Lovisa Lundholm

Mechanisms of antidiabetogenic and body weight-lowering effects of estrogen in high-fat diet-fed mice

The high-fat diet (HFD)-fed mouse is a model of obesity, impaired glucose tolerance, and insulin resistance. The main objective of this study was to elucidate the molecular mechanisms underlying the antidiabetogenic and weight-lowering effects of 17beta-estradiol (E(2)) in this mouse model. C57BL/6 female mice (8 wk old) were fed on a HFD for 10 mo. E(2), given daily (50 microg/kg s.c.) during the last month of feeding, decreased body weight and markedly improved glucose tolerance and insulin sensitivity. Plasma levels of insulin, leptin, resistin, and adiponectin were decreased. We demonstrated that E(2) treatment decreased the expression of genes encoding resistin and leptin in white adipose tissue (WAT), whereas adiponectin expression was unchanged. Furthermore, in WAT we demonstrated decreased expression levels of sterol regulatory element-binding protein 1c (SREBP1c) and its lipogenic target genes, such as fatty acid synthase and stearoyl-CoA desaturase 1 (SCD1). In the liver, the expression levels of transcription factors such as liver X receptor alpha and SREBP1c were not changed by E(2) treatment, but the expression of the key lipogenic gene SCD1 was reduced. This was accompanied by decreased hepatic triglyceride content. Importantly, E(2) decreased the hepatic expression of glucose-6-phosphatase (G-6-Pase). We conclude that E(2) treatment exerts antidiabetic and antiobesity effects in HFD mice and suggest that this is related to decreased expression of lipogenic genes in WAT and liver and suppression of hepatic expression of G-6-Pase. Decreased plasma levels of resistin probably also play an important role in this context.

The estrogen receptor α-selective agonist propyl pyrazole triol improves glucose tolerance in ob/ob mice; potential molecular mechanisms

The aim of this study was to validate the role of estrogen receptor alpha (ERalpha) signaling in the regulation of glucose metabolism, and to compare the molecular events upon treatment with the ERalpha-selective agonist propyl pyrazole triol (PPT) or 17beta-estradiol (E(2)) in ob/ob mice. Female ob/ob mice were treated with PPT, E(2) or vehicle for 7 or 30 days. Intraperitoneal glucose and insulin tolerance tests were performed, and insulin secretion was determined from isolated islets. Glucose uptake was assayed in isolated skeletal muscle and adipocytes. Gene expression profiling in the liver was performed using Affymetrix microarrays, and the expression of selected genes was studied by real-time PCR analysis. PPT and E(2) treatment improved glucose tolerance and insulin sensitivity. Fasting blood glucose levels decreased after 30 days of PPT and E(2) treatment. However, PPT and E(2) had no effect on insulin secretion from isolated islets. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissue were similar in PPT and vehicle-treated ob/ob mice. Hepatic lipid content was decreased after E(2) treatment. In the liver, treatment with E(2) and PPT increased and decreased the respective expression levels of the transcription factor signal transducer and activator of transcription 3, and of glucose-6-phosphatase. In summary, our data demonstrate that PPT exerts anti-diabetic effects, and these effects are mediated via ERalpha.

Key lipogenic gene expression can be decreased by estrogen in human adipose tissue

OBJECTIVE To characterize the molecular mechanisms by which estrogens reduce adipose tissue mass, with particular focus on genes involved in lipogenesis. DESIGN This study involves one arm of an open randomized clinical study with parallel group comparison. SETTING Womens health clinical research unit at a university hospital and a university research laboratory. PATIENT(S) Samples from ten naturally postmenopausal women are included in the study. INTERVENTION(S) The participants were studied before and after 3 months of treatment with estradiol valerate (2 mg daily). MAIN OUTCOME MEASURE(S) Affymetrix gene chips were used to study changes in gene expression upon estrogen treatment in subcutaneous abdominal adipose tissue. RESULT(S) Genes involved in fatty acid synthesis, such as stearoyl-CoA desaturase, fatty acid synthase, acetyl-coenzyme A carboxylase alpha, and fatty acid desaturase 1 were decreased by estrogen treatment in a subgroup of women. Changes in the expression of these genes were correlated to changes in plasma triglyceride levels. Another gene decreased by estrogen treatment was peroxisome proliferator activated receptor gamma (PPARG). CONCLUSION(S) Key lipogenic genes and the important adipogenic gene PPARG can be regulated by estrogen in human abdominal adipose tissue, which could be relevant for increased adiposity following menopause.

The estrogen receptor alpha-selective agonist PPT improves glucose tolerance in ob/ob mice; potential molecular mechanisms

The aim of this study was to validate the role of estrogen receptor alpha (ERalpha) signaling in the regulation of glucose metabolism, and to compare the molecular events upon treatment with the ERalpha-selective agonist propyl pyrazole triol (PPT) or 17beta-estradiol (E(2)) in ob/ob mice. Female ob/ob mice were treated with PPT, E(2) or vehicle for 7 or 30 days. Intraperitoneal glucose and insulin tolerance tests were performed, and insulin secretion was determined from isolated islets. Glucose uptake was assayed in isolated skeletal muscle and adipocytes. Gene expression profiling in the liver was performed using Affymetrix microarrays, and the expression of selected genes was studied by real-time PCR analysis. PPT and E(2) treatment improved glucose tolerance and insulin sensitivity. Fasting blood glucose levels decreased after 30 days of PPT and E(2) treatment. However, PPT and E(2) had no effect on insulin secretion from isolated islets. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissue were similar in PPT and vehicle-treated ob/ob mice. Hepatic lipid content was decreased after E(2) treatment. In the liver, treatment with E(2) and PPT increased and decreased the respective expression levels of the transcription factor signal transducer and activator of transcription 3, and of glucose-6-phosphatase. In summary, our data demonstrate that PPT exerts anti-diabetic effects, and these effects are mediated via ERalpha.

miRNA-214 is related to invasiveness of human non-small cell lung cancer and directly regulates alpha protein kinase 2 expression.

The prognosis of non‐small cell lung cancer (NSCLC) is poor, since it has often metastasized to distant organs by the time of diagnosis. Therefore, biomarkers predicting metastasis are crucial. miRNAs play important roles in the regulation of different tumor cell processes, including metastasis. We recently showed that miRNA‐214 is linked to a radioresistant phenotype of NSCLC. miRNA‐214 has been linked to metastasis in other tumor types. Therefore, we examined the role of miRNA‐214 in the metastatic potential of NSCLC. We showed that downregulation of miRNA‐214 increased invasive potential, and conversely, overexpression of miRNA‐214 decreased invasiveness of NSCLC cells in vitro. Gene expression and bioinformatic analyses of NSCLC cells with ablated miRNA‐214, identified a number of metastasis‐related target genes, including pregnancy‐associated plasma protein A (PAPP‐A), alpha protein kinase 2 (ALPK2), cyclin‐dependent kinase 6 (CDK6) and tumor necrosis‐factor alpha‐induced protein 3 (TNFAIP3). These were validated on mRNA and protein level to be regulated by miRNA‐214. Through immunoprecipitation we showed that only ALPK2 is directly regulated by miRNA‐214. We also examined the protein expression of these four genes in NSCLC tumors with respect to metastatic potential. These results showed that NSCLC tumors express these proteins at moderate‐high levels in the nucleus, cytoplasm and/or plasma membrane although with no significant correlation to the overall survival or the metastatic potential of the patients. However, we also showed that the membrane‐localized PAPP‐A had a higher expression level compared to the cytoplasm‐localized. In conclusion, we show that low miRNA‐214 expression is linked to a higher invasive potential of NSCLC cells.

Effects of estrogen on gene expression profiles in mouse hypothalamus and white adipose tissue: target genes include glutathione peroxidase 3 and cell death-inducing DNA fragmentation factor, α-subunit-like effector A

Obesity has become a major health problem in many parts of the world. Estrogens are known to reduce adipose tissue mass in both humans and animals but the molecular mechanisms are not well characterized. We used gene expression profiling to study long-term effects of estrogen on gene expression in mouse white adipose tissue and hypothalamus. Overall, the effects of estrogen on hypothalamic gene expression were much smaller than the corresponding effects on white adipose tissue gene expression. We characterize in detail estrogenic regulation of glutathione peroxidase 3 (GPX3). Our studies suggest that GPX3 is a direct estrogen receptor alpha target gene in white adipose tissue. Since obesity is correlated with oxidative stress, and GPX3 has been demonstrated to be lower in obesity and higher after weight loss, we hypothesize that GPX3 is one important mediator of effects of estrogen in relation to fat mass. Additional genes that were affected by estrogen in adipose tissue include cell death-inducing DNA fragmentation factor, alpha-subunit-like effector A (CIDEA), a gene shown to be related to body fat in mice. We conclude that estrogen has large effects on gene expression in white adipose tissue and hypothesize that GPX3 and CIDEA could be important mediators of the effects of estrogen on fat mass.

Abstract B60: Phenothiazines inhibits repair of chemotherapy-induced DNA double-strand break in tumor but not in normal fibroblasts by modulating chromatin configuration.

Acquisition of drug resistance mechanisms by tumor cells poses severe limitations on the efficacy of conventional chemotherapy. Since most clinically useful chemotherapeutic agents cause DNA damage, suppression of tumor DNA repair capacity is expected to enhance tumor eradication. Based on earlier observations that phenothiazine compounds confer chemosensitization in lung cancer cells, we investigated whether these compounds could modulate DNA repair and the molecular details of their mechanisms of action. Colony formation and MTT assays were used to demonstrate phenothiazine-mediated chemosensitization. Immunoblotting, immunofluorescence and FACS studies were used to probe the effects of phenothiazines on DNA repair. Modulation of chromatin structure was achieved by increasing or decreasing the salt content in the culture media. In silico predictions of drug action based on gene signatures was performed by Connectivity Mapping. Our data revealed that phenothiazine co-treatment along with DNA damaging agents (e.g., bleomycin, cisplatin) resulted in sustained activation of, DNA-PK and FancD2, both of which are involved in DSB repair, even many hours after the removal of the DNA damaging agent. This effect was observed in a variety of human tumor cell lines, including those derived from lung-, breast- and ovarian cancer. Notably, repair of DNA damage by non-transformed fibroblasts and epithelial cells was not antagonized by phenothiazines, revealing a potential selectivity for tumor cells. Modulation of DSB repair by phenothiazines was sensitive to osmolality of the culture media. In addition, gene signature induced by phenothiazine treatment showed similarities to gene signatures induced by HDAC inhibitors. Taken together, phenothiazines may represent a novel of class of chemosensitizers which disrupt DSB repair by altering chromatin configuration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B60.

Abstract 163: mRNA and miRNA profiling of platinum-resistant non-small cell lung cancer cell clones identifies potential targets involved in DNA metabolism and DNA replication

Non small cell lung cancer (NSCLC) constitutes the majority of lung cancer diagnoses and platinum-based chemotherapy regimen is the standard treatment. Unfortunately, a large proportion do not respond or relapse on such treatment regimens for yet only partly revealed mechanisms. We profiled residual clones of NSCLC cells after treatment with cisplatin. The mRNA and miRNA pattern was compared between untreated and cisplatin-resistant NSCLC clones using Affymetrix gene and miRNA expression arrays, respectively. We found that around 1100 genes were up regulated (at least two fold) and about 1200 genes were down regulated (at least two fold) in the cisplatin-resistant clones compared with untreated NSCLCs. We then used the Gene Set Enrichment Analysis (GSEA) to find the over- and underrepresented gene sets in large datasets like, Gene Trail and Gene Ontology. We found that cisplatin-refractory NSCLCs showed increased expression of genes involved in different cellular mechanisms including DNA metabolic processes, DNA replication and DNA-dependent DNA replication initiation for example, XRCC2, FANCI, FANCD2, RRM1, MRE11A, BLM, RRM2 and TOP2A which were validated on RNA, protein and functional level for their role in cisplatin sensitivity of NSCLC. We also found a few miRNAs that were up regulated in cisplatin refractory NSCLC by a least 1.3-fold e.g. miRNA-25 star and miRNA-1290. Interestingly, only 14 miRNAs were down regulated (by at least 1.3 fold) in the surviving cisplatin refractory NSCLC cells. Among these miRNAs were (miR-29b, miR-210, miR-486-3p, miR-324-5p, miR-572, miR-30a, e, miR-424, miR-449a, b, miR-301a, miR-153, miR-150 star and miR-34c-5p). We then looked for the predicted targets of these miRNAs using Target scan and microcosm and we found a large list of genes to be potential targets of the miRNAs among them are genes involved in apoptosis, cell cycle regulation, Wnt signaling pathway and other cellular processes (ex. VDAC, SMAD2, RAN, etc.). Correlation analysis between miRNA gene targets and gene expression data is ongoing with the aim to identify and validate putative targets on RNA, protein and functional level. In conclusion, our data show that cisplatin refractory NSCLC tumors have dysregulated mRNAs and miRNAs. Among the dysregulated genes, are certain genes involved in DNA metabolism, DNA replication and DNA-dependent DNA replication initiation which may be novel targets for improved platinum-based therapy response of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 163. doi:10.1158/1538-7445.AM2011-163

Abstract B187: Role of aminopeptidase N in the activation of the alkylating prodrug J1

Aminopeptidase N is a zinc‐dependent metalloprotease which is involved in cancer invasion, metastasis and angiogenesis, and considered as a suitable target for anti‐cancer drug design. This in vitro study demonstrates a role of APN in the activation of J1, a prodrug of the alkylating agent melphalan, and subsequently, its cytotoxic activity. J1 (melphalanyl‐L‐p‐fluorophenylalanyl ethyl ester) is currently in a phase I/II clinical trial. Previous studies have shown that J1‐mediated cytotoxicity is dependent on hydrolytic activity of tumor cells. In this report we have analyzed potential peptidases and esterases of importance for release of free melphalan from J1. Exposure of tumor cell lines to J1 resulted in a significant increased level of free intracellular melphalan, at least ten‐fold at Cmax, compared to exposure to melphalan at the same molar concentration. This efficient intracellular delivery could be inhibited in both magnitude and in time by bestatin, a broad spectrum inhibitor of the aminopeptidases, including the metalloproteinase aminopeptidase N (APN) and ebelactone A, an esterase inhibitor. These effects resulted, as expected, in decreased cytotoxic effects of J1. A specific role of APN in hydrolyzing J1 releasing free melphalan was demonstrated in vitro with pure APN enzyme. By using plasmid‐based overexpression of APN or down regulation of endogenous APN with siRNA in different tumor cell lines we confirmed the involvement of APN in J1‐mediated cytotoxic and apoptotic signaling. Given that APN is shown to be overexpressed in several solid tumors our data suggest that J1 may be activated in a tumor selective manner. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B187.