and butylated hydroxyanisole. We analyzed the expression and activation of endoplasmic reticulum (ER) stress-associated molecules and show that 4HPR-induced ER stress is a consequence of ROS generation. Salubrinal, an ER stress inhibitor, abrogated 4HPR-induced PLAB upregulation and protected the cells from apoptosis. Downstream of ROS generation and ER stress, 4HPR activated c-Jun N-terminal kinase (JNK), which was inhibited by vitamin C and salubrinal. The JNK inhibitor SP600125 reduced 4HPR-induced PLAB upregulation, by decreasing PLAB mRNA half-life, and protected the cells from apoptosis. These data indicate that 4HPR-induced PLAB upregulation occurs downstream of a signaling cascade involving ROS generation, ER stress induction and JNK activation and that these steps are mediators of 4HPR-induced apoptosis.

Plasma retinol and prognosis of postmenopausal breast cancer patients.

BACKGROUND: The role of retinol (vitamin A) in breast cancer prognosis has never been investigated in postmenopausal women. We prospectively assessed the long-term prognostic role of retinol plasma levels in a cohort of postmenopausal breast cancer patients. PATIENTS AND METHODS: We investigated 208 women self-reported as postmenopausal operated on for T(1-2)N(0)M(0) breast cancer who participated in a chemoprevention trial as controls and never received chemotherapy or hormone therapy. Plasma samples were collected 3 months (median) after surgery and assayed within 3 weeks for retinol. Minimum and median potential follow-up were 12 and 15 years, respectively. The main analyses were on all women and on a subgroup ages >or=55 years, assumed too old to be in perimenopause. The main end point was breast cancer death. Breast cancer survival was estimated by the Kaplan-Meier method. The hazard ratios of breast cancer death by retinol level were estimated by Cox models stratified for age, where relevant, and recruitment period, and adjusted for tumor size and histology. RESULTS: At 12 years, patients with low retinol (<2.08 micromol/L, median of distribution) had lower breast cancer survival than those with high retinol (log-rank P = 0.052); the difference was significant for women >or=55 years (log-rank P = 0.006). The adjusted hazard ratios for low versus high retinol were 2.11 (95% confidence interval, 1.08-4.14) for all women and 3.58 (95% confidence interval, 1.50-8.57) for those >or=55 years. CONCLUSIONS: Low plasma retinol strongly predicts poorer prognosis in postmenopausal breast cancer patients. Retinol levels should be determined as part of the prognostic workup.

Valproic acid enhances bosutinib cytotoxicity in colon cancer cells.

Unbalanced histone deacetylase (HDAC) hyperactivity is a common feature of tumor cells. Inhibition of HDAC activity is often associated with cancer cell growth impairment and death. Valproic acid (VPA) is a HDAC inhibitor used for the treatment of epilepsy. It has recently been recognized as a promising anticancer drug. We investigated the effects of VPA on growth and survival of colon cancer cells. VPA caused growth inhibition and programmed cell death that correlated with histone hyperacetylation. VPA modulated the expression of various factors involved in cell cycle control and apoptosis and induced caspase activation. Interestingly, VPA induced downregulation of c-Src and potentiated the cytotoxic effects of the c-Src inhibitor bosutinib, both in vitro and in vivo. The combination of sublethal doses of VPA and bosutinib led to massive apoptosis of colon cancer cells, irrespective of their genetic background. These results suggest that VPA may be employed as a positive modulator of bosutinib antitumor activity in colorectal cancer.

Effect of fenretinide and low-dose tamoxifen on insulin sensitivity in premenopausal women at high risk for breast cancer.

The prevalence of metabolic syndrome is increasing along with breast cancer incidence worldwide. Because fenretinide improves insulin action and glucose tolerance in insulin-resistant obese mice and because tamoxifen has shown to regulate several markers involved in metabolic syndrome, we sought to investigate the effect of fenretinide or tamoxifen at low dose on features linked to insulin resistance in premenopausal women at risk for breast cancer. We randomized 235 women to low-dose tamoxifen (5 mg/daily), fenretinide (200 mg/daily), or their combination or placebo for 2 years. We used the homeostasis model assessment (HOMA; fasting insulin x glucose/22.5) to estimate insulin sensitivity. Women were considered to improve insulin sensitivity when they shifted from a HOMA >/=2.8 to <2.8. There was no effect of fenretinide or tamoxifen on HOMA overall, but overweight women (body mass index, >or=25 kg/m(2)) had a 7-fold greater probability to normalize HOMA after 2 years of fenretinide treatment [odds ratio (OR), 7.0; 95% confidence interval (95% CI), 1.2-40.5], with 25% of women improving their insulin sensitivity, whereas tamoxifen decreased insulin sensitivity by almost 7 times compared with subjects not taking tamoxifen (OR, 0.15; 95% CI, 0.03-0.88). In this group only, 5% improved their insulin sensitivity. Interestingly, women with intraepithelial or microinvasive neoplasia had higher HOMA (3.0) than unaffected subjects (2.8; P = 0.07). Fenretinide can positively balance the metabolic profile in overweight premenopausal women and this may favorably affect breast cancer risk. Furthermore, features of the metabolic syndrome should be taken into consideration before proposing tamoxifen for breast cancer prevention. The clinical implications of these results require further investigations.

Characterization of compound 584, an Abl kinase inhibitor with lasting effects.

BACKGROUND: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). DESIGN AND METHODS: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. RESULTS: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. CONCLUSIONS: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.

Metallothionein 1G acts as an oncosupressor in papillary thyroid carcinoma.

The molecular pathogenesis of tumors arising from the thyroid follicular epithelial cells, including papillary (PTC) and follicular thyroid carcinoma (FTC), is only partially understood, and the role of tumor suppressor genes has not yet been assessed. The metallothionein (MT) gene family encodes a class of metal-binding proteins involved in several cellular processes, and their expression is often deregulated in human tumors. Recently, downregulation of MT gene expression in PTC has been reported, suggesting a possible oncosuppressor role of this gene family in the pathogenesis of thyroid tumors. To further explore this possibility, we performed expression and functional studies. Analysis of microarray data of thyroid tumors of different histologic types showed that several MT genes were downregulated with respect to normal tissue. The microarray data were corroborated by quantitative PCR experiments, showing downregulation of MTs in PTC and FTC, but to a greater extent in papillary carcinoma. The expression of MTs was also investigated at the protein level by immunohistochemistry; the results were consistent with the microarray data, showing general downregulation in tumor samples, which was more evident in PTC. The functional consequence of MT downregulation was addressed employing an experimental model made of the PTC-derived K1 cell line in which MT1G expression is repressed by promoter methylation. Restoration of MT1G expression by cDNA transfection affected growth rate and in vivo tumorigenicity of K1 cells, indicating an oncosuppressor role for MT1G in thyroid papillary tumorigenesis.

Pharmacokinetics of oral fenretinide in neuroblastoma patients: indications for optimal dose and dosing schedule also with respect to the active metabolite 4-oxo-fenretinide.

PURPOSE: 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). EXPERIMENTAL DESIGN: 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. RESULTS: 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). CONCLUSIONS: 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.

Plasma testosterone and prognosis of postmenopausal breast cancer patients.

PURPOSE: High endogenous testosterone is associated with increased breast cancer (BC) risk. We designed this study specifically to assess the long-term prognostic role of testosterone in a cohort of postmenopausal BC patients. PATIENTS AND METHODS: We considered 194 postmenopausal women, operated on for early BC (T1-2N0M0), who never received chemotherapy or hormonal therapy, and who participated in a fenretinide BC prevention trial as untreated controls. Blood samples were collected 3 months (median) after surgery; plasma samples, stored at -80 degrees C, were radioimmunoassayed for testosterone. Median follow-up was 14 years. The main end point was any cancer event. Event-free survival was estimated by the Kaplan-Meier method. Hazard ratios (HRs) of events by testosterone level were estimated by the Cox model, adjusting for age, tumor size, and histology. RESULTS: Patients with high testosterone (> or = 0.40 ng/mL, median of distribution) had significantly lower event-free survival than those with low testosterone (log-rank P = .004). The adjusted HR of patients with high versus low testosterone was 2.05 (95% CI, 1.28 to 3.27). High testosterone was also associated with a significantly higher risk of BC events (relapse and second primary) with an adjusted HR of 1.77 (95% CI, 1.06 to 2.96). Eleven second primaries (non-BC) occurred in the high-testosterone group, four in the low-testosterone group. CONCLUSION: High plasma testosterone strongly predicts poorer prognosis in postmenopausal BC patients not administered adjuvant therapy. Testosterone levels should be determined as part of the prognostic work-up.

IFN-gamma enhances the antimyeloma activity of the fully human anti-human leukocyte antigen-DR monoclonal antibody 1D09C3.

To investigate the therapeutic activity of the fully human anti-HLA-DR antibody 1D09C3 in multiple myeloma (MM), we reevaluated HLA-DR expression on CD138(+) cells, analyzed the capacity of IFN-gamma to up-regulate HLA-DR expression on MM cell lines, and tested the in vitro and in vivo activity of 1D09C3 alone or in combination with IFN-gamma. CD138(+)HLA-DR(+) cells were detected in 31 of 60 patients, with 15 of 60 patients having >/=20% CD138(+)HLA-DR(+) cells (median, 50%; range, 23-100). Because primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin V/propidium iodide (PI) staining showed that 1D09C3-induced cell death correlated with constitutive HLA-DR expression. Induction of HLA-DR by IFN-gamma restored the sensitivity of HLA-DR dim cell lines to 1D09C3. In vivo, the combined IFN-gamma/1D09C3 treatment significantly increased the median survival of nonobese diabetic/severe combined immunodeficient mice xenografted with KMS-11 cell line, compared with controls (147 versus 48 days, P

Placental growth factor-1 potentiates hematopoietic progenitor cell mobilization induced by granulocyte colony-stimulating factor in mice and nonhuman primates.

The complex hematopoietic effects of placental growth factor (PlGF) prompted us to test in mice and nonhuman primates the mobilization of peripheral blood progenitor cells (PBPCs) elicited by recombinant mouse PlGF-2 (rmPlGF-2) and recombinant human PlGF-1 (rhPlGF-1). PBPC mobilization was evaluated by assaying colony-forming cells (CFCs), high-proliferative potential-CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). In mice, both rmPlGF-2 and rhPlGF-1 used as single agents failed to mobilize PBPCs, whereas the combination of rhPlGF-1 and granulocyte colony-stimulating factor (rhG-CSF) increased CFCs and LTC-ICs per milliliter of blood by four- and eightfold, respectively, as compared with rhG-CSF alone. rhPlGF-1 plus rhG-CSF significantly increased matrix metalloproteinase-9 plasma levels over rhG-CSF alone, suggesting a mechanistic explanation for rhPlGF-1/rhG-CSF synergism. In rhesus monkeys, rhPlGF-1 alone had no mobilization effect, whereas rhPlGF-1 (260 microg/kg per day) plus rhG-CSF (100 microg/kg per day) increased rhG-CSF-elicited mobilization of CFCs, HPP-CFCs, and LTC-ICs per milliliter of blood by 5-, 7-, and 15-fold, respectively. No specific toxicity was associated with the administration of rhPlGF-1 alone or in combination. In conclusion, our data demonstrate that rhPlGF-1 significantly increases rhG-CSF-elicited hematopoietic mobilization and provide a preclinical rationale for evaluating rhPlGF-1 in the clinical setting.