Overexpression of p49/STRAP alters cellular cytoskeletal structure and gross anatomy in mice.

BACKGROUND: The protein p49/STRAP (SRFBP1) is a transcription cofactor of serum response factor (SRF) which regulates cytoskeletal and muscle-specific genes. RESULTS: Two conserved domains were found in the p49/STRAP protein. The SRF-binding domain was at its N-terminus and was highly conserved among mammalian species, xenopus and zebrafish. A BUD22 domain was found at its C-terminus in three sequence databases. The BUD22 domain was conserved among mammalian p49/STRAP proteins, and yeast cellular morphogenesis proteins, which is involved in ribosome biogenesis that affects growth rate and cell size. The endogenous p49/SRAP protein was localized mainly in the nucleus but also widely distributed in the cytoplasm, and was in close proximity to the actin. Transfected GFP-p49/STRAP protein co-localized with nucleolin within the nucleolus. Overexpression of p49/STRAP reduced actin content in cultured cells and resulted in smaller cell size versus control cells. Increased expression of p49/STRAP in transgenic mice resulted in newborns with malformations, which included asymmetric abdominal and thoracic cavities, and substantial changes in cardiac morphology. p49/STRAP altered the expression of certain muscle-specific genes, including that of the SRF gene, which is a key regulator of cardiac genes at the developmental, structural and maintenance level and has two SRE binding sites. CONCLUSIONS: Since p49/STRAP is a co-factor of SRF, our data suggest that p49/STRAP likely regulates cell size and morphology through SRF target genes. The function of its BUD22 domain warrants further investigation. The observed increase in p49/STRAP expression during cellular aging may contribute to observed morphological changes in senescence.

Glutamine affects glutathione recycling enzymes in a DMBA-induced breast cancer model.

Malignancy depletes host glutathione (GSH) levels to increase treatment-related toxicity and increases itself to resist the treatments. Our previous studies have shown that dietary glutamine (GLN) prevented 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors through enhancing gut GSH release and reducing tumor GSH level. In addition, GSH synthesis, metabolism, and recycling are accomplished in gamma-glutamyl cycle. We hypothesized that the GLN prevention might be through a differential regulation of the gamma-glutamyl cycle enzymes. Female Sprague-Dawley rats were randomized into DMBA-tumor bearing, DMBA-treated, and control groups subdivided into GLN and water groups. GLN supplementation was given at 1 g/kg/day by gastric gavage. The activities and messenger RNA levels of gamma-glutamyl transpeptidase (GTP), gamma-glutamylcysteine synthetase (GCS), 5-oxo-L-prolinase (OPase), gamma-glutamyl transferase (GTF), and glutaminase (GLNase) were determined in gut mucosa and breast tumor using specific enzyme assays and semiquantitative reverse transcription polymerase chain reaction. GLN upregulated gut GTP, GCS, OPase, and GLNase in DMBA-tumor bearing, DMBA-treated, and/or control rats; however, it downregulated these enzymes in the tumor. The paradoxical effect of GLN on key GSH recycling enzymes in the gut versus tumor suggests that dietary supplemental GLN could be used in the clinical practice to increase the therapeutic index of cancer treatments by protecting normal tissues from, and sensitizing tumor cells to, chemotherapy and radiation-related injury.

Oral glutamine (AES-14) supplementation inhibits PI-3k/Akt signaling in experimental breast cancer.

BACKGROUND: 7,12-dimethylbenz [a] anthracene (DMBA) administration to pubertal rats causes breast tumors and inhibits glutathione (GSH) production. Our previous results have established that oral glutamine (GLN) supplementation significantly reduced tumor development, restored the depressed GSH production, and caused a significant decrease in the circulating levels of insulinlike growth factor-1 (IGF-1). The present study was designed to investigate the involvement of the IGF-1-activated phosphatidylinositol 3 kinase (PI-3K)/Akt apoptotic signaling pathway. MATERIALS AND METHODS: Forty female Sprague-Dawley rats were randomly divided into 4 groups: DMBA+GLN (n = 16), DMBA+water (n = 8), Oil+GLN (n = 8) and Oil+water (n = 8). At the age of 50 days, rats received a single dose of 100 mg/kg DMBA (n = 24) or sesame oil (n = 16) and were gavaged with a GLN suspension formulation (AES-14) or water for the duration of the entire experiment. The animals were killed 11 weeks after the DMBA application, and the levels of IGF-1, IGF-1 receptor (IGF-IR), Akt, Bcl-2 and Bad in tumorous and nontumorous breast tissue samples were measured by Western blot analysis. RESULTS: GLN supplementation resulted in a significant decrease in the levels of IGF-1, IGF-IR, Akt, and Bcl-2 in nontumorous samples. At the same time, the levels of pro-apoptotic protein Bad were significantly elevated. The samples collected from tumor tissues showed lower levels of IGF-1, Akt, Bcl-2, Bad, and IGF-IR in comparison with nontumorous tissues. CONCLUSIONS: GLN supplementation inhibited the PI-3K/Akt pathway that is thought to be important in increasing cell survival during tumorigenesis. These results are in agreement with our hypothesis that GLN counteracts the effects of DMBA and blocks carcinogenesis in vivo.

Exposure to high or low glucose levels accelerates the appearance of markers of endothelial cell senescence and induces dysregulation of nitric oxide synthase.

To test the hypothesis that aging impairs endothelial cell response to glucose stress, we utilized a human umbilical vein endothelial cell in vitro model in which clinically relevant concentrations of normal (5.5 mM), high (25 mM), and low (1.5mM) glucose were tested. With advancing population doubling, exposure to normal glucose gradually decreased endothelial nitric oxide synthase expression and activity, resulting in slow, progressive development of markers of cell senescence (by population doubling level [PDL] 44). High or low glucose treatment accelerated the appearance of markers of senescence (by ~PDL 35) along with declines in endothelial nitric oxide synthase expression and activity. Human umbilical vein endothelial cells exposed to alternating low and high glucose gave even more rapid acceleration in the appearance of markers of senescence (by ~PDL 18) and reduction in endothelial nitric oxide synthase levels. Thus, exposure to low and high glucose induces earlier appearance of markers of endothelial cell senescence and dysregulation of the nitric oxide synthase gene and protein expression and function. These findings will help to elucidate endothelial dysfunction associated with glucose intolerance and improve future therapy for diabetic seniors.

Tamoxifen and raloxifene suppress the proliferation of estrogen receptor-negative cells through inhibition of glutamine uptake.

PURPOSE: Modulation of estrogen receptor (ER) plays a central role in selective estrogen receptor modulators (SERMs) molecular mechanism of action, although studies have indicated that additional, non-ER-mediated mechanisms exist. It has been suggested that the induction of oxidative stress by SERM could be one of the non-ER-mediated mechanisms held responsible for their pro-apoptotic role in ER-negative cells. Tumor cells are known for their high requirement of glutamine (Gln) that serves multiple functions within the cells, including nutritional and energy source, as well as one of the precursors for the synthesis of natural antioxidant glutathione (GSH). We hypothesized that one of the mechanisms responsible for ER-independent anti-neoplastic properties of SERMs and also for their adverse side effects could be dependent on the inhibition of Gln uptake. METHODS: Human ER-negative MDA-MB231 breast cancer cells were treated with different doses of Tam and Ral. Gln uptake was monitored by using [(3)H]Gln assay. The effect of Tam and Ral on Gln transporter ASCT2 expression, glutathione (GSH) levels and cellular proliferation was determined. RESULTS: Tam and Ral inhibited Gln uptake in a dose-dependent manner through inhibition of ASCT2 Gln transporter. This effect of the anti-estrogens was associated with inhibition of GSH production and apoptosis. Treatment of cells with N-acetyl L-cysteine and 17 beta-estradiol 2 reversed the effects of Ral and Tam. CONCLUSIONS: Our results indicate that one of the mechanisms of action (and possibly some of the side effects) of TAM and RAL is associated with inhibition of cellular Gln uptake, oxidative stress and induction of apoptosis.

Estrogen replacement attenuates exaggerated neointimal hyperplasia following carotid endarterectomy in rats.

BACKGROUND: To investigate whether estrogen may attenuate neointima formation in hyperhomocysteinemic rat carotid endarterectomy. METHODS: Rats were divided into 6 groups: ovariectomized estradiol-treated homocysteine or chow; ovariectomized placebo-treated homocysteine or chow; intact placebo-treated homocysteine or chow. Chow served as controls while homocysteine served as exaggerated intimal hyperplasia. Prior to endarterectomy, rats were implanted with estradiol mini-pump or placebo, diets given 2 weeks before and after surgery. Homocysteine, estrogen, and neointimal hyperplasia were determined. RESULTS: Homocysteine was elevated in homocysteine groups versus controls except in estradiol-treated group. Intimal hyperplasia increased in placebo-treated ovariectomized homocysteine versus intact group. Exaggerated intimal hyperplasia in placebo-treated ovariectomized homocysteine was reduced by estrogen and so was homocysteine. Estrogen replacement in ovariectomized homocysteine group reduced intimal hyperplasia to that of intact or ovariectomized controls. CONCLUSION: Estradiol treatment in this ovariectomized hyperhomocysteinemia carotid endarterectomy and resultant attenuation of homocysteine and neointima may have relevance to the beneficial effects of estrogen on hyperplastic response.

The effects of acarbose treatment on intimal hyperplasia in a rat carotid endarterectomy model of diet-induced insulin resistance.

BACKGROUND: Increased carotid restenosis due to revascularization therapy is associated with insulin resistance. We hypothesize that glucose control using acarbose may attenuate intimal hyperplasia in rat carotid endarterectomy model of diet-induced insulin resistance. METHODS: Rats were fed low-fat complex carbohydrate (control) or high-fat sucrose (insulin resistance) for 4 months. Three days preoperatively, some high-fat-sucrose rats were on acarbose, remainder of the rats received placebo. Rat carotids were assessed with duplex pre-and postoperatively. Acarbose and placebo continued for 2 weeks. Glucose, insulin, blood flow velocities and intimal hyperplasia were determined. RESULTS: High-fat sucrose plus acarbose attenuated intimal hyperplasia. Post-drug high-fat sucrose glucose decreased. Blood flow velocities postoperatively elevated above baseline. High-fat sucrose increased blood flow velocities postoperatively, which was attenuated with acarbose. CONCLUSION: Glucose control by acarbose in rat carotid endarterectomy model of diet-induced insulin resistance resulted in attenuation of intimal hyperplasia.

Modulation of p53 and c-myc in DMBA-induced mammary tumors by oral glutamine.

Previous studies established that oral glutamine (GLN) reduced tumor development in implantable and 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer models. This finding was associated with a decrease in tumor glutathione (GSH) levels, while maintaining normal gut, blood, and breast GSH. Alterations in GSH levels contribute to the control of apoptotic and cell cycle-regulating signaling. The aim of this study was to examine the role of dietary GLN on activation of p53 and c-myc, which play critical roles in cancer development and sensitivity to radiation and chemotherapy. Mammary gland carcinomas were induced in rats by DMBA. The rats were gavaged daily with GLN or water (controls), starting 1 wk prior DMBA-application and throughout the duration of the experiment (11 wk after DMBA). Tumor DNA was examined for mutations in p53 exons 5 and 6. Protein and mRNA levels of p53, p21(WAF1/CIP1), PTEN, IGF-IR, mdm2, and c-myc in tumors of GLN-supplemented rats were compared with those of the control rats (received water). The sequencing of p53 showed that it was wild type. Increased phosphorylation of p53, as well as higher mRNA and protein levels of p21(WAF1/CIP1), PTEN, and mdm2, and lower levels of IGF-IR were detected in tumors of GLN-supplemented rats vs. controls. Both phosphorylated c-myc and c-myc mRNA levels were reduced by GLN. The up-regulation of tumor p53 signaling and down-regulation of c-myc, in addition to previously established inhibition of Akt signaling in DMBA-breast cancer model, suggest that dietary GLN could be a useful approach for increasing the effectiveness of cancer treatment.

Timing of oral glutamine on DMBA-induced tumorigenesis.

INTRODUCTION: A single dose of oral 7,12-dimethylbenz(a)anthracene (DMBA) in pubertal rats causes breast tumors by 11 weeks and is associated with ablation of the normal gut glutathione (GSH) production for up to 4 weeks. We hypothesized that glutamine (GLN), known to restore the gut GSH production inhibited by DMBA, given only during this 4-week period, would prevent breast cancer initiation. METHODS: 160 Female Sprague-Dawley rats were divided to 10 groups (n = 16/group): Long Term (LT): DMBA + GLN, DMBA + FA, DMBA + H2O, OIL + GLN, OIL + FA, OIL + H2O; Short Term (ST): DMBA + GLN, DMBA + FA, OIL + GLN, OIL + FA At age 50 days old, rats received a one-time dose of 100 mg/kg DMBA or sesame oil. LT rats were gavaged daily with isonitrogenous GLN, (FA), or water (H2O) the entire study. ST rats were gavaged with GLN, freamine, or H2O the first 4 weeks and then H2O the remaining 7 weeks. All rats were pair-fed defined chow. Rats were sacrificed at 11 weeks, observed for tumors, blood assayed for GLN, GSH, gut GLN and GSH and uptake or production calculated using labeled C-14-PAH. RESULTS: ST and LT GLN were equally effective in preventing tumor formation. GLN doubled gut GSH production in LT animals as compared to all other groups (P < 0.05). Control rats developed no tumors and had superior gut GSH production as compared with tumor-bearing rats. CONCLUSIONS: Oral GLN when given only during the 4 weeks of known gut GSH ablation had the same tumor prevention efficacy as prolonged GLN administration. Not previously reported, GLN appears to affect the initiation of tumor formation in this model.

Effect of glutamine on glutathione, IGF-I, and TGF-beta 1.

BACKGROUND: Our previous results have showed that oral glutamine (GLN) supplementation decreased carcinogenesis in 7,12-dimethylbenz[a]antracene (DMBA) breast cancer model. We also have found that GLN raises blood glutathione (GSH) levels in an implantable breast cancer model. The process of tumor growth was accompanied by depressed GSH production and increased levels of insulin-like growth factor-I (IGF-I) and transforming growth factor beta1 (TGF-beta 1). GSH is counter-regulatory to IGF-I. We therefore hypothesized that in DMBA model of breast cancer, the increased GSH levels seen with oral GLN would be associated with lowered levels of IGF-I &TGF-beta(1). METHODS: Time-dated pubertal Sprague-Dawley rats were gavaged at time 0 with 1 g/kg/day glutamine (GLN) (n = 18), isonitrogenous Freamine (FA) (n = 18), or water (H(2)O) (n = 18). Rats were further randomized on day 7 to 100 mg/kg DMBA or oil. After 14 days, the animals were sacrificed and blood GSH, IGF-1, TGF-beta 1, breast tissue, and gut mucosa GSH levels were measured. RESULTS: Oral GLN increased significantly blood, breast tissue, and gut mucosa levels of GSH in both DMBA and control groups in comparison with the control groups not treated with GLN. At the same time, the levels of blood IGF-I and TGF-beta 1 decreased significantly in both DMBA-treated and control groups. DMBA did not significantly affect any of these levels. CONCLUSIONS ;Oral GLN increased GSH levels and lowered IGF-I and TGF-beta 1 in a range that is considered clinically significant. However, the effect of GLN in maintaining normal gut GSH production in the presence of DMBA was much more significant. Inconsistent with our hypothesis, reduction in IGF and TGF-beta 1 levels did not correlate with DMBA's effect on gut GSH production.

Gut glutathione metabolism and changes with 7,12-DMBA and glutamine.

INTRODUCTION: The mechanism by which oral glutamine (GLN) prevents 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer is unknown. While GLN triples the negative extraction of gut glutathione (GSH) in rats, DMBA significantly disrupts it. Actual gut GSH flux has not been reported. We hypothesized that the gut is a producer of GSH; DMBA blocks gut GSH production and supplemental oral GLN antagonizes this effect. MATERIALS AND METHODS: Eighty Sprague-Dawley rats were randomized to four groups (n = 20/group): DMBA + GLN, DMBA + FA, OIL + GLN, OIL + FA. Rats (age 50 days) were gavaged with a one-time dose of 100 mg/kg DMBA or oil. Rats were gavaged with AES-14 as GLN (1 gm/kg/day) or an isonitrogenous amount of Freamine (FA) from 1 week before till sacrifice at 1 week after DMBA (greatest effect on gut GSH extraction). Arterial and portal blood was taken for GLN and GSH levels, and blood flow was measured using (14)C-PAH. Gut GLN and GSH fluxes (uptake or production) were calculated. RESULTS: DMBA abrogated the normal GSH production (negative flux) in OIL + FA while not affecting GLN metabolism. GLN maintained GSH production in DMBA + GLN. CONCLUSIONS: Oral GLN restores to normal GSH production in DMBA-treated animals suggesting one of the mechanism(s) by which GLN prevents breast cancer in this model. Unchanged uptake of GLN in the DMBA-treated animals may indicate a block in GSH transport rather than actual intracellular production.

Effect of dietary glutamine on tumor glutathione levels and apoptosis-related proteins in DMBA-induced breast cancer of rats.

Glutamine (GLN) is a non-essential amino acid that is present in nearly every biochemical pathway and is the major intraorgan nitrogen carrier. GLN via glutamate, is one of the precursors for the synthesis of glutathione (GSH), the major endogenous antioxidant in mammalian cells, which protects them from oxidative injury and cell death. Cancer cells have higher GSH levels than the surrounding normal cells, which attributes to a higher rate of cell proliferation and resistance to chemotherapy. Therefore, selective tumor depletion of GSH presents a promising strategy in cancer treatment. Experimental studies have associated decreased GSH levels with inhibition of proliferation and stimulation of apoptosis. Previous results of our laboratory have provided evidence that dietary GLN diminished tumor development in implantable as well as 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer and elevated GSH in the host tissues. In this study we examined the effects of GLN on GSH levels in DMBA-induced mammary tumors and correlated the results with protein and mRNA expression of apoptosis-related proteins Bcl-2, Bax and caspase-3 in tumor cells. The results have shown that GLN supplementation caused a significant decrease in the tumor GSH levels and the ratio GSH/oxidized GSH (GSSG), accompanied by up-regulation of Bax and caspase-3, and down-regulation of Bcl-2. These findings suggest that dietary GLN supplementation suppresses mammary carcinogenesis by activation of apoptosis in tumor cells and this probably is a result of GSH down-regulation.