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Ovarian cancer has a unique tumor microenvironment (TME) that enables cancer-associated fibroblasts (CAFs) to interact with cellular and matrix constituents and influence tumor development and migration into the peritoneal cavity. Collagen type XI alpha 1 (COL11A1) is overexpressed in CAFs; therefore this study examines its role during CAF activation in epithelial ovarian cancer (EOC). Coculturing human ovarian fibroblasts (HOFs) with high COL11A1-expressing EOC cells or exposure to the conditioned medium of these cells prompted the expression of COL11A1 and CAF phenotypes. Conversely, coculturing HOFs with low COL11A1-expressing EOC cells or COL11A1-knockdown abrogated COL11A1 overexpression and secretion, in addition to CAF activation. Increased p-SP1 expression attributed to COL11A1-mediated extracellular signal-regulated kinase activation (ERK) induced p65 translocation into the nucleus and augmented its binding to the insulin-like growth factor binding protein 2 (IGFBP2) promoter, ultimately inducing TGF-β3 activation. The CAF-cancer cell crosstalk triggered interleukin-6 release, which in turn promoted EOC cell proliferation and invasiveness. These in vitro results were confirmed by in vivo findings in a mouse model, showing that COL11A1 overexpression in EOC cells promoted tumor formation and CAF activation, which was inhibited by TGF-β3 antibody. Human tumors with high TGF-β3 levels showed elevated expression of COL11A1 and IGFBP2, which was associated with poor survival. Our findings suggest the possibility that anti-TGF-β3 treatment strategy may be effective in targeting CAFs in COL11A1-positive ovarian tumors.

Simple Summary High collagen type XI alpha 1 (COL11A1) levels are associated with tumor progression, chemoresistance, and poor patient survival in several cancer types. We examined the role of miRNAs in regulating COL11A1 expression. This study highlighted the importance of miR-335 in downregulating COL11A1-mediated ovarian tumor progression, chemoresistance, and poor survival and suggested its potential application as a therapeutic target. Abstract High collagen type XI alpha 1 (COL11A1) levels are associated with tumor progression, chemoresistance, and poor patient survival in several cancer types. MicroRNAs (miRNAs) are dysregulated in multiple cancers, including epithelial ovarian carcinoma (EOC); however, the regulation of COL11A1 by miRNAs in EOC remains unclear. We examined the role of miRNAs in regulating COL11A1 expression. We identified miR-509 and miR-335 as the candidate miRNAs through an online database search. EOC cell treatment with miR-335 mimics abrogated COL11A1 expression and suppressed cell proliferation and invasion, besides increasing the sensitivity of EOC cells to cisplatin. Conversely, treatment with miR-335 inhibitors prompted cell growth/invasiveness and chemoresistance of EOC cells. miR-335 inhibited COL11A1 transcription, thus reducing the invasiveness and chemoresistance of EOC cells via the Ets-1/MMP3 and Akt/c/EBPβ/PDK1 axes, respectively. Furthermore, it did not directly regulate PDK1 but increased PDK1 ubiquitination and degradation through COL11A1 inhibition. In vivo findings highlighted significantly decreased miR-335 mRNA expressions in EOC samples. Furthermore, patients with low miR335 levels were susceptible to advanced-stage cancer, poor response to chemotherapy, and early relapse. This study highlighted the importance of miR-335 in downregulating COL11A1-mediated ovarian tumor progression, chemoresistance, and poor survival and suggested its potential application as a therapeutic target.

Background Focal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca 2 + /calpain-dependent proteolysis and disassembly. Here, we investigated how Ca 2 + influx induces cascades of FA turnover in living cells. Methods Images obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca 2 + ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover. Results Vinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA. Conclusions These results suggest that Ca 2 + -mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca 2 + -mediated FA turnover. General significance This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca 2 + .

Additional file 4 : Fig. S4. SOCE inhibitors decrease migration ability of IGROV1-WT cells. In vitro (a, b) transwell migration assay and (c, d) wound healing assay were performed to evaluate the effect of SOCE inhibitors (2 μΜ SKF-96365, 10 μΜ YM-58483, 0.1 μM 2-APB) on IGROV1-WT cells. a Representative photomicrographs of cells that penetrated a filter of pore size 8 μm. Scale bars, 200 μm. b Migrated cells were counted in 15 random fields on the lower surface of the filters and expressed as a percentage (%) of SOCE inhibitor pretreated cells compared with DMSO control (Ctrl). c Cells were seeded into silicon inserts with 10% FBS medium. Following cell adhesion, inserts were removed and incubated for 96 h. Phase images were captured every 24 h and wound spaces were analyzed using ImageJ. d Cellular migratory ability is presented as the percentage of wound closure. Each bar represents mean ± SEM from three independent experiments. *: significant difference between cells treated with SOCE inhibitors and DMSO control (Ctrl). *: p

Additional file 2 : Fig. S2. Chemoresistant IGROV1 sublines exhibit high adhesive ability. Cell adhesion assay was performed 30 min after seeding to evaluate the adhesion ability of IGROV1 parental (WT) and chemoresistant cells (CP, SRT). a Nuclear staining by DAPI showed remaining cells after PBS washing. b Quantitative analyses of adherent cells per field. Each bar represents mean ± SEM from at least 300 cells of three independent experiments. *: significant difference between chemoresistant (CP, SRT) and parental (WT) cells. ***: p

Additional file 3 : Fig. S3. Effect of SOCE inhibitors on Ca2+ elevation in IGROV1-WT cells. a Pre-incubation of IGROV1-WT cells with 2 μM fura-2/AM and SOCE inhibitors (2 μΜ SKF-96365, 10 μΜ YM-58483, 0.1 μM 2-APB) at 37 °C for 30 min for cytosolic Ca2+ measurement using a single-cell fluorimeter. Depletion of ER lumen-resident Ca2+ was induced by treating cells in Ca2+-free buffer with 2 μM thapsigargin for 10 min. Representative tracings show the subsequent elevation of Ca2+, indicating that SOCE occurred during the exchange of Ca2+-free buffer to 2 mM Ca2+ buffer for 5 min. The data in representative curves for the measurement of SOCE from three independent experiments. b SOCE-mediated total Ca2+ elevation was calculated from area under the curve. a.u., arbitrary unit. Each bar represents mean ± SEM from at least 120 cells. *: significant difference between cells treated with SOCE inhibitors and DMSO control (Ctrl). ***: p

Additional file 1 : Fig. S1. Chemoresistant IGROV1 sublines exhibit characteristics of epithelial-to-mesenchymal transition (EMT). a Phase contrast images of parental (WT) and chemoresistant (CP, SRT) IGROV1 cells. Scale bars, 100 μm. b Epithelial markers (E-cadherin, ZO-1; blue rectangles) and mesenchymal marker (vimentin, fibronectin, and N-cadherin; red rectangles) were detected using immunoblotting in IGROV1 parental (WT) and chemoresistant (CP, SRT) cells. β-actin served as the internal control.

Ca2+ plays a significant role in linking the induction of apoptosis. The key anti-apoptotic protein, Bcl-2, has been reported to regulate the movement of Ca2+ across the ER membrane, but the exact effect of Bcl-2 on Ca2+ levels remains controversial. Store-operated Ca2+ entry (SOCE), a major mode of Ca2+ uptake in non-excitable cells, is activated by depletion of Ca2+ in the ER. Depletion of Ca2+ in the ER causes translocation of the SOC channel activator, STIM1, to the plasma membrane. Thereafter, STIM1 binds to Orai1 or/and TRPC1 channels, forcing them to open and thereby allow Ca2+ entry. In addition, several anti-cancer drugs have been reported to induce apoptosis of cancer cells via the SOCE pathway. However, the detailed mechanism underlying the regulation of SOCE by Bcl-2 is not well understood. In this study, a three-amino acid mutation within the Bcl-2 BH1 domain was generated to verify the role of Bcl-2 in Ca2+ handling during ER stress. The subcellular localization of the Bcl-2 mutant (mt) is similar to that in the wild-type Bcl-2 (WT) in the ER and mitochondria. We found that mt enhanced thapsigargin and tunicamycin-induced apoptosis through ER stress-mediated apoptosis but not through the death receptor- and mitochondria-dependent apoptosis, while WT prevented thapsigargin- and tunicamycin-induced apoptosis. In addition, mt depleted Ca2+ in the ER lumen and also increased the expression of SOCE-related molecules. Therefore, a massive Ca2+ influx via SOCE contributed to caspase activation and apoptosis. Furthermore, inhibiting SOCE or chelating either extracellular or intracellular Ca2+ inhibited mt-mediated apoptosis. In brief, our results explored the critical role of Bcl-2 in Ca2+ homeostasis and the modulation of ER stress.

// Wen-Tai Chiu 1,2 , Yu-Fang Huang 3 , Huei-Yu Tsai 4 , Chien-Chin Chen 5,6 , Chang-Hwa Chang 2 , Soon-Cen Huang 7 , Keng-Fu Hsu 3,4 and Cheng-Yang Chou 3,4 1 Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan 2 Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan 3 Department of Obstetrics and Gynecology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan 4 Cancer Research Center, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan 5 Department of Pathology, Chia-Yi Christian Hospital, Chiayi, Taiwan 6 Department of Cosmetic Science, Chia Nan University of Pharmacy and Science, Tainan, Taiwan 7 Department of Obstetrics and Gynecology, Chi Mei Medical Center, Liouying Campus, Tainan, Taiwan Correspondence: Cheng-Yang Chou, email: // Keywords : ovarian cancer, FOXM1, β-CATENIN, chemoresistance, stemness Received : October 15, 2014 Accepted : December 09, 2014 Published : December 10, 2014 Abstract Chemoresistance to anti-cancer drugs substantially reduces survival in epithelial ovarian cancer. In this study, we showed that chemoresistance to cisplatin and paclitaxel induced the epithelial-mesenchymal transition (EMT) and a stem cell phenotype in ovarian cancer cells. Chemoresistance was associated with the downregulation of epithelial markers and the upregulation of mesenchymal markers, EMT-related transcription factors, and cancer stem cell markers, which enhanced invasion and sphere formation ability. Overexpression of FOXM1 increased cisplatin-resistance and sphere formation in cisplatin-sensitive and low FOXM1-expressing ovarian cancer cells. Conversely, depletion of FOXM1 via RNA interference reduced cisplatin resistance and sphere formation in cisplatin-resistant and high FOXM1-expressing cells. Overexpression of FOXM1 also increased the expression, nuclear accumulation, and activity of β-CATENIN in chemoresistant cells, whereas downregulation of FOXM1 suppressed these events. The combination of cisplatin and the FOXM1 inhibitor thiostrepton inhibited the expression of stem cell markers in chemoresistant cells and subcutaneous ovarian tumor growth in mouse xenografts. In an analysis of 106 ovarian cancer patients, high FOXM1 levels in tumors were associated with cancer progression and short progression-free intervals. Collectively, our findings highlight the importance of FOXM1 in chemoresistance and suggest that FOXM1 inhibitors may be useful for treatment of ovarian cancer.

Focal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by CaImages obtained with a total internal reflection fluorescence microscope (TIRFM) showed that CaVinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA.These results suggest that CaThis study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca

Hypoxia and acidosis are microenvironmental selection forces during somatic evolution in breast carcinogenesis. The effect of cobalt chloride (CoCl(2))-induced hypoxia on the expression of hypoxia-inducible factor (HIF)-1alpha, glucose transporter 1 (GLUT1), and carbonic anhydrase IX (CAIX) was assessed in breast cancer cells derived from primary sites (HCC1395 and HCC1937) and metastatic sites (MCF-7 and MDA-MB-231) by reverse transcriptase-polymerase chain reaction and immunoblotting. We analyzed these proteins' expression in tissue samples from normal breast tissue, usual ductal hyperplasia (DH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) using immunohistochemistry. CAIX mRNA was expressed constitutively in MDA-MB-231 cells but not in the other three cell lines. CAIX mRNA expression was increased after CoCl(2)-induced hypoxia in all four breast cancer cell lines. The expression of HIF-1alpha and GLUT1 proteins was increased after CoCl(2)-induced hypoxia in all breast cancer cell lines tested. Hypoxia significantly increased CAIX protein expression in primary cancer cells but not in metastatic ones. HIF-1alpha was not expressed in benign breast tissue, whereas it was significantly expressed in DH, ADH, DCIS, and IDC (p0.001). GLUT1 and CAIX were expressed only in DCIS (56.8% and 25.0%) and IDC (44.1% and 30.5%), with higher expression in high grade DCIS than low/intermediate grade DCIS (79.2% vs. 30.0%, p = 0.001 and 37.5% vs. 10.0%, p = 0.036, respectively). High CAIX expression was significantly associated with poor histological grade of IDC (p = 0.005). During breast carcinogenesis, the role of HIF-1alpha changes from response to proliferation to tumor progression. GLUT1 expression (glycolytic phenotype) and CAIX expression (acid-resistant phenotype) may result in a powerful adaptive advantage and represent an aggressive phenotype.

This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia-mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute-2 (MDM2) interaction. In addition, ISL-mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL-mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl-2 and Bcl-X(L), and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase-9 inhibitor blocked ISL-induced apoptosis, indicating that caspase-9 activation is involved in ISL-mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer.

Objectives. Malignant phenotypic traits are caused by microenvironmental selection pressures during carcinogenesis. Hypoxia can drive a tumor toward a more aggressive malignant phenotype. The objective was to better understand the role of the hypoxia-regulated genes in cervical carcinogenesis. Methods. We analyzed the expression of the hypoxia-regulated genes, including hypoxia-inducible factor-1α (HIF-1α), erythropoietin (Epo), vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), carbonic anhydrase IX (CAIX), and MET, in cervical cell lines and human tissue samples of cervical intraepithelial neoplasia (CIN I–III) and invasive squamous cell carcinoma (ISCC). Results. CAIX and MET were expressed in cervical carcinoma cell lines, but not in normal or human papillomavirus-immortalized cervical cells. In clinical tissue samples, Epo, VEGF, GLUT1, and CAIX were not detected in normal squamous epithelia. GLUT1 was expressed in nearly all cases of CIN and ISCC, however, CAIX was expressed only in CIN III and ISCC. HIF-1α and MET expression was confined to the basal cells in normal squamous epithelia and was detected in the dysplastic cells of CIN and ISCC. Conclusions. The role of HIF-1α and MET changes from response to proliferation to tumor progression during cervical carcinogenesis. GLUT1 expression, a glycolytic phenotype adaptive to glycolysis, occurs early during cervical carcinogenesis and is a specific marker for dysplasia or carcinoma. MET and CAIX may contribute tumor progression in later stage. CAIX expression, an acid-resistant phenotype, may be a powerful adaptive advantage during carcinogenesis. Successful adaptation to the hypoxia–glycolysis–acidosis sequence in a microenvironment is crucial during carcinogenesis.

Summary Objective To report a rare fetal congenital lung anomaly characterized by overinflation of a pulmonary lobe. Case Report A 28-year-old systemic lupus erythematous mother, gravida 1 para 0, who had normal prenatal care in our department, was admitted for labor pain and an abnormal fetal heart location was noted incidentally during labor. The baby showed rib retraction in room air but no obvious cyanotic change after delivery. Both the fetus chest X-ray and ultrasound showed a hyperechogenic tumor in the left thoracic cavity with a right-side-shifted heart and trachea. Computed tomography showed a hypodense and multiseptal tumor in the left thoracic cavity with right-sided shift of the heart and trachea. It was a soft, solid tumor in the parenchyma of the left lung and the histopathology confirmed it to be benign congenital lobar emphysema. Conclusion The favorable outcome in both asymptomatic and mildly symptomatic children suggests that a nonsurgical approach should be considered for these patients.

Squamous cell carcinoma antigen (SCCA) is a tumor marker for patients with squamous cell carcinoma of uterine cervix, lung, and esophagus. It was encoded by two highly homologous genes, SCCA1 and SCCA2. However, the relevance of SCCA genes to squamous cell carcinogenesis and patient outcome remains far from clear. In this study, by using laser microdissection and real-time quantitative polymerase chain reaction procedures, the messenger RNA (mRNA) expression of the SCCA1 and SCCA2 genes in normal, dysplastic, and malignant squamous epithelia from uterine cervical tissues were analyzed and correlated with outcome of cancer patients. We found that the SCCA2/A1 mRNA ratios were progressively increased from normal, dysplastic, to cancer cells, and the mean ratio was significantly higher in cancer tissues than that in normal epithelium (P= 0.02). The SCCA2/A1 mRNA ratios were not significantly associated with types of human papillomavirus infection (P > 0.05). High SCCA2/SCCA1 mRNA ratios (ratio >1) were an independent predictor of disease recurrence (relative risk: 3.58; P= 0.003). Of the 38 patients with cervical cancer, 12 patients with high SCCA2/SCCA1 mRNA ratios had a significant lower 2-year disease-free survival of only 50%, while it was 92% in those with low SCCA2/SCCA1 mRNA ratios (P < 0.001). In conclusion, our study indicated that the ratios of SCCA2 to SCCA1 RNA were increased during the process of cervical carcinogenesis, and patients with elevated SCCA2/A1 ratio carried a higher risk for recurrence in early-stage uterine cervical cancer.

Summary Objective To investigate the change in volume of uterine fibroids after GnRH analog (GnRHa) treatment. Materials and Methods Twenty-five patients who had a uterine leiomyoma were included in this study. Four of them were lost to follow-up during the study, and a myomectomy was performed on the remaining subjects. All of these patients received two doses, 4 weeks apart, each of 3.75 mg leuprorelin acetate subcutaneously (Leuplin depot, a GnRHa) before the operation. The vascularization index (VI), flow index (FI), vascularization-flow index (VFI), pulsative index (PI), resistance index (RI), vascular patterns (histogram), blood flows, and sizes (volume and largest diameter) of each fibroid were measured with power Doppler by the same technician every month before the operation. In addition, the total blood loss and time required for each operation were also recorded. Results Results of this study showed that the volume of the uterus and the fibroids, but not the vascularity, including VI, FI, VFI, PI and RI, decreased significantly after two doses of GnRHa treatment. In addition, blood loss during the operation decreased significantly compared to an untreated group. Conclusion We found that the volumes of the uterus and fibroids decreased significantly after treatment with two consecutive doses (given a month apart) of GnRHa. The 3D color Doppler including a histogram and blood flow parameters is another useful tool for fibroid evaluation.

A 58-year-old woman presented with pelvic, para-aortic masses and two isolated nodules in the right lung 6 years after hysterectomy and bilateral salpingo-oophorectomy for uterine leiomyoma. Laparotomy was carried out and all the intra-abdominal tumors were excised; pathology showed metastatic low-grade leiomyosarcomas. Immunohistochemical staining revealed a high expression level of estrogen and progesterone receptors in these tumors. The pulmonary nodules were left and the patient was given oral medroxyprogesterone acetate 200 mg daily after the operation. One of the pulmonary masses regressed progressively and had disappeared 7 months later on chest X-ray examination. However, the other was persistent. She then received wedge resection to excise the pulmonary nodule, which showed the same histologic pattern as the abdomen masses. However, the immunohistochemical staining on this nodule showed positive estrogen receptor expression but was negative for progesterone receptor expression. After the operation, she maintained progestin treatment and was tumor free for the following 12 months.

GnRH agonist (GnRH-a) therapy is known to shrink uterine leiomyoma, although the molecular mechanisms responsible for this effect remain poorly understood. Conflicting results exist as to whether GnRH-a treatment increases apoptosis in leiomyoma cells. The aim of this study is to investigate the effects of GnRH-a on uterine leiomyomas by profiling the expression levels of apoptosis-related molecules such as Fas/Fas ligand (FasL), caspases 3, 6, 7, 8, 9, and 10, and Bcl-2 from specimens of 20 patients receiving Leuplin Depot (LA), a long-acting GnRH-a, of 3 doses before myomectomy, as well as 24 controls. We found that uterine leiomyomas had up-regulated expressions of FasL and caspase 3 as compared with their homologous normal myometrium control. Both leiomyomas and myometria from LA-treated patients, however, presented a significant decrease in the expressions of FasL and caspase 3 as compared with those from LA-naive control patients. In addition, it was at the posttranscription level that the tumorigenesis of leiomyoma modulated the expressions of FasL and caspase 3 higher, whereas LA suppressed them at gene transcription. Unlike the case of FasL and caspase 3 mentioned above, no significant difference was found between leiomyomas and homologous myometria in the expressions of Fas and caspases 6, 7, 8, 9, and 10. The LA effect made drug-treated leiomyomas produce less Fas and caspases 7, 9, and 10 as compared with nontreated leiomyomas. Immunohistochemical analysis showed that both Fas and FasL were localized predominantly in cytosol. Moreover, leiomyomas had an up-regulated Bcl-2 level, which remained high even in the LA-treated cells. Our findings provide molecular evidence to support our previous observations that GnRH-a therapy fails to increase apoptosis in uterine leiomyomas.

Objective To examine whether gonadotrophin-releasing hormone agonist or oestradiol can directly affect DNA in leiomyoma cells. Design In vitro explant culture of leiomyoma cells. Setting University research group. Sample Leiomyoma cells were cultured from the specimens of four premenopausal women at myomectomy. Methods The presence of gonadotrophin-releasing hormone receptor in leiomyoma cells was determined by reverse transcriptase–olymerase chain reaction. Leiomyoma cells were treated with gonadotrophin-releasing hormone agonist or cultured in different concentrations of oestrogen, progesterone or fetal calf serum for one, four or seven days. Main outcome measures Cell number, expression of proliferating cell nuclear antigen, and DNA damage after one, four or seven days of treatment. Results Gonadotrophin-releasing hormone receptor messenger ribonucleic acid was detected on cultured leiomyoma cells. Leiomyoma cell growth was not affected by the addition of gonadotrophin-releasing hormone agonist or progesterone, but increased with oestrogen or fetal calf serum supplementation. Overexpression of proliferating cell nuclear antigen was prevented in cultures added with oestrogen or fetal calf serum, but not related to gonadotrophin-releasing hormone agonist treatment. Significant decreases in DNA damage as indicated by decreased comet number were found in the leiomyoma cultures treated with oestrogen or fetal calf serum for four and seven days but not with gonadotrophin-releasing hormone agonist or progesterone. Furthermore, 5% fetal calf serum supplementation was more growth supporting and more significantly reduced the comet number than 250 pM 17 β-oestradiol. Conclusion Cell growth, proliferating cell nuclear antigen expression and DNA damage are dependent on oestrogen or fetal calf serum, but independent of gonadotrophin-releasing hormone agonist or progesterone. Our findings suggest that gonadotrophin-releasing hormone agonist-induced leiomyoma shrinkage may be due in part to a mechanism involving DNA damage, and support the hypothesis that gonadotrophin-releasing hormone agonist exerts its action indirectly through oestrogen action on the tumour level.

Smooth muscle tumor of uncertain malignant potential (STUMP) presenting as pseudo-Meigs' syndrome with low vascular endothelial growth factor (VEGF) expression has not been reported in previous literature. Here, we report a case of uterine STUMP associated with ascites and pleural effusion, which was resolved completely after hysterectomy. A 47-year-old woman presented to the clinic with a complaint of progressive abdominal distension for several months. A large movable, painless pelvic mass located upward above the umbilical level was palpated. Sonography and computed tomography showed a hypervascular solid pelvic mass measuring 20 x 17 x 15 cm in size associated with ascites and right pleural effusion. Laparotomy revealed a large uterine mass with ascites in the abdomen. Total hysterectomy and left-side salpingo-oophorectomy were performed. The final pathologic report revealed a STUMP tumor with low expression of VEGF by immunohistochemistry. A follow-up chest X-ray revealed that the pleural effusion was resolved completely 1 week postoperatively. The patient is doing well without recurrence in the following 2 years. Uterine STUMP tumor may cause pseudo-Meigs' syndrome. However, the ascites or the pleural effusion may not be induced by VEGF, known as vascular permeability factor, in our case.

Our purpose was to investigate the histopathologic changes in uterine leiomyomas in cell proliferation, proliferating cell nuclear antigen expression, angiogenesis, and apoptosis after treatment with gonadotropin-releasing hormone agonist.Fifteen consecutive patients who had undergone gonadotropin-releasing hormone agonist treatment before surgery and 44 patients who did not were studied. The volumes of myomas were determined ultrasonographically, and in patients receiving gonadotropin-releasing hormone agonist therapy measurements were done again after administration of the gonadotropin-releasing hormone agonist to evaluate the response to treatment. Paraffin sections were stained with hematoxylin and eosin, PC 10 for proliferating cell nuclear antigen expression, MIB 1 for measurement of cell proliferation, ApopTag for apoptosis, and factor VIII for quantitation of microvessel density. A deoxyribonucleic acid fragmentation test was also done on nine cases with available frozen tissues.Most of the leiomyomas showed substantial expression of proliferating cell nuclear antigen. Gonadotropin-releasing hormone agonist therapy further induced significant overexpression of proliferating cell nuclear antigen (p = 0.0004, chi 2 test). All three leiomyomas that failed to respond to therapy showed less proliferating cell nuclear antigen staining compared with the good responders. In contrast, data from MIB 1 immunostaining showed that0.3% of leiomyoma cells were proliferating. However, positive-staining cells were more frequently detected in the treatment group (0.075% +/- 0.091% vs 0.002% +/- 0.010%, p = 0.0002, Mann-Whitney U test). Apoptosis developed spontaneously in leiomyoma cells independent of gonadotropin-releasing hormone agonist therapy. No significant change in apoptosis but a significant increase in microvessel density was observed in the treatment group compared with the control group.Enhanced deoxyribonucleic acid damage or repair with cell growth arrest may be responsible for the action of gonadotropin-releasing hormone agonist in shrinking uterine leiomyomas. Moreover, the extent of proliferating cell nuclear antigen expression seems to be associated with the response to gonadotropin-releasing hormone agonist therapy.

To investigate the differences of biological aggressiveness in terms of proliferating cell nuclear antigen (PCNA) expression, cell proliferation, and microvessel density between uterine leiomyosarcoma and leiomyoma.All patients with uterine leiomyosarcoma undergoing surgery at National Cheng Kung University Hospital were eligible. Forty-four patients with uterine myoma were also studied as the benign counterpart. The paraffin-embedded slides were stained with hematoxylin and eosin to confirm the presence of tumor and to quantitate mitoses, PC 10 for measurement of PCNA expression, MIB 1 for measurement of cell proliferation, and factor VIII for quantitation of microvessel density. The immunohistochemical findings of the slides were correlated with clinocopathologic findings of the patients, and the data were analyzed by either chi2 or unpaired t test.Six patients with uterine leiomyosarcoma and 44 patients with uterine leiomyoma were studied. Statistically significant higher mean levels of PCNA and MIB 1 were observed in uterine leiomyosarcoma compared with those of uterine myoma (for PCNA expression, P = 0.0001; for MIB 1, 11.61 +/- 11.42% vs 0.45 +/- 0.21%, P0.0001). No significant difference of microvessel density was observed between these two groups (65.73 +/- 48.62 vs 41.97 +/- 28.20, P = 0.084). Among the six patients with leiomyosarcoma, two patients with a higher percentage of MIB 1-positive tumor cells died of recurrent disease. In contrast, two patients with lower MIB 1 counts were disease-free for 3 years or more.Deregulated cell growth in uterine leiomyosarcoma may account for the biological aggressiveness of this tumor. Furthermore, the percentage of MIB 1-positive tumor cells seems to be associated with the prognosis or extent of uterine leiomyosarcoma.

Presacral hemorrhage during pelvic surgery is usually difficult to control. We here report our experience in controlling intractable presacral hemorrhage by using bipolar electrocauterization in four patients with cervical carcinoma. During radical hysterectomy, presacral hemorrhage occurred in these four patients and failed to be controlled by conventional maneuvers, such as compression with digit or gauze or suture ligation of the bleeding areas or both. The amount of estimated blood loss exceeded 3000 ml in each patient. Satisfactory hemostasis was achieved by bipolar electrocoagulation of the bleeding areas with Kleppinger forceps. Presacral hemorrhage was well controlled in all cases, without obvious complications. (J GYNECOL SURG 13:89, 1997)

Objective To investigate the correlation of intratumoral blood flow in uterine myoma with cell proliferation, angiogenesis, tumor size, and tumor volume. Methods Thirty-nine patients who had been scheduled for surgery because of symptomatic uterine myomas were evaluated by transvaginal sonography and color Doppler ultrasound before surgery. The largest dimension of each tumor and the volumes of myomas were determined ultrasonographically. Pulsatility index (PI) was determined by color Doppler ultrasound according to the maximum systolic, end-diastolic, and the mean flow velocities measured within the uterine nodules. After surgery, the paraffinembedded slides containing representative leiomyoma tissues were stained with hematoxylin and eosin, proliferating cell nuclear antigen for measurement of cell proliferation, and factor VIII for quantitation of microvessel density. The ultrasonographic findings were correlated postoperatively with pathologic findings, and the data were analyzed by simple linear regression and Fisher r to z transformation. Results Simple regression analysis of the intratumoral PI values on the sizes of myomas showed a negative correlation ( r = −0.47, P = .003; n = 39), whereas a less significant correlation between PI values and tumor volumes was observed ( r = −0.42, P = .008). In contrast, no statistically significant correlation was observed between the intratumoral PI values and the values of the proliferating cell nuclear antigen index ( r = 0.10, P = .547) or microvessel density counts ( r = 0.18, P = .282). Conclusion The intratumoral blood flow by transvaginal color Doppler ultrasound correlated with a reduced tumor size and tumor volume, but did not correlate with cell proliferation or angiogenesis.

The clinical utility of transvaginal ultrasonography in the differentiation of adenomyoma from leiomyoma was evaluated in 147 patients who had been scheduled for surgery due to symptomatic uterine masses. In all subjects, ultrasonographic images obtained preoperatively were correlated postoperatively with surgicopathological findings. Pathological findings showed that 110 patients proved to have fibroids, while 30 had adenomyomata. For the diagnosis of adenomyoma, transvaginal ultrasonography attained a sensitivity of 80%, a specificity of 94.3%, a positive predictive value of 85.7% and a negative predictive value of 90.9%, compared with a sensitivity of 94.3%, a specificity of 80%, a positive predictive value of 90.9% and a negative predictive value of 85.7% for leiomyoma diagnosis. Further to assess which characteristic used in ultrasonography was useful in the differential diagnosis, five characteristics were analyzed and compared by chi 2 test. These were position, number, margin and echogenicity of the uterine masses and the presence or absence of hypoechoic spaces (lacunae). Margin, echogenicity, mass number and lacunae were significantly different between both conditions. A stepwise logistic regression procedure revealed that margin, lacunae and echogenicity were good parameters for differentiating adenomyoma from leiomyoma. If we selected the features of distinct margin and absence of hypoechoic lacunae within the masses for analysis, leiomyoma could be correctly predicted in 97% of patients.

Tumor hypoxia promotes cancer progression. Matrix metalloproteinases (MMPs) are required for breast cancer cell invasion.The effect of cobalt chloride (CoCl(2))-stimulated hypoxia on invasion potential and the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) were investigated in four breast cancer cell lines, derived from primary sites (HCC1395 and HCC1937) and metastatic sites (MCF-7 and MDA-MB-231).CoCl(2)-induced hypoxia induced HIF-1alpha protein expression in all four cell lines. Hypoxia significantly increased the invasiveness of HCC1395 cells, which did not correlate with a change of any one MMP. Constitutive MMP expression was different between primary and metastatic breast cancer cells. MMP-2 and MMP-9 measured by RT-PCR and zymography were notably expressed in primary cancer cells but not apparent in metastatic ones. MMP-7 was also highly expressed in primary cancer cells. Hypoxia increased the expression of MMP-1, -10 and -13 in metastatic breast cancer cells, whereas only MMP-13 was up-regulated in primary HCC1937 cells by hypoxic stimulation. TIMPs were not altered by hypoxia, except for TIMP-4 which was down-regulated in MDA-MB-231 cells.This study demonstrated a cell line-specific effect of hypoxia on invasive potential and differential expression of constitutive MMPs in primary versus metastatic breast cancer cells, i.e. primary cancer cells expressed a wider range of MMPs, in particular MMP-2, -7 and -9, than the metastatic ones. The data suggest that MMPs play no crucial roles in hypoxia-induced tumor progression in primary breast cancer cells.

Cathepsins have long been considered as housekeeping molecules. However, specific functions have also been attributed to each of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wortmannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an antiapoptotic role through anti-cat L activity.