OBJECTIVETo investigate the effect of estrogen on heat shock protein (HSP) 70 expression in rat masseter muscle.

METHODSSixty twelve-week-old female Sprague-Dawley rats were randomly divided into three groups: Sham surgery group (control group), ovariectomy group (OVX group), ovariectomy with estradiol valerate replacement treatment group (OVX/EV group). Half of the animals were sacrificed at 4 and 8 weeks respectively, then the masseter muscle was removed. Immunohistochemistry (IHC) method was employed to study the HSP70 expression in masseter muscle.

RESULTSCompared to control group and OVX/EV group, the expression of HSP70 was significantly lower at 8 weeks in OVX group (P < 0.05). There were no significantly difference between the HSP70 expression of control group and that of OVX/EV group.

CONCLUSIONEstrogen may affect HSP70 expression in rat masseter muscle, and estrogen replacement therapy may prevent HSP70 reduction.

Animals ; Estradiol ; analogs & derivatives ; Estrogens ; Female ; HSP70 Heat-Shock Proteins ; Humans ; Masseter Muscle ; Ovariectomy ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the effect of 2-methoxyestradiol (2-ME2) on apoptosis of human acute T lymphoblastic leukemia cells, and its underlying mechanism.

METHODSThe growth inhibition of CEM cells was detected by MTT assay; apoptotic cells were detected by DNA laddering analysis; the expressions of P53 mRNA and protein were detected by RT-PCR and Western blot respectively.

RESULTS2-ME2 remarkably inhibited the CEM cell growth and the 50% growth inhibitory concentration (IC50) at 48 h was 2 µmol/L. The DNA ladder could be detected in CEM cells after treating with 2 µmol/L 2-ME2 for 24, 48 and 72 hours; after treating with 2 µmol/L 2-ME2 for 24, 48 and 72 hours, a time-dependent reduction of P53 mRNA and protein expressions was found in CEM cells.

CONCLUSIONThe anti-leukemia effect of 2-ME2 is completed through the induction of cell apoptosis. Down-regulation of P53 gene expression may be an underlying mechanism.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Estradiol ; analogs & derivatives ; Genes, p53 ; Humans ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma

The fragmentation pathways of five estrogens (estradiol, estrone, equilin sulfate, 17 a-dihydroequilin sulfate and equilenin sulfate) have been studied with high resolution and high mass accuracy using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF/MS) in the negative ion mode. Molecular weights were obtained from [M-H](-) ions in the product ion spectra. The results indicate that the five structurally similar estrogens have similar fragmentation pathways. Using their stable isotope forms as internal reference compounds, the accurate mass and composition of the fragment ions were determined. During collision-induced dissociation (CID), cleavage is initiated by loss of oxygen atoms from carbon-17, after which D and C rings cleave sequentially and rearrange to finally form stable conjugate structures with highly abundant characteristic fragment ions at m/z 183 (accompanied by m/z 181), m/z 169 and m/z 145 (accompanied by m/z 143). Understanding these characteristic fragmentation pathways of estrogens will be helpful in identifying the structures of steroid hormones in general.
Chemical Fractionation ; methods ; Equilenin ; chemistry ; Equilin ; analogs & derivatives ; chemistry ; Estradiol ; chemistry ; Estrogens ; chemistry ; Estrone ; chemistry ; Ions ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVETo investigate the influence of parathyroid hormone and estrogen on alveolar bone metabolism of castrated female rats.

METHODSSixty-six female Wistar rats which were healthy and 4 months old were divided into two groups, with group SHAM (n = 18) and group ovariectomy (OVX) (n = 48). After 8 weeks of ovariectomy, the osteoporosis model was confirmed by examing 8 ovariectomized and sham-operated rats. The rest 10 rats in group SHAM were the control group (group A). The rest 40 rats in group OVX were divided into ovariectomized group (group B), ovariectomized and treated with estrogen (group C), ovariectomized and treated with parathyroid hormone (group D), ovariectomized and treated with estrogen and parathyroid hormone (group E) at random with 10 in each group. Group A and B injected physiological saline (1 mL x kg(-1)), group C injected estradiol benzoate (10 microg x kg(-1)), group D injected parathyroid hormone (20 microg x kg(-1)), group E injected parathyroid hormone (20 microg x kg(-1)) and estradiol benzoate (10 microg x kg(-1)). The intraperitoneal injection were maken every other day to rats in each group, which continued for 8 weeks. The bone mineral density (BMD), bone histomorphology and serum Ca, P, alkaline phosphatase (ALP) were measured after therapy.

RESULTSAfter 8 weeks of ovariectomy, the lumbar BMD of ovariectomized rats were significantly declined compared with those of the sham-operated rats (P < 0.05). Eight weeks later after the drug use, the BMD, %Tb.Ar, Tb.Th, Tb.N in group C, D, E were slightly elevated compared to group B, especially the group E (P < 0.05). Serum calcium and phosphorus values did not change significantly (P > 0.05). ALP values in group B was significantly higher than that in group A (P < 0.05).

CONCLUSIONIntermittent application of parathyroid hormone in small doses can increase alveolar BMD of castration rats and improve their bone structure. And it can have synergy effects on the treatment of osteoporosis if it is used combining with estrogen.

Alkaline Phosphatase ; Animals ; Bone Density ; Estradiol ; analogs & derivatives ; Estrogens ; Female ; Osteoporosis ; Ovariectomy ; Parathyroid Hormone ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar

OBJECTIVETo study the effect of 2-methoxyestradiol(2-ME) on telomerase activity expression and apoptosis in human myelodysplastic syndrome cells line MUTZ-1.

METHODSMUTZ-1 cells were incubated with different concentrations of 2-ME, apoptosis rate and cell cycle were measured by flow cytometry (FCM). Telomerase activity in MUTZ-1 cells was examined by telomeric repeat amplification protocol-Enzyme linked immunosorbent assay (TRAP-ELISA).

RESULTSThe FCM analysis showed that cells in G0/G1 phase and S phase were decreased, while in G2/M phase increased after exposed to 1,2 and 4 micromol/L of 2-ME for 12 hours (P < 0.05). 1 and 2 micromol/L of 2-ME had no notable effect on MUTZ-1 cells as compared with the control group (P > 0. 05). Cells incubated with 1, 2 and 4 micromol/L of 2-ME for 36 hours were induced apoptosis, the percentage of apoptosis was between (12.87 +/- 0.86)% and (21.82 +/- 1.71)% with a dose- and time- dependent manner. Telomerase activity was significantly inhibited in these concentration and negatively correlated with cell number in G2/M phase (r = -0.979, P = 0.021) and increased apoptosis (r = -0.970, P = 0.030 ), respectively. Moreover, the inhibition effect of telomerase activity was enhanced in a dose- and time- dependent manner.

CONCLUSIONS2-ME-induced apoptosis and inhibition of telomerase activity provide a possible mechanism for explaining the 2-ME's anticancer activity.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line ; Estradiol ; analogs & derivatives ; pharmacology ; Humans ; Myelodysplastic Syndromes ; enzymology ; pathology ; Telomerase ; metabolism

The aim of this study was to investigate the effects of 2-methoxyestradiol (2-ME) on expressions of caspase-3 and survivin in chronic myelocytic leukemia (CML) K562 cells. The experiment was divided into 3 groups: control group, in which K562 cells were cultured in medium without 2-ME; the experimental group, in which K562 cells were cultured in medium containing different concentrations of 2-ME (1, 2, 4, 8 and 16 micromol/L) for 36 hours; the negative control group, in which K562 cells were replaced by distilled water without RNase in medium. The apoptosis rate, the protein and its mRNA expressions of caspase-3 and survivin of K562 cells was detected by TUNEL, flow cytometry (FCM), half-quantitative RT-PCR respectively. The results showed that the apoptosis rate of K562 cells in experimental group was significantly higher than that in control group (p < 0.05). The apoptosis rate of K562 cells detected by FCM was almost the same as that detected by TUNEL method (p < 0.01). The result detected by TUNEL methods was positively correlated with that detected by FCM (gamma = 0.845, p = 0.034). The expression of caspase-3 protein increased in a concentration-dependent manner, and also this expression level in the experimental group was higher than that in the control group (p < 0.05); the expression of survivin protein decreased along with the increasing of 2-ME concentration. and the difference between the experimental group and the control group was statistically significant (p < 0.05). The expression of caspase-3 mRNA was higher in the experimental group than that in the control group (p < 0.01), and the expression of survivin mRNA was lower in the experimental group than that in the control group (p < 0.01). The expression level of caspase-3 mRNA was negatively correlated with that of survivin (gamma = -0.966, p = 0.001). It is concluded that the 2-ME can induce apoptosis of K562 cells in a concentration-dependent manner and indicate its promising potential in the treatment of CML patients.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Estradiol ; analogs & derivatives ; pharmacology ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism

OBJECTIVETo study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment.

METHODSAn eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed.

RESULTSA stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed.

CONCLUSIONExogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Estradiol ; pharmacology ; Estrogen Antagonists ; pharmacology ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Humans ; Tamoxifen ; analogs & derivatives ; pharmacology ; Transfection

OBJECTIVETo establish a mouse model of hypospadias induced by benzoate estradiol to further the studies on the molecular mechanisms of hypospadias.

METHODSA total of pregnant mice were randomly divided into 5 groups, Group A, B, C, D and E, and injected subcutaneously (sc) with estradiol benzoate at the dose of 0, 0.2, 1, 5 and 25 mg x kg(-1) d(-1) respectively from the 12th to the 16th gestational day. The mortality of the newborn mice was recorded and the male neonates of 2 pregnant mice from each group were anatomized to observe the testis position and prostate agenesis on the delivery day. Examinations were made for urethra and cryptorchidism on the 28th postnatal day.

RESULTSThe death rates of the neonates in Group A, B, C, D and E were 21.6%, 21.5%, 41.4%, 56. 6% and 75.0%, respectively. Hypospadias was detected in Group C (3.3%, 1/30), D (20.0%, 4/20) and E (23.0%, 3/13), with significant difference between Group D and A (P < 0.05) and E and A (P < 0.05), but not between Group D and E (P > 0.05). Cryptorchidism was found in Group C (6.6%, 2/30) , D (30.0%, 6/20) and E (61.6%, 8/13), with significant difference between Group D and A (P < 0.05) and E and A (P < 0.05) , but not between Group D and E (P >0.05).

CONCLUSIONExposure of pregnant mice to large dose of estradiol benzoate can induce hypospadias and cryptorchidism in their neonates. And the right dose of estradiol benzoate for the establishment of the mouse model of hypospadias should be 5 mg x kg(-1) x d(-1).

Animals ; Animals, Newborn ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Estradiol ; analogs & derivatives ; toxicity ; Female ; Hypospadias ; chemically induced ; Male ; Mice ; Mice, Inbred ICR ; Pregnancy ; Random Allocation

OBJECTIVETo observe the effect of estrogen and progestin on the blood levels of nitric oxide and angiotensin II in aid of the application of hormone replacement therapy in postmenopausal women.

METHODSThe serum nitric oxide and plasma angiotensin II levels in postmenopausal women were determined before and 3 months after oral intake of estradiol valerate 1 mg/day (n = 10) or estradiol valerate, 1 mg/d plus medroxyprogesterone acetate, 2 mg/d (n = 30).

RESULTSThe serum nitric oxide levels of postmenopausal women were significantly increased by 3 months of oral estradiol valerate 1 mg/d (P < 0.05), whereas the plasma levels of angiotensin II tended to decrease. The positive correlation between the increases of nitric oxide and the changes of estradial 3 months after oral intake of estradiol valerate 1 mg/d was significant. Compared with the baseline, no significant changes were observed in both serum nitric oxide levels and plasma angiotensin II levels 3 months after oral intake of estradiol valerate, 1 mg/d plus medroxyprogesterone acetate, 2 mg/d (P < 0.05).

CONCLUSIONSThe vascular functions can be improved through increasing the serum nitric oxide level after 3-month oral intake of estradiol valerate, 1 mg/d in postmenopausal women, and estradiol valerate plus medroxyprogesterone acetate intake may attenuate the beneficial effects.

Adult ; Angiotensin II ; blood ; Estradiol ; analogs & derivatives ; therapeutic use ; Estrogen Replacement Therapy ; Female ; Humans ; Medroxyprogesterone Acetate ; therapeutic use ; Middle Aged ; Nitric Oxide ; blood ; Postmenopause ; blood

This study was aimed to explore the synergistic effect of 2-methoxyestradiol (2-ME2) and bortezomib (Bor) on the proliferative inhibition and apoptosis of U266 cell line and its possible mechanism. The cells were treated with 2-ME2, Bor alone and 2-ME2 combined with Bor, respectively. The cell viability and proliferative curve were detected by CCK8, the cell apoptosis was detected by caspase 3/7 activity test, cell cycle status was analyzed by flow cytometry, and real-time PCR was used to detect the mRNA expression of P21, BAX and BCL-2. The results showed that compared with cells treated with 2-ME2 or Bor alone, the proliferative potential of cells in combination group was significantly inhibited (p < 0.05), and apoptosis rate markedly increased (p < 0.05), cell cycle was arrested at G(1)-S phase, the mRNA expressive level of P21 and BAX increased, while the expression of BCL-2 decreased. It is concluded that 2-ME2 combined with Bor synergistically inhibits cell proliferation and induces apoptosis in U266 cell line. The possible mechanism may be associated with its effect of up-regulating P21 and BAX expressions.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Drug Synergism ; Estradiol ; analogs & derivatives ; pharmacology ; Humans ; Pyrazines ; pharmacology