OBJECTIVETo establish a bioluminescent MDA-MB-231 cell line which can stably express luciferase and green fluorescent protein to allow bioluminescent imaging in nude mouse models bearing human triple-negative breast cancer xenografts.

METHODSThe lentivirus carrying luc2, eGFP and neo fusion genes were packaged in 293T cells via calcium phosphate co-precipitation. Human triple-negative breast cancer cell line MDA-MB-231 was infected by the lentivirus, and the positive cell clones were tested for eGFP and luc2 expressions by fluorescence microscopy and Xenogen IVIS200 bioluminescent imaging system, respectively. MTT assay, transwell invasion assay and wound healing assay were performed to evaluate the changes in the proliferation, invasion and migration abilities of the infected cells. The cells were then orthotopically implanted into the right second mammary fat pat of female BALB/c nude mice. The tumor growth was monitored by the in vivo imaging system every week, and the tumor tissues were harvested to evaluate the in vivo stability and tumorigenicity of the modified cells using cryosection and HE staining.

RESULTSThe lentivirus-infected MDA-MB-231cells could stably express luc2 and eGFP, and the luciferase activity reached 9689 phontons/s/per cell. No significant changes occurred in the biological activities of the lentivirus-infected MDA-MB-231 cells. We successfully established the nude mouse model bearing orthotopically implanted human triple-negative breast cancer cells.

CONCLUSIONThe modified MDA-MB-231 cell line can be detected sensitively at the primary implantation site and distant metastasis site in nude mice, which provides a convenient and sensitive platform for the research of metastatic mechanism and new antitumor drugs of human triple-negative breast cancer. The combination of eGFP and luc2 is superior to single reporter gene.

Animals ; Brain Neoplasms ; secondary ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Genes, Reporter ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; biosynthesis ; genetics ; Luminescent Measurements ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism

Steroid cell tumor, not otherwise specified (NOS), are rare ovarian tumor, in addition, it is more rare in children. The majority of these tumors produce several steroid hormones, particularly testosterone. Estrogen also secreted by steroid cell tumor, NOS, but it is uncommon. Furthermore, hypertension is an infrequent sign in steroid cell tumor, NOS. An 8.5-yr-old girl with hypertension and frequent vaginal spotting visited at our clinic. On laboratory evaluation, secondary hypertension due to an elevated plasma renin level and isosexual pseudoprecocious puberty was diagnosed. Right solid ovarian mass was detected in radiologic tests. She underwent a right ooporectomy and it revealed renin and progesterone receptor positive steroid cell tumor, NOS. After operation, her blood pressure returned to normal level and vaginal bleeding disappeared. Even though this case is very rare, when hypertension coincides with virilization or feminization, a renin-secreting ovarian steroid cell tumor, NOS, should be considered.
Child ; Female ; Humans ; Hypertension/*etiology ; Ovarian Neoplasms/complications/*diagnosis/pathology ; Puberty, Precocious/enzymology/*etiology ; Receptors, Cell Surface/metabolism ; Receptors, Progesterone/metabolism ; Renin/blood ; Sex Cord-Gonadal Stromal Tumors/complications/*diagnosis/pathology ; Steroids/biosynthesis ; Tomography, X-Ray Computed ; Vacuolar Proton-Translocating ATPases/metabolism

OBJECTIVETo study the regulatory role of BRCA1 in the expression of progesterone receptors A and B (PRA and PRB) in breast cancer cells.

METHODSBreast cancer MCF-7 cells were transfected with pFlag-CMV2-BRCA1 wt plasmid containing a full-length BRCA1 cDNA or with BRCA1-specific siRNA via lipofectamine 2000 to induce overexpression or suppressed expression of BRCA1, respectively. Twenty-four hours after the transfection, the cells were incubated in fresh culture medium containing 100 nmol/L progesterone for 24 h. The total RNA extract or whole cell lysate was prepared for detecting BRCA1, PRA and PRB expressions using RT-PCR and Western blotting.

RESULTSThe protein expressions of PRA and PRB were significantly decreased whereas their mRNA expressions remained unchanged in MCF-7 cells overexpressing BRCA1. In MCF-7 cells with BRCA1 knock-down, in contrast, the PRA and PRB protein expressions were markedly increased.

CONCLUSIONIn breast cancer cells, exogenous and endogenous BRCA1 can both down-regulate the expressions of PRA and PRB at the protein level.

BRCA1 Protein ; biosynthesis ; genetics ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Receptors, Progesterone ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo explore the regulatory effect and mechanism of Ningxin Hongqi Capsule on local ovarian autocrine and paracrine factors in peri-menopausal rats.

METHODSSD female rats aged 4 months were allocated in a normal control group (A) and those aged 14 months with vagino-cytologic figure of oestrus elongation were allocated in a senile female rat model group (B). Rats in Group B were subdivided into 5 groups randomly as the B1, B2 and B3 subgroups treated respectively with high, moderate and low dose Ningxin Hongqi Capsule, the B4 subgroup treated with estradiol and the B5 subgroup untreated for control. Rats' ovaries were obtained at the end of the experiment for observing the conditions of ovarian growing follicles and corpus luteum by HE staining, determining expressions of ovarian estradiol receptor (ER), progesterone receptor (PR), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin alpha (INHalpha), activin (ACT) alpha-beta, follistatin (FS), and insulin-like growth factor (IGF-1).

RESULTSAs compared with Group B5, the ovary index, number of growing follicle were higher and levels of FSH and LH were lower in Group B2 and B3, expression of ER was higher in Group B1 and B4, IGF-1 and INHalpha was higher in Group B2 and B3, and ACTalpha-beta and FS were lower (all P < 0.05).

CONCLUSIONNirigxin Hongqi Capsule could adjust and balance the local ovarian autocrine and paracrine factors to improve the ovarian function.

Animals ; Autocrine Communication ; drug effects ; physiology ; Capsules ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Models, Animal ; Ovary ; drug effects ; metabolism ; physiology ; Paracrine Communication ; drug effects ; physiology ; Perimenopause ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Estradiol ; biosynthesis ; Receptors, FSH ; biosynthesis ; Receptors, Progesterone ; biosynthesis

To investigate the role of progesterone receptor (PR) expression in malignant melanoma (MM), PR and proliferative cell nuclear antigen (PCNA) expression were immunohistochemistrically evaluated in a series of 35 specimens of MM, and the correlation between the immunohistochemistrical findings and clinicopathological data was also analyzed. PR expression was detected in 25.7% (9/35) of the patients with MM. No PR expression was observed in nevi. PR expression was inversely correlated with PCNA expression (r=-0.353, P=0.026). PR expression was slightly increased in females, subjects aged under 55 y, those with ulceration, non-acral subtype and diagnosis delay longer than 1 y, but the difference was not statistically significant. Selective expression of progesterone receptor in malignant melanoma might be correlated with inhibited tumor growth.
Gene Expression Regulation, Neoplastic ; Immunohistochemistry ; Melanoma/*metabolism ; Models, Biological ; Prognosis ; Proliferating Cell Nuclear Antigen/*metabolism ; Receptors, Progesterone/*biosynthesis ; Receptors, Progesterone/genetics ; Skin/metabolism ; Skin Neoplasms/metabolism

OBJECTIVETo observe the influence of acupuncture on embryo implantation in rat model of embryo implantation dysfunction, and to primarily explore its possible mechanisms.

METHODSPregnant rats were randomly allocated into the control group, the model group and the acupuncture group. The pregnancy rate and average number of blastocyst were observed, the serum levels of estrodiol (E2), progesterone (P4) and prolactin (PRL) were detected by RIA, and the protein and mRNA expression of progesterone receptor (PR) and prolactin receptor (PRLR) in endometrial tissue of implantation site were determined using immunohistochemical assay and RT-PCR respectively.

RESULTSThe pregnancy rate and average number of blastocyst were significantly higher in the acupuncture group than those in the control group respectively (P <0.01). The serum levels of P4 and PRL as well as the protein and mRNA expression levels of PR and PRLR in the model group were significantly lower than those in the other two groups (P<0.05).

CONCLUSIONAcupuncture can promote embryo implantation in rats to a certain degree, and its mechanism might be related with the effects of acupuncture in mediating the sexual hormone levels and the receptor expression of rats.

Acupuncture Therapy ; Animals ; Embryo Implantation ; physiology ; Embryonic Development ; physiology ; Estradiol ; blood ; Female ; Immunohistochemistry ; Male ; Pregnancy ; Progesterone ; blood ; RNA, Messenger ; genetics ; metabolism ; Radioimmunoassay ; Random Allocation ; Rats ; Rats, Wistar ; Receptors, Progesterone ; biosynthesis ; genetics ; Receptors, Prolactin ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the effect of bushen tiaochong recipe (BSTCR) on rats' ovarian granulosa cell (GC) proliferation, steroidogenesis and follicle-stimulating hormone receptor (FSHR), and insulin-like growth factor-1 (IGF-1) mRNA expression using serum pharmacological method.

METHODSRats' GCs were incubated with 10% blank serum (as negative control group), follicle-stimulating hormone (FSH)-containing serum (S-FSH, as positive control group), or BSTCR (in different dosages) containing serum (S-BSTCR, as the BSTCR groups) for 48 h. 3H-TdR incorporation was then performed; DNA was measured to analyze the distribution of GCs in the cell cycle and their proliferation index (PI) using a flow cytometer; estradiol (E2) and progesterone (P) content in the culture fluid were examined by radioimmunoassay; and levels of FSHR and IGF-1 mRNA expression in GCs were measured by real-time RT-PCR.

RESULTSA dose-dependent increase of 3H-TdR incorporation in GC was shown in the BSTCR groups. Cells in G0/G1 phase had markedly less, while those in S phase had a significantly higher increase in the BSTCR groups compared with the negative control group. A high value of PI was also shown in the BSTCR groups, especially in the high dose group where the influence of cell proliferation was stronger than that in the positive control group. The levels of E2 and P in the BSTCR groups of all dosages were significantly higher than those in the negative control group, and did not show any significant difference compared with those in the positive control group. Levels of FSHR and IGF-1 mRNA expression in the BSTCR groups increased in a dose-dependent manner at levels higher than those in the negative control group.

CONCLUSIONS-BSTCR can obviously stimulate the proliferation and steroidogenesis of ovarian GCs. It is speculated that BSTCR could play a regulatory action on ovarian function through two different pathways of endocrine and autocrine by promoting FSHR and IGF-1 mRNA expression.

Animals ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; DNA ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; Estradiol ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Granulosa Cells ; cytology ; drug effects ; Humans ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Progesterone ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, FSH ; genetics ; metabolism ; Steroids ; biosynthesis ; Tritium

OBJECTIVETo explore the estrogenic activity of ethanol extract from adzuki bean Phaseolus angularis and its effect on progesterone receptor (PR) level of human breast cancer MCF-7 cells.

METHODThe modified MCF-7 cell proliferation assay was used to evaluate the estrogenic activity of adzuki bean extract. And its effect on PR mRNA and protein were addressed using RT-PCR measurements and western blotting analysis respectively in which pure estrogen receptor antagonist ICI 182,780 was employed as the tool.

RESULTThe estrogenic activity of adzuki bean extract at various concentrations (10-200 microg x mL(-1)), expressed as proliferative effect (PE) relative to that of solvent control, was examined. The results indicated that the extract of adzuki bean was able to induce MCF-7 cell growth with a maximum at 100 microg x mL(-1). The results of RT-PCR and western blotting showed that PR mRNA and protein could be significantly induced by adzuki bean extract in MCF-7 cells. Moreover, the specific estrogen receptor antagonist ICI 182,780 could block these reactions.

CONCLUSIONEthanol extract of adzuki bean has the estrogen-like activities through the estrogen response pathway.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Estradiol ; analogs & derivatives ; pharmacology ; Estrogen Antagonists ; pharmacology ; Female ; Humans ; Phaseolus ; chemistry ; Phytoestrogens ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Progesterone ; biosynthesis ; genetics ; Seeds ; chemistry

OBJECTIVETo investigate the expression of interleukin-8 (IL-8) and its prognostic significance in breast cancer.

METHODSExpression of IL-8 in 113 breast cancers, 19 breast benign tumors and 20 breast normal tissues was examined by tissue microarray using immunohistochemistry, and the association of IL-8 expression with patient's clinico-pathological characteristics and prognosis was further analyzed.

RESULTSThe positive rate of IL-8 expression in breast cancer was 27.4%, which was significantly higher than that in benign tumor and normal tissue of breast (P = 0.002). IL-8 expression related to histological type (P = 0.040) and lymph node status (P = 0.021). The expression of IL-8 was observed to correlate negatively with ER and PR status (P = 0.015 and P = 0.034), and correlate positively with C-erbB-2 status (P = 0.002). In addition, Kaplan-Meier curves of disease-free survival analysis showed a significant difference between IL-8 positive groups and negative group (P = 0.031).

CONCLUSIONSIL-8 might be a poor prognostic factor for human breast cancer, and also might be a novel molecular marker to predicate the occurrence and progression of breast cancer.

Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; diagnosis ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Interleukin-8 ; biosynthesis ; Lymphatic Metastasis ; Middle Aged ; Prognosis ; Receptor, ErbB-2 ; biosynthesis ; Receptors, Estrogen ; biosynthesis ; Receptors, Progesterone ; biosynthesis

OBJECTIVETo investigate the correlation between the lack of estrogen receptor (ER) gene expression and hypermethylation of ER gene, and detect whether re-expressed ER protein is activated.

METHODSThe methylation status of ER gene promoter in the ER-negative breast cancer cells was evaluated by methylation specific PCR (MSP) and genomic sequencing. The expression of ER and progesterone receptor (PR) mRNA as well as the production of ER protein were detected by RT-PCR and Western blot method, respectively. MTI assay was used to examine the function of re-expressed ER protein.

RESULTSThe ER gene promoter was highly methylated, while ER mRNA and ER protein were not expressed in the ER-negative breast cell line MDA-MB-231. The ER-negative breast cells treated with demethylating agent 5 -aza-2'-deoxycytidine (5-AZA-2'-deoxyC) restored the expression of ER mRNA and ER protein. Expression of the endogenous ER-responsive PR gene was activated and the methylation of ER gene was simultaneously decreased. After MDA-MB-231 was treated with 5-AZA-2'-deoxyC, the protein of ER was re-expressed and the growth of cells treated with tamoxifen were inhibited significantly (P < 0.05).

CONCLUSIONinactivation of ER gene has a close relationship with the abnormal methylation of ER gene promoter. 5-AZA-2'-deoxyC may effectively cause demethylation and restore functional expression of ER silenced by aberrant hypermethylation. The result may offer a new measure and theory for breast cancer patients with ER-negative expression to receive endocrine therapies.

Antimetabolites, Antineoplastic ; pharmacology ; Antineoplastic Agents, Hormonal ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Base Sequence ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Methylation ; Estrogen Receptor alpha ; biosynthesis ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Progesterone ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Selective Estrogen Receptor Modulators ; pharmacology ; Tamoxifen ; pharmacology