Comparison of cytologic findings between specimens with and without high-risk HPV infection in those with ASC-US judgement.

The objective of the study was to realize whether specific signatures for high-risk human papilloma virus (HPV) infection exist in the cytologic specimens with ASC-US judgement or not. The materials are 132 cytologic specimens with the diagnosis of ASC-US, including 56 cases with positive and 76 cases with negative HPV infection. Cytological findings are compared between two groups. Immature squamous metaplastic cells with nuclear atypia, SFT/IMT dyskaryotic cells, atypical parakeratosis, smudgy nuclei and multinucleated cells are the signature of high-risk HPV infection, whereas in the HPV(-) group immature metaplastic cells without atypia, moderately mature metaplastic cells without nuclear atypia and atrophic background are more popular. Instead, there are no differences on SFT/IMT background, microorganism infection and koilocytosis with or without nuclear atypia in both groups. The specific findings to confirm high-risk HPV infection are realized and the present results will contribute to decrease an unnecessary ASC-US judgement.

Anti-Tumor Effect of Inhibition of DNA Damage Response Proteins, ATM and ATR, in Endometrial Cancer Cells.

: While the incidence of endometrial cancer continues to rise, the therapeutic options remain limited for advanced or recurrent cases, and most cases are resistant to therapy. The anti-tumor effect of many chemotherapeutic drugs and radiotherapy depends on the induction of DNA damage in cancer cells; thus, activation of DNA damage response (DDR) pathways is considered an important factor affecting resistance to therapy. When some DDR pathways are inactivated, inhibition of other DDR pathways can induce cancer-specific synthetic lethality. Therefore, DDR pathways are considered as promising candidates for molecular-targeted therapy for cancer. The crosstalking ataxia telangiectasia mutated and Rad3 related and checkpoint kinase 1 (ATR-Chk1) and ataxia telangiectasia mutated and Rad3 related and checkpoint kinase 2 (ATM-Chk2) pathways are the main pathways of DNA damage response. In this study, we investigated the anti-tumor effect of inhibitors of these pathways in vitro by assessing the effect of the combination of ATM or ATR inhibitors and conventional DNA-damaging therapy (doxorubicin (DXR), cisplatin (CDDP), and irradiation) on endometrial cancer cells. Both the inhibitors enhanced the sensitivity of cells to DXR, CDDP, and irradiation. Moreover, the combination of ATR and Chk1 inhibitors induced DNA damage in endometrial cancer cells and inhibited cell proliferation synergistically. Therefore, these molecular therapies targeting DNA damage response pathways are promising new treatment strategies for endometrial cancer.

Kaempferol, a natural dietary flavonoid, suppresses 17ß-estradiol-induced survivin expression and causes apoptotic cell death in endometrial cancer.

Endometrioid endometrial carcinoma, commonly known as type 1 endometrial cancer, accounts for >80% of endometrial carcinomas and is dependent on estrogen. We recently reported on the prognostic significance of the BIRC5 survivin gene in endometrial cancer. Estradiol induces survivin expression in estrogen receptor-positive, but not in estrogen receptor-negative, cancer cells. Kaempferol, a bioflavonoid, reportedly inhibits estrogen receptor-α (ERα) in hormone receptor-positive breast cancer cells. However, whether kaempferol-mediated inhibition of ERα suppresses survivin and induces cell death in endometrial cancer remains unclarified. The present study evaluated the antitumor effects of kaempferol on endometrial cancer cells. Cell viability assays, flow cytometry analysis, western blotting and annexin V analyses were used to analyze the antitumor effects of kaempferol. The results demonstrated that kaempferol successfully suppressed the viability of two ER-positive endometrial cancer cell lines, with IC50 values of 83 and 65 µM. In addition, kaempferol induced sub-G1 cell accumulation and apoptotic cell death (P<0.01) in a dose-dependent manner. Treatment of cells with estradiol significantly induced co-expression of nuclear ERα and survivin proteins (P<0.001). Further evaluation revealed that kaempferol causes apoptotic cell death largely by suppressing ERα, survivin and Bcl-2 protein. Therefore, the results of the present study suggested that targeting ERα and survivin with kaempferol may be a novel therapeutic option against endometrial carcinoma.

Oncogenic histone methyltransferase EZH2: A novel prognostic marker with therapeutic potential in endometrial cancer.

The histone methyltransferase EZH2, a key epigenetic modifier, is known to be associated with human tumorigenesis. However, the physiological importance of EZH2 and its clinical relevance in endometrial cancer remain unclear. Hence, in the present study, we investigated the expression and function of EZH2 in endometrial cancer. In a quantitative real-time PCR analysis of 11 endometrial cancer cell lines and 52 clinical endometrial cancer specimens, EZH2 was significantly overexpressed in cancer cells and tissues compared to that in corresponding normal control cells and tissues. Kaplan-Meier survival analysis using data of the TCGA RNA-seq database and tissue microarrays (TMAs) indicated that EZH2 overexpression is associated with endometrial cancer prognosis. In addition, knockdown of EZH2 using specific siRNAs resulted in growth suppression and apoptosis induction of endometrial cancer cells, accompanied by attenuation of H3K27 trimethylation. Consistent with these results, treatment with GSK126, a specific EZH2 inhibitor, suppressed endometrial cancer cell growth and decreased the number of cancer cell colonies. Furthermore, GSK126 showed additive effects with doxorubicin or cisplatin, which are conventional drugs for treatment of endometrial cancer. Further studies should explore the therapeutic potential of inhibiting EZH2 in patients with endometrial cancer.

Cytological Characteristics of Endometrial Phasing Using the Specimens Prepared with the Liquid-Based Procedure: Comparison with the Conventional Procedure.

OBJECTIVE: To evaluate whether or not the liquid-based procedure (LBP) for endometrial cytology is as worthwhile for endometrial phasing as conventional slides. MATERIALS AND METHODS: The subjects were 81 women who underwent endometrial cytology and were defined as negative. The specimens obtained by either Endocyte® or Masubuchi aspiration tube® were processed first with the conventional procedure and then with LBP using TACAS™. RESULTS: (1) The number of subjects diagnosed by the conventional method as having proliferative, mid-, middle-secretory and late-secretory and atrophic phases was 40, 11, 10, 0 and 20, respectively. The rate of agreement with those using LBP was 87.7%. (2) Incidences of large clusters, ductal clusters, palisade arrangement, uneven staining and dirty mucous background detected were significantly higher with the conventional method, whereas with LBP clean background, inconspicuous bonding of cells, scattered solitary glandular cells, clear well-stained cytoplasm and cell compactness were higher. (3) Especially in the proliferative phase, clusters tended to be smaller and lose their architectural structures, and scattered solitary columnar cells were present. (4) Cells in the mid-phase tended to have loose contact and to mimic other phases. CONCLUSIONS: Cytodiagnosis of endometrial phasing prepared with LBP is feasible to perform when some modifications are implemented.

Significance of survivin as a prognostic factor and a therapeutic target in endometrial cancer.

INTRODUCTION: Survivin is an anti-apoptotic protein encoded by the baculoviral inhibitor of apoptosis repeat-containing (BIRC5) gene and is upregulated in 83% of endometrial cancers. We aimed to elucidate the prognostic importance of BIRC5 expression, and evaluate survivin as a therapeutic target for endometrial cancer, by knock-down of BIRC5 and using the survivin inhibitor-YM155. METHODS: RNA sequencing data in 234 patients with endometrial carcinoma was obtained from The Cancer Genome Atlas database, and analyzed using Kaplan-Meier method, log-rank test and Cox proportional hazard model. Expressions of survivin in 16 endometrial cancer cell lines were analyzed by western blotting. Knocking down effect on survivin expression was evaluated using a small interfering RNA (siRNA). The anti-proliferative and pro-apoptotic effects of YM155 were assessed with cell viability, flow cytometry, and annexin V/propidium iodide assays. RESULTS: High expression of BIRC5 was associated with poor progression free survival (P=0.006), and shown to be an independent prognostic factor (HR=1.97, 95% CI=1.29-4.5, P=0.045). Survivin was upregulated in 14 of 16 (87.5%) endometrial cancer cell lines, compared with endometrial immortalized cells. Apoptosis was induced by knockdown of BIRC5 in all 3 cell lines examined. YM155 showed increased population of sub-G1 cells (P<0.001) in all 16 cell lines, and IC50 values to YM155 were <50nm in 15 cell lines. YM155 dose-dependently and significantly increased the apoptotic cell population in all 16 cell lines (P<0.001). CONCLUSIONS: Present study indicated that survivin expression is a significant prognostic factor and that survivin is a promising therapeutic target for endometrial cancer.

Phenylethanoid and phenylpropanoid glycosides with melanogenesis inhibitory activity from the flowers of Narcissus tazetta var. chinensis.

A methanol extract of the flowers of Narcissus tazetta var. chinensis Roem. (Amaryllidaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the extract, four new phenylethanoid glycosides, tazettosides A­D (1­4), and a new phenylpropanoid glycoside, tazettoside E (5), were isolated along with 23 known compounds (6­28). Of the isolates, 1 (IC50 = 22.0 µM) and 4 (82.5 µM), 3-methoxy-8,9-methylenedioxy-3,4-dihydrophenanthridine (13, IC50 = 28.5 µM), 5,6-dihydrobicolorine (14, 23.7 µM), tazettine (16, 60.8 µM), benzyl ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranoside (18, 27.8 µM), 2-(3,4-dimethoxyphenyl)ethyl ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranoside (21, 74.6 µM), 3-phenylpropyl ß-D-glucopyranoside (22, 59.0 µM), and cinnamyl ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranoside (24, 88.0 µM) showed inhibitory effects without notable cytotoxicity at the effective concentrations.

Antitumor activity of a combination of dual PI3K/mTOR inhibitor SAR245409 and selective MEK1/2 inhibitor pimasertib in endometrial carcinomas.

OBJECTIVE: We aimed to clarify whether dual inhibition of PI3K/MAPK and MAPK pathways synergistically suppresses cell growth in endometrial cancer cells. METHODS: We exposed a panel of 12 endometrial cancer cell lines to a PI3K/mTOR inhibitor (voxtalisib, SAR245409) and/or a MEK inhibitor (pimasertib). The effect of each drug singly or in combination was evaluated by MTT assay, flow cytometry, and immunoblotting. Combination indexes (CIs) were calculated using the Chou-Talalay method to evaluate the synergy. RESULTS: The IC50 values for SAR245409 and pimasertib varied from 0.5 µM to 7 µM and from 0.1 µM to >20 µM, respectively. A combination of both compounds (1 µM SAR245409 and 30 nM pimasertib) caused a synergistic antitumor effect in 6 out of 12 endometrial cell lines (CI, 0.07-0.46). The synergistic effect was exclusively observed in 6 pimasertib-sensitive cell lines (IC50 of pimasertib, ≤5 µM). We found that 30 nM pimasertib, a concentration much lower than the IC50 for each cell line, was sufficient to cause a synergistic effect with SAR245409. Flow cytometric analysis showed that this combination significantly increased the population of G1 cells. However, a combination of rapamycin (an mTOR inhibitor) and pimasertib did not induce a synergistic effect in endometrial cancer cells, except for HEC-1B cells. CONCLUSIONS: The combination of a PI3K/mTOR inhibitor and a MEK inhibitor induced a synergistic antitumor effect in certain endometrial cancer cells. This study underscores the importance of using optimized doses of antitumor agents, singly or in combination, in treating endometrial cancer.

Human Papillomavirus Test for Triage of Japanese Women With Low-Grade Squamous Intraepithelial Lesions.

We evaluated high-risk human papillomavirus (HR-HPV) DNA testing for high-grade cervical intraepithelial neoplasia (CIN) lesions by cobas HPV test and diagnostic HPV16/18 genotyping in Japanese women with low-grade squamous intraepithelial lesions. Of 357 patients, HR-HPV positivity prevalence was 75.6%, and 21.3% had grade 2 or higher CIN lesions (CIN2+), with the highest prevalence at 30 to 34 years. Negative predictive values of HR-HPV for CIN2+ in our patients were 93.1% (any age) and 94.9% (40-50 years). Absolute risk for CIN2+ in HR-HPV positive and HPV16/18 positive individuals was 25.9 and 35.1, respectively. Relative risk for CIN2+ lesions was 5.1 for HPV16/18 positive versus HR-HPV negative, and 3.8 for HR-HPV positive versus HR-HPV negative women. Predictive values of CIN2+ positive were higher for HPV16/18 positive women (any age) than 12 other HPV positive-genotypes, and highest (50%) at 40-50 years. The HPV16/18 genotyping might prevent women (>40 years) at risk of high-grade CIN lesions from undergoing unnecessary colposcopy/overtreatment of nonprogressive lesions.

Evaluation of a New Automated Processing System (TACAS™ Pro) for Liquid-Based Procedures.

OBJECTIVE: To evaluate a fully automated processing system (TACAS™ Pro) for liquid-based procedures (LBPs). METHODS AND MATERIALS: Materials were 3,483 and additionally 502 specimens that were taken at Kanagawa Health Service Association. Specimens obtained with a Cervex-Brush® were first smeared to glass slides using one side of the brush and then processed to TACAS Pro. RESULTS: (1) The microscopy watching time per normal case was 3.65 ± 0.85 min in the conventional procedure, whereas in the LBP it was 1.95 ± 0.60 min, and the latter reduced workload to 53%. (2) The handling time of TACAS Pro per day was 2 h and 25.8 min. The workload at a laboratory offset it and revealed the work saving to be 63.8%. (3) Unsatisfactory rates were 0% in the conventional procedure, whereas in the LBP it was 1.88% at first. The latter rate decreased to 0.5% after system improvement. (4) Specimens which may disturb microscopy analysis were found in 1.06%, including 3 cases of possible carry-over of cells to the following slides. An additional study with the revised system confirmed no carry-over. (5) Incidences of abnormal cytology were consistent between the two methods. CONCLUSIONS: The revised automated processing system TACAS Pro is a feasible and useful LBP and reduces the workload of cytology laboratories.

PI3K/mTOR pathway inhibition overcomes radioresistance via suppression of the HIF1-α/VEGF pathway in endometrial cancer.

Radiation therapy is a key therapeutic strategy for endometrial carcinomas. However, biomarkers that predict radiosensitivity and drugs to enhance this sensitivity have not yet been established. We aimed to investigate the roles of TP53 and MAPK/PI3K pathways in endometrial carcinomas and to identify appropriate radiosensitizing therapeutics. D10 values (the irradiating dose required to reduce a cell population by 90%) were determined in eight endometrial cancer cell lines with known mutational statuses for TP53, PIK3CA, and KRAS. Cells were exposed to ionizing radiation (2-6Gy) and either a dual PI3K/mTOR inhibitor (NVP-BEZ235) or a MEK inhibitor (UO126), and their radiosensitizing effects were evaluated using clonogenic assays. The effects of silencing hypoxia-inducible factor-1 α (HIF-1α) expression with small interfering RNAs (siRNAs) were evaluated following exposure to ionizing radiation (2-3Gy). D10 values ranged from 2.0 to 3.1Gy in three cell lines expressing wild-type TP53 or from 3.3 to more than 6.0Gy in five cell lines expressing mutant TP53. NVP-BEZ235, but not UO126, significantly improved radiosensitivity through the suppression of HIF-1α/vascular endothelial growth factor-A expression. HIF-1α silencing significantly increased the induction of the sub-G1 population by ionizing radiation. Our study data suggest that TP53 mutation and PI3K pathway activation enhances radioresistance in endometrial carcinomas and that targeting the PI3K/mTOR or HIF-1α pathways could improve radiosensitivity.

Dipeptidyl peptidase-IV inhibitory activity of dimeric dihydrochalcone glycosides from flowers of Helichrysum arenarium.

A methanol extract of everlasting flowers of Helichrysum arenarium L. Moench (Asteraceae) was found to inhibit the increase in blood glucose elevation in sucrose-loaded mice at 500 mg/kg p.o. The methanol extract also inhibited the enzymatic activity against dipeptidyl peptidase-IV (DPP-IV, IC50 = 41.2 µg/ml), but did not show intestinal α-glucosidase inhibitory activities. From the extract, three new dimeric dihydrochalcone glycosides, arenariumosides V-VII (2-4), were isolated, and the stereostructures were elucidated based on their spectroscopic properties and chemical evidence. Of the constituents, several flavonoid constituents, including 2-4, were isolated, and these isolated constituents were investigated for their DPP-IV inhibitory effects. Among them, chalconaringenin 2'-O-ß-D-glucopyranoside (16, IC50 = 23.1 µM) and aureusidin 6-O-ß-D-glucopyranoside (35, 24.3 µM) showed relatively strong inhibitory activities.

The anti-malarial chloroquine suppresses proliferation and overcomes cisplatin resistance of endometrial cancer cells via autophagy inhibition.

OBJECTIVE: The anti-malarial drug chloroquine (CQ) is also known as an autophagy inhibitor. Autophagy can promote tumor growth by fueling the necessary energy metabolism and inducing resistance to chemotherapy and/or irradiation in various human cancers. However, the role of autophagy in endometrial cancer has not yet been established. We investigated the anti-tumor effects and autophagy inhibition caused by CQ in endometrial cancer cells. METHODS: Cell proliferation and cell cycle were assessed in response to CQ in six endometrial cancer cell lines by using an MTT assay and/or flow cytometry. To assess the level of autophagy, western blotting and an immunofluorescence assay were used to measure LC3 expression. The effects of knockdown of either ATG5 or ATG7, both of which are indispensable for induction of autophagy, were assessed via an MTT assay. Sensitivity to CQ was compared between parental and cisplatin-resistant (CP-r) Ishikawa endometrial cancer cells. RESULTS: CQ suppressed proliferation in all six endometrial cancer cell lines in a dose-dependent manner, whereas it increased the population of apoptotic cells. Inhibition of autophagy via knockdown of either ATG5 or ATG7 decreased the sensitivity to CQ. Additionally, sensitivity to cisplatin was improved by knocking down ATG5 or ATG7. Finally, CP-r Ishikawa cells, with a high basal level of autophagy, were more sensitive to CQ than parental Ishikawa cells. CONCLUSIONS: Our data suggest that autophagy is involved in endometrial tumor growth and cisplatin resistance. Furthermore, our data support a therapeutic role for CQ in endometrial cancer cells with upregulation of autophagy.

Performance of p16INK4a/Ki-67 immunocytochemistry for identifying CIN2+ in atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion specimens: a Japanese Gynecologic Oncology Group study.

BACKGROUND: p16(INK4a) immunohistochemistry has revealed a high rate of positivity in cervical intraepithelial neoplasia grade 2 (CIN2) and more severe conditions (CIN2+). The Lower Anogenital Squamous Terminology Standardization project proposed p16(INK4a) immunohistochemistry as an ancillary test for CIN. Immunocytochemistry involving dual staining for p16(INK4a) and Ki-67 in the triage of atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) is reported to be useful in the identification of CIN2+. However, it is unclear whether p16(INK4a)/Ki-67 immunocytochemistry is of practical relevance for the triage of ASCUS and LSIL in the Japanese screening system. METHODS: From 427 women fulfilling the eligibility criteria, 188 ASCUS and 239 LSIL specimens were analyzed. The accuracy of p16(INK4a)/Ki-67 immunocytochemistry and genotyping of high-risk human papillomaviruses (HPVs) in detecting CIN2+ were compared. RESULTS: p16(INK4a)/Ki-67 immunocytochemistry was positive in 33.5 % (63/188) of ASCUS, and 36.8 % (88/239) of LSIL specimens. The sensitivity and specificity of p16(INK4a)/Ki-67 immunocytochemistry was 87.3 % (95 % confidence interval 78.0-93.8 %) and 76.4 % (71.6-80.8 %), respectively. The positive and negative predictive values were 45.7 % (37.6-54.0 %) and 96.4 % (93.4-98.3 %), respectively; positive and negative likelihood ratios were 3.71 and 0.17, respectively. Using the McNemar test, p16(INK4a)/Ki-67 immunocytochemistry showed equivalent sensitivity but higher specificity than the HPV genotyping test CONCLUSIONS: Compared with high-risk HPV genotyping, p16(INK4a)/Ki-67 immunocytochemistry was a more accurate triage test for identifying CIN2+ in ASCUS and LSIL specimens.

Anti-tumor activity of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in cultured endometrial carcinoma cells.

BACKGROUND: PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines. METHODS: The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. The effects of siRNA to PTEN were analyzed in cells with wild-type PTEN. RESULTS: The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 ± 838 nM; Wild-type [n = 4]: 215 ± 85 nM, p = 0.26 by Student's t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN + cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN + cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN. CONCLUSIONS: Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. Inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer.

Optimal sampling devices for liquid-based procedure in screening for cervical cancer: comparison between cotton stick/Cytobrush and Cervex-Brush.

OBJECTIVE: To find an appropriate sampling device for a liquid-based procedure in the population screening for cervical cancer, focusing on bleeding at sampling and the amount of cells smeared. METHODS AND MATERIALS: 1,000 consecutive women who underwent primary screening were studied. The specimens were obtained with the cotton stick/Cytobrush® method in the first 500 cases or with the Cervex-Brush® in the following 500 subjects, and were processed using the Thinlayer Advanced Cytology Assay System (TACAS™) following the manufacturer's instructions. RESULTS: (1) Bleeding at cellular sampling using the cotton stick/Cytobrush and Cervex-Brush methods occurred in 1.2 and 8.8% of the cases, respectively (p < 0.0001). (2) The incidences of cells obtained with the two methods which covered the whole area, <1/2 and ≥1/4, and <1/4 of the observation fields were 55.4 versus 62.2% (p < 0.05), 14.6 versus 9.4% (p < 0.05), and 2.0 versus 4.0% (p < 0.05), respectively. (3) The incidences of endocervical or metaplastic cells obtained with ≥500 and <10 were 34.6 versus 20.0% (p < 0.01) and 9.4 versus 18.4% (p < 0.01), respectively. In cases of cells covering <1/4, incidences with <10 were 0 and 0.6% (n = 3), respectively. (4) Detection rates of abnormal cytology were 3.4 and 5.2% (n.s.), including atypical squamous cells of undetermined significance in 2.4 and 3.2%. CONCLUSIONS: The cotton stick/Cytobrush is superior to the Cervex-Brush as a cellular sampling device for the TACAS liquid-based procedure.

Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVP-BEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas.

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235--a dual PI3K/mTOR inhibitor--and RAD001--an mTOR inhibitor--in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n = 9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n = 4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas.

Application of a new liquid-based procedure (TACAS) for the screening of cervical cancer: a preliminary study.

OBJECTIVE: To evaluate the usefulness of a new liquid-based cytological procedure in a population screening program for cervix cancer. SUBJECTS AND METHODS: Subjects were 1,000 women who underwent primary screening at the Kanagawa Health Service Association. The cytological specimens obtained by either cotton stick and Cytobrush® or Cervex-Brush® were processed using the Thinlayer Advanced Cytology Assay System (TACAS™), following the manufacturer's instructions. RESULTS: (1) Cells were evenly distributed on specimens and stained evenly; (2) shrinkage of cells was 5% based on measurement of the nuclear diameters of granulocytes in comparison with those of the conventional procedure; (3) incidences of cells that occupied the whole area, 1/20≤, 1/4≤, 1/4> of the observation fields were 58.8, 26.2, 12.0 and 3.0%, respectively; (4) number of the squamous cells in cases with 1/4> was <5,000, in which specimen cells were correctly obtained from the squamocolumnar junction except in 3 cases (0.3%); (5) bleeding at cellular sampling was 5%, but did not disturb cell analysis; (6) inflammation caused by organisms was easily diagnosed; (7) detection rate of abnormal cytology was 4.3%, including ASC-US in 2.8% and ASC-H in 0.1%. CONCLUSION: TACAS is a feasible and useful cytological procedure.

Significance of nuclear p-Akt in endometrial carcinogenesis: rapid translocation of p-Akt into the nucleus by estrogen, possibly resulting in inhibition of apoptosis.

INTRODUCTION: Endometrial endometrioid adenocarcinoma (endometrial cancer) develops through endometrial hyperplasia caused by estrogenic hyperstimulation. Estrogen is known to activate growth factor signaling pathways, resulting in cellular proliferation, but precisely how has not been clarified. The aim of this study was to investigate the significance of estrogen and downstream factors such as the MAPK (MEK, ERK) and Akt pathways in endometrial carcinogenesis. METHODS: The expression of p-MEK, p-ERK, and p-Akt was analyzed immunohistochemically in normal, hyperplastic, and neoplastic endometria. The estrogenic effect on p-Akt was examined using an endometrial cancer cell line (Ishikawa cells). The estrogenic effect on the apoptosis of Ishikawa cells was assessed by the TUNEL method. RESULTS: Phospho-MEK (p-MEK) and p-ERK expression levels were similar among histological types but correlated with each other. The nuclear p-Akt labeling index (LI) was higher in cancer than in normal endometrium and hyperplasia. The nuclear p-Akt LI of well-differentiated cancer (G1) was higher than that of moderately (G2) or poorly (G3) differentiated cancers. The nuclear expression of p-Akt was correlated with that of estrogen receptor α (ER-α). The nuclear p-Akt level was significantly correlated with prognosis in cases of G1. In Ishikawa cells transfected with ERα, p-Akt was translocated into the nucleus from the cytoplasm in 1 to 3 hours after estrogenic stimulation. Further, apoptosis induced by H2O2 was inhibited by estrogen in the ER-α-positive cells. CONCLUSIONS: The translocation of p-Akt into the nucleus by estrogen may be related to the suppression of apoptosis by estrogen and consequently to endometrial carcinogenesis and prognosis in G1 endometrial cancer.

Liquid-based preparation for endometrial cytology--usefulness for predicting the prognosis of endometrial carcinoma preoperatively.

BACKGROUND: The authors evaluated the applicability and usefulness of immunocytochemical staining for cyclin A, p53, estrogen receptor alpha (ER-alpha), and progesterone receptor B (PR-B) as a preoperative prognostic indicators for endometrial carcinoma using endometrial cytology with the liquid-based cytology (LBC) method. METHODS: Cytologic specimens from 44 patients who had endometrial carcinoma were prepared with the LBC method. The results of immunocytochemical and immunohistochemical staining for cyclin A, p53, ER-alpha, and PR-B were compared with clinicopathologic parameters and prognosis. RESULTS: Patients who had positive results for cyclin A and p53 and negative results for ER-alpha and PR-B appeared to have unfavorable clinicopathologic characteristics, such as high-grade histology, advanced clinical stage, lymphovascular space involvement (LVSI), and deeper myometrial invasion (MI), and had a poor prognosis. In contrast, patients who had positive results for ER-alpha and PR-B, and negative results for cyclin A and p53 had favorable characteristics, such well differentiated tumor, early clinical stage, negative LVSI, and less MI, and had a good prognosis. Immunostaining results from cytologic specimens obtained in the clinic and at surgery and from histologic specimens obtained at surgery were correlated positively. CONCLUSIONS: Consistent specimens that were prepared using the LBC method facilitated multiple immunocytochemical analyses. Endometrial cytology with the LBC method was useful for predicting the prognosis of patients with endometrial carcinoma before therapy.