Chemoresistant ovarian cancer enhances its migration abilities by increasing store-operated Ca2+ entry-mediated turnover of focal adhesions.

BACKGROUND: Among gynecological cancers, ovarian carcinoma has the highest mortality rate, and chemoresistance is highly prevalent in this cancer. Therefore, novel strategies are required to improve its poor prognosis. Formation and disassembly of focal adhesions are regulated dynamically during cell migration, which plays an essential role in cancer metastasis. Metastasis is intricately linked with resistance to chemotherapy, but the molecular basis for this link is unknown. METHODS: Transwell migration and wound healing migration assays were used to analyze the migration ability of ovarian cancer cells. Real-time recordings by total internal reflection fluorescence microscope (TIRFM) were performed to assess the turnover of focal adhesions with fluorescence protein-tagged focal adhesion molecules. SOCE inhibitors were used to verify the effects of SOCE on focal adhesion dynamics, cell migration, and chemoresistance in chemoresistant cells. RESULTS: We found that mesenchymal-like chemoresistant IGROV1 ovarian cancer cells have higher migration properties because of their rapid regulation of focal adhesion dynamics through FAK, paxillin, vinculin, and talin. Focal adhesions in chemoresistant cells, they were smaller and exhibited strong adhesive force, which caused the cells to migrate rapidly. Store-operated Ca2+ entry (SOCE) regulates focal adhesion turnover, and cell polarization and migration. Herein, we compared SOCE upregulation in chemoresistant ovarian cancer cells to its parental cells. SOCE inhibitors attenuated the assembly and disassembly of focal adhesions significantly. Results of wound healing and transwell assays revealed that SOCE inhibitors decreased chemoresistant cell migration. Additionally, SOCE inhibitors combined with chemotherapeutic drugs could reverse ovarian cancer drug resistance. CONCLUSION: Our findings describe the role of SOCE in chemoresistance-mediated focal adhesion turnover, cell migration, and viability. Consequently, SOCE might be a promising therapeutic target in epithelial ovarian cancer.

Revealing the three dimensional architecture of focal adhesion components to explain Ca2+-mediated turnover of focal adhesions.

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 Ca2+/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca2+ influx induces cascades of FA turnover in living cells. METHODS: Images obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca2+ 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 Ca2+-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 Ca2+-mediated FA turnover. GENERAL SIGNIFICANCE: This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca2+.

The downregulation of miR-509-3p expression by collagen type XI alpha 1-regulated hypermethylation facilitates cancer progression and chemoresistance via the DNA methyltransferase 1/Small ubiquitin-like modifier-3 axis in ovarian cancer cells.

BACKGROUND: MicroRNAs are a group of small non-coding RNAs that are involved in development and diseases such as cancer. Previously, we demonstrated that miR-335 is crucial for preventing collagen type XI alpha 1 (COL11A1)-mediated epithelial ovarian cancer (EOC) progression and chemoresistance. Here, we examined the role of miR-509-3p in EOC. METHODS: The patients with EOC who underwent primary cytoreductive surgery and postoperative platinum-based chemotherapy were recruited. Their clinic-pathologic characteristics were collected, and disease-related survivals were determined. The COL11A1 and miR-509-3p mRNA expression levels of 161 ovarian tumors were determined by real-time reverse transcription-polymerase chain reaction. Additionally, miR-509-3p hypermethylation was evaluated by sequencing in these tumors. The A2780CP70 and OVCAR-8 cells transfected with miR-509-3p mimic, while the A2780 and OVCAR-3 cells transfected with miR-509-3p inhibitor. The A2780CP70 cells transfected with a small interference RNA of COL11A1, and the A2780 cells transfected with a COL11A1 expression plasmid. Site-directed mutagenesis, luciferase, and chromatin immunoprecipitation assays were performed in this study. RESULTS: Low miR-509-3p levels were correlated with disease progression, a poor survival, and high COL11A1 expression levels. In vivo studies reinforced these findings and indicated that the occurrence of invasive EOC cell phenotypes and resistance to cisplatin are decreased by miR-509-3p. The miR-509-3p promoter region (p278) is important for miR-509-3p transcription regulation via methylation. The miR-509-3p hypermethylation frequency was significantly higher in EOC tumors with a low miR-509-3p expression than in those with a high miR-509-3p expression. The patients with miR-509-3p hypermethylation had a significantly shorter overall survival (OS) than those without miR-509-3p hypermethylation. Mechanistic studies further indicated that miR-509-3p transcription was downregulated by COL11A1 through a DNA methyltransferase 1 (DNMT1) stability increase. Moreover, miR-509-3p targets small ubiquitin-like modifier (SUMO)-3 to regulate EOC cell growth, invasiveness, and chemosensitivity. CONCLUSION: The miR-509-3p/DNMT1/SUMO-3 axis may be an ovarian cancer treatment target.

The Downregulation of miR-509-3p Expression by Collagen Type XI Alpha 1-Regulated Hypermethylation Facilitates Cancer Progression and Chemoresistance via the DNA Methyltransferase 1/Small Ubiquitin-like Modifier-3 Axis in Ovarian Cancer Cells.

Background MicroRNAs are a group of small non-coding RNAs that are involved in development and diseases such as cancer. Previously, we demonstrated that miR-335 is crucial for preventing collagen type XI alpha 1 (COL11A1)-mediated epithelial ovarian cancer (EOC) progression and chemoresistance. Here, we examined the role of miR-509-3p in EOC. Methods The patients with EOC who underwent primary cytoreductive surgery and postoperative platinum-based chemotherapy were recruited. Their clinic-pathologic characteristics were collected, and disease-related survivals were determined. The COL11A1 and miR-509-3p mRNA expression levels of 161 ovarian tumors were determined by real-time reverse transcription-polymerase chain reaction. Additionally, miR-509-3p hypermethylation was evaluated by sequencing in these tumors. The A2780CP70 and OVCAR-8 cells transfected with miR-509-3p mimic, while the A2780 and OVCAR-3 cells transfected with miR-509-3p inhibitor. The A2780CP70 cells transfected with a small interference RNA of COL11A1, and the A2780 cells transfected with a COL11A1 expression plasmid. Site-directed mutagenesis, luciferase, and chromatin immunoprecipitation assays were performed in this study. Results Low miR-509-3p levels were correlated with disease progression, a poor survival, and high COL11A1 expression levels. In vivo studies reinforced these findings and indicated that the occurrence of invasive EOC cell phenotypes and resistance to cisplatin are decreased by miR-509-3p. The miR-509-3p promoter region (p278) is important for miR-509-3p transcription regulation via methylation. The miR-509-3p hypermethylation frequency was significantly higher in EOC tumors with a low miR-509-3p expression than in those with a high miR-509-3p expression. The patients with miR-509-3p hypermethylation had a significantly shorter overall survival (OS) than those without miR-509-3p hypermethylation. Mechanistic studies further indicated that miR-509-3p transcription was downregulated by COL11A1 through a DNA methyltransferase 1 (DNMT1) phosphorylation and stability increase. Moreover, miR-509-3p targets small ubiquitin-like modifier (SUMO)-3 to regulate EOC cell growth, invasiveness, and chemosensitivity. Conclusion The miR-509-3p/DNMT1/SUMO-3 axis may be an ovarian cancer treatment target.

COL11A1 activates cancer-associated fibroblasts by modulating TGF-ß3 through the NF-κB/IGFBP2 axis in ovarian cancer cells.

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-ß3 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-ß3 antibody. Human tumors with high TGF-ß3 levels showed elevated expression of COL11A1 and IGFBP2, which was associated with poor survival. Our findings suggest the possibility that anti-TGF-ß3 treatment strategy may be effective in targeting CAFs in COL11A1-positive ovarian tumors.

miR-335 Restrains the Aggressive Phenotypes of Ovarian Cancer Cells by Inhibiting COL11A1.

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/EBPß/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.

Roles for hypoxia-regulated genes during cervical carcinogenesis: somatic evolution during the hypoxia-glycolysis-acidosis sequence.

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-1alpha (HIF-1alpha), 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-1alpha 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-1alpha 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.

Association of extremely skewed X-chromosome inactivation with Taiwanese women presenting with recurrent pregnancy loss.

X-chromosome inactivation (XCI) is a phenomenon that occurs in female mammals. Typically, maternally and paternally-derived X chromosomes are inactivated at approximately the same frequency. If preferential inactivation occurs, the person is considered to have skewed XCI. Skewed XCI has been reported to occur more frequently in women who experience recurrent pregnancy loss (RPL). In this study, we sought to investigate if there is an association between skewed XCI and unexplained RPL in Taiwanese women. A total of 194 women who had experienced unexplained RPL were recruited into the study. Human androgen receptor or DXS6673E and DX15-134 loci were used in the XCI assay. The results of our study suggested that a cut-off point less than 90% may not be justified for skewed XCI. Only extremely skewed (more than 95%) XCI is associated with RPL. Extremely skewed XCI occurs in a subset of Taiwanese women with RPL.

Bcl-2 regulates store-operated Ca2+ entry to modulate ER stress-induced apoptosis.

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.

Chromosomal gain of 3q and loss of 11q often associated with nodal metastasis in early stage cervical squamous cell carcinoma.

BACKGROUND/PURPOSE: Cervical cancer remains a health problem among women worldwide. Delineation of genetic changes is critical to understanding the molecular basis of tumor progression, as well as for identifying genetic markers for early identification of patients at high risk for a poor outcome. METHODS: To provide comparative genomic hybridization data for cervical squamous cell carcinoma in Taiwan, and to gain further insight into genetic markers associated with lymph node metastasis of this disease, we performed comparative genomic hybridization analysis of 30 consecutive cases of cervical squamous cell carcinoma (24 stage IB and 6 stage IIB). RESULTS: The results disclosed that higher staged tumors or those with lymph node metastasis had more chromosomal imbalances. The commonly recurrent chromosomal imbalances were gains of 3q (46.7%), 1q (36.7%) and 8q (20.0%) and losses of 11q (36.7%), 3p (33.3%), 6q (23.3%), and 2q (20.0%). The frequencies of these chromosomal imbalances in stage IB and IIB tumors did not differ significantly. However, when compared with tumors without lymph node metastasis, the loss of 11q14-q22 (5/9 vs. 3/21, p = 0.019) and gains of 3q11-q22 and 3q26-qter (6/9 vs. 5/21, p = 0.026) were significantly more prevalent in tumors with lymph node metastasis. CONCLUSION: The results suggest that certain tumor-associated genes residing on 3q and 11q warrant further investigation to elucidate their role in the progression of this disease.

FOXM1 confers to epithelial-mesenchymal transition, stemness and chemoresistance in epithelial ovarian carcinoma cells.

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 ß-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.

Fas and its ligand, caspases, and bcl-2 expression in gonadotropin-releasing hormone agonist-treated uterine leiomyoma.

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.

Enhanced polyadenosine diphosphate-ribosylation in gonadotropin-releasing hormone agonist-treated uterine leiomyoma.

This study aimed to examine the activation of poly(ADP-ribose) polymerase (PARP) and the accumulation of its end product, poly(ADP-ribose) (PAR), in uterine leiomyoma specimens obtained from 25 patients receiving Leuplin depot [leuprorelin acetate, depot (LA)] treatment and 46 control patients and explore their correlation with tumor shrinkage and degeneration caused by the therapy. Immunoblotting analysis showed that specimens from LA-treated patients had higher PARP expression. The numbers of both PARP- and PAR-immunolabeled cells were higher in leiomyoma with LA treatment. This was correlated with the clinical response of LA therapy that LA induced more leiomyoma degeneration. The analysis of power Doppler sonography indicated a progressive decrease in blood supply to tumor following LA treatment. In vitro experiments using primarily cultured leiomyoma cells exhibited that the deprivation of serum or ovarian hormones or LA directly failed to induce PARP and PAR production. Our results suggested that reduced blood flow and subsequent ischemic damages in leiomyoma could be responsible for PARP overexpression and PAR accumulation, clinical response, and tumor degeneration caused by LA treatment.

Clinical significance of serum human papillomavirus DNA in cervical carcinoma.

OBJECTIVE: To study the association between serum human papillomavirus (HPV) deoxyribonucleic acid (DNA) and clinicopathologic prognostic factors and the clinical usefulness of serum HPV DNA in early-stage cervical cancer. METHODS: Deoxyribonucleic acids extracted from cervical tissues and sera of patients with stage IB or IIA cervical cancer and 40 controls including patients with cervical carcinoma in situ or benign disease were examined for HPV DNA with L1 consensus and types 16- and 18-specific E7 primers. Multivariable logistic regression was used to determine significant correlates of positive serum HPV DNA, and the receiver operating characteristic curve was applied in risk-factor assessment. RESULTS: Human papillomavirus DNA was not detected in sera from patients with carcinoma in situ or benign disease. Among the 112 patients with cervical cancer, we detected 27 positive samples (24.1%) in serum. Positive HPV DNA in serum was significantly associated with lymphovascular invasion and deep stromal invasion with or without parametrial extension (P <.001 for both conditions), pelvic lymph nodal metastasis (P =.001), large tumor size, and elevated levels of serum squamous cell carcinoma antigen (P <.001 for both conditions). When serum HPV DNA was used to predict high-risk patients who require adjuvant therapy, a sensitivity of 45.2%, a specificity of 88.6%, a positive predictive value of 70.4%, and a negative predictive value of 72.9% were obtained. CONCLUSION: The presence of serum HPV DNA in patients with early-stage cervical cancer was correlated with poor prognosis factors that warrant adjuvant therapy.

Ascites and elevated androgen level in a pregnant patient with an ovarian sclerosing stromal tumor.

We report the case of a 31-year-old woman with an androgen-producing sclerosing stromal tumor found during the eighth week of gestation in association with ascites and elevated serum androgen and cancer antigen (CA)-125 levels. The combined features of ascites, elevated serum androgen and elevated CA-125 in association with this rare type of ovarian tumor is unusual. Surgical removal of the tumor resulted in relief of symptoms, resolution of ascites, and decreases in serum androgen and CA-125 levels. The pregnancy was uneventful.

Enhanced myometrial autophagy in postpartum uterine involution.

OBJECTIVE: To understand the mechanisms of postpartum uterine involution, we investigated the uterine myometrial changes during pregnancy and the postpartum period. MATERIALS AND METHODS: Nine groups of uterine myometrial samples from mice (n = 4) were collected on gestational Day 0 (nonpregnant), Day 1, Day 2, Day 7, Day 14, and Day 21 and on postpartum Day 1, Day 2, and Day 7. Human samples of uterine myometrium on term (n = 1) and postpartum Day 1 (n = 2) were also collected. Ki-67 immunostaining was used to determine myometrial proliferation. For cell hypertrophy analysis, organelle proteins, ß-actin, prohibin, calnexin, and golgin-97 were analyzed by Western blotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and evaluation of activated caspase-3 expression by Western blot analysis assay were used to detect apoptosis. Autophagy was assayed via the evaluation of LC3 expression by Western blotting, immunohistochemistry, and autophagosomes by electron microscopy. RESULTS: Uterine myocytes proliferated during the early stage of gestation with a peak at Day 2, whereas myocyte hypertrophy with increased cellular organelle production occurred gradually in later stages of pregnancy. Postpartum autophagy developed abruptly in uterine myocytes without obvious apoptosis. CONCLUSION: Autophagy of myocytes may play an important role in uterine involution. These results have implications for our understanding of myometrial functional adaptations during pregnancy and the physiological role of autophagy in the uterine remodeling events in the postpartum period.

Hypoxia and metabolic phenotypes during breast carcinogenesis: expression of HIF-1alpha, GLUT1, and CAIX.

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 (p < 0.001). 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.

Cobalt chloride-induced hypoxia modulates the invasive potential and matrix metalloproteinases of primary and metastatic breast cancer cells.

BACKGROUND: Tumor hypoxia promotes cancer progression. Matrix metalloproteinases (MMPs) are required for breast cancer cell invasion. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells.

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.