A novel role for progesterone and progesterone receptor membrane component 1 in regulating spindle microtubule stability during rat and human ovarian cell mitosis

The present studies were designed to assess the roles of progesterone (P4) and Progesterone Receptor Membrane Component 1 (PGRMC1) in regulating mitosis of spontaneously immortalized granulosa cells (SIGCs) and ovarian cancer cells, SKOV-3 cells. Because PGRMC1 has been detected among the proteins of the human mitotic spindle, we theorized that P4 and PGRMC1 could affect mitosis through a microtubule-dependent process. The present study confirms that SIGC growth is slowed by either P4 treatment or transfection of a PGRMC1 antibody. In both cases, slower cell proliferation was accompanied by an increased percentage of mitotic cells, which is consistent with a P4-induced prolongation of the M phase of the cell cycle. In addition, P4 increased the stability of the spindle microtubules, as assessed by the rate of beta-tubulin disassembly in response to cooling. Also, P4 increased spindle microtubule stability of SKOV-3 cells. This effect was mimicked by the depletion of PGRMC1 in these cells. Importantly, P4 did not increase the stability of the microtubules over that observed in PGRMC1-depleted SKOV-3 cells. Immunofluorescent analysis revealed that PGRMC1 is distributed to the spindle apparatus as well as to the centrosomes at metaphase. Further in situ proximity ligation assay revealed that PGRMC1 interacted with beta-tubulin. Taken together, these results suggest that P4 inhibits mitosis of ovarian cells by increasing the stability of the mitotic spindle. Moreover, P4's actions appear to be dependent on PGRMC1's function within the mitotic spindle.     

Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer

Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.     

Expression of CD40 and growth-inhibitory activity of CD40 agonist in ovarian carcinoma cells

The CD40 receptor is a member of the tumour necrosis factor receptor family and is widely expressed on various cell types. The antitumour activity of CD40 agonist antibody has been observed in B-cell-derived malignancies, but its activity on ovarian cancer remains unclear. However, in this paper, we first confirmed that the anti-CD40 agonist antibody could inhibit the growth of ovarian cancer cells and induce apoptosis. This study investigated the expression of CD40 by ovarian carcinoma tissues and cell lines, at the same time, we evaluated the effect of a recombinant soluble human CD40L (rshCD40L) and an anti-CD40 agonist antibody on cell growth and apoptosis. Flow cytometry and immunohistochemistry assay demonstrated that CD40 was expressed on ovarian carcinoma cell lines and primary ovarian carcinoma cells derived from ascites, as well as on ovarian carcinoma tissues. The growth inhibition of rshCD40L and the anti-CD40 agonist antibody on ovarian carcinoma cells was examined by MTT assay, and the proportion of apoptotic tumour cells was analysed by flow cytometry and Hoechst staining. Our study showed that CD40 was expressed on all ovarian carcinoma cell lines and was examined in 86.2% (162/188) of ovarian cancer tissue samples, but not in normal ovarian tissues (n?=?20). Treatment with rshCD40L or anti-CD40 agonist antibody significantly inhibited ovarian carcinoma cell growth and induced apoptosis. Theses results suggest that CD40 is expressed on ovarian carcinoma cells, moreover, that rshCD40L and anti-CD40 agonist antibody have therapeutic potential to inhibit human ovarian cancer growth.     

Vinculin presents unfavorable prediction in ovarian cancer and prevents proliferation and migration of ovarian cancer cells

The influences of Vinculin on many cancers were blurry, including ovarian cancer. Thus, we concentrated on the efficient role of Vinculin in ovarian cancer and explored the potential mechanism(s). Expression of Vinculin in ovarian cancer tissues and cell lines was investigated by real‐time polymerase chain reaction, immunohistochemistry, and Western blot. The Kaplan–Meier manner with the logrank was performed to assess overall survival. We further evaluated the relations between Vinculin expression and clinicopathological features of ovarian cancer. Moreover, Vinculin was overexpressed or silenced by respectively transfection with pcDNA‐Vinculin or small interfering (si‐Vinculin) into human ovarian cancer cell line Caov3 or human ovarian epithelial cell line (HOEpiC). Thereafter, cell viability, cell apoptosis, and migration were checked by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide, flow cytometer, and scratch assay, respectively. Likewise, the apoptosis‐ and migration‐related proteins were distinguished by Western blot. Compared to the nontumor tissues or HOEpiC cells, Vinculin was significantly lower expressed in the ovarian cancer tissues and cells. Furthermore, we found out that Vinculin was primarily distributed at the cell membrane and cytoplasm. Moreover, Vinculin was negatively associated with International Federation of Gynecology and Obstetrics stage, grade, and distant metastasis. Overexpression of Vinculin dramatically weakened cell viability and migration and stimulated apoptosis. Conversely, suppression of Vinculin showed opposite results. Vinculin presents unfavorable prediction in ovarian cancer and inhibits ovarian cancer proliferation and migration.     

Iron(III)-salophene: an organometallic compound with selective cytotoxic and anti-proliferative properties in platinum-resistant ovarian cancer cells

Background

In this pioneer study to the biological activity of organometallic compound Iron(III)-salophene (Fe-SP) the specific effects of Fe-SP on viability, morphology, proliferation, and cell-cycle progression on platinum-resistant ovarian cancer cell lines were investigated.

Methodology/Principal Findings

Fe-SP displayed selective cytotoxicity against SKOV-3 and OVCAR-3 (ovarian epithelial adenocarcinoma) cell lines at concentrations between 100 nM and 1 µM, while the viability of HeLa cells (epithelial cervix adenocarcinoma) or primary lung or skin fibroblasts was not affected. SKOV-3 cells in contrast to fibroblasts after treatment with Fe-SP revealed apparent hallmarks of apoptosis including densely stained nuclear granular bodies within fragmented nuclei, highly condensed chromatin and chromatin fragmentation. Fe-SP treatment led to the activation of markers of the extrinsic (Caspase-8) and intrinsic (Caspase-9) pathway of apoptosis as well as of executioner Caspase-3 while PARP-1 was deactivated. Fe-SP exerted effects as an anti-proliferative agent with an IC₅₀ value of 300 nM and caused delayed progression of cells through S-phase phase of the cell cycle resulting in a complete S-phase arrest. When intra-peritoneally applied to rats Fe-SP did not show any systemic toxicity at concentrations that in preliminary trials were determined to be chemotherapeutic relevant doses in a rat ovarian cancer cell model.

Conclusion/Significance

The present report suggests that Fe-SP is a potent growth-suppressing agent in vitro for cell lines derived from ovarian cancer and a potential therapeutic drug to treat such tumors in vivo.     

Aromatase expression in ovarian epithelial cancers

Our study focused on aromatase cytochrome P450 (CYP19) expression in ovarian epithelial normal and cancer cells and tissues. Aromatase mRNA expression was analyzed by real-time PCR in ovarian epithelial cancer cell lines, in human ovarian surface epithelial (HOSE) cell primary cultures, and in ovarian tissue specimens (n=94), including normal ovaries, ovarian cysts and cancers. Aromatase mRNA was found to be expressed in HOSE cells, in BG1, PEO4 and PEO14, but not in SKOV3 and NIH:OVCAR-3 ovarian cancer cell lines. Correlation analysis of aromatase expression was performed according to clinical, histological and biological parameters. Aromatase expression in ovarian tissue specimens was higher in normal ovaries and cysts than in cancers (P<0.0001). Using laser capture microdissection in normal postmenopausal ovaries, aromatase was found to be predominantly expressed in epithelial cells as compared to stromal component. Using immunohistochemistry (IHC), aromatase was also detected in the epithelium component. There was an inverse correlation between aromatase and ERalpha expression in ovarian tissues (P<0.001, r=-0.34). In the cancer group, no significant differences in aromatase expression were observed according to tumor histotype, grade, stage and survival. Aromatase activity was evaluated in ovarian epithelial cancer (OEC) cell lines by the tritiated water assay and the effects of third-generation aromatase inhibitors (AIs) on aromatase activity and growth were studied. Letrozole and exemestane were able to completely inhibit aromatase activity in BG1 and PEO14 cell lines. Interestingly, both AI showed an antiproliferative effect on the estrogen responsive BG1 cell line co-expressing aromatase and ERalpha. Aromatase expression was found in ovarian epithelial normal tissues and in some ovarian epithelial cancer cells and tissues. This finding raises the possibility that some tumors may respond to estrogen and provides a basis for ascertaining an antimitogenic effect of AI in a subgroup of ovarian epithelial cancers.     

Association of expression of XIAP-associated factor 1 (XAF1) with clinicopathologic factors, overall survival, microvessel density and cisplatin-resistance in ovarian cancer

XIAP-associated factor 1 (XAF1) was identified as a novel X-linked inhibitor of apoptosis (XIAP) binding partner that can reverse the anti-apoptotic effect of XIAP. XAF1 levels are greatly decreased in many cancer tissues and cell lines. The aim of this study was to investigate the expression of XAF1 and XIAP in advanced epithelial ovarian cancer and role of XAF1 in cisplatin resistance of ovarian cancer cells. Tissues from 94 patients with advanced epithelial ovarian cancer (EOC) and 30 ovarian cystadenomas were obtained. We analyzed the association of the immunohistochemical-determined expression of these two factors and clinicopathologic variables, overall survival, and angiogenesis. We established SKOV3 cells stably overexpressing XAF1 and explored the possible functions of XAF1 in ovarian cancer cells in vitro and in vivo. The protein expression of XAF1 was significantly lower and that of XIAP higher in malignant than nonmalignant tissues. Low XAF1 expression was associated with high-grade tumors and poor overall survival for patients. XAF1 expression was associated with microvessel density. Overexpression of XAF1 suppressed cell proliferation and enhanced SKOV3 cells sensitivity to cisplatin, as well as inhibited tumor growth and decreased MVD in vivo. Overexpression of XAF1 induced XIAP inactivation, caspase-3 activation and cytosolic expression of cytochrome c. These results suggested that XAF1 may be involved in ovarian cancer development and up-regulation of XAF1 may confer sensitivity of ovarian cancer cells to cisplatin-mediated apoptosis.     

Targeting cleavage and polyadenylation specific factor 1 via shRNA inhibits cell proliferation in human ovarian cancer

Cleavage and polyadenylation specificity factor 1 (CPSF1), a member of CPSF complex, has been reported to play a key role in pre-mRNA 3′-end formation, but its possible role in ovarian cancer remains unclear. In the present study, we found the mRNA level of CPSF1 was overexpressed in ovarian cancer tissues using Oncomine Cancer Microarray database. Then the loss-of-function assays, including CCK-8, colony formation and flow cytometry assays, were performed to determine the effects of CPSF1 on cell viability, proliferation, cell cycle and apoptosis of human ovarian cancer cell lines (SKOV-3 and OVCAR-3). The results indicated that depletion of CPSF1 suppressed cell viability, impaired colony formation ability, induced cell cycle arrest at G0/G1 phase and promoted cell apoptosis in ovarian cancer cells. Furthermore, knockdown of CPSF1 upregulated the expression of cleaved caspase-3 and PARP and downregulated CDK4/cyclin D1 expression. These data suggested that CPSF1 could promote ovarian cancer cell growth and proliferation in vitro and its depletion might serve as a potential therapeutic target for human ovarian cancer.     

A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells

Background

Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays.

Principal Findings

LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis.

Conclusion

LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.     

Synthetic retinoids as inducers of apoptosis in ovarian carcinoma cell lines

Apoptosis is also known as programmed cell death. Apoptosis plays an essential role in maintaining normal tissue and cell physiology in multicellular organisms. Clearance of aberrant or pre-cancerous cells occurs through the induction of apoptosis. It has been reported that many tumors and tumor cell lines have dysfunctional apoptosis signaling, causing these tumors to escape immune monitoring and internal cellular control mechanisms. One potential cause of this dysfunctional apoptosis is the tumor suppressor p53, an important regulator of growth arrest and apoptosis that is mutated in over 50% of all cancers. Retinoids have great potential in the areas of cancer therapy and chemoprevention. While some tumor cells are sensitive to the growth inhibitory effects of natural retinoids such as all-trans-retinoic acid (ATRA), many ovarian tumor cells are not. 6-[3-(1-Admantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and fenretinide N-[4-hydroxyphenyl] retinamide (4-HPR) are conformationally restricted synthetic retinoids that induce growth arrest and apoptosis in both ATRA-sensitive and ATRA-resistant ovarian tumor cell lines. Recently, we have identified the molecular pathways of apoptosis induced by treatment of ovarian carcinoma cells with mutated p53 by CD437 and 4-HPR.     

Oestradiol inhibits spontaneous and cisplatin-induced apoptosis in epithelial ovarian cancer cells: relationship to DNA repair capacity

A prospective role of sex steroid hormones in the pathogenesis of common epithelial ovarian cancer remains equivocal. We hypothesized that oestradiol can protect ovarian cells from apoptosis by augmenting their DNA repair capacity. Two established oestrogen receptor-positive human cancer cell lines of ovarian surface epithelial origin (OVCAR-3, SKOV-3) were studied during short-term (24 h) subculture in the absence or presence of oestradiol-17β and/or the DNA-damaging chemotherapeutic agent cisplatin. Apoptosis was monitored among individual cells by in situ DNA fragmentation analysis. Basal rates of apoptosis were diminished by exposure to oestradiol (progesterone or testosterone were without effect). Oestradiol also suppressed apoptosis induced by cisplatin and enhanced the repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene transfected into ovarian cells. The ability of oestrogen-responsive ovarian cancer cells to efficiently repair DNA and thereby avoid apoptosis may be related to propensity for clonal expansion and drug resistance. This revised version was published online in November 2006 with corrections to the Cover Date.     

Mechanisms regulating the constitutive activation of the extracellular signal-regulated kinase (ERK) signaling pathway in ovarian cancer and the effect of ribonucleic acid interference for ERK1/2 on cancer cell proliferation

The ERK1/2 MAPK pathway is a critical signaling system that mediates ligand-stimulated signals for the induction of cell proliferation, differentiation, and cell survival. Studies have shown that this pathway is constitutively active in several human malignancies and may be involved in the pathogenesis of these tumors. In the present study we examined the ERK1/2 pathway in cell lines derived from epithelial and granulosa cell tumors, two distinct forms of ovarian cancer. We show that ERK1 and ERK2 are constitutively active and that this activation results from both MAPK kinase-dependent and independent mechanisms and is correlated with elevated BRAF expression. MAPK phosphatase 1 (MKP-1) expression, which is involved in ERK1/2 deactivation, is down-regulated in the cancer cells, thus further contributing to ERK hyperactivity in these cells. Treatment of these cancer cell lines with the proteasome inhibitor ZLLF-CHO increased MKP-1 but not MKP-2 expression and decreased ERK1/2 phosphorylation. More importantly, silencing of ERK1/2 protein expression using RNA interference led to the complete suppression of tumor cell proliferation. These results provide evidence that the ERK pathway plays a major role in ovarian cancer pathogenesis and that down-regulation of this master signaling pathway is highly effective for the inhibition of ovarian tumor growth.     

Apoptosis induced by progesterone in human ovarian cancer cell line SNU-840

Progesterone has been used as an ingredient of anticancer drug for patients with ovarian carcinoma. However, the mechanism of anticancer effects by progesterone has not been understood. In this study, the effects of progesterone on ovarian cancer cells, SNU-840, were investigated. After the incubation with progesterone, the viability of the cells was evaluated by MTT assay. As a result, 45% of the cells were viable after 48 h of incubation with 100 microM progesterone. In addition, [(3)H]thymidine incorporation assay showed that the proliferation of the cells was completely inhibited by progesterone after 48 h of incubation at 100 microM concentration. Colorimetric TUNEL assay revealed the fragmentation of the chromosomal DNA, suggesting that the process of the cell death was apoptosis. The level of the p53 mRNA was determined by northern blotting assay, since many apoptosis processes are mediated by up-regulation of the p53 expression. The level of the p53 mRNA reached its maximum at 12 h and decreased after 24 h of incubation with progesterone. In conclusion, progesterone inhibits the proliferation and elicits apoptosis of SNU-840 cells. Also, it up-regulates the p53 mRNA transiently.     

Nerve growth factor induces the expression of chaperone protein calreticulin in human epithelial ovarian cells

Epithelial ovarian cancer is highly angiogenic and high expression of Nerve Growth Factor (NGF), a proangiogenic protein. Calreticulin is a multifunctional protein with anti-angiogenic properties and its translocation to the tumor cell membrane promotes recognition and engulfment by dendritic cells. The aim of this work was to evaluate calreticulin expression in human normal ovaries, benign and borderline tumors, and epithelial ovarian cancer samples and to evaluate whether NGF regulates calreticulin expression in human ovarian surface epithelium and in epithelial ovarian cancer cell lines. Calreticulin mRNA and protein levels were analyzed using RT-PCR, Western blot and immunohistochemistry in 67 human ovarian samples obtained from our Institution. Calreticulin expression induced by NGF stimulation in cell lines was evaluated using RT-PCR, Western blot and immunocytochemistry. We found a significant increase of calreticulin mRNA levels in epithelial ovarian cancer samples as compared to normal ovaries, benign tumors, and borderline tumors. Calreticulin protein levels, evaluated by Western blot, were also increased in epithelial ovarian cancer with respect to benign and borderline tumors. When HOSE and A2780 cell lines were stimulated with Nerve Growth Factor, we found an increase in calreticulin protein levels compared to controls. This effect was reverted by GW441756, a TRKA specific inhibitor. These results suggest that NGF regulates calreticulin protein levels in epithelial ovarian cells through TRKA receptor activation.     

Overexpression of the lncRNA FER1L4 inhibits paclitaxel tolerance of ovarian cancer cells via the regulation of the MAPK signaling pathway

To determine how the lncRNA FER1L4 in ovarian cancer cells influences paclitaxel (PTX) resistance, we examined the expression level of FER1L4 in human ovarian epithelial cell lines IOSE80 and HOSEpiC and human ovarian cancer cell lines OVCAR-3, Caov-3, and SKOV3 through RNA isolation and quantitative polymerase chain reaction (qRT-PCR). SKOV3 cell lines were treated with PTX. The cell survival rate and apoptosis rate of SKOV3 and SKOV3-PR at different PTX dose levels were evaluated. Next, qRT-PCR was performed to detect the expression of FER1L4 in SKOV3 and SKOV3-PR cell lines. SKOV3-PR cell lines were transfected with pcDNA3.1 as the control group (SKOV3-PR/pcDNA3.1) or pcDNA3.1-FER1L4 to upregulate the expression level of FER1L4 (SKOV3-PR/pcDNA3.1-FER1L4). The level of cell survival, apoptosis, and colony formation were compared between the two groups using MTT, flow cytometry analysis, and colony formation assay. To reveal the molecular mechanism, we measured the relative protein phosphorylation level of ERK and MAPK in SKOV3, SKOV3-PR, SKOV3-PR/pcDNA3.1, and SKOV3-PR/pcDNA3.1-FER1L4 groups using an enzyme-linked immunosorbent assay. The effects of SB203580 (a p38 MAPK inhibitor) on PTX were also investigated to reveal the function of the MAPK pathway on the PTX tolerance of SKOV3. In comparison with normal ovarian epithelial cells, FER1L4 was downregulated. The FER1L4 level was decreased in human ovarian cancer cells with drug resistance than in common ovarian cancer cells. The upregulation of FER1L4 could promote the PTX sensitivity of ovarian cancer cells. The increased level of FER1L4 could suppress the PTX resistance of ovarian cancer cells through the inhibition of the MAPK signaling pathway.     

Glucocorticoid regulation of SLIT/ROBO tumour suppressor genes in the ovarian surface epithelium and ovarian cancer cells

The three SLIT ligands and their four ROBO receptors have fundamental roles in mammalian development by promoting apoptosis and repulsing aberrant cell migration. SLITs and ROBOs have emerged as candidate tumour suppressor genes whose expression is inhibited in a variety of epithelial tumours. We demonstrated that their expression could be negatively regulated by cortisol in normal ovarian luteal cells. We hypothesised that after ovulation the locally produced cortisol would inhibit SLIT/ROBO expression in the ovarian surface epithelium (OSE) to facilitate its repair and that this regulatory pathway was still present, and could be manipulated, in ovarian epithelial cancer cells. Here we examined the expression and regulation of the SLIT/ROBO pathway in OSE, ovarian cancer epithelial cells and ovarian tumour cell lines. Basal SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 expression was lower in primary cultures of ovarian cancer epithelial cells when compared to normal OSE (P<0.05) and in poorly differentiated SKOV-3 cells compared to the more differentiated PEO-14 cells (P<0.05). Cortisol reduced the expression of certain SLITs and ROBOs in normal OSE and PEO-14 cells (P<0.05). Furthermore blocking SLIT/ROBO activity reduced apoptosis in both PEO-14 and SKOV-3 tumour cells (P<0.05). Interestingly SLIT/ROBO expression could be increased by reducing the expression of the glucocorticoid receptor using siRNA (P<0.05). Overall our findings indicate that in the post-ovulatory phase one role of cortisol may be to temporarily inhibit SLIT/ROBO expression to facilitate regeneration of the OSE. Therefore this pathway may be a target to develop strategies to manipulate the SLIT/ROBO system in ovarian cancer.     

MicroRNA-181a suppresses norethisterone-promoted tumorigenesis of breast epithelial MCF10A cells through the PGRMC1/EGFR–PI3K/Akt/mTOR signaling pathway

BackgroundResearch suggests that hormone replacement therapy may increase the risk of breast cancer, and progestins such as norethisterone (NET) play a key role in this phenomenon. We have demonstrated that microRNA-181a (miR-181a) suppresses NET-promoted breast cancer cell survival. Nonetheless, the effects of NET and miR-181a on the tumorigenesis of human breast epithelial cells have not yet been elaborated.MethodsAssays of cell viability, proliferation, migration, apoptosis, and colony formation were performed to investigate the pro-tumorigenesis effect of NET and the effects of miR-181a on human breast epithelial MCF10A cells. The expressions of cell-proliferation-related genes and apoptotic factors were analyzed by quantitative RT-PCR and Western blot in MCF10A cells treated with NET and miR-181a.ResultsNET significantly increased MCF10A cell viability, proliferation, migration, and colony formation, but reduced cellular apoptosis. In addition, NET increased the expression of progesterone receptor membrane component 1 (PGRMC1), EGFR, B-cell lymphoma 2, cyclin D1, and proliferating cell nuclear antigen, but decreased the expression of pro-apoptosis factors, such as Bax, caspase-7, and caspase-9. Overexpression of miR-181a strongly inhibited the effects of NET on MCF10A cells and abrogated NET-stimulated PGRMC1, EGFR, and mTOR expression.ConclusionsActivation of the PGRMC1/EGFR–PI3K/Akt/mTOR signaling pathway is the primary mechanism underlying the pro-tumorigenesis effects of NET on human breast epithelial MCF10A cells. Additionally, miR-181a can suppress the effects of NET on these cells. These data suggest a therapeutic potential for miR-181a in reducing or preventing the risk of breast cancer in hormone replacement therapy using NET.     

Toll-like receptor expression in normal ovary and ovarian tumors

Recent studies have implicated inflammation in the initiation and progression of ovarian cancer, though the mechanisms underlying this effect are still not clear. Toll-like receptors (TLRs) allow immune cells to recognize pathogens and to trigger inflammatory responses. Tumor cell expression of TLRs can promote inflammation and cell survival in the tumor microenvironment. Here we sought to characterize the expression of TLRs in normal human ovaries, benign and malignant ovarian tumors from patients, and in established ovarian tumor cell lines. We report that TLR2, TLR3, TLR4, and TLR5 are strongly expressed on the surface epithelium of normal ovaries. In contrast to previous studies of uterus and endocervix, we found no cyclic variation in TLR expression occurred in murine ovaries. TLR2, TLR3, TLR4, and TLR5 are expressed in benign conditions, epithelial tumors, and in ovarian cancer cell lines. Variable expression of TLR6 and TLR8 was seen in benign and malignant epithelium of some patients, while expression of TLR1, TLR7, and TLR9 was weak. Normal and malignant ovarian stroma were negative for TLR expression. Vascular endothelial cells, macrophages, and occasional fibroblasts in tumors were positive. Functional activity for TLRs was demonstrated by stimulation of cell lines with specific ligands and subsequent activation and translocation of NFκB and release of the proinflammatory cytokines interleukin-6 and CCL-2. These studies demonstrate expression of multiple TLRs in the epithelium of normal ovaries and in ovarian tumor cells, and may indicate a mechanism by which epithelial tumors manipulate inflammatory pathways to facilitate tumor progression.