Realgar, a traditional Chinese medicine, induces apoptosis of HPV16-positive cervical cells through a HPV16 E7-related pathway.

PURPOSE: In this study, we investigated the effect of Realgar on the apoptosis of HPV16-positive cervical cells in vitro. METHODS: The effect of Realgar on the apoptosis of HPV16-positive cervical cells was investigated by annexin V-fluorescein isothiocyanate/propidium iodide staining and growth inhibition assays using HPV16-positive cervical cancer cell line SiHa and HPV16-positive immortalized cervical epithelial cell line S12. The expression of genes was measured by real-time PCR, and the expression of corresponding proteins was detected by Western blotting. The adhesion and invasion of cells were detected by adhesion assay and Transwell invasion assay, respectively. RESULTS: The Realgar inhibited the proliferation and induced the apoptosis of SiHa and S12 cells in a dose- and time-dependent manner. The Realgar suppressed the expression of HPV16 E7 and caspase-3. The Realgar suppressed the adhesion and invasion of both cells. CONCLUSION: The Realgar induced apoptosis, inhibited the proliferation of HPV16-positive cell lines through a HPV16 E7-dependent pathway, and inhibited cell adhesion and invasion.

Lentiviral vector-mediated down-regulation of Notch1 in endometrial stem cells results in proliferation and migration in endometriosis.

The recent characterization of stem/progenitor cells in the endometrium has shed new light for pathogenesis of endometriosis. The present study was undertaken to investigate the role of Notch1, known as a cell fate regulator, in the mechanism of endometriosis. Influence of Notch1 on endometrial stem cells proliferation and migration was evaluated by knocking down Notch1 expression using shRNA. Furthermore, human endometrial stromal and epithelial stem cells with or without LV-Notch1-shRNA were injected into the peritoneal cavity of nude mice, to assess the in vivo effects of a specific antagonist of Notch1 on the progression of endometriosis. The results showed that LV-Notch1-shRNA led to a significant decline of clonogenicity and migration in human endometrial stem cells in vitro, as well as the size of endometriotic lesions in murine models. These data provide evidence that specific inhibition of Notch1 alters endometriotic tissue growth and progression, and may represent a promising potential therapeutic avenue.

Analysis of WNT9B mutations in Chinese women with Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer­Rokitansky­Küster­Hauser (MRKH) syndrome is a rare congenital female genital anomaly, which is caused by aplasia of the caudalportion of the Müllerian duct. The WNT9B gene encodes a secretory glycoprotein essential for the caudal extension of the Müllerian duct during embryonic development in mice. Coding regions and exon/intron boundaries of the WNT9B gene were amplified and sequenced in 42 Chinese women with MRKH syndrome and 42 controls. Two novel heterozygous mutationswere identified,which were absent in controls. Onewas amissensemutation in exon 1, and the other was located in the 30-untranslated region. Both variants were detected in one out of 42 patients. The two novel mutations may be pathogenic variants in MRKH patients and warrant further functional study.

HOXA10 expression is decreased by testosterone in luteinized granulosa cells in vitro.

Polycystic ovary syndrome (PCOS) is perhaps the most prevalent endocrine disorder in women of reproductive age, characterized by elevated levels of circulating androgens or clinical manifestations of androgen excess. The specific cytokine profile of PCOS patients is probably related to the lower implantation rate, since follicular fluid appears to function as an embryotrophic agent. For a better understanding of the local regulation of human follicles, the present study investigated the protein expression levels and cellular localization of HOXA10 in granulosa cells (GCs) from women with normal ovarian function undergoing IVF due to their husbands suffering from azoospermia. We demonstrated by immunohistochemical studies that the expression of HOXA10 was mainly localized in the cytoplasm of GCs. Our data indicate that these alterations were associated with changes in the expression of ovarian transcription factors of HOXA10. GC dose-responsive decreases in HOXA10 protein were observed in response to physiological or supraphysiological concentrations (10-4 to 10-7 M) of testosterone. These data reveal that testosterone may be involved in HOXA10 gene regulation in GCs. Decreased HOXA10 expression in GCs treated with testosterone suggest that this androgen is responsible for the decreased expression of HOXA10 in PCOS patients.

Realgar-induced apoptosis of cervical cancer cell line Siha via cytochrome c release and caspase-3 and caspase-9 activation.

OBJECTIVE: To explore the molecular mechanism of realgar-induced apoptosis of cervical cancer cells. METHODS: The cervical cancer cell line Siha was used to determine the cell viability and apoptosis after treatment with realgar using MTT assay and flow cytometry. The activities of caspase-3, -8, and -9 were detected by fluorescence resonance energy transfer technology and colorimetric assay, while the levels of Bcl-2, cytochrome c, and Bax were detected by Western blot method. RESULTS: Induction of apoptosis by realgar was detected in Siha cell line in a dose-dependent manner. The apoptosis was accompanied by a significant increase in cytochrome c release and activation of caspase-3 and caspase-9 but not caspase-8. Further, the realgar-induced apoptosis was inhibited by a broad-spectrum caspase inhibitor, a caspase-3 inhibitor, and a caspase-9 inhibitor but not by a caspase-8 inhibitor. Bcl-2 and Bax protein expressions were not changed by realgar. CONCLUSION: The induction of apoptosis by realgar is mediated through a cytochrome c-dependent pathway, which sequentially activates caspase-9 and caspase-3.

Change profiles in matrix metalloproteinase-2 and -9 in induced endometriosis in mice.

To examine the changes in matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in the development and progression of endometriosis, real time quantitative polymerase chain reaction, enzyme-linked immunoabsorbent assay and gelatin zymography were employed to determine the mRNA and protein levels and activities of MMP-2 and MMP-9 from the first day to the 21(st) day after the induction in mice with induced endometriosis (experimental group) and sham-operated animals (controls). The results showed that the mRNA and protein levels and activities of the MMP-2 and MMP-9 were significantly increased on the first day after the induction and the level of MMP-2 stayed at a level higher than that in the control group. MMP-9 had two or three peaks during the 21 days, taking place at day 1, 4 and 15. It is concluded that the changes in the MMP-2 and MMP-9 might be involved in pathogenesis of endometriosis.

15-Epi-lipoxin A(4) inhibits the progression of endometriosis in a murine model.

OBJECTIVE: To examine the pro-resolution actions of 15-epi-lipoxin A(4) (LXA(4)) on endometriotic lesions, on the concentrations and activities of matrix metalloproteinases (MMP-2 and MMP-9) in murine endometriosis. DESIGN: Prospective, vehicle-controlled experimental study. SETTING: Animal research facility. ANIMAL(S): BALB/c mice. INTERVENTION(S): Endometriosis (EM) was induced in 30 mice. Fifteen of them were administered LXA(4) for 24 days (LXA(4) group), whereas the other 15 served as a control group (EM group). Another 15 sham-operated mice (sham-operated group) were treated with vehicles. MAIN OUTCOME MEASURE(S): The weight of the endometriotic lesions was measured. The concentrations, mRNA, and activities of MMP-2 and MMP-9 were determined by enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and gelatin zymography, respectively. RESULT(S): Compared with EM group, the weight of the endometriotic lesions was decreased, the concentrations of MMP-2 and MMP-9 dropped, the mRNA levels of MMP-2 and MMP-9 in the peritoneal fluid cells and the endometriotic lesions were reduced, and the activities of MMP-2 and MMP-9 were inhibited in the LXA(4) group. CONCLUSION(S): LXA(4) may inhibit the progression of endometriosis possibly by lowering the concentrations and the activities of MMP-2 and MMP-9.

Change Profiles in Matrix Metalloproteinase-2 and-9 in Induced Endometriosis in Mice / 华中科技大学学报（医学）（英德文版）

To examine the changes in matrix metalloproteinase-2(MMP-2)and-9(MMP-9)in the development and progression of endometriosis,real time quantitative polymerase chain reaction,enzyme-linked immunoabsorbent assay and gelatin zymography were employed to determine the mRNA and protein levels and activities of MMP-2 and MMP-9 from the first day to the 21st day after the induction in mice with induced endometriosis(experimental group)and sham-operated animals (controls).The results showed that the mRNA and protein levels and activities of the MMP-2 and MMP-9 were significantly increased on the first day after the induction and the level of MMP-2 stayed at a level higher than that in the control group.MMP-9 had two or three peaks during the 21 days,taking place at day 1,4 and 15.It is concluded that the changes in the MMP-2 and MMP-9 might be involved in pathogenesis of endometriosis.

The inhibitory effect of 15-R-LXA4 on experimental endometriosis.

OBJECTIVE: To determine the pro-resolution actions of 15-R-LXA(4) (LXA(4)) on endometriotic lesions and on the expression of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in the murine endometriosis model. STUDY DESIGN: In a prospective, placebo-controlled experimental study, endometriosis was induced in thirty BALB/c mice, and fifteen sham-operated mice served as negative controls. Among the thirty mice with induced endometriosis, fifteen were administered 15-R-LXA(4) and acted as study group (LXA(4) group) and the other fifteen were used as positive controls (EM group). Then all the mice were sacrificed and the endometriotic lesions were weighed. The mRNA and protein levels of IL-1beta and TNF-alpha in the peritoneal fluid were quantified by using real-time polymerase chain reaction (PCR) and enzyme-linked immunoabsorbent assay (ELISA). RESULT(S): Compared with the positive controls, 15-R-LXA(4) reduced the weight of the endometriotic lesions, decreased the concentrations of IL-1beta and TNF-alpha, and lowered the mRNA levels of IL-1beta and TNF-alpha in peritoneal fluid cells. CONCLUSION(S): These findings suggest that 15-R-LXA(4) inhibits the progression of endometriosis possibly by suppressing the gene and protein expression of IL-1beta and TNF-alpha.

Induction of SiHa cells apoptosis by nanometer realgar suspension and its mechanism.

The effects of nanometer realgar suspension on proliferation and apoptosis of human uterine cervix cancer cell line SiHa cells and oncogenic genes HPV16E6/E7 were investigated. A "micro-jet efflux" strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations (6.25, 12.5, 25 and 50 mg/L) for different durations (12, 24, 48 and 72 h). The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy (TEM) and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry (FCM). The expression of HPV16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25 50 mg/L nanometer realgar suspension for 48 h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time-and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group (P<0.01), and G(0)/G(1) phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50 mg/L for 48 h. RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

[Realgar nanometer suspension inducing apoptosis of Siha cell and its effect on expression of HPVE6/E7 oncogene].

OBJECTIVE: To study the growth-inhibitory and apoptosis-inducing effects of realgar nanometer suspension in human carcinoma cervical cell Siha line, and the effect on HPV16E6/E7 oncogene expression. METHOD: A " micro-jet efflux" strategy was used for the preparation of realgar nanometer suspension. Siha cells were treated with various concentrations (6.25, 12.5, 25, 50 mg x L(-1)) of realgar nanometer suspension for different hours (12, 24, 48, 72 h). The effect of realgar nanometer suspension on Siha cell growth suppression was detected by MTT method. Special morphological changes of apoptosis were observed by light and transmission electron microscopy (TEM) and DNA fragments electrophoresis. The apoptotic rates were quantified by flow cytometry (FCM). The expression of HPV16E6/E7 mRNA was assayed by RT-PCR. RESULT: After being treated with 25-50 mg x L(-1) realgar nanometer suspension for 48, 72 h, the survival of Siha cells decreased, and the rate of apoptosis markedly increased. With TEM and DNA electrophoresis, the special morphological changes were found. The apoptotic rates of Siha cells treated with realgar nanometer suspension were significantly higher than those in the control group (P < 0.01). G0-G1 phase arrest appeared following the treatment with realgar nanometer suspension in 25 and 50 mg x L(-1) 48 h. RT-PCR assay revealed that realgar nanometer suspension reduced HPV16E6/E7 gene expression. CONCLUSION: Realgar nanometer suspension can inhibit the proliferation of human carcinoma cervical cell Siha line and induce the cell apoptosis. The mechanism may be related to the down-regulation of HPV16E6/E7 oncogene expression.

Induction of SiHa Cells Cells Apoptosis by Nanometer Realgar Suspension and Its Mechanism / 华中科技大学学报（医学）（英德文版）

The effects of nanometer realgar suspension on proliferation and apoptosis of human uterine cervix cancer cell line SiHa cells and oncogenic genes HPV16E6/E7 were investigated. A "micro-jet efflux" strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations (6.25,12.5,25 and 50mg/L) for different durations (12,24,48 and 72h). The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MIT method. Special morphological changes of apoptosis were observed by transmission electron microscopy (TEM) and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry (FCM). The expression of HPV16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25-50mg/L nanometer realgar suspension for 48h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group (P<0.01), and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50mg/L for 48h.RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

[Influence of hypoxia inducible factor-1alpha on cervical cancer cell line HeLa in vitro].

OBJECTIVE: To explore the direct influence of hypoxia inducible factor-1alpha (HIF-1alpha) on the development of invasive cervical cancer and the possible molecular mechanism. METHODS: Recombinant antisense targeting HIF-1alpha eukaryotic expression vector was constructed and transfected into cultured human cervical cancer cell line HeLa to reduce the expression of HIF-1alpha and its effect on cell proliferation, apoptosis, invasion and the cascade downstream gene expression of HIF-1alpha, including vascular endothelial growth factor (VEGF), glucose transport 1 (GLUT1) and multidrug resistance 1 (MDR1) genes was observed. The chemical method using CoCl(2) to induce hypoxia environment of growing cell was performed. Cells were divided into six groups, NN (normal non-transfected), NI (normal invalid transfected), NT (normal transfected), HN (hypoxia non-transfected), HI (hypoxia invalid transfected), and HT (hypoxia transfected). Methyl thiazolyl tetrazolium (MTT), flow cytometry, and Transwell methods were performed to evaluate the proliferation, invasion and apoptosis, and RT-PCR method was used to detect the gene expression of HIF-1alpha, VEGF, GLUT1 and MDR1. RESULTS: After induction of hypoxia by CoCl(2), the change of gene expression of HIF-1alpha in HN (or HI) group compared to that in NN (or NI) group was not obvious (P > 0.05), but expression of VEGF, GLUT1 and MDR1 were all enhanced and overall proliferation was promoted, apoptosis inhibited [(11.46 +/- 0.28)% vs (29.27 +/- 0.18)%, (15.77 +/- 0.49)% vs (31.13 +/- 0.08)%], and transmembrane behavior enhanced [(37 +/- 12)% vs (26 +/- 7)%, (40 +/- 9)% vs (28 +/- 5)%], and the variations were significant (P < 0.05). On the contrary, transfection with pcDNA3.0/HIF-1alpha was companied by declined gene expression of HIF-1alpha (NT: 0.05 +/- 0.12, HT: 0.04 +/- 0.16), and all the variations were significant (P < 0.05). CONCLUSIONS: HIF-1alpha may participate in malignant biological behaviors of cervical cancer such as anti-apoptosis, accelerating proliferation, increasing supply of blood and energy, increased resistance to chemotherapy through upregulation of its downstream genes. Suppression of HIF-1alpha expression in vitro can inhibit cervical cancer cell line.

The expression of cyclooxygenase-2 in cervical cancers and Hela cells was regulated by estrogen/progestogen.

To investigate the relationship between the expression of cyclooxygenase-2 (COX-2) and menstrual cycle, the regulatory effects of 17-beta-estradiol (E(2)) and medroxyprogesterone acetate (MPA) on the expression of COX-2 in cervical cancer Hela cells were examined. Cervical cancer specimens were obtained from 47 pre-menopausal patients. The phase of menstrual cycle was determined by case history and HE staining of uterine endometrium. COX-2 was immunohistochemically stained by SABC staining and the staining intensity was determined with computerized image analysis system. Hela cells were incubated with alcohol, E(2), E(2)+MPA, MPA for 12, 24 and 48 h respectively. The expression of COX-2 in Hela cells was detected by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Our results showed that the expression of COX-2 was significantly higher during proliferative phase than secretory phase (P<0.05), but there was no difference in the positive rate between proliferative phase and secretory phase (P>0.05). Incubation with E(2) could significantly enhance the expression of COX-2 continually. On the contrary, E(2)+MPA and MPA alone could decrease the expression of COX-2 as compared with the control and E(2) group (P<0.05 and P<0.01 respectively). It is concluded that the expression of COX-2 in cervical cancer of pre-menopausal patients and Hela cells was regulated by estrogen/progestogen.

[Expressions of receptor-binding cancer antigen expressed on SiSo cells, vascular endothelial growth factor, and matrix metalloproteinase-9 in cervical carcinoma and correlation thereof with the invasion and metastasis of the cancerous tissues].

OBJECTIVE: To investigate the expression of receptor-binding cancer antigen expressed on SiSo cells (RCAS1), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9) in early invasive cervical carcinoma and their relationship with the invasion and metastasis of the cervical cancerous tissues. METHODS: Samples of cervical tissues were obtained from 95 cases of early invasive carcinoma of cervix (ICC) and 82 cases of cervical intraepithelial neoplasm (CIN) during operation. 24 samples of normal cervical epithelium (NCE) were obtained during resection of hysteromyomas. Immunohistochemistry (S-P method) was used to detect the expression of RCAS1, VEGF, and MMP-9 in these samples. The relationship between those indexes and the factors related to clinical pathology of cervical carcinoma were analyzed statistically. RESULTS: The positive protein expression rates of RCAS1, VEGF, and MMP-9 in different samples all increased in the sequence of NCE, CIN, and ICC (0, 39.0%, and 72.6% respectively for RCAS1, 16.7%, 41.5%, and 67.4% respectively for VEGF, and 18.2%, 53.7%, and 78.9% respectively for MMP-9), with a significant difference between any 2 groups (all P < 0.001). The expression of RCAS1 protein was correlated with pelvic lymph node metastasis, intravascular and stromal infiltration, and histological grading (P = 0.001 or 0.000); but not correlated with patient's age, clinical stage, and histological types (all P > 0.05). The expression of VEGF and MMP-9 was correlated with pelvic lymph node metastasis, intravascular and stromal infiltration, and clinical stage (all P < 0.05); but not correlated with patient's age, histological grading, and histological types (all P > 0.05). There was an obviously positive correlation between the RCAS1 expression and VEGF expression (r = 0.882, P < 0.01), between the RCAS1 expression and MMP-9 expression (r = 0.868, P < 0.01), as well as between the VEGF and MMP-9 expression (r = 0.765, P < 0.01) in the cervical cancer. CONCLUSION: The positive expression of RCAS1, VEGF, and MMP-9 in invasive cervical carcinoma may play an important role in the development, lymph node metastasis, intravascular and stromal involvement of cancer cells. The expression of some metastasis related genes can be used to estimate the metastasis potentiality and is helpful for the treatment.

[Correlation of RCAS1 expression to human papillomavirus 16 (HPV16) infection in cervical cancer].

BACKGROUND & OBJECTIVE: Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is highly expressed in various human cancer cells and is related to tumor escape from host immune surveillance. This study was to investigate the correlation of RCAS1 expression to human papillomavirus 16(HPV16) infection in cervical carcinoma, and explore their clinical significance. METHODS: The expression of RCAS1 and HPV16 E7 protein in 71 specimens of cervical carcinoma, 76 specimens of cervical intraepithelial neoplasia, and 20 specimens of normal cervical tissue was detected by SP immunohistochemistry. RESULTS: RCAS1 was mainly expressed on cellular membrane and in cytoplasm of tumor cells, whereas the expression of HPV16 E7 was mainly confined to the nuclei. The positive rate of RCAS1 was 0.00% in normal cervical tissue, 39.47% in cervical intraepithelial neoplasia, and 77.46% in cervical cancerû the positive rate of HPV16 E7 was 0.05% in normal cervical tissue, 28.94% in cervical intraepithelial neoplasia, and 61.97% in cervical cancer. The positive rates of RCAS1 and HPV16 E7 tended to increase along with tumor progression (P<0.05). The expression of RCAS1 in cervical cancer was significantly related to histological grade (P=0.002), but had no correlation to age, clinical stage, and histological classification (P>0.05). The expression of HPV16 E7 in cervical cancer was significantly related to histological classification (P<0.001), but had no correlation to age, clinical stage, and histological grade (P>0.05). RCAS1 expression was positively correlated to HPV16 infection in cervical carcinoma (r=0.780, P<0.001). CONCLUSIONS: The expression of RCAS1 in cervical cancer is significantly increased, and has correlation with malignant degree of cervical carcinoma. Some RCAS1-positive cervical cancer tissues are infected by HPV16.

Matrix metalloproteinase-2 and -9 expression correlated with angiogenesis in human adenomyosis.

BACKGROUND: Adenomyosis is an important cause of dysmenorrhea and infertility for women all over the world, however, the pathogenesis has not been completely elucidated. The purpose of the study was to investigate the role of matrix metalloproteinases (MMPs) and their relation to angiogenesis in human adenomyosis. METHODS: Adenomyotic endometrial specimens were removed by hysterectomy from 68 women with adenomyosis. The control group consisted of 26 normal endometrial specimens. Immunohistochemistry was used to demonstrate expression of MMP-2, -9, vascular endothelial growth factor (VEGF) and microvessel density (MVD). Staining intensity was analyzed by computerized image analysis system. RESULTS: In both eutopic and ectopic endometrium of adenomyosis, the expression of MMP-2, -9 as well as VEGF was significantly greater than in normal endometrium (p < 0.05). MVD was higher in ectopic endometrium than eutopic endometrium with or without adenomyosis (p < 0.05). In adenomyosis, a positive correlation was observed between VEGF expression and MMP-2 (p < 0.001, r = 0.583) as well as MMP-9 expression (p = 0.002,r = 0.490). A positive correlation was also found between MVD and MMP-2 (p < 0.001,r = 0.589) or MMP-9 expression (p < 0.001,r = 0.589). CONCLUSION: Our results indicate that the elevation of MMP-2, -9 expression may have an important role in the development of adenomyosis, probably through contributing to invasion of endometrial tissues into the myometrium and angiogenesis in adenomyotic implants.