Oral Administration Of Tamoxifen Research Articles

P-372 The role of the immune system in the physiopathology of infertility in case of adenomyosis: a mouse model study

Abstract Study question Are there any fertility disorders and related local and/or systemic immune changes during the early implantation period in a mouse model of adenomyosis? Summary answer An increase in fertility disorders was observed in adenomyosis mice and coincide with local and systemic immune changes observed during the period of implantation. What is known already Adenomyosis is as a common pathology that could be responsible for fertility alteration. Immune changes in uterus are implicated in adenomyosis physiopathology. One hypothesis is that the physiological immune environment necessary for a successful implantation can be altered in adenomyosis, leading to fertility disorders. Study design, size, duration Randomly selected CD-1 female neonatal pups were orally dosed with oral administration of tamoxifen in order to induce adenomyosis (TAM group), while other received solvent only (control group). At 3 months, CD-1 mice (F1) of both group were put into mating. 36 pregnant mice were included in the TAM group and 30 in the control group. Participants/materials, setting, methods Ultrasounds were performed during pregnancy at E(E=embryonic day)7.5 and E12.5 to evaluate fertility outcomes in mice of the TAM and control group. Mice were sacrificed at E18.5 and histological,morphometric and functional analysis were performed on the placentas. In order to identify local and/or systemic immune changes in the uterus, mice of both group were sacrificed at E4.5 of pregnancy, during the implantation period. Uterine horns and spleen were collected for flow cytometry and RT-qPCR analyzes. Main results and the role of chance We observed a significantly lower number of implantation sites and a significantly higher number of resorption (3.88±2.36 versus 1.00±0.82(p < 0.001)) in TAM compared to control group. Analysis of placentas showed a significantly higher junctional/labyrinthic area ratio (0.60±0.09 versus 0.30±0.05(p = 0.0052)) and a significantly lower expression of Vascular Endothelial Growth-Factor(GF), Platelet endothelial cell adhesion molecule, Insulin-like GF2 and Placental GF in the TAM group compared to the control group, indicating an altered placental vascularization compared to controls. To characterize the immune change during the early implantation period, we analyzed some immune cells populations in the uteri and the spleen: In the TAM group, the number of macrophages(F4/80), Natural Killers(NK) cells(NKP46+/NKG2D+) and dendritic cells (DC)(CD11b+) were significantly decreased compared to control uteri. However, the number of M1 macrophages(Ly6c+hight) and their activation were significantly increased. DC activation was also increased in TAM group. In the spleen, a significant increase in the activation macrophages and DC was observed in adenomyosis group compared to control. In the uteri, the number of LT4(CD4+) cells and number of LTreg cells(CD25+/FOXP3+) were significantly increased in the TAM group compared to control group. In the spleen, a significant increase in LT4 cells count was observed in TAM compared to control group. Limitations, reasons for caution This study is limited by the use of an animal model and the lack of intervention. Wider implications of the findings This study provides evidence that adenomyotic lesions in mice induced fertility disorders and immune modifications at a local and at a systemic level during the early implantation period. These data support the involvement of innate and adaptive immune cells in implantation failure observed in the mouse model of adenomyosis. Trial registration number not applicable

The role of PTEN in primary sensory neurons in processing itch and thermal information in mice.

PTEN is known as a tumor suppressor and plays essential roles in brain development. Here, we report that PTEN in primary sensory neurons is involved in processing itch and thermal information in adult mice. Deletion of PTEN in the dorsal root ganglia (DRG) is achieved in adult Drg11-CreER: PTENflox/flox (PTEN CKO) mice with oral administration of tamoxifen, and CKO mice develop pathological itch and elevated itch responses on exposure to various pruritogens. PTEN deletion leads to ectopic expression of TRPV1 and MrgprA3 in IB4+ non-peptidergic DRG neurons, and the TRPV1 is responsive to capsaicin. Importantly, the elevated itch responses are no longer present in Drg11-CreER: PTENflox/flox: TRPV1flox/flox (PTEN: TRPV1 dCKO) mice. In addition, thermal stimulation is enhanced in PTEN CKO mice but blunted in dCKO mice. PTEN-involved regulation of itch-related gene expression in DRG neurons provides insights for understanding molecular mechanism of itch and thermal sensation at the spinal level.

Cystic endometrial hyperplasia-pyometra complex in a mastectomized Boerboel bitch following tamoxifen administration


 
 
 
 Tamoxifen is a non-steroidal, anti-estrogenic and selective estrogen receptor modulator commonly used as adjuvant chemotherapy in humans with breast cancer and occasionally in dogs following mastectomy. This report presents a case of cystic endometrial hyperplasia-pyometra in a dog following oral administration of the tamoxifen. A 4-year-old intact nulliparous Boerboel bitch presented at Veterinary Teaching Hospital, Federal University of Agriculture, Abeokuta. It was diagnosed with mammary carcinoma of the right cranial lumbar mammary gland. Mastectomy was performed followed by oral administration of Tamoxifen (10mg daily) for six weeks as adjunct chemotherapy. Eight weeks after, the owner reported that the bitch was lethargic, anorexic and had a purulent vaginal discharge. Abdominal ultrasound revealed cystic endometrial hyperplasia. Results of complete blood counts showed neutrophilic (absolute neutrophil count = 19.5×103/L) leukocytosis (total leukocyte count = 24.9X103/L), while bacterial culture yielded moderate growth of Staphylococcus aureus, which was sensitive to Ciprofloxacin, Ofloxacin, Sparfloxacin and Gentamycin. The bitch was treated with 400μg of Misoprostol and 500mg of Ciprofloxacin twice daily for two weeks. Ovariohysterectomy (OVH) was performed using a ventral midline approach when no significant improvement was observed from medical treatment. The dog improved significantly after ovariohysterectomy and was discharged one week after OVH. It was concluded that, although tamoxifen is routinely used as adjuvant chemotherapy following mastectomy, more research is required to evaluate its safety in intact bitches.
 
 
 

Hypoxia and its possible relationship with endometrial receptivity in adenomyosis: a preliminary study

BackgroundAdenomyosis (AM) is an important cause of female infertility. However, the underlying mechanism remains unclear. This report describes a preliminary study of hypoxia and its possible association with endometrial receptivity in AM.MethodsThe study was divided into in vitro and in vivo experiments. In vitro, expression levels of the endometrial receptivity markers HOXA10 and HOXA11 in the implantation period were examined using real-time PCR and western blotting. Endometrial expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, and HIF-3α was determined using immunohistochemistry. In vivo, using an AM mouse model established by oral administration of tamoxifen, we inhibited expression of HIF-2α using an HIF-2α antagonist (PT2399; 30 mg/kg body weight, twice daily by oral gavage for 2 days) and then examined expression levels of Hoxa10 and Hoxa11 using real-time PCR and western blotting.ResultsEndometrial mRNA and protein expression levels of HOXA10 and HOXA11 were significantly lower in patients with AM than in control patients. Expression of HIF-2α was significantly higher in the AM group than in the control group, whereas that of HIF-1α and HIF-3α was equivalent in both groups. In vivo analysis showed that administration of the HIF-2α antagonist resulted in increased expression of Hoxa10 and Hoxa11 at both the mRNA and protein levels in AM model mice.ConclusionsHIF-2α overexpression may be one reason for decreased endometrial receptivity in AM. The current findings provide insight into HIF-2α-mediated AM-related infertility and suggest that PT2399 has potential as a treatment for AM.Trial registrationThis trial was retrospectively registered.

Mediator Med23 Regulates Adult Hippocampal Neurogenesis.

Mammalian Mediator (Med) is a key regulator of gene expression by linking transcription factors to RNA polymerase II (Pol II) transcription machineries. The Mediator subunit 23 (Med23) is a member of the conserved Med protein complex and plays essential roles in diverse biological processes including adipogenesis, carcinogenesis, osteoblast differentiation, and T-cell activation. However, its potential functions in the nervous system remain unknown. We report here that Med23 is required for adult hippocampal neurogenesis in mouse. Deletion of Med23 in adult hippocampal neural stem cells (NSCs) was achieved in Nestin-CreER:Med23flox/flox mice by oral administration of tamoxifen. We found an increased number of proliferating NSCs shown by pulse BrdU-labeling and immunostaining of MCM2 and Ki67, which is possibly due to a reduction in cell cycle length, with unchanged GFAP+/Sox2+ NSCs and Tbr2+ progenitors. On the other hand, neuroblasts and immature neurons indicated by NeuroD and DCX were decreased in number in the dentate gyrus (DG) of Med23-deficient mice. In addition, these mice also displayed defective dendritic morphogenesis, as well as a deficiency in spatial and contextual fear memory. Gene ontology (GO) analysis of hippocampal NSCs revealed an enrichment in genes involved in cell proliferation, Pol II-associated transcription, Notch signaling pathway and apoptosis. These results demonstrate that Med23 plays roles in regulating adult brain neurogenesis and functions.

Dysregulation of humoral immunity in Foxp3 conditional-knockout mice

Foxp3+ regulatory T cells (Tregs) are crucial for maintaining tolerance to self-antigens and preventing autoimmune diseases. Loss of Foxp3 expression leads to autoimmunity and disrupts humoral immune responses, including hyperproduction of immunoglobulin E (IgE). Elucidation of how Tregs control antibody production can lead to the development of new therapies for autoimmune and allergic diseases. However, premature death of Foxp3-deficient mice makes it difficult to analyze the roles of Tregs in humoral immunity of adult mice. In this study, we developed Foxp3 conditional-knockout mice (Foxp3floxR26CreERT2) in which the Foxp3 gene was inducibly deleted by tamoxifen administration. After oral administration of tamoxifen, titers of immunoglobulins, particularly IgG2c and IgE, were increased in Foxp3floxR26CreERT2 mice compared with that in controls. Under these conditions, CD4+ T cells from Foxp3floxR26CreERT2 mice had increased expression of several activation markers, including inducible costimulator and CD40 ligand, as well as the cytokines interleukin 4 and interferon gamma. In addition, the proportions of T follicular helper (Tfh) cells and germinal center (GC) B cells were increased in Foxp3floxR26CreERT2 mice compared with those in controls. These results indicated that Tregs controlled excessive or pathogenic antibody production by suppressing Tfh cell differentiation and GC formation. Furthermore, these data suggested that Foxp3floxR26CreERT2 mice could be a useful tool for screening therapeutic agents.

Abstract 101: Endothelial Arnt Regulates Microvascular Endothelial Barrier Function in Heart Failure Through a Novel Mmp3 Pathway

Background: Increased microvascular permeability and cardiac interstitial edema formation are major contributors to progressive myocyte dysfunction during ischemia/reperfusion (IR) injury. Effective clinical therapies for IR injury are lacking, primarily because the mechanisms linking IR to cardiac microvascular permeability and edema are largely unknown. Results from my current studies indicate that vascular permeability is limited by the expression of Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT), a transcription factor that regulates gene expression by homodimerizing or by heterodimerizing with hypoxia-inducible factor (HIF) 1α, HIF2α, or other cofactors. We hypothesize that endothelial ARNT mediates cardiac remodeling following IR. Methods and Results: We have recently generated inducible VE-cadherin-Cre ERT2 endothelial cell (EC)-specific ARNT knockout mice (ecARNT -/- ), which, after ecARNT -/- induction by oral tamoxifen administration, display increased cardiac endothelial permeability with preserved contractile function of the heart. However, after 24 hours of ischemia-reperfusion, ecARNT -/- mice exhibit severe cardiac blood vessel leakage and impaired cardiac function. Following one month of IR, ecARNT -/- mice have a 50% mortality rate, and impaired cardiac function, as determined by EF% and FS%. Only 10% mortality is observed in the control group (ARNTf/f mice treated with tamoxifen). Furthermore, data from microarray analysis show that matrix metalloproteinases (MMPs) are significantly upregulated in ARNT deficient mouse cardiac microvascular endothelial cells (CMVECs), with a particularly significant increase in MMP3. MMP3 can cleave most extracellular matrix constituents and its inhibition attenuates the increase in cell permeability observed in ecARNT -/- CMVECs. Finally, use of an MMP inhibitor in vivo rescues heart failure induced by IR in ecARNT -/- mice. Taken together, these results suggest that ecARNT may mediate heart failure during IR through an MMP3 pathway. Conclusion: Endothelial ARNT is a critical regulator of endothelial function and could potentially serve as a therapeutic target for heart failure in response to IR injury.

Abstract 5092: Establishment of an induction protocol for medulloblastoma during prenatal stages of a floxed-cre inducible model

Abstract Advances in mouse genetics in the last decades have improved the site-specific and conditional control of specific genes in vivo. Despite the availability of different recombinases, the most popular and best-characterized model is the Cre-loxP system. To achieve the recombination in specific tissues or cell types, Cre expression is regulated by a cell-specific promoter and it is also possible to use a tamoxifen-inducible form of Cre for temporal control of the recombination process. Different models have been described using this technology. However, the recombination in the central nervous system tissues is still problematic. Here, we describe a pilot study to induce the recombination process in the brain at prenatal stages in a Cre-loxP system. To test the temporal control of the recombination in the brain, we used a Ptch1fl/fl mouse line in combination with the previously described Math1-creERT2 mouse line that can be induced by tamoxifen administration. All experimental procedures on animals, together with mouse breeding, were performed in accordance with the guidelines of the animal ethics committee of the Karolinska Institutet. The detection of the floxed allele and the cre gene was carried out by PCR at each breeding step. In order to control the specific time point of the induction, the presence of the males for mating was controlled for two days. After this, females were monitored and the induction of the model was carried out at E14.5 (embryonic days) for two days by oral administration of tamoxifen. Issues due to tamoxifen administrations as problems in delivery and mortality/toxicity in pups were not detected. The litters obtained were genotyped to detect the correct combination, Ptch1fl/fl; Math1creERT2 +/-. Mice with the correct genotype developed neurological symptoms between 7 and 9 weeks old, and once the brain was dissected, an abnormal size of the cerebellum was detected. Cerebellum was dissected and the cells obtained were grown and maintained in different specific culture media. The deleted allele of Ptch1 was detected by PCR in the tumoral tissue and in the cell cultures. Here, we described a protocol to induce a Cre-loxP system during embryonic stages. The present protocol avoids the problems described for extensive tamoxifen administration. We also described new culture media to grow and expand the tumor cells obtained. Our findings could help to improve the induction of this kind of systems and study mutations during embryonic stages related with the development and progression of medulloblastoma. The culture of cells derived from these systems, can be used to establish therapeutic mouse models and to evaluate novel treatment strategies for this tumor. Citation Format: Gabriel Gallo-Oller, Ani Azatyan, Ninib Baryawno, Per Kogner, John Inge Johnsen. Establishment of an induction protocol for medulloblastoma during prenatal stages of a floxed-cre inducible model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5092.

A novel mouse model for tracking the fate of CXCR5-expressing T cells

The germinal center (GC) reaction, a critical process in the humoral immune response, requires follicular helper T (Tfh) cells. Tfh cells express the master transcription factor Bcl6 and chemokine receptor CXCR5, which enable them to migrate from the T cell zone to B cell follicles and interact with GC B cells. However, CXCR5 is downregulated when Tfh cells become memory cells. Therefore, it is difficult to track Tfh cells continuously in vivo. In this study, we generated a mouse strain, Cxcr5CreERT2R26Tomato, in which the fluorescent protein tdTomato is inducibly expressed in CXCR5+ cells by tamoxifen administration. After the oral administration of tamoxifen, most Tfh cells in Peyer's patches (PP) from Cxcr5CreERT2R26Tomato mice were tdTomato+. To track antigen-specific Tfh cells in vivo, OVA-specific OT-II T cells derived from Cxcr5CreERT2R26Tomato mice were transferred to wild-type mice, and the recipient mice were immunized with OVA followed by tamoxifen administration. CXCR5+ T cells became tdTomato+ and were mainly located in B cell follicles and GC areas 8 days after immunization. Four weeks after immunization, tdTomato+ OT-II T cells migrated from B cell follicles to the T-B border area and T cell zone after CXCR5 downregulation and CCR7 upregulation. These results indicate that Cxcr5CreERT2R26Tomato mice are a useful tool for studying the cell fate of differentiated Tfh cells in vivo and therefore have implications for the development of therapeutic strategies for infectious and autoimmune diseases.

Host regulation of liver fibroproliferative pathology during experimental schistosomiasis via interleukin-4 receptor alpha.

Interleukin-4 receptor (IL-4Rα) is critical for the initiation of type-2 immune responses and implicated in the pathogenesis of experimental schistosomiasis. IL-4Rα mediated type-2 responses are critical for the control of pathology during acute schistosomiasis. However, type-2 responses tightly associate with fibrogranulomatous inflammation that drives host pathology during chronic schistosomiasis. To address such controversy on the role of IL-4Rα, we generated a novel inducible IL-4Rα-deficient mouse model that allows for temporal knockdown of il-4rα gene after oral administration of Tamoxifen. Interrupting IL-4Rα mediated signaling during the acute phase impaired the development of protective type-2 immune responses, leading to rapid weight loss and premature death, confirming a protective role of IL-4Rα during acute schistosomiasis. Conversely, IL-4Rα removal at the chronic phase of schistosomiasis ameliorated the pathological fibro-granulomatous pathology and reversed liver scarification without affecting the host fitness. This amelioration of the morbidity was accompanied by a reduced Th2 response and increased frequencies of FoxP3+ Tregs and CD1dhiCD5+ Bregs. Collectively, these data demonstrate that IL-4Rα mediated signaling has two opposing functions during experimental schistosomiasis depending on the stage of advancement of the disease and indicate that interrupting IL-4Rα mediated signaling is a viable therapeutic strategy to ameliorate liver fibroproliferative pathology in diseases like chronic schistosomiasis.

Abstract 306: Endothelial Arnt Mediates Cardiac Remodeling After Ischemia Reperfusion Injury Through an MMP Pathway

Background: Prevention of myocardial injury following heart attack is a challenge due to lack of effective treatments and the potential to develop chronic heart failure (HF). Impaired endothelial function is thought to contribute to the progression of HF after cardiac ischemia-reperfusion injury (IR). However, the underlying mechanisms are unknown. Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT), also known as hypoxia-inducible factor 1-beta (HIF1-β), plays a major role in vascular endothelial function and is implicated in ischemic diseases. We hypothesize that endothelial ARNT mediates cardiac remodeling following IR. Methods and Results: We generated inducible endothelial specific ARNT knockout mice (ecARNT -/- ) by crossing mice with loxP sequences flanking exon 6 of ARNT with Cre ERT2 mice under the VE-cadherin promoter. A 90% deletion of ecARNT was achieved following two weeks of oral tamoxifen administration. ecARNT -/- mice exhibit severe cardiac blood vessel leakage and impaired cardiac function after IR. Following 1 month of IR, ecARNT -/- mice had a 50% mortality rate, while no mortality was observed in the control group (ARNTf/f mice treated with tamoxifen). To determine the underlying mechanisms by which ARNT regulates endothelial function, we performed DNA sequencing on cardiac microvascular endothelial cells (CMECs) isolated from control and ecARNT -/- hearts. Data suggest a significant increase in the matrix metalloproteinases (MMP) family of proteins following ARNT knockdown. An increase in MMP activity in the serum of ecARNT -/- CMECs after IR was also detected. Moreover, use of an MMP inhibitor in vivo rescues heart failure induced by IR in ecARNT -/- mice. Taken together, these results suggest that ecARNT may mediate heart failure during IR through an MMP pathway. Conclusion: Endothelial ARNT is a critical regulator of endothelial function and could potentially serve as a therapeutic target for HF in response to IR injury.

Abstract 45: Endothelial Aryl Hydrocarbon Receptor Nucleatranslator (Arnt) is a Novel Regulator of Cardiac Endothelial Barrier Integrity in Diabetes

Background: Diabetes leads to endothelial barrier dysfunction and altered endothelial permeability, which results in increased cardiovascular risk. ARNT, also known as HIF-1β, a transcription factor that functions as a master regulator of glucose homeostasis, has been implicated in diabetes. Endothelial-specific ARNT deletion (ArntΔEC) in mice is embryonically lethal, with hemorrhage occurring in the heart during the embryonic stage. However, the particular role of endothelial ARNT(ecARNT) in diabetes is largely unknown. We have found a significant decrease in ARNT expression in both diabetic rodent endothelial cells and diabetic human hearts. We hypothesize that a loss of ecARNT mediates endothelial barrier dysfunction during diabetes. Methods and Results: We generated inducible endothelial specific ARNT knockout mice (ecARNT-/-) by crossing mice with loxP sequences flanking exon 6 of ARNT with Cre ERT2 mice under the VE-cadherin promoter. A 90% deletion of ecARNT was achieved following two weeks of oral tamoxifen administration. ecARNT-/- mice exhibit severe blood vessel leakage, which is restricted to the heart, suggesting a distinct function for ecARNT in different tissues. Cardiomyopathy is evident 6 months after ARNT deletion. In vitro , trans-endothelial electrical resistance (TER) and transwell assays have confirmed endothelial barrier disruption in cardiac microvascular endothelial cells (CMEC) isolated from both ecARNT-/- hearts and diabetic (DB/DB) mouse hearts. To determine the underlying mechanisms by which ARNT may regulate endothelial barrier function, we performed DNA sequencing on CMEC isolated from control, ecARNT-/-, and DB/DB mice. Data suggest a significant increase in TNFa signaling, including ELAM-1 and ICAM-1 in CMEC isolated from ecARNT-/- CMEC and diabetic CMEC. Moreover, use of anti-TNFa antibody rescues endothelial barrier dysfunction in CMEC isolated from ecARNT-/- mice. Taken together, these results suggest that a reduction in ecARNT during diabetes may mediate endothelial barrier dysfunction through a TNFa signaling pathway. Conclusion: ecARNT is a critical mediator of endothelial barrier function and could potentially serve as a therapeutic target for diabetic cardiovascular diseases.

Effect of Melatonin and Calmodulin in an Idiopathic Scoliosis Model.

Background. To explore influence of continuous illumination, luzindole, and Tamoxifen on incidence of scoliosis model of rats. Methods. Thirty-two one-month-old female rats were rendered into bipedal rats. The bipedal rats were divided into 4 groups: group A by intraperitoneal injection of luzindole and continuous illumination; group B by intraperitoneal injection of luzindole only; group C by intraperitoneal injection of luzindole and oral administration of Tamoxifen; and group D by intraperitoneal injection of equivalent saline. Radiographs were taken at 8th week and 16th week, and incidence and the Cobb angles of scoliosis were calculated. At 16th week, all rats were sacrificed. Before the sacrifice, the levels of calmodulin were measured in each group. Results. At 8th week, scoliosis occurred in groups A and B, with an incidence of 75% and 12.5%, respectively, while rats in group C or D had no scoliosis. At 16th week, scoliosis incidences in groups A and B were 57% and 62.5%, respectively. No scoliosis occurred in group C or D. Calmodulin in platelets in group B was significantly different, compared with groups A and D. There was no significant difference in calmodulin in platelets in groups B and C. Conclusion. By intraperitoneal injection of luzindole in bipedal rats, scoliosis rat models could be successfully made. Under light, incidence of scoliosis may be increased at an early period but it is reversible. Tamoxifen can suppress natural process of scoliosis.

Hepatoprotection by active fractions from Desmostachya bipinnata stapf (L.) against tamoxifen-induced hepatotoxicity.

Objective:The aim was to evaluate the effect of the polyphenolic fraction of Desmostachya bipinnata Stapf (PFDB) (Poaceae) on tamoxifen (TAM)-induced liver damage in female Sprague-Dawley rats.Materials and Methods:The roots of Desmostachya bipinnata were extracted in 70% methanol, and the polyphenolic fraction was isolated. Protection of BRL3A cells against ethanol-induced damage was determined by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Hepatotoxicity was induced in rats by oral administration of TAM (45 mg/kg/day) for 21 days. The PFDB was administered to experimental animals at two selected doses (100 and 200 mg/kg/day) during the treatment. The serum levels of various biochemical parameters and the antioxidant enzymes were examined by standard procedures.Results:A dose-dependent increase in percentage viability was observed when ethanol-exposed BRL3A cells were treated with PFDB. Both the treatment groups upon pretreatment with PFDB exhibited a significant (P ≤ 0.05) protective effect by lowering serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, triglycerides, cholesterol, urea, uric acid, bilirubin and creatinin levels and improving protein level in serum in dose-dependent manner, which was comparable to that of silymarin group. In addition, PFDB prevented elevation of reduced glutathione, glutathione peroxidase, superoxide dismutase and catalase in the TAM-intoxicated rats in concentration-dependent manner and significantly (P < 0.05) reduced the lipid peroxidation in the liver tissue. The biochemical observations were supplemented with histopathological reports, which showed the attenuation of hepatocellular necrosis.Conclusions:The results of this study strongly indicate that the polyphenolic fraction of the plant roots has a potent hepatoprotective action against TAM-induced hepatic damage in rats.

Abstract 2448: Meiosis activating sterols counteract KRas-driven epithelial carcinogenesis via an LXR-dependent mechanism

Abstract Sterols are structural components of lipid membranes which can exert potent biological activities by regulating cell surface receptor trafficking and stability. Arrest of the sterol pathway at the level of SC4MOL (sterol C4-methyl oxydase-like) or NSDHL (NADPH-dependent steroid dehydrogenase-like) specifically antagonizes signaling and trafficking of the epidermal growth factor receptor (EGFR) (Sukhanova, 2013). SC4MOL and NSDHL catalyze two sequential steps of oxidative decarboxylation of the C4-methyl groups of meiosis activating sterols (MAS). The protein binding MAS has not been identified. We investigated whether the anti-EGFR activities of MAS metabolites are mediated via their interaction with the Liver X Receptor (LXR) so that LXR and its transcriptional targets induce subsequent deregulation of intracellular cholesterol homeostasis. In EGFR-positive carcinoma cell, inhibition of SC4MOL or NSDHL induces upregulation of two canonical LXR targets: the ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1). In keeping with the LXR activation, expression of low density lipoprotein receptor (LDLR) is markedly reduced in SC4MOL or NSDHL-deficient cells. These effects were completely reversible with inactivation of LXR alpha, or via inactivation of an upstream enzyme, CYP51A1, which eliminated MAS. Inactivation of SC4MOL and NSDHL sterol pathway enzymes and upregulation of cholesterol efflux via ABCA1 depleted cholesterol from cellular membranes as evidenced by marked increase of nuclear form of SREBP2. Direct manipulation of SREBP, LXR or LDLR produced concordant synergistic effects with anti-EGFR drugs in carcinoma cell lines suggesting novel combinatorial strategies to treat EGFR-positive carcinomas. In in vivo experiments, SC4MOL or NSDHL-depleted A431 carcinoma xenografts showed exquisite sensitivity to cetuximab. This phenotype was associated with upregulation of LXR target ABCA1 and loss of LDLR. Conditional inactivation of a “floxed” Nsdhl allele via K14-Cre transgene triggered ABCA1 expression in the skin, arrested keratinocytes proliferation and caused lethality in newborn male pups. The anti-proliferative effect of NSDHL deficiency was tested in vivo against KRAS-driven tumors. Skin tumors induced by KRAS in transgenic LSL-KRasG12D mice are EGFR-dependent. Tamoxifen-inducible Tg(K5-CreERTam) mice were crossed with LSL-KRasG12D;NsdhlloxP/loxP and tumors induced by oral tamoxifen administration. Both, NsdhlloxP/Y (NSDHL-null) males carrying only the “floxed” allele and NsdhlloxP/+ heterozygous mosaic females formed skin tumors at similar rate. However, the growth of tumors in NSDHL-null males was completely abrogated. In sum, specific sterol metabolites play a fundamental role in regulating activity of oncogenic EGFR. Inhibition of SC4MOL or NSDHL is a highly effective strategy to counteract oncogenic signaling in carcinomas with activated EGFR-KRAS axis. Citation Format: Linara Gabitova, Andrey Gorin, Diana Restifo, Dong-Hua Yang, David Cunningham, Gail E. Herman, Igor A. Astsaturov. Meiosis activating sterols counteract KRas-driven epithelial carcinogenesis via an LXR-dependent mechanism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2448. doi:10.1158/1538-7445.AM2014-2448

Dendron‐Based Micelles for Topical Delivery of Endoxifen: A Potential Chemo‐Preventive Medicine for Breast Cancer

Endoxifen (EDX) is an active metabolite of tamoxifen that has been proven effective in the prevention and treatment of estrogen‐positive breast cancer; however, oral administration of tamoxifen often causes severe side effects. Here, the topical delivery of EDX is explored using polymeric micelles to achieve localized drug delivery with potentially minimal side effects. EDX is encapsulated into dendron micelles (DM) with various surface groups (‐NH2, ‐COOH, or ‐Ac) and into cationic liposomes as a control. End‐group modification significantly affects the drug loading, where the DM‐COOH micelles allow the most efficient encapsulation. Furthermore, unlike the burst release from the liposomes, all DMs show sustained release of EDX over 6 days. Each formulation is evaluated for its potential to deliver EDX across the skin layers. DMs substantially enhance the permeation of EDX through both mouse (up to 20‐fold) and human (up to 4‐fold) skin samples relative to ethanol, a chemical penetration enhancer. Franz diffusion cell experiments reveal that DM‐COOH induces the highest flux of EDX among all groups. The enhanced drug loading, controlled release profiles, and enhanced skin permeation all demonstrate that DMs are a useful platform for the topical delivery of EDX, offering a potential alternative administration route for chemoprevention.

Effect of administration of tamoxifen citrate on xanthine oxidase activity of liver during 3-methylcholanthrene induced carcinogenesis in female albino mice

Tamoxifen citrate, the non-steroidal anti-estrogen and the first selective estrogen receptor modulator is the treatment of choice for patients with all stages of estrogen receptor positive breast cancer. Although Cytochrome P-450s (CYP) are well known to be responsible for much of its metabolism, fragmentary studies have been carried out to analyze the activity of xanthine oxidase, a non-CYP drug metabolizing enzyme, in tamoxifen metabolism. So, the present investigation is aimed to ascertain the effect of tablet and nano-formulation of tamoxifen on xanthine oxidase expression during 3-methylcholanthrene-induced carcinogenesis. It is found that oral administration of tamoxifen in both the formulations significantly (p < 0.01) decreases the xanthine oxidase activity in liver tissue of female mice. We speculate our results to be beneficial for the better understanding of drug metabolism and intensity, which in turn will pave the way for new and efficient nano-formulations of drug coming into therapeutic use.

Abstract 16841: Cardiac Deletion of Aryl Hydrocarbon Nuclear Translocator (Hypoxia Inducible Factor 1-beta) in the Adult Heart Leads to Cardiomyopathy Due to Lipid Accumulation via Peroxisome Proliferator Activated Receptor Activation

Background: Aryl hydrocarbon receptor nuclear translocator (ARNT), also known as hypoxia-inducible factor 1-beta (HIF1-beta), serves as a binding partner for numerous basic helix-loop-helix Per/ARNT/Sim proteins, including HIF1alpha. Although HIF1alpha is known to be required for normal cardiac development, the role of ARNT in basal cardiac function in the adult is not known. We hypothesized that ARNT is required for normal cardiac physiology, and that ARNT deletion in the adult heart results in cardiomyopathy. Methods and Results: Cardiac-specific ARNT deletion was achieved by crossing ARNT flox/flox with αMHC-MCM (tamoxifen-inducible heart specific Cre) transgenic mice, followed by oral administration of tamoxifen. MCM/ARNT +/+ (WT) littermates were used as a control. Echocardiography of the ARNT knockout (KO) mice showed enlarged left ventricle with a reduction in ejection fraction (KO vs. WT: 35.8 ± 3.6% vs. 61.2 ± 2.8%, n=12, p&lt;0.01). Closed-chest catheterization indicated that +dP/dt (KO vs. WT: 4355 ± 538 vs. 9426 ± 180 mmHg, p&lt;0.01) was reduced in KO mice but not in WT mice. Electron microscopy revealed lipid droplets in ARNT KO heart tissues that were confirmed by oil-red staining. Furthermore, genes involved in fatty acid oxidation were up-regulated in KO hearts, as shown by microarray analysis and qRT-PCR. We further confirmed these results in vitro with siRNA-mediated knock down of ARNT in H9c2 cardiac myoblasts. Compared to control siRNA transfection, ARNT knockdown led to increases in PPAR-alpha (1.7-fold, p&lt;0.001), fatty acid translocase (1.4-fold, P&lt;0.01), and acyl-coenzyme A dehydrogenase (1.6-fold, p&lt;0.0001) mRNA levels and to enhanced triglyceride accumulation (p&lt;0.05). Moreover, insulin resistance was noted in ARNT knockdown cells, as demonstrated by significantly reduced glucose uptake in response to insulin stimulation. These results suggest that impaired fatty acid oxidation leads to lipid accumulation in response to a reduction in ARNT levels. Conclusion: ARNT is essential for normal cardiac function, and a reduction in ARNT levels leads to cardiomyopathy by increasing triglyceride accumulation through a PPAR-alpha pathway.

Effects of baicalein on the pharmacokinetics of tamoxifen and its main metabolite, 4-hydroxytamoxifen, in rats: Possible role of cytochrome p450 3A4 and P-glycoprotein inhibition by baicalein

The purpose of this study was to investigate the effects of baicalein on the pharmacokinetics of tamoxifen and its active metabolite, 4-hydroxytamoxifen, in rats. Tamoxifen and baicalein interact with cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp), and the increase in the use of health supplements may result in baicalein being taken concomitantly with tamoxifen as a combination therapy to treat orprevent cancer diseases. Pharmacokinetic parameters of tamoxifen and 4-hydroxytamoxifen were determined in rats after an oral administration of tamoxifen (10 mg/kg) to rats in the presence and absence of baicalein (0.5, 3, and 10 mg/kg). Compared to the oral control group (given tamoxifen alone), the area under the plasma concentration-time curve and the peak plasma concentration of tamoxifen were significantly increased by 47.6-89.1% and 54.8-100.0%, respectively. The total body clearance was significantly decreased (3 and 10 mg/kg) by baicalein. Consequently, the absolute bioavailability of tamoxifen in the presence of baicalein (3 and 10 mg/kg) was significantly increased by 47.5-89.1% compared with the oral control group (20.2%). The metabolite-parent AUC ratio of tamoxifen was significantly reduced, implying that the formation of 4-hydroxytamoxifen was considerably affected by baicalein. Baicalein enhanced the oral bioavailability of tamoxifen, which may be mainly attributable to inhibition of the CYP3A4-mediated metabolism of tamoxifen in the small intestine and/or in the liver, and inhibition of the P-gp efflux pump in the small intestine and/or reduction of total body clearance by baicalein.

P-Glycoprotein (ABCB1) Transports the Primary Active Tamoxifen Metabolites Endoxifen and 4-Hydroxytamoxifen and Restricts Their Brain Penetration

P-glycoprotein (P-gp, ABCB1) is a highly efficient drug efflux pump expressed in brain, liver, and small intestine, but also in tumor cells, that affects pharmacokinetics and confers therapy resistance for many anticancer drugs. The aim of this study was to investigate the impact of P-gp on tamoxifen and its primary active metabolites, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen. We used in vitro transport assays and Abcb1a/1b(-/-) mice to investigate the impact of P-gp on the oral availability and brain penetration of tamoxifen and its metabolites. Systemic exposure of tamoxifen and its metabolites after oral administration of tamoxifen (50 mg/kg) was not changed in the absence of P-gp. However, brain accumulation of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were modestly, but significantly (1.5- to 2-fold), increased. Endoxifen, however, displayed a 9-fold higher brain penetration at 4 h after administration. Endoxifen was transported by P-gp in vitro. Upon direct oral administration of endoxifen (20 mg/kg), systemic exposure was slightly decreased in Abcb1a/1b(-/-) mice, but brain accumulation of endoxifen was dramatically increased (up to 23-fold at 4 h after administration). Shortly after high-dose intravenous administration (5 or 20 mg/kg), endoxifen brain accumulation was increased only 2-fold in Abcb1a/1b(-/-) mice compared with wild-type mice, suggesting a partial saturation of P-gp at the blood-brain barrier. Endoxifen, the clinically most relevant metabolite of tamoxifen, is a P-gp substrate in vitro and in vivo, where P-gp limits its brain penetration. P-gp might thus be relevant for tamoxifen/endoxifen resistance of P-gp-positive breast cancer and tumors positioned behind a functional blood-brain barrier.