Proteomic analysis in mammary glands of rat offspring exposed in utero to bisphenol A

Bisphenol A (BPA) is a ubiquitous environmental contaminant with established endocrine disruptor properties. The objective of our study was to determine the effects of prenatal exposure to BPA on the rat mammary gland proteome in postnatal rats as a first step toward the investigation of translational biomarkers of susceptibility in the human population. Pregnant rats were treated orally with 0, 25 or 250 µg BPA/kg body weight from days 10 to 21 post-conception. Female offspring were euthanized at 21 and 50 days, and mammary glands were collected. Proteomic analysis was conducted using 2-DE, followed by a combination of MALDI-TOF–TOF and LC–MS/MS, which led to the identification of 21 differentially abundant proteins including vimentin, SPARC and 14–3–3. Western blot analysis of key downstream signaling proteins demonstrated increased phospho-AKT, c-Raf, phospho-ERKs-1 and 2, but decreased TGF-β in mammary glands of 50 day old rats exposed prenatally to BPA. Our studies indicate for the first time that key proteins involved in signaling pathways such as cellular proliferation are regulated at the protein level by BPA. This data is expected to aid in the understanding of how BPA may be influencing the susceptibility of the mammary gland to cancer transformation.     

2D difference gel electrophoresis of prepubertal and pubertal rat mammary gland proteomes

Rat mammary gland proteomes at day 21 (prepubertal) and day 50 (late puberty) were compared by 2D difference gel electrophoresis. Two-hundred fifty-one spots were significantly different ( p < 0.05) in abundance. Peptide mass fingerprint analysis of a subset of these proteins identified two significantly over-represented classes including structural and blood proteins (increased), and metabolism-relevant proteins (reduced) in day 50 relative to day 21 glands. This is a first report of mammary gland proteome differences at these important breast cancer-relevant time-points.     

Heavy water labeling method for measuring the effect of genistein on mammary gland carcinogenesis

Heavy water labeling method was applied to measure the effect of genistein on mammary gland carcinogenesis by incoporating ²H from ²H₂O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. In the present study, we followed the study design of Lamartiniere group to evaluate the efficacy of ²H₂O labeling on the measurement of mammary gland carconogenesis. Female Sprague–Dawley rats were fed estrogen-free AIN-93G diet starting 1 week before breeding and continuing through pregnancy and lactation. Female pups were assigned to the following groups on postnatal day 16 and fed AIN-93G diet: vehicle (dimethylsulfoxide) (DMSO), genistein, and estradiol benzoate (EB). On postnatal days 16, 18, and 20, female pups were injected subcutaneously with 500 μg genistein/g body wt, 500 ng EB/g body wt, or an equivalent volume of the vehicle. At day 50 postpartum, half of each group were gavaged with 60 mg dimethylbenz[a]anthracene (DMBA) in perila oil. After 1 week of DMBA treatment, all animals were labeled with ²H₂O by administration of 4% ²H₂O in drinking water after single intraperitonial bolus injection with 99.9% ²H₂O until sacrifice on postnatal day 81. The time-dependent weight gains were observed in all groups throughout the experimental period. The enrichment of body ²H₂O was attained at 1.84–2.47% through oral administration of ²H₂O. Mammary epithelial cell proliferation was measured by enrichment (EM1) of dA from rats. DMBA-treated groups showed higher fractional synthesis than DMBA non-treated groups. The group exposed only to genistein showed significantly lower EM1 (1.46 ± 0.87%) than those of control groups, i.e., the DMBA non-treated group (2.28 ± 0.29%) and the DMBA-treated group (2.32 ± 0.28%). Bromodeoxyuridine (BrdU) immunostaining of mammary tissue revealed that genistein reduced proliferation of the mammary epithelial, and the number of cells stained positive for BrdU both in DMBA-treated groups and DMBA non-treated groups. H&E staining of mammary epithelium also showed that the exposure to genistein decreased proliferation of the mammary epithelium. The epithelium in the rats treated with DMBA showed mostly multiple cell layers, in contrast to the mostly double layer shown in the DMBA non-treated rats. The exposure to genistein in the prepubertal period inhibited mammary epithelial cell proliferation. In conclusion, the ²H₂O labeling results were in good agreement with the results of BrdU incorporation and histomorphometry, which demonstrates that ²H₂O labeling can be used as a tool to measure carcinogenesis.     

Prepubertal octylphenol exposure up-regulate BRCA1 expression,down-regulate ERα expression and reduce rat mammary tumorigenesis

Background: Endogenous estrogens play an important role in the development of breast cancer. Octylphenol (OP) and genistein (GEN) are estrogen-like chemicals. Prepubertal estradiol and genistein exposure can up-regulate BRCA1 mRNA in mammary gland and reduce futuer breast cancer risk. In the present study, the effects of prepubertal exposure to high-dose OP and GEN on mammary carcinogenesis and the association with the expression of BRCA1 and ERα were investigated. Methods: Prepubertal female Sprague–Dawley rats were exposed to 20, 40, 80 mg/kg OP daily from postnatal day (PND) 22–28, subsequently, the rats were given a single dose of 100 mg/kg 7,12-dimethylbenz [a] anthracene (DMBA) on PND42 to induce mammary tumor. Results: The incidence of DMBA-induced mammary tumors significantly decreased when rats were treated with 40 mg/kg OP. BRCA1 mRNA and protein expression were found up-regulated and ERα expression was down-regulated in the mammary tumor when rats were exposed to 40 mg/kg octylphenol. Conclusion: Exposure 40 mg/kg octylphenol can reduce later breast cancer risk in prepubertal Sprague–Dawley rats, the protective effect of OP is associated with persistent up-regulation of BRCA1 and down-regulation of ERα in the mammary tumor.     

Chemoprevention of mammary tumorigenesis and chemomodulation of the antioxidative enzymes and peroxidative damage in prepubertal Sprague Dawley rats by Biochanin A

Although chemopreventive action of Biochanin A against various cancers including that of prostate, breast, colon, and fore-stomach has been reported earlier, none of the studies was made in prepubertal subjects. The present study appears to be the first one on prepubertal rats that indicates the efficacy of the test compound in the prevention of tumorigenesis. The antioxidative status and xenobiotic metabolism were also evaluated to understand the mechanism of Biochanin A induced prevention of cancer. For the tumorigenesis study 500 μg/g bwt of Biochanin A or vehicle dimethyl sulfoxide (DMSO) s.c, was injected at 16th, 18th, and 20th days post-partum followed by the administration of dimethylbenz[a]nthracene (DMBA) (80 μg/g bwt) at 50th day. In another set of experiments, to study the involvement of peroxidative process in the mechanism of action of test compound, different antioxidant parameters were studied following the administration of two different doses of Biochanin A (0.5 and 50 mg/kg bwt, through oral gavage for 10 days) in the prepubertal rats from day 16 post-partum. Results showed a significant reduction in the mammary tumors (more than 40%) in Biochanin A treated animals, as compared to animals treated with DMBA only. Spectrophotometric enzyme estimations revealed that the specific activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione transferase (GST), DT-diaphorase (DTD), and reduced glutathione (GSH) levels were increased, whereas specific activities of lactate dehydrogenase (LDH) and lipid peroxidation (LPO) were decreased significantly, both in liver as well as in mammary gland, in animals treated with Biochanin A prepubertally. These results reveal the possible involvement of the antioxidative and metabolic enzymes in the suppression of cancer burden and incidence in a prepubertal rat model suggesting that the intake of this phytoestrogen at an early stage may help in lowering the risk of mammary tumor.     

Different effects of genistein on molecular markers related to apoptosis in two phenotypically dissimilar breast cancer cell lines

The association between consumption of genistein-containing soybean products and lower risk of breast cancer suggests a cancer chemopreventive role for genistein. Consistent with this suggestion, exposing cultured human breast cancer cells to genistein inhibits cell proliferation, although this is not completely understood. To better understand how genistein works, the ability of genistein to induce apoptosis was compared in phenotypically dissimilar MCF-7 and MDA-MB-231 human breast cancer cells that express the wild-type and mutant p53 gene, respectively. After 6 days of incubation with 50 microM genistein, MCF-7 but not MDA-MB-231 cells, showed morphological signs of apoptosis. Marginal proteolytic cleavage of poly-(ADP-ribose)-polymerase and significant DNA fragmentation were also detected in MCF-7 cells. In elucidating these findings, it was determined that after 2 days of incubation with genistein, MCF-7 but not MDA-MB-231 cells, had significantly higher levels of p53. Accordingly, the expression of certain proteins modulated by p53 was studied next. Levels of p21 increased in both of the genistein-treated cell lines, suggesting that p21 gene expression was activated but in a p53-independent manner, whereas no significant changes in levels of the pro-apoptotic protein, Bax, were found. In MCF-7 cells, levels of the anti-apoptotic protein, Bcl-2, decreased slightly at 18-24 h but then increased considerably after 48 h. Hence, the Bax:Bcl-2 ratio initially increased but later decreased. These data suggest that at the genistein concentration tested, MCF-7 cells in contrast to MDA-MB-231 cells were sensitive to the induction of apoptosis by genistein, but Bax and Bcl-2 did not play clear roles.     

Influence of melatonin on mammary gland growth: in vivo and in vitro studies

Our objective was to determine whether melatonin influenced mammary growth in response to mammogenic hormones. Prepubertal female BALB/c mice were injected for 9 days with 1 microgram of 17 beta-estradiol and 1 mg of progesterone or 17 beta-estradiol/progesterone plus 50, 100, or 200 micrograms of melatonin. Area of the parenchyma and total DNA content of the second thoracic gland were similar between controls and melatonin-injected mice. However, micrograms of DNA/100 mg of mammary tissue were lower in animals treated with 17 beta-estradiol/progesterone plus 200 micrograms of melatonin than in controls. Triglyceride content of mammary glands from animals treated with 100 or 200 micrograms of melatonin/day increased relative to controls. In an in vitro experiment, thoracic mammary glands of 21-day-old mice were cultured for 6 days in a mammogenic milieu of hormones (17 beta-estradiol/progesterone, aldosterone, bovine prolactin, growth hormone, and insulin) with 0 (control), 10(-6), 10(-9), or 10(-12) M melatonin. Relative to controls, 10(-12) M melatonin increased and 10(-6) M melatonin decreased mammary DNA and uptake of [methyl-3H]thymidine. We conclude that high doses of melatonin reduce mammary development in normal mice and that some of this effect may be mediated directly at the mammary tissue.     

Effect of melengestrol acetate on the organ weight & the mammary lobulo-alveolar development in rats

The effect of melengestrol acetate (MGA) on the organ weight and the mammary lobulo-alveolar development in rats was studied. 33 adult female rats were divided into 4 groups: 1) 5 mcg MGA/gm feed; 2) a normal diet; 3) ovariectomized and fed 5 mcg MGA/gm feed; and 4) ovairectomized and fed a normal diet for 30 days when the rats were sacrificed. In the second experiment, 21 primiparous female rats were ovariectomized, and 10 days later 1 group was injected with 2 mcg estradiol for 10 days, while the 2nd group was injected with 50 mcg MGA/day, and the 3rd group with estradiol plus MGA in the above doses. The animals were sacrificed after 10 days of treatment. MGA decreased anterior pituitary, ovary, uterus, and adrenal weight, but enhanced (p less than .01) mammary lobulo-alveolar development in intact rats. No effect on mammary development in ovariectomized rats was noticed whether the drug was given orally or by injection; however uterine and adrenal weights were reduced. MGA plus estradiol caused significant (p less than .01) mammary growth in ovariectomized rats as compared with that in rats given MGA or estradiol alone. Uterine weight was increased slightly after supplementation with estradiol, but adrenal weight did not show improvement. It is suggested that MGA is without any estrogenic activity and therefore requires the presence of ovaries or estrogen to exhibit development of mammary growth.     

Mammary gland morphogenesis is enhanced by exposure to flaxseed or its major lignan during suckling in rats

The exposure of rats to 10% flaxseed (FS) or an equivalent level of its major lignan, secoisolariciresinol diglucoside (SDG), during suckling enhances mammary gland differentiation, which protects against mammary carcinogenesis at adulthood. We determined whether this diet-induced mammary gland differentiation is mediated through the estrogenic pathway via epidermal growth factor receptor (EGFR) and estrogen receptor (ER) signaling. Rats were fed the AIN-93G basal diet (BD) from day 7 of pregnancy until delivery and then randomized to consume BD, FS, or SDG during lactation. After weaning, female offspring were fed BD throughout the experiment. At postnatal day (PND) 21 and the proestrus phase on PND 49-51, mammary glands of offspring were analyzed for morphology, cell proliferation, and expression of EGFR, epidermal growth factor (EGF), transforming growth factor-alpha, ER-alpha, and ER-beta. At PND 21, compared with the BD control, the number of terminal end buds (TEBs) and terminal ducts were increased by FS, whereas mammary epithelial cell proliferation was increased by both FS and SDG, suggesting that mammary morphogenesis was enhanced. Epithelial EGFR and stromal fibroblast EGF were increased by SDG, whereas epithelial ER-beta was decreased by FS. Conversely, at PND 49-51, a lower number of TEBs but a higher ratio of lobules to TEBs with decreased expression of EGFR or EGF was observed in both treatment groups. EGFR expression was positively associated with EGF expression and cell proliferation in TEB epithelium at PND 21. Urinary lignans of lactating dams were related to their offspring's indices of mammary gland development. In conclusion, exposure to FS or SDG during suckling enhanced mammary gland morphogenesis by modulation of EGFR and ER signaling, which led to more differentiated mammary glands at PND 49-51. The physiological outcomes of FS and SDG were similar, which suggests that SDG is partly responsible for the mammary gland differentiation effect.     

Effects of iron deficiency and training on mitochondrial enzymes in skeletal muscle

We measured mitochondrial enzyme activities in skeletal muscle under conditions of iron deficiency and endurance training to assess the effects of these interventions on the contents and proportions of non-iron-containing and iron-dependent enzymes and proteins. Male Sprague-Dawley rats, 21 days of age, received a diet containing either 6 (iron deficient) or 50 mg iron/kg diet (iron sufficient). At 35 days of age animals were subdivided into sedentary and endurance training groups (running at 0.7 mph, 0% grade, 45 min/day, 6 days/wk). By 70 days of age, iron deficiency had decreased gastrocnemius muscle cytochrome c by 62% in sedentary animals. In contrast, the activities of tricarboxylic acid cycle enzymes were increased, remained unchanged or were slightly decreased, indicating that iron deficiency markedly altered mitochondrial composition. Endurance training increased cytochrome c (35%), tricarboxylic acid cycle enzymes (approximately 15%), and manganese superoxide dismutase (33%) in iron-deficient rats, whereas the same exercise regimen had no effect on the skeletal muscle of iron-sufficient animals. The interactive effect of dietary iron deficiency and mild exercise on mitochondrial enzymes suggests that adaptation to a training stimulus is, to some extent, geared to the relationship between the energy demand of exercise and the capacity for O2 transport and utilization.     

Effect of dietary polyunsaturated fat and 7,12-dimethylbenz(a)-anthracene on rat splenic natural killer cells and prostaglandin E synthesis

Summary Dietary polyunsaturated fat has been shown to stimulate mammary tumorigenesis induced in rats by 7,12-dimethylbenz(a)anthracene (DMBA). Studies were undertaken to investigate the effect of polyunsaturated fat and DMBA on splenic natural killer (NK) activity and prostaglandin E (PGE) synthesis. In a first experiment, splenic NK activity at 33, 55, 75, and 110 days of age was measured in Sprague-Dawley rats fed 0.5% low fat (LF), 5% normal fat (NF), or 20% high fat (HF) corn oil diets from 23 days of age. At 55 days of age, half of the rats from the 75 and 110 day age groups were given 5 mg DMBA. Ten days after the initiation of the diets splenic NK activity against YAC-1 lymphoma was decreased from 50% cytotoxicity in rats fed NF diet to 21% cytotoxicity in rats fed HF diet, but was not affected by LF feeding. No difference in NK activity was observed among the groups at the later time periods. DMBA had no effect on NK activity at 20 or 55 days after its administration. In a second experiment, where DMBA (15 mg/rat) was given to half of the rats at 50 days of age and NF or HF diets were started 3 days later, NK activity was 35% in rats fed NF diet and 21% in rats fed HF diet, 5 days after the diets were started. No difference in NK activity in rats fed either diet was observed at later time periods. DMBA decreased both NK activity and spleen cellularity transiently. In both experiments, PGE synthesis by spleen cells cultured for 18 h was not affected by dietary fat intake, but was slightly increased 3 days after DMBA administration. Results from these experiments suggest that the stimulation of DMBA-induced mammary tumorigenesis by polyunsaturated fat and by DMBA itself may possibly be mediated by a transient decrease in splenic NK cell activity.This work was supported by grants CA-35641, CA-33240, CA-13038 and Core Grant CA-24538 from the National Cancer Institute     

The effect of dietary zinc - and polyphenols intake on DMBA-induced mammary tumorigenesis in rats

Background

The aim of the study was to investigate the effect of dietary supplementation with zinc and polyphenol compounds, i.e. resveratrol and genistein, on the effectiveness of chemically induced mammary cancer and the changes in the content of selected elements (Zn, Cu, Mg, Fe, Ca) in tumors as compared with normal tissue of the mammary gland.

Methods

Female Sprague-Dawley rats were divided into study groups which, apart from the standard diet and DMBA (7,12-dimethyl-1,2- benz[a]anthracene), were treated with zinc ions (Zn) or zinc ions + resveratrol (Zn + resveratrol) or zinc ions + genistein (Zn + genistein) via gavage for a period from 40 days until 20 weeks of age. The ICP-OES (inductively coupled plasma optical emission spectrometry) technique was used to analyze the following elements: magnesium, iron, zinc and calcium. Copper content in samples was estimated in an atomic absorption spectrophotometer.

Results

Regardless of the diet (standard; Zn; Zn + resveratrol; Zn + genistein), DMBA-induced breast carcinogenesis was not inhibited. On the contrary, in the Zn + resveratrol supplemented group, tumorigenesis developed at a considerably faster rate. On the basis of quantitative analysis of selected elements we found - irrespectively of the diet applied - great accumulation of copper and iron, which are strongly prooxidative, with a simultaneous considerable decrease of the magnesium content in DMBA-induced mammary tumors. The combination of zinc supplementation with resveratrol resulted in particularly large differences in the amount of the investigated elements in tumors as compared with their content in normal tissue.

Conclusions

Diet supplementation with zinc and polyphenol compounds, i.e. resveratrol and genistein had no effect on the decreased copper level in tumor tissue and inhibited mammary carcinogenesis in the rat. Irrespectively of the applied diet, the development of the neoplastic process in rats resulted in changes of the iron and magnesium content in the cancerous tissue in comparison with the healthy mammary tissue. The application of combined diet supplementation with zinc ions and resveratrol considerably promoted the rate of carcinogenesis and increased the number of DMBA-induced mammary tumors.     

Exposure to lard-based high-fat diet during fetal and lactation periods modifies breast cancer susceptibility in adulthood in rats

The present study investigated whether early life exposure to high levels of animal fat increases breast cancer risk in adulthood in rats. Dams consumed a lard-based high-fat (HF) diet (60% fat-derived energy) or an AIN93G control diet (16% fat-derived energy) during gestation or gestation and lactation. Their 7-week-old female offspring were exposed to 7,12-dimethyl-benzo[a]anthracene to induce mammary tumors. Pregnant dams consuming an HF diet had higher circulating leptin levels than pregnant control dams. However, compared to the control offspring, significantly lower susceptibility to mammary cancer development was observed in the offspring of dams fed an HF diet during pregnancy (lower tumor incidence, multiplicity and weight), or pregnancy and lactation (lower tumor multiplicity only). Mammary epithelial elongation, cell proliferation (Ki67) and expression of NFκB p65 were significantly lower and p21 expression and global H3K9me3 levels were higher in the mammary glands of rats exposed to an HF lard diet in utero. They also tended to have lower Rank/Rankl ratios (P=.09) and serum progesterone levels (P=.07) than control offspring. In the mammary glands of offspring of dams consuming an HF diet during both pregnancy and lactation, the number of terminal end buds, epithelial elongation and the BCL-2/BAX ratio were significantly lower and serum leptin levels were higher than in the controls. Our data confirm that the breast cancer risk of offspring can be programmed by maternal dietary intake. However, contrary to our expectation, exposure to high levels of lard during early life decreased later susceptibility to breast cancer.     

Interaction of zearalenone and soybean isoflavone on the development of reproductive organs, reproductive hormones and estrogen receptor expression in prepubertal gilts

The aim of the present study was to investigate the interactive effect of zearalenone (ZEA) and soybean isoflavone (ISO) on the development of reproductive organs, reproductive hormones and estrogen receptor expression in prepubertal gilts. Ninety 75-day-old female pigs (Duroc × Landrace × Yorkshire, 26.50 ± 0.60 kg) were randomly allocated to nine diet treatments during the 21 day study. The experiment employed a 3 × 3 factorial design using a non-soybean meal diet with addition of 0, 0.5 or 2.0mg/kg ZEA and 0, 300 or 600 mg/kg ISO. The results indicated that diets supplemented with 600 mg/kg ISO could reduce the increased weight of the reproductive organs induced by ZEA at 2mg/kg (P<0.05) while feed containing ISO and 0.5mg/kg ZEA increased the weight of the reproductive organs compared with pigs fed diets with 0.5mg/kg ZEA alone. Diets with ISO at 600 mg/kg reduced the large width of vulvas induced by diets with 2mg/kg ZEA (P<0.05). Simultaneous provision of ZEA and ISO to prepubertal gilts increased the level of E2 at days 7 and 14, but decreased it at day 21 (P<0.05). Pigs simultaneously fed 2mg/kg ZEA and 600 mg/kg ISO had the highest level of FSH (P<0.05). There was a significant interaction (P<0.05) between ZEA and ISO supplementation on the level of LH, and pigs offered diets with 2mg/kg ZEA and 600 mg/kg ISO had the lowest level of LH on days 14 and 21. Animals supplemented simultaneously with ZEA and ISO showed higher ERα/ERβ mRNA expression compared to those offered diets containing 0.5mg/kg ZEA alone or basal diets. However, this simultaneous supplementation resulted in a lower level of ERα/ERβ mRNA expression compared to offering diets with 2mg/kg ZEA alone. It appears that ISO can counteract the estrogenic influence of a high dosage of ZEA (2mg/kg). This affect might be attributed to competitive binding with estrogen receptors, thereby weakening the estrogenic effect of ZEA. Meanwhile, interactions between ZEA and ISO may interfere with the functioning of E2, FSH and LH in prepubertal gilts.     

Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M(r) proteins and was complemented by the 2-DE/MS method, which showed a preference for low M(r) proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied.     

Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.     

Proteomics of Medicago sativa cell walls

A method for the sequential extraction and profiling by two-dimensional gel electrophoresis (2-DE) of Medicago sativa (alfalfa) stem cell wall proteins is described. Protein extraction included freezing, grinding in a sodium acetate buffer, separation by filtration of cell walls from cytosolic contents, and extensive washing. Cell wall proteins were then extracted sequentially with a solution containing 200 mM CaCl2 and 50 mM sodium acetate, followed by extraction with 3.0 M LiCl and 50 mM sodium acetate. Cell wall proteins from both the CaCl2 and LiCl fractions were profiled by 2-DE. Approximately 150 protein spots were extracted from these two gels, digested with trypsin, and analyzed using nanoscale HPLC coupled to a hybrid quadrupole time-of-flight (Q-tof) tandem mass spectrometer (LC/MS/MS). More than 100 proteins were identified and used in conjunction with the 2-DE profiles to generate proteomic reference maps for cell walls of this important legume. Identified proteins include classical cell wall proteins as well as proteins traditionally considered as non-secreted. Two unique extracellular proteins were also identified.     

Genistein regulation of transforming growth factor-alpha, epidermal growth factor (EGF), and EGF receptor expression in the rat uterus and vagina.

Epidemiological reports and laboratory data have associated soy and genistein with reduced incidence of uterine, breast, and prostate cancers, cardiovascular disease and osteoporosis, and lower total blood cholesterol. The aim of this study was to investigate the effect of genistein in the uterus and vagina of rats, focusing our attention on the distribution of transforming growth factor (TGF) alpha, epidermal growth factor (EGF), and EGF receptor. A pharmacological dose of genistein (500 microg/g body weight) injected in rats on days 16,18, and 20 postpartum resulted in significant uterine wet weight gain, with hypertrophy of the luminal and glandular epithelium of the uteri, and squamous epithelium of the vagina in 21-day-old animals. At 50 days of age, hypertrophy was no longer evident in the uterus and vagina. Prepubertal genistein treatment resulted in significantly increased EGF immunostaining in individual stromal cells and reduced EGF receptor immunostaining in blood vessels of the uterus. Genistein-treated rats had decreased TGF-alpha immunostaining in glandular and luminal epithelium and a slight increase in EGF receptor immunostaining in stromal cells of the uterus. This suggests paracrine interaction between cells elevating the level of EGF ligand in the stroma and the EGF receptor in the luminal and glandular epithelium, resulting in uterine hypertrophy. In the vagina, genistein did not cause significant alterations to the EGF-signaling pathway in 21- and 50-day-old rats. We conclude that pharmacological doses of genistein during the prepubertal period can modulate the EGF-signaling pathway in the uterus and exert a uterotrophic response in a short-term manner.