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1.  The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells. 
Molecular and Cellular Biology  1994;14(12):8356-8364.
The biological response to progesterone is mediated by two distinct forms of the human progesterone receptor (hPR-A and hPR-B). In most cell contexts, hPR-B functions as a transcriptional activator of progesterone-responsive genes, whereas hPR-A functions as a transcriptional inhibitor of all steroid hormone receptors. We have created mutations within the carboxyl terminus of hPR which differentially effect the transcriptional activity of hPR-B in a cell- and promoter-specific manner. Analogous mutations, when introduced into hPR-A, have no effect on its ability to inhibit the transcriptional activity of other steroid hormone receptors. The observed differences in the structural requirements for hPR-B and hPR-A function suggest that transcriptional activation and repression by PR are mediated by two separate pathways within the cell. In support of this hypothesis, we have shown that hPR-A mediated repression of human estrogen receptor (hER) transcriptional activity is not dependent on hER expression level but depends largely on the absolute expression level of hPR-A. Thus, it appears that hPR-A inhibits hER transcriptional activity as a consequence of a noncompetitive interaction of hPR-A with either distinct cellular targets or different contact sites on the same target. We propose that hPR-A expression facilitates a ligand-dependent cross-talk among sex steroid receptor signaling pathways within the cell. It is likely, therefore, that alterations in the expression level of hPR-A or its cellular target can have profound effects on the physiological or pharmacological responses to sex steroid hormone receptor ligands.
PMCID: PMC359374  PMID: 7969170
2.  Modulation of the insulin-like growth factor-I system by N-(4-hydroxyphenyl)-retinamide in human breast cancer cell lines. 
British Journal of Cancer  1998;77(12):2138-2147.
The potent mitogenic activity of insulin-like growth factor I (IGF-I) on breast epithelium is inhibited by retinoic acid in oestrogen receptor-positive (ER+) breast cancer cell lines. We studied and compared the effects of N-(4-hydroxyphenyl)-retinamide (4-HPR) in terms of growth inhibition and modulation of the IGF-I system in ER+ (MCF-7) and oestrogen receptor-negative (ER-) (MDA-MB231) breast cancer cell lines. Treatment with 1-10 microM 4-HPR for up to 96 h induced a dose- and time-dependent inhibition of proliferation in both breast cancer cell lines. Induction of apoptosis was much more evident in MCF-7 than in MDA-MB231 cells (30-40% compared with 0-5% respectively at 5 microM for 48 h). Exogenous human recombinant IGF-I (hr-IGF-I)-stimulated cell proliferation was abolished by 1 microM 4-HPR in MCF-7 cells. Immunoreactive IGF-I-like protein concentration in conditioned medium was reduced by 38% in MCF-7 and by 90% in MDA-MB231 cell lines following treatment for 48 h with 5 microM 4-HPR. Western ligand blot analysis showed a reduction of IGF-binding protein 4 (BP4) and BP5 by 67% and 87%, respectively, in MCF-7, whereas IGF-BP4 and -BP1 were reduced by approximately 20% in MDA-MB231 cells. Exposure to 5 microM 4-HPR for 48 h inhibited [125I]IGF-I binding and Scatchard analysis revealed a decrease of more than 50% in maximum binding capacity (Bmax) and a reduced receptor number/cell in both cancer cell lines. Steady-state type I IGF-receptor mRNA levels were reduced by approximately 30% in both tumour cell lines. We conclude that 4-HPR induces a significant down-regulation of the IGF-I system in both ER+ (MCF-7) and ER- (MDA-MB231) breast cancer cell lines. These findings suggest that, in our model, interference with the ER signalling pathway is not the only mechanism of breast cancer growth inhibition by 4-HPR.
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PMCID: PMC2150424  PMID: 9649125
3.  Infectious salmon anaemia virus (ISAV) in Chilean Atlantic salmon (Salmo salar) aquaculture: emergence of low pathogenic ISAV-HPR0 and re-emergence of virulent ISAV-HPR∆: HPR3 and HPR14 
Virology Journal  2013;10:344.
Abstact
Infectious salmon anaemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae. ISA is caused by virulent ISAV strains with deletions in a highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein (designated virulent ISAV-HPR∆). This study shows the historic dynamics of ISAV-HPR∆ and ISAV-HPR0 in Chile, the genetic relationship among ISAV-HPR0 reported worldwide and between ISAV-HPR0 and ISAV-HPR∆ in Chile, and reports the 2013 ISA outbreak in Chile. The first ISA outbreak in Chile occurred from mid-June 2007 to 2010 and involved the virulent ISAV-HPR7b, which was then replaced by a low pathogenic ISAV-HPR0 variant. We analyzed this variant in 66 laboratory-confirmed ISAV-HPR0 cases in Chile in comparison to virulent ISAV-HPR∆ that caused two new ISA outbreaks in April 2013. Multiple alignment and phylogenetic analysis of HE sequences from all ISAV-HPR0 viruses allowed us to identify three genomic clusters, which correlated with three residue patterns of ISAV-HPR0 (360PST362, 360PAN362 and 360PAT362) in HPR. The virus responsible for the 2013 ISAV-HPR∆ cases in Chile belonged to ISAV-HPR3 and ISAV-HPR14, and in phylogenetic analyses, both clustered with the ISAV-HPR0 found in Chile. The ISAV-HPR14 had the ISAV-HPR0 residue pattern 360PAT362, which is the only type of ISAV-HPR0 variant found in Chile. This suggested to us that the 2013 ISAV-HPR∆ re-emerged from ISAV-HPR0 that is enzootic in Chilean salmon aquaculture and were not new introductions of virulent ISAV-HPR∆ to Chile. The clinical presentations and diagnostic evidence of the 2013 ISA cases indicated a mixed infection of ISAV with the ectoparasite Caligus rogercresseyi and the bacterium Piscirickettsia salmonis, which underscores the need for active ISAV surveillance in areas where ISAV-HPR0 is enzootic, to ensure early detection and control of new ISA outbreaks, as it is considered a risk factor. This is the first report of ISA linked directly to the presence of ISAV-HPR0, and provides strong evidence supporting the contention that ISAV-HPR0 shows a strong relationship to virulent ISAV-HPR∆ viruses and the possibility that it could mutate to virulent ISAV-HPR∆.
doi:10.1186/1743-422X-10-344
PMCID: PMC4222741  PMID: 24268071
Low pathogenic infectious salmon anaemia virus; ISAV-HPR0; Virulent ISAV; ISAV-HPR∆; Virulence; Salmo salar
4.  Activation of p53, inhibition of telomerase activity and induction of estrogen receptor beta are associated with the anti-growth effects of combination of ovarian hormones and retinoids in immortalized human mammary epithelial cells 
Background
A full-term pregnancy has been associated with reduced risk for developing breast cancer. In rodent models, the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P). However, the effects of pregnancy levels of E/P in humans and their underlying mechanisms are not fully understood. In this report, we investigated the growth inhibitory effects of pregnancy levels of E/P and both natural and synthetic retinoids in an immortalized human mammary epithelial cell line, 76N TERT cell line.
Results
We observed that cell growth was modestly inhibited by E/P, 9-cis-retinoic acid (9-cis RA) or all-trans-retinoic acid (ATRA), and strongly inhibited by N-(4-hydroxyphenyl) retinamide (HPR). The growth inhibitory effects of retinoids were further increased in the presence of E/P, suggesting their effects are additive. In addition, our results showed that both E/P and retinoid treatments resulted in increased RARE and p53 gene activity. We further demonstrated that p53 and p21 protein expression were induced following the E/P and retinoid treatments. Furthermore, we demonstrated that while the telomerase activity was moderately inhibited by E/P, 9-cis RA and ATRA, it was almost completely abolished by HPR treatment. These inhibitions on telomerase activity by retinoids were potentiated by co-treatment with E/P, and correlated well with their observed growth inhibitory effects. Finally, this study provides the first evidence that estrogen receptor beta is up-regulated in response to E/P and retinoid treatments.
Conclusion
Taken together, our studies show that part of the anti-growth effects of E/P and retinoids is p53 dependent, and involve activation of p53 and subsequent induction of p21 expression. Inhibition of telomerase activity and up-regulation of estrogen receptor beta are also associated with the E/P- and retinoid-mediated growth inhibition. Our studies also demonstrate that the potency of retinoids on cell growth inhibition may be increased through combination of estrogen and progesterone treatment.
doi:10.1186/1475-2867-5-6
PMCID: PMC555559  PMID: 15755327
5.  In vitro activities of novel 4-HPR derivatives on a panel of rhabdoid and other tumor cell lines 
Background
Rhabdoid tumors (RTs) are aggressive pediatric malignancies with poor prognosis. N-(4-hydroxy phenyl) retinamide (4-HPR or fenretinide) is a potential chemotherapeutic for RTs with activity correlated to its ability to down-modulate Cyclin D1. Previously, we synthesized novel halogen-substituted and peptidomimetic-derivatives of 4-HPR that retained activity in MON RT cells. Here we analyzed the effect of 4-HPR in inhibiting the growth of several RT, glioma, and breast cancer cell lines and tested their effect on cell cycle, apoptosis and Cyclin D1 expression.
Methods
Effect of compounds on RT cell cycle profiles, and cell death were assessed by MTS cell survival assays and FACS analysis. The effects of treatment on Cyclin D1 expression were determined by immunoblotting. The efficacy of these compounds on glioma and breast cancer cell lines was also determined using MTS assays.
Results
Low micromolar concentrations of 4-HPR derivatives inhibited cell survival of all RT cells tested. The 4-HPR derivatives altered RT cell cycle profiles and induced high levels of cell death that was correlated with their potency. ATRA exhibited high IC50 values in all cell lines tested and did not cause cell death. In MON RT cells, the iodo-substituted compounds were more active than 4-HPR in inducing cell cycle arrest and apoptosis. Additionally, the activity of the compounds correlated with their ability to down-modulate Cyclin D1: while active compounds reduced Cyclin D1 levels, inactive ATRA did not. In glioma and breast cancer cell lines, 4-HPR and 4-HPR derivatives showed variable efficacy.
Conclusions
Here we demonstrate, for the first time, that the inhibitory activities of novel halogen-substituted and peptidomimetic derivatives of 4-HPR are correlated to their ability to induce cell death and down-modulate Cyclin D1. These 4-HPR derivatives showed varied potencies in breast cancer and glioma cell lines. These data indicate that further studies are warranted on these derivatives of 4-HPR due to their low IC50s in RT cells. These derivatives are of general interest, as conjugation of halogen radioisotopes such as 18F, 124I, or 131I to 4-HPR will allow us to combine chemotherapy and radiotherapy with a single drug, and to perform PET/SPECT imaging studies in the future.
doi:10.1186/1475-2867-11-34
PMCID: PMC3204277  PMID: 21951911
6.  Identification of mammalian target of rapamycin as a direct target of fenretinide both in vitro and in vivo  
Carcinogenesis  2012;33(9):1814-1821.
N-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) is a synthetic retinoid that has been tested in clinical trials as a cancer therapeutic and chemopreventive agent. Although 4HPR has been shown to be cytotoxic to many kinds of cancer cells, the underlying molecular mechanisms are only partially understood. Until now, no direct cancer-related molecular target has been reported to be involved in the antitumor activities of 4HPR. Herein, we found that 4HPR inhibited mammalian target of rapamycin (mTOR) kinase activity by directly binding with mTOR, which suppressed the activities of both the mTORC1 and the mTORC2 complexes. The predicted binding mode of 4HPR with mTOR was based on a homology computer model, which showed that 4HPR could bind in the ATP-binding pocket of the mTOR protein through hydrogen bonds and hydrophobic interactions. In vitro studies also showed that 4HPR attenuated mTOR downstream signaling in a panel of non-small-cell lung cancer cells, resulting in growth inhibition. Moreover, knockdown of mTOR in cancer cells decreased their sensitivity to 4HPR. Results of an in vivo study demonstrated that i.p. injection of 4HPR in A549 lung tumor-bearing mice effectively suppressed cancer growth. The expression of mTOR downstream signaling molecules in tumor tissues was also decreased after 4HPR treatment. Taken together, our results are the first to identify mTOR as a direct antitumor target of 4HPR both in vitro and in vivo, providing a valuable rationale for guiding the clinical uses of 4HPR.
doi:10.1093/carcin/bgs234
PMCID: PMC3515856  PMID: 22798378
7.  Fenretinide metabolism in humans and mice: utilizing pharmacological modulation of its metabolic pathway to increase systemic exposure 
British Journal of Pharmacology  2011;163(6):1263-1275.
BACKGROUND AND PURPOSE
High plasma levels of fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] were associated with improved outcome in a phase II clinical trial. Low bioavailability of 4-HPR has been limiting its therapeutic applications. This study characterized metabolism of 4-HPR in humans and mice, and to explore the effects of ketoconazole, an inhibitor of CYP3A4, as a modulator to increase 4-HPR plasma levels in mice and to increase the low bioavailability of 4-HPR.
EXPERIMENTAL APPROACH
4-HPR metabolites were identified by mass spectrometric analysis and levels of 4-HPR and its metabolites [N-(4-methoxyphenyl)retinamide (4-MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR)] were quantified by high-performance liquid chromatography (HPLC). Kinetic analysis of enzyme activities and the effects of enzyme inhibitors were performed in pooled human and pooled mouse liver microsomes, and in human cytochrome P450 (CYP) 3A4 isoenzyme microsomes. In vivo metabolism of 4-HPR was inhibited in mice.
KEY RESULTS
Six 4-HPR metabolites were identified in the plasma of patients and mice. 4-HPR was oxidized to 4-oxo-4-HPR, at least in part via human CYP3A4. The CYP3A4 inhibitor ketoconazole significantly reduced 4-oxo-4-HPR formation in both human and mouse liver microsomes. In two strains of mice, co-administration of ketoconazole with 4-HPR in vivo significantly increased 4-HPR plasma concentrations by > twofold over 4-HPR alone and also increased 4-oxo-4-HPR levels.
CONCLUSIONS AND IMPLICATIONS
Mice may serve as an in vivo model of human 4-HPR pharmacokinetics. In vivo data suggest that the co-administration of ketoconazole at normal clinical doses with 4-HPR may increase systemic exposure to 4-HPR in humans.
doi:10.1111/j.1476-5381.2011.01310.x
PMCID: PMC3144539  PMID: 21391977
fenretinide; metabolism; ketoconazole; paediatric cancers
8.  Fenretinide metabolism in humans and mice: utilizing pharmacological modulation of its metabolic pathway to increase systemic exposure 
British Journal of Pharmacology  2011;163(6):1263-1275.
BACKGROUND AND PURPOSE
High plasma levels of fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] were associated with improved outcome in a phase II clinical trial. Low bioavailability of 4-HPR has been limiting its therapeutic applications. This study characterized metabolism of 4-HPR in humans and mice, and to explore the effects of ketoconazole, an inhibitor of CYP3A4, as a modulator to increase 4-HPR plasma levels in mice and to increase the low bioavailability of 4-HPR.
EXPERIMENTAL APPROACH
4-HPR metabolites were identified by mass spectrometric analysis and levels of 4-HPR and its metabolites [N-(4-methoxyphenyl)retinamide (4-MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR)] were quantified by high-performance liquid chromatography (HPLC). Kinetic analysis of enzyme activities and the effects of enzyme inhibitors were performed in pooled human and pooled mouse liver microsomes, and in human cytochrome P450 (CYP) 3A4 isoenzyme microsomes. In vivo metabolism of 4-HPR was inhibited in mice.
KEY RESULTS
Six 4-HPR metabolites were identified in the plasma of patients and mice. 4-HPR was oxidized to 4-oxo-4-HPR, at least in part via human CYP3A4. The CYP3A4 inhibitor ketoconazole significantly reduced 4-oxo-4-HPR formation in both human and mouse liver microsomes. In two strains of mice, co-administration of ketoconazole with 4-HPR in vivo significantly increased 4-HPR plasma concentrations by > twofold over 4-HPR alone and also increased 4-oxo-4-HPR levels.
CONCLUSIONS AND IMPLICATIONS
Mice may serve as an in vivo model of human 4-HPR pharmacokinetics. In vivo data suggest that the co-administration of ketoconazole at normal clinical doses with 4-HPR may increase systemic exposure to 4-HPR in humans.
doi:10.1111/j.1476-5381.2011.01310.x
PMCID: PMC3144539  PMID: 21391977
fenretinide; metabolism; ketoconazole; paediatric cancers
9.  Polymorphic repeat in AIB1 does not alter breast cancer risk 
Breast Cancer Research : BCR  2000;2(5):378-385.
We assessed the association between a glutamine repeat polymorphism in AIB1 and breast cancer risk in a case-control study (464 cases, 624 controls) nested within the Nurses' Health Study cohort. We observed no association between AIB1 genotype and breast cancer incidence, or specific tumor characteristics. These findings suggest that AIB1 repeat genotype does not influence postmenopausal breast cancer risk among Caucasian women in the general population.
Introduction:
A causal association between endogenous and exogenous estrogens and breast cancer has been established. Steroid hormones regulate the expression of proteins that are involved in breast cell proliferation and development after binding to their respective steroid hormone receptors. Coactivator and corepressor proteins have recently been identified that interact with steroid hormone receptors and modulate transcriptional activation [1]. AIB1 (amplified in breast 1) is a member of the steroid receptor coactivator (SRC) family that interacts with estrogen receptor (ER)α in a ligand-dependent manner, and increases estrogen-dependent transcription [2]. Amplification and overexpression of AIB1 has been observed in breast and ovarian cancer cell lines and in breast tumors [2,3]. A polymorphic stretch of glutamine amino acids, with unknown biologic function, has recently been described in the carboxyl-terminal region of AIB1 [4]. Among women with germline BRCA1 mutations, significant positive associations were observed between AIB1 alleles with 26 or fewer glutamine repeats and breast cancer risk [5]
Aim:
To establish whether AIB1 repeat alleles are associated with breast cancer risk and specific tumor characteristics among Caucasian women.
Patients and methods:
We evaluated associations prospectively between AIB1 alleles and breast cancer risk in the Nurses' Health Study using a nested case-control design. The Nurses' Health Study was initiated in 1976, when 121 700 US-registered nurses between the ages of 30 and 55 years returned an initial questionnaire reporting medical histories and baseline health-related exposures. Between 1989 and 1990 blood samples were collected from 32 826 women. Eligible cases in this study consisted of women with pathologically confirmed incident breast cancer from the subcohort who gave a blood specimen. Cases with a diagnosis anytime after blood collection up to June 1, 1994, with no previously diagnosed cancer except for nonmelanoma skin cancer were included. Controls were randomly selected participants who gave a blood sample and were free of diagnosed cancer (except nonmelanoma skin cancer) up to and including the interval in which the cases were diagnosed, and were matched to cases on year of birth, menopausal status, postmenopausal hormone use, and time of day, month and fasting status at blood sampling. The nested case-control study consisted of 464 incident breast cancer cases and 624 matched controls. The protocol was approved by the Committee on Human Subjects, Brigham and Womens' Hospital, Boston, Massachusetts USA. Information regarding breast cancer risk factors was obtained from the 1976 baseline questionnaire, subsequent biennial questionnaires, and a questionnaire that was completed at the time of blood sampling. Histopathologic characteristics, such as stage, tumor size and ER and progesterone receptor (PR) status, were ascertained from medical records when available and used in case subgroup analyses.
AIB1 repeat alleles were determined by automated fluorescence-based fragment detection from polymerase chain reaction (PCR)-amplified DNA extracted from peripheral blood lymphocytes. Fluorescent 5' -labeled primers were utilized for PCR amplification, and glutamine repeat number discrimination was performed using the ABI Prism 377 DNA Sequencer (Perkin-Elmer, Foster City, CA, USA). Genotyping was performed by laboratory personnel who were blinded to case-control status, and blinded quality control samples were inserted to validate genotyping identification procedures (n = 110); concordance for the blinded samples was 100%. Methods regarding plasma hormone assays have previously been reported [6]. Conditional and unconditional logistic regression models, including terms for the matching variables and other potential confounders, were used to assess the association of AIB1 alleles and breast cancer characterized by histologic subtype, stage of disease, and ER and PR status. We also evaluated whether breast cancer risk associated with AIB1 genotype differed within strata of established breast cancer risk factors, and whether repeat length in AIB1 indirectly influenced plasma hormone levels.
Results:
The case-control comparisons of established breast cancer risk factors among these women have previously been reported [7], and are generally consistent with expectation. The mean age of the women was 58.3 (standard deviation [SD] 7.1) years, ranging from 43 to 69 years at blood sampling. There were 188 premenopausal and 810 postmenopausal women, with mean ages of 48.1 (SD 2.8) years and 61.4 (SD 5.0) years, respectively, at blood sampling. Women in this study were primarily white; Asians, African-Americans and Hispanics comprised less than 1% of cases or controls.
The distribution of AIB1 glutamine repeat alleles and AIB1 genotypes for cases and controls are presented in Table 1. Women with AIB1 alleles of 26 glutamine repeats or fewer were not at increased risk for breast cancer (odds ratio [OR] 1.01, 95% confidence interval [CI] 0.75-1.36; Table 2). Results were also similar by menopausal status and in analyses additionally adjusting for established breast cancer risk factors. Among premenopausal women, the OR for women with at least one allele with 26 glutamine repeats or fewer was 0.82 (95% Cl 0.37-1.81), and among postmenopausal women the OR was 1.09 (95% Cl 0.78-1.52; Table 2). We did not observe evidence of a positive association between shorter repeat length and advanced breast cancer, defined as women with breast cancer having one or more involved nodes (OR 1.07, 95% Cl 0.64-1.78), or with cancers with a hormone-dependent phenotype (ER-positive: OR 1.16, 95% Cl 0.81-1.65; Table 3). No associations were observed among women who had one or more alleles with 26 glutamine repeats or fewer, with or without a family history of breast cancer (family history: OR 1.09; 95% Cl 0.46-2.58; no family history: OR 0.94; 95% Cl 0.68-1.31; test for interaction P = 0.65). We also did not observe associations with breast cancer risk to be modified by other established breast cancer risk factors. Among postmenopausal controls not using postmenopausal hormones, geometric least-squared mean plasma levels of estrone sulfate and estrone were similar among carriers and noncarriers of AIB1 alleles with 26 glutamine repeats or fewer (both differences: ≤ +3.5%; P >0.50). Mean levels of estradiol were slightly, but nonsignificantly elevated among carriers of alleles with 26 glutamine repeats or fewer (+11.6%; P = 0.08).
Discussion:
In this population-based nested case-control study, women with at most 26 repeating glutamine codons (CAG/CAA) within the carboxyl terminus of AIB1 were not at increased risk for breast cancer. We did not observe shorter repeat alleles to be positively associated with breast cancer grouped by histologic subtype, stage of disease, or by ER and PR status. These data suggest that AIB1 repeat length is not a strong independent risk factor for postmenopausal breast cancer, and does not modify the clinical presentation of the tumor among Caucasian women in the general population.
PMCID: PMC13920  PMID: 11056690
AIB1 polymorphism; breast cancer; genetic susceptibility; molecular epidemiology
10.  Decrease in drug accumulation and in tumour aggressiveness marker expression in a fenretinide-induced resistant ovarianumour cell line 
British Journal of Cancer  2001;84(11):1528-1534.
We investigated whether the efficacy of fenretinide (HPR) against ovarian tumours may be limited by induction of resistance. The human ovarian carcinoma cell line A2780, which is sensitive to a pharmacologically achievable HPR concentration (IC 50= 1 μM), became 10-fold more resistant after exposure to increasing HPR concentrations. The cells (A2780/HPR) did not show cross-resistance to the synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and were not sensitive, similarly to the parent line, to all- trans -retinoic acid, 13- cis -retinoic acid or N-(4-methoxyphenyl)retinamide. A2780/HPR cells showed, compared to parental cells, a 3-fold reduction in colony-forming ability in agar. The development of HPR resistance was associated with a marked increase in retinoic acid receptor β (RARβ) mRNA and protein levels, which decreased, together with drug resistance, after drug removal. The expression of cell surface molecules associated with tumour progression including HER-2, laminin receptor and β1 integrin was markedly reduced. The increase in the levels of reactive oxygen species is not involved in HPR-resistance because it was similar in parental and resistant cells. Conversely differences in pharmacokinetics may account for resistance because, in A2780/HPR cells, intracellular peak drug levels were 2 times lower than in A2780 cells and an as yet unidentified polar metabolite was present. These data suggest that acquired resistance to HPR is associated with changes in marker expression, suggestive of a more differentiated status and may be explained, at least in part, by reduced drug accumulation and increased metabolism. © 2001 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2001.1826
PMCID: PMC2363672  PMID: 11384104
retinoids; ovarian tumour; fenretinide-resistance; drug uptake; differentiation; RARβ
11.  Relationships of hHpr1/p84/Thoc1 Expression to Clinicopathologic Characteristics and Prognosis in Non-small Cell Lung Cancer 
Nuclear matrix proteins (NMPs) are important diagnostic and prognostic markers in various human cancers. The hHpr1/p84/Thoc1 protein, a key NMP, resides in the nuclear matrix and is involved in the human TREX complex, which is required for regulation of transcription elongation, pre-RNA splicing, and mRNA export of a subset of human genes. Depletion of hHpr1/p84/Thoc1 decreases growth rates in multiple cancer cell lines, and the expression levels of hHpr1/p84/Thoc1 are strongly associated with tumor size and aggressiveness of several human cancers. Little is known about the expression of this protein in human non-small cell lung cancer (NSCLC) and its association with patients’ clinicopathologic characteristics and prognosis. We evaluated hHpr1/p84/Thoc1 expression in 133 NSCLC patients by immunohistochemistry of tissue microarrays using paraffin-embedded tumor tissue and we confirmed the tissue staining by Western blot analysis. The prognostic significance of hHpr1/p84/Thoc1 expression in tumor tissue was assessed by the Cox proportional hazards regression model. Expression of hHpr1/p84/Thoc1 was found in 51% of patients, and was more prevalent in males than females (59% vs 43%, p = 0.07) and in blacks than whites (91% vs 48%, p = 0.009). In survival analysis, hHpr1/p84/Thoc1 expression appeared to be weakly associated with elevated risk of death among patients with stage I tumors (RR = 1.53, 95% CI = 0.85-2.77, p = 0.16), squamous cell carcinomas (RR = 1.75, 95% CI = 0.73-4.21, p = 0.21), and family histories of lung cancer (RR = 1.55, 95% CI = 0.81-2.97, p=0.18), although none of these associations was statistically significant. Thus elevated expression of hHpr1/p84/Thoc1 is common in NSCLC and may have prognostic significance in subgroups of patients. Further studies with larger sample size are needed to elucidate the role of this critical nuclear matrix protein in NSCLC prognosis.
PMCID: PMC2606038  PMID: 18469354
nuclear matrix proteins; hHpr1/p84/Thoc1; non-small cell lung cancer; tissue microarray
12.  4-oxo-N-(4-hydroxyphenyl)retinamide: Two Independent Ways to Kill Cancer Cells 
PLoS ONE  2010;5(10):e13362.
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.
doi:10.1371/journal.pone.0013362
PMCID: PMC2954786  PMID: 20976277
13.  AF1q: A Novel Mediator of Basal and 4-HPR-Induced Apoptosis in Ovarian Cancer Cells 
PLoS ONE  2012;7(6):e39968.
Background
Fenretinide (4-HPR) is a synthetic retinoid that exhibits potent antitumor and chemopreventive activities against different malignancies, including ovarian tumors. We previously showed that in ovarian cancer cells, 4-HPR induces apoptosis through a signaling cascade starting from reactive oxygen species (ROS) generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and induction of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Since recent studies have shown that the oncogene ALL1-fused from chromosome 1q (AF1q), a retinoic acid target gene, is implicated in apoptosis induction by several therapeutic agents, we investigated its possible involvement in the apoptosis induced by 4-HPR in ovarian cancer cells.
Methodology/Principal Findings
Protein expression analysis, performed in ovarian cancer cells and extended to other histotypes (breast, neuroblastoma, and cervical), revealed that 4-HPR enhanced AF1q expression in cancer cells sensitive to the retinoid but not in resistant cells. Through gene silencing, AF1q was found functionally involved in 4-HPR-induced apoptosis in A2780, an ovarian cancer cell line highly sensitive to retinoid growth inhibitory and apoptotic effects. Inhibition of the signaling intermediates of the 4-HPR apoptotic cascade showed that AF1q upregulation was depended on prior generation of ROS, induction of ER stress response, JNK activation, and PLAB upmodulation. Finally, we found that direct overexpression of AF1q, in the absence of external stimuli, increased apoptosis in ovarian cancer cell lines.
Conclusions/Significance
The study expands the knowledge of the 4-HPR mechanism of action, which has not yet been completely elucidated, identifying AF1q as a novel mediator of retinoid anticancer activity. In addition, we demonstrate, for the first time, that AF1q plays a role in the onset of basal apoptosis in ovarian cancer cells, thus providing new information about the activity of this protein whose biologic functions are mostly unknown.
doi:10.1371/journal.pone.0039968
PMCID: PMC3383705  PMID: 22761939
14.  Phosphoenolpyruvate-sugar phosphotransferase transport system of Streptococcus mutans: purification of HPr and enzyme I and determination of their intracellular concentrations by rocket immunoelectrophoresis. 
Infection and Immunity  1985;50(3):817-825.
Enzyme I and HPr, the general proteins of the phosphoenolpyruvate-sugar phosphotransferase system, play a pivotal role in the control of sugar utilization in gram-negative and gram-positive bacteria. To determine whether growth conditions could modify the rate of biosynthesis of these proteins in Streptococcus mutans, we first purified to homogeneity enzyme I and HPr from S. mutans ATCC 27352. Using specific antibodies obtained against these proteins, we determined by rocket electrophoresis the intracellular levels of enzyme I and HPr in cells of S. mutans 27352 grown under various batch culture conditions and in a number of glucose-grown cells of other strains of S. mutans. HPr was purified by the procedure reported by Gauthier et al. (L. Gauthier, D. Mayrand, and C. Vadeboncoeur, J. Bacteriol. 160:755-763, 1984) and displayed a single band with a molecular weight of 6,650 when analyzed by sodium dodecyl sulfate-urea gel electrophoresis. Enzyme I was purified by DEAE-cellulose chromatography, affinity chromatography on an anti-Streptococcus salivarius column, and preparative electrophoresis. The protein migrated as a single band in native and denaturating gel electrophoresis. The subunit molecular weight of enzyme I determined by electrophoresis under denaturating conditions was 68,000. In gel filtration chromatography at 4 degrees C, the enzyme migrated as a 135,000- to 160,000-molecular-weight species, suggesting that enzyme I is a dimer. In double immunodiffusion experiments, antibodies against HPr reacted with several oral streptococci, Streptococcus lactis, Streptococcus faecium, and Lactobacillus casei, but not with Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. Antibodies against enzyme I of S. mutans 27352 cross-reacted with enzyme I from all the other oral streptococci tested. No cross-reaction was observed with other gram-positive and gram-negative bacteria. The levels of enzyme I and HPr determined by rocket electrophoresis in S. mutans 27352 varied at the most by twofold, depending on the growth conditions. Glucose-grown cells of other S. mutans strains contained levels of enzyme I and HPr which were similar to those found in S. mutans 27352.
Images
PMCID: PMC261154  PMID: 4066033
15.  Induction of Heparanase-1 Expression by Mutant B-Raf Kinase: Role of GA Binding Protein in Heparanase-1 Promoter Activation1 
Neoplasia (New York, N.Y.)  2010;12(11):946-956.
Heparanase-1 (HPR1), an endoglycosidase that specifically degrades heparan sulfate (HS) proteoglycans, is overexpressed in a variety of malignancies. Our present study sought to determine whether oncogene BRAF and RAS mutations lead to increased HPR1 expression. Reverse transcription-polymerase chain reaction analysis revealed that HPR1 gene expression was increased in HEK293 cells transiently transfected with a mutant BRAF or RAS gene. Flow cytometric analysis revealed that B-Raf activation led to loss of the cell surface HS, which could be blocked by two HPR1 inhibitors: heparin and PI-88. Cotransfection of a BRAF or RAS mutant gene with HPR1 promoter-driven luciferase reporters increased luciferase reporter gene expression in HEK293 cells. Knockdown of BRAF expression in a BRAF-mutated KAT-10 tumor cell line led to the suppression of HPR1 gene expression, subsequently leading to increased cell surface HS levels. Truncational and mutational analyses of the HPR1 promoter revealed that the Ets-relevant elements in the HPR1 promoter were critical for BRAF activation-induced HPR1 expression. Luciferase reporter gene expression driven by a four-copy GA binding protein (GABP) binding site was significantly lower in BRAF siRNA-transfected KAT-10 cells than in the control siRNA-transfected cells. We further showed that BRAF knockdown led to suppression of the expression of the GABPβ, an Ets family transcription factor involved in regulating HPR1 promoter activity. Taken together, our study suggests that B-Raf kinase activation plays an important role in regulating HPR1 expression. Increased HPR1 expression may contribute to the aggressive behavior of BRAF-mutated cancer.
PMCID: PMC2978917  PMID: 21076620
16.  Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells 
BMC Cancer  2011;11:477.
Background
N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model.
Methods
CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling.
Results
No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells.
Conclusions
In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.
doi:10.1186/1471-2407-11-477
PMCID: PMC3218121  PMID: 22061047
17.  Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis 
British Journal of Cancer  2009;101(10):1758-1768.
Background:
The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested.
Methods:
The antibody was modified using EBNA cells cotransfected with β-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes.
Results:
The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcγRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcγRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcγRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model.
Conclusion:
The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.
doi:10.1038/sj.bjc.6605355
PMCID: PMC2778542  PMID: 19904275
PR1A3; CEA; ADCC; ADCP; colorectal cancer; glycoengineering; NK cells; monocyte-derived macrophages
18.  Profiling of zinc altered gene expression in human prostate normal versus cancer cells: a time course study 
We have demonstrated that zinc exposure induces apoptosis in human prostate cancer cells (PC-3) and benign hyperplasia cells (BPH), but not in normal prostate cells (HPR-1). However, the mechanisms underlying the effects of zinc on prostate cancer cell growth and zinc homeostasis remain unclear.
To explore the zinc effect on gene expression profiles in normal (HPR-1) and malignant prostate cells (PC-3), we conducted a time course study of Zn treatment with microarray analysis. Microarray data were evaluated and profiled using computational approach for the primary and secondary data analyses. Final analyses were focused on the genes: 1. highly sensitive to zinc, 2. associated with zinc homeostasis, i.e. metallothioneins (MTs), solute zinc carriers (ZIPs) and zinc exporters (ZnTs), 3. relevant to several oncogenic pathways. Zinc-mediated mRNA levels of MT isotypes were further validated by semi-quantitative RT-PCR.
Results showed that zinc effect on genome-wide expression patterns was cell type specific, and zinc appeared to have mainly down-regulatory effects on thousands of genes (1,953 in HPR-1; 3,534 in PC-3) with a threshold of ±2.5-fold, while fewer genes were up-regulated (872 in HPR-1; 571 in PC-3). The patterns of zinc effect on functional MT genes’ expression provided evidence for the cell-type dependent zinc accumulation and zinc-induced apoptosis in prostate cells. In PC-3 cells, zinc significantly up-regulated the expression of MT-1 isotypes -J and -M, denoted previously as “non-functional” MT genes, and now a depictive molecular structure of MT-1J was proposed. Examination of genes involved in oncogenic pathways indicated that certain genes, e.g. Fos, Akt1, Jak3 and PI3K were highly regulated by zinc with cell type specificity.
This work provided an extensive database on zinc related prostate cancer research. The strategy of data analysis was devoted to find genes highly sensitive to Zn, and the genes associated with zinc accumulation and zinc-induced apoptosis. The results indicate that zinc regulation of gene expression is cell-type specific, and MT genes play important roles in prostate malignancy.
doi:10.1016/j.jnutbio.2008.09.004
PMCID: PMC2821158  PMID: 19071009
microarray; zinc; prostate malignant cells; gene isoform; metallothionein; zinc transporter
19.  Effects of retinoic acid and fenretinide on the c-erbB-2 expression, growth and cisplatin sensitivity of breast cancer cells. 
British Journal of Cancer  1998;78(1):79-87.
We investigated the effects of all-trans retinoic acid (ATRA) and fenretinide (4-HPR) on c-erbB-2 expression in SK-BR-3, BT-474 and MCF-7 breast cancer cells and on the growth, differentiation, apoptosis and cisplatin (CDDP) sensitivity of SK-BR-3 cells. It has been reported that oestrogen inhibits c-erbB-2 in oestrogen receptor-positive breast cancer cells. Using ELISA, Western and Northern analysis we have demonstrated that ATRA and 4-HPR exert similar effects down-regulating c-erbB-2 protein and mRNA in c-erbB-2-overexpressing SK-BR-3 and BT-474 and in normally expressing MCF-7 cells. Both retinoids inhibit SK-BR-3 cell growth. ATRA induces cellular enlargement and flattening, suggesting epithelial differentiation. 4-HPR causes nuclear and cytoplasmic condensation, DNA fragmentation and externalization of phosphatidylserine, indicating apoptosis. c-erbB-2 expression/activity has been linked to sensitivity against CDDP. Therefore, combinations of ATRA or 4-HPR with CDDP were tested for their anti-proliferative activity. Retinoid-conditioned cells were either exposed to retinoid and CDDP (schedule I, 'continuous retinoid treatment') or to CDDP alone (schedule II, 'retinoid pretreatment'). This retinoid-conditioning followed by CDDP +/- retinoid yields stronger growth inhibition compared with unconditioned cells, which were exposed to CDDP +/- retinoid (schedule III, 'no retinoid pretreatment'). The inefficacy of schedule III indicates that retinoid-conditioning is essential for the improvement of the antiproliferative effect. The interactions in schedules I and II are synergistic for ATRA and CDDP, but slightly antagonistic for 4-HPR and CDDR However, 4-HPR + CDDP is more effective in growth inhibition than each drug alone.
Images
PMCID: PMC2062943  PMID: 9662255
20.  Survivin knockdown and concurrent 4-HPR treatment controlled human glioblastoma in vitro and in vivo 
Neuro-Oncology  2010;12(11):1088-1101.
Survivin is highly expressed in most cancers, including glioblastoma, and it plays a significant role in inhibiting apoptosis and promoting tumor growth. Treatment of cancer cells with N-(4-hydroxyphenyl) retinamide (4-HPR) induces apoptosis through destabilization of mitochondrial membrane and activation of caspase-mediated apoptotic pathways. We studied the efficacy of a combination of survivin knockdown and 4-HPR treatment to induce apoptosis and inhibit invasion, angiogenesis, and growth of human glioblastomas in vitro and in vivo. Using a plasmid encoding survivin shRNA, we downregulated survivin in glioblastoma U251MG and U118MG cells and simultaneously treated with 1 µM 4-HPR for 48 hours. Cells following treatments were subjected to the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and invasion assays. In vivo angiogenesis and tumor regression studies were performed in nude mice. TUNEL assay demonstrated apoptosis in more than 80% of cells after survivin knockdown and 4-HPR treatment. Matrigel invasion assays demonstrated marked decreases in tumor cell invasion. In vivo angiogenesis studies depicted a remarkable inhibition of neovascularization due to the knockdown of survivin and 4-HPR treatment. Imaging of intracerebral tumorigenesis and longitudinal studies on subcutaneous solid tumor formation showed dramatic decreases in tumorigenesis and solid tumor progression, respectively, after treatment with the combination. Studies to elucidate the molecular mechanisms of the inhibition of angiogenesis and tumor regression demonstrated marked decreases in proliferating cell nuclear antigen, metalloproteinase-9, vascular endothelial growth factor, basic fibroblast growth factor, and CD31 in solid tumors. Our data demonstrated that survivin knockdown and concurrent 4-HPR treatment could be a novel therapeutic strategy for controlling growth of human glioblastomas.
doi:10.1093/neuonc/noq079
PMCID: PMC3098031  PMID: 20679253
4-HPR; glioblastoma; intracerebral tumors; survivin; U118MG; U251MG
21.  Premenopausal endogenous steroid hormones and breast cancer risk: results from the Nurses' Health Study II 
Introduction
Prior research supports an association between endogenous sex steroids and breast cancer among postmenopausal women; the association is less clear among premenopausal women.
Methods
We evaluated the associations between estrogens, androgens, progesterone and sex hormone binding globulin (SHBG) and breast cancer in a nested case-control study in the Nurses' Health Study II. Between 1996 and 1999, 29,611 participants provided blood samples; 18,521 provided samples timed in early follicular and mid-luteal phases of the menstrual cycle. A total of 634 women, premenopausal at blood collection, developed breast cancer between 1999 and 2009 and were matched to 1,264 controls (514 cases and 1,030 controls with timed samples). We used conditional logistic regression controlling for breast cancer risk factors for overall analyses; unconditional logistic regression additionally controlling for matching factors was used for subgroup analyses.
Results
In analyses of premenopausal estrogens including breast cancers diagnosed both before and after menopause, there was no association between follicular estradiol, estrone and free estradiol and risk of either total or invasive breast cancer. Luteal estradiol was positively associated with estrogen receptor positive (ER+)/progesterone receptor positive (PR+) cancers (5th vs. 1st quintile odds ratio (OR): 1.7 (95% confidence interval (CI): 1.0 to 2.9), Ptrend = 0.02). Luteal estrone, free estradiol and progesterone were not associated with risk. Androgens were suggestively or significantly associated with risk when the sample was restricted to invasive tumors (for example, testosterone: OR: 1.4 (1.0 to 2.0), Ptrend = 0.23) and ER+/PR+ disease (testosterone: OR: 1.7 (1.1 to 2.6) Ptrend = 0.10; dehydroepiandrosterone sulfate (DHEAS) OR: 1.3 (0.8 to 2.0) Ptrend = 0.05). SHBG was not associated with breast cancer risk. The results varied by menopausal status at diagnosis, with follicular estradiol suggestively positively associated with breast cancers in women premenopausal at diagnosis (OR: 1.1 (0.9 to 1.3) and significantly inversely associated with postmenopausal disease (OR: 0.6 (0.4 to 0.9); Pheterogeneity < 0.01).
Conclusions
Androgens were associated with modestly increased risk of breast cancer in this population, with stronger associations for invasive and ER+/PR+ disease. Luteal phase estradiol levels were suggestively associated with ER+/PR+ tumors but no other strong associations were observed with estrogens. Associations with follicular phase estrogens may vary by menopausal status at diagnosis, but case numbers were limited. Additional studies to confirm the role of premenopausal hormones in the etiology of both premenopausal and postmenopausal breast cancer are needed.
doi:10.1186/bcr3394
PMCID: PMC3672790  PMID: 23497468
22.  Inhibition of aromatase activity and expression in MCF-7 cells by the chemopreventive retinoid N -(4-hydroxy-phenyl)-retinamide 
British Journal of Cancer  2000;83(3):333-337.
The effect of the chemopreventive synthetic retinoid N -(4-hydroxyphenyl)-retinamide (4-HPR) on aromatase activity and expression was examined. 4-HPR caused a dose-dependent inhibition of aromatase activity in microsomes isolated from JEG-3 human placental carcinoma cells. The kinetics of inhibition were analysed by double-reciprocal plot. The K m of the substrate increased and the V max of the reaction decreased in the presence of 4-HPR, indicating that enzyme inhibition involved both competition for the substrate-binding site and non-competitive mechanisms. To determine whether 4-HPR would also inhibit aromatase activity in intact cells, MCF-7 human breast cancer cells were incubated with or without cAMP in the presence of 4-HPR. 4-HPR inhibited both basal and cAMP-induced aromatase activity in intact MCF-7 cells. The induction of aromatase mRNA expression in MCF-7 cells by cAMP was inhibited in cells treated with 4-HPR. These results indicate that 4-HPR inhibits both the enzymatic activity and expression of aromatase. These activities may play an important role in the known chemopreventive effect of 4-HPR towards breast cancer. © 2000 Cancer Research Campaign
doi:10.1054/bjoc.2000.1269
PMCID: PMC2374555  PMID: 10917548
aromatase; 4-HPR; MCF-7; cAMP
23.  The hydroxyl functional group of N-(4-hydroxyphenyl)retinamide mediates cellular uptake and cytotoxicity in premalignant and malignant human epithelial cells 
Free radical biology & medicine  2010;49(12):2001-2009.
In a previous study, we demonstrated that the anticancer synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) redox cycles at the mitochondrial enzyme dihydroorotate dehydrogenase to trigger anomalous reactive oxygen species (ROS) production and attendant apoptosis in transformed human epithelial cells. Furthermore, we speculated that the hydroxyl functional group of 4HPR was required for this prooxidant property. In this study, we investigated the role of the hydroxyl functional group in 4HPR's in vitro cytotoxicity. Using 4HPR, its primary in vivo metabolite N-(4-methoxyphenyl)retinamide (4MPR), and the synthetic derivative N-(4-trifluromethylphenyl)retinamide (4TPR), we examined the prooxidant and apoptotic effects, as well as the cellular uptake, of these three N-(4-substituted-phenyl)retinamides in premalignant and malignant human skin, prostate, and breast epithelial cells. Compared to 4HPR, both 4MPR and 4TPR were ineffective in promoting conspicuous cellular ROS production, mitochondrial disruption, or DNA fragmentation in these transformed cells. Interestingly, both 4MPR and 4TPR were not particularly cell permeant relative to 4HPR in skin or breast epithelial cells, which implicated an additional role for the hydroxyl functional group in 4HPR's cellular uptake. Moreover, the short-term uptake of 4HPR was directly proportional to cell size, but this characteristic, in obvious contrast to cellular bioenergetic status and/or dihydroorotate dehydrogenase expression, was not fundamentally influential in the overall sensitivity to the promotion of cellular ROS production and apoptosis induction by this agent. Together, these results strongly implicate the hydroxyl functional group in the cytotoxic effects of 4HPR.
doi:10.1016/j.freeradbiomed.2010.09.032
PMCID: PMC3005946  PMID: 20923701
N-(4-hydroxyphenyl)retinamide; 4HPR; fenretinide; N-(4-methoxyphenyl)retinamide; cellular uptake; alkylphenol; reactive oxygen species; dihydroorotate dehydrogenase; apoptosis
24.  Regulation of ATP-dependent P-(Ser)-HPr formation in Streptococcus mutans and Streptococcus salivarius. 
Journal of Bacteriology  1995;177(10):2751-2759.
Sugar transport via the phosphoenolpyruvate (PEP) phosphotransferase system involves PEP-dependent phosphorylation of the general phosphotransferase system protein, HPr, at histidine 15. However, gram-positive bacteria can also carry out ATP-dependent phosphorylation of HPr at serine 46 by means of (Ser)HPr kinase. In this study, we demonstrate that (Ser)HPr kinase in crude preparations of Streptococcus mutans Ingbritt and Streptococcus salivarius ATCC 25975 is membrane associated, with pH optima of 7.0 and 7.5, respectively. The latter organism possessed 7- to 27-fold-higher activity than S. mutans NCTC 10449, GS-5, and Ingbritt strains. The enzyme in S. salivarius was activated by fructose-1,6-bisphosphate (FBP) twofold with 0.05 mM ATP, but this intermediate was slightly inhibitory with 1.0 mM ATP at FBP concentrations up to 10 mM. Similar inhibition was observed with the enzyme from S. mutans Ingbritt. A variety of other glycolytic intermediates had no effect on kinase activity under these conditions. The activity and regulation of (Ser)HPr kinase were assessed in vivo by monitoring P-(Ser)-HPr formation in steady-state cells of S. mutans Ingbritt grown in continuous culture with limiting glucose (10 and 50 mM) and with excess glucose (100 and 200 mM). All four forms of HPr [free HPr, P approximately (His)-HPr, P-(Ser)-HPr, and P approximately (His)-P-(Ser)-HPr] could be detected in the cells; however, significant differences in the intracellular levels of the forms were apparent during growth at different glucose concentrations. The total HPr pool increased with increasing concentrations of glucose in the medium, with significant increases in the P-(Ser)-HPr and P approximately HHis)-P-(Ser)-HPr concentrations. For example, while total PEP-dependent phosphorylation [P approximately(His)-HPr plus P approximately (His)-P-(Ser)-HPr] varied only from 21.5 to 52.5 microgram mg of cell protein (-1) in cells grown at the four glucose concentrations, the total ATP-dependent phosphorylation [P-(Ser)-HPr plus P approximately (His)-P-(Ser)-HPr] increased 12-fold from the 10 mM glucose-grown cells (9.1 microgram mg of cell protein (-1) to 106 and 105 microgram mg(-1) in the 100 and 200 mM glucose-grown cultures, respectively. (Ser)HPr kinase activity in membrane preparations of the cells varied little between the 10, 50, and 100 mM glucose-grown cells but increased threefold in the 200 mM glucose-grown cells. The intracellular levels of ATP, glucose-6-phosphate, and FBP increased with external glucose concentration, with the level of FBP being 3.8-fold higher for cells grown with 200 mM glucose than for those grown with 10 mM glucose. However, the variation in the intracellular levels of FBP, particularly between cells grown with 100 and 200 mM glucose, did not correlate with the extent of P-(Ser)-HPr formation, suggesting that the activity of (Ser)HPr kinase is not critically dependent on the availability of intracellular FBP.
PMCID: PMC176946  PMID: 7751285
25.  Anti-tumor activity of fenretinide complexed with human serum albumin in lung cancer xenograft mouse model 
Oncotarget  2014;5(13):4811-4820.
Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and Acsvl3 mainly connected with tumor growth.
Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and Acsvl3 suggested a reduction of tumor growth.
In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary.
PMCID: PMC4148101  PMID: 25015569
Fenretinide; caveolin-1; ACSVL3; Albumin; lung cancer

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