[Microcystic, elongated and fragmented invasive pattern in endometrial adenocarcinoma: a clinicopathologic analysis of 72 cases].

Objective: To investigate the clinicopathologic features of microcystic, elongated and fragmented (MELF) pattern invasion of endometrial adenocarcinoma. Methods: HE and immunohistochemistry staining method were used to analysis morphologic features and immunophenotype of 72 patients of endometrial adenocarcinoma with MELF pattern invasion, and chi-square test was used to analysis the clinicopathologic features. Results: The mean age of 72 patients was 54 years (40 to 70 years). Thirty-two patients were pre-menopausal and 40 were post-menopausal. According to the FIGO staging system (2014), 32 cases(44.4%)were at stage Ⅰ, 22 cases(30.6%)at stage Ⅱ, 17 cases(23.6%)at stage Ⅲ and 1 case(1.4%) at stage Ⅳ. Microscopically, MELF invasion showed microcystic, elongated slit-like or fragmented glands in myometrium and their lining cells usually were cube or flat, as well as the single or clusters of eosinophilic tumor cells mimicking histocytes. In addition, a fibromyxoid or inflammatory stromal response was often present.Immunohistochemical staining showed that MELF invasion was positive for p16, CA125 and CA19-9, but negative for ER, PR and p53.Compared with non-MELF pattern invasion, significant differences were noted in menopause pausimenia, FIGO stages, deep invasion into myometrium, lymph metastasis, lymphovascular space invasion (LVSL), serum CA125 and CA19-9 in patients with MELF pattern invasion (all P<0.05). Conclusions: MELF pattern invasion of endometrial adenocarcinoma is characterized by advanced FIGO stage, deep myoinvasion, high metastasis rate to lymph node and LVSL. Pathologists should recognize the MELF invasion and evaluate the depth of myometrium of infiltration and LVSL with special attention to the presence of MELF invasion with necessary immunohistochemistry for more accurate pathological diagnosis.

Autophagy orchestrates adaptive responses to targeted therapy in endometrial cancer.

Targeted therapies in endometrial cancer (EC) using kinase inhibitors rarely result in complete tumor remission and are frequently challenged by the appearance of refractory cell clones, eventually resulting in disease relapse. Dissecting adaptive mechanisms is of vital importance to circumvent clinical drug resistance and improve the efficacy of targeted agents in EC. Sorafenib is an FDA-approved multitarget tyrosine and serine/threonine kinase inhibitor currently used to treat hepatocellular carcinoma, advanced renal carcinoma and radioactive iodine-resistant thyroid carcinoma. Unfortunately, sorafenib showed very modest effects in a multi-institutional phase II trial in advanced uterine carcinoma patients. Here, by leveraging RNA-sequencing data from the Cancer Cell Line Encyclopedia and cell survival studies from compound-based high-throughput screenings we have identified the lysosomal pathway as a potential compartment involved in the resistance to sorafenib. By performing additional functional biology studies we have demonstrated that this resistance could be related to macroautophagy/autophagy. Specifically, our results indicate that sorafenib triggers a mechanistic MAPK/JNK-dependent early protective autophagic response in EC cells, providing an adaptive response to therapeutic stress. By generating in vivo subcutaneous EC cell line tumors, lung metastatic assays and primary EC orthoxenografts experiments, we demonstrate that targeting autophagy enhances sorafenib cytotoxicity and suppresses tumor growth and pulmonary metastasis progression. In conclusion, sorafenib induces the activation of a protective autophagic response in EC cells. These results provide insights into the unopposed resistance of advanced EC to sorafenib and highlight a new strategy for therapeutic intervention in recurrent EC.

Apoptosis-Promoting Effects of Hematoporphyrin Monomethyl Ether-Sonodynamic Therapy (HMME-SDT) on Endometrial Cancer.

OBJECTIVE: The aim of the present study was to examine the apoptosis-promoting effects and mechanisms of hematoporphyrin monomethyl ether (HMME)-sonodynamic therapy (SDT) on endometrial cancer cells in vitro. METHODS: Endometrial cancer cell samples were divided into four groups: 1) untreated control group, 2) HMME group, 3) pure ultrasound group, and 4) HMME combined with ultrasound, i.e. SDT group. CCK-8 method was utilized to assess the inhibiting effect of SDT on the proliferation of endometrial cancer cells. Optical microscope and field emission transmission electron microscopy were used to characterize the morphology changes of the cancer cells induced by the treatments. Apoptosis rate, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were examined by flow cytometer. Fluorescence intensity measured by laser scanning confocal microscopy was used to explore the variation of intracellular calcium ion (Ca2+) concentration. Apoptosis-related proteins involved in both intrinsic and extrinsic apoptosis signallings were analyzed by western blot. RESULTS: SDT can effectively induce the apoptosis of endometrial cancer cells. Compared with ultrasound which is known as an effective anti-tumor method, SDT leads to a significant improvement on suppression of cell viability and induction of apoptosis, together with more remarkable modifications on the morphology and substructure in both ultrasound sensitive and resistant endometrial cancer cells. Further studies reveals that SDT promotes ROS production, induces loss of MMP and increases intracellular Ca2+ concentration more efficiently than HMME or ultrasound alone. SDT groups also show a rather high expression of apoptosis-promoting proteins, including Bax, Fas and Fas-L, and a significant low expression of apoptosis-suspending proteins including Bcl-2 and Survivin. Meanwhile, both cleaved caspse-3 and caspase-8 are dramatically enhanced in SDT groups. Multiple pathways has been proposed in the process, including the intrinsic activation by excessive ROS and overloaded Ca2+, silencing survivin gene, and the extrinsic pathway mediated by the death receptor. CONCLUSION: Given its considerable effectivity in both ultrasound sensitive and resistant cells, SDT may therefore be a promising therapeutic method for treating endometrial cancers.

Transvaginal/transrectal ultrasound for assessing myometrial invasion in endometrial cancer: a comparison of six different approaches.

OBJECTIVE: To compare the diagnostic performance of six different approaches for assessing myometrial infiltration using ultrasound in women with carcinoma of the corpus uteri. METHODS: Myometrial infiltration was assessed by two-dimensional (2D) transvaginal or transrectal ultrasound in 169 consecutive women with well (G1) or moderately (G2) differentiated endometrioid type endometrial carcinoma. In 74 of these women three-dimensional (3D) ultrasound was also performed. Six different techniques for myometrial infiltration assessment were evaluated. The impression of examiner and Karlsson's criteria were assessed prospectively. Endometrial thickness, tumor/uterine 3D volume ratio, tumor distance to myometrial serosa (TDS), and van Holsbeke's subjective model were assessed retrospectively. All subjects underwent surgical staging within 1 week after ultrasound evaluation. Definitive histopathological data regarding myometrial infiltration was used as gold standard. Sensitivity and specificity for all approaches were calculated and compared using McNemar test. RESULTS: The impression of examiner and subjective model performed similarly (sensitivity 79.5% and 80.5%, respectively; specificity 89.6% and 90.3%, respectively). Both methods had significantly better sensitivity than Karlsson's criteria (sensitivity 31.8%, p<0.05) and endometrial thickness (sensitivity 47.7%, p<0.05), and better specificity than tumor/uterine volume ratio (specificity 28.3%, p<0.05) and TDS (specificity 41.5%, p<0.05). CONCLUSION: Subjective impression seems to be the best approach for assessing myometrial infiltration in G1 or G2 endometrioid type endometrial cancer by transvaginal or transrectal ultrasound. The use of mathematical models and other objective 2D and 3D measurement techniques do not improve diagnostic performance.

8-oxo-7,8-dihydroguanine level - the DNA oxidative stress marker - recognized by fluorescence image analysis in sporadic uterine adenocarcinomas in women.

OBJECTIVES: In the case of carcinogenesis in human endometrium no information exists on tissue concentration of 8-oxo-7,8-dihydroguanine, the DNA oxidative stress marker This was the main reason to undertake the investigation of this DNA modification in human uterine estrogen-dependent tissue cancers. MATERIAL AND METHODS: In order to estimate the level of oxidative damage, 8-oxo-7,8-dihydroguanine was determined directly in cells of tissue microscope slides using OxyDNA Assay Kit, Fluorometric. Cells were investigated under confocal microscope. Images of individual cells were captured by computer-interfaced digital photography and analyzed for fluorescence intensities (continuous inverted 8-bit gray-scale = 0 [black]-255 [white]). Fluorescence scores were calculated for each of 13 normal endometrial samples and 31 uterine adenocarcinoma specimens. Finally the level of the oxidative stress marker was also analyzed according to histological and clinical features of the neoplasms. RESULTS: The obtained data revealed that: 8-oxo-7,8-dihydroguanine levels were higher in uterine adenocarcinomas than in normal endometrial samples (48,32 vs. 38,64; p<0,001); in contrast to normal endometrium there was no correlation between age and DNA oxidative modification content in uterine cancer; highest mean fluorescence intensity was recognized in G2 endometrial adenocarcinomas; level of 8-oxo-7,8-dihydroguanine does not depend on Body Mass Index (BMI) and cancer uterine wall infiltration or tumor FIGO stage. CONCLUSIONS: Our study indicates that accumulation of the oxidized DNA base may contribute to the development of endometrial neoplasia, however oxidative DNA damage does not seem to increase with tumor progression.

Sister Mary Joseph nodule in endometrial carcinoma: detection with FDG PET/CT.

Sister Mary Joseph nodules are very rare. They are commonly caused by gastrointestinal tract and ovarian cancers, but they are seen rarely in uterine carcinoma, lung carcinoma, breast carcinoma, and others. We present F-FDG PET/CT findings in a 55-year-old woman with endometrial adenocarcinoma, who presented with a mass protruding from the umbilicus after surgery and pelvic radiotherapy.

Nuclear characteristics of the endometrial cytology: liquid-based versus conventional preparation.

The aim of this study was to assess the utility of liquid-based cytologic preparation (LP) compared with conventional preparation (CP) for the assessment of nuclear findings in endometrial glandular and stromal breakdown (EGBD) which may be misdiagnosed as carcinoma in EGBD cases. The material consists of cytologic smears including 20 cases of proliferative endometrium (PE), 20 cases of EGBD, and 20 cases of endometrioid adenocarcinoma grade1 (G1) for which histopathological diagnosis was obtained by endometrial curettage at the JA Suzuka General Hospital. Nuclear findings were examined in PE cells, EGBD-stromal cells, EGBD-metaplastic cells, and G1 cells, respectively. It was examined about the following items; (1) nuclear shape; (2) A long/minor axis ratio in cell nuclei; (3) an area of cell nuclei; (4) overlapping nuclei. Results are as follows: (1) nuclear shape; as for the reniform shape of EGBD-stromal cells and spindle shape of EGBD-metaplastic cells, the ratio of the LP method was a higher value than the CP method. (2) The long axis and area of cell nuclei; LP in all groups was a recognizable tendency for nuclear shrinkage. (3) The long/minor axis ratio in cell nuclei; only EGBD-metaplastic cells recognize a significant difference between CP and LP. (4) Overlapping nuclei; LP was a higher value in comparison with CP in the other groups except PE cells, and the degree of overlapping nuclei was enhanced about three times. Therefore, although a cell of LP has a shrinking tendency, (1) it is excellent that LP preserves a characteristic of nuclear shape than CP; (2) a cellular characteristic becomes clearer, because three-dimensional architecture of LP is preserved of than CP. As for the standard preparation method for endometrial cytology samples, we considered that a concrete introduction of the LP method poses no problems.

Quantitative analysis with atomic force microscopy of cisplatin-induced morphological changes in HeLa and Ishikawa cells.

Because the cell membrane is an important regulator of cell function, its morphological changes are important markers of cell apoptosis. These changes can differ for each cell type, and depend on the treatment conditions, including the drug, doses, and treatment time. To quantify morphological changes, HeLa and Ishikawa cells were investigated with atomic force microscopy. Both cells were treated with cisplatin (1 mM) for 24 hours. The viability and proliferation of the cells were analyzed with methylthiazol tetrazolium method. The proliferation rates of both cells treated with cisplatin decreased more than 50%. The morphological changes induced by cisplatin were dependent on the cell type, and the results were determined quantitatively. The surface of HeLa cells became rougher with cisplatin treatment, whereas cisplatin-treated Ishikawa cells were smoother than untreated cells. In both cases, cell height was decreased with cisplatin treatment. These results suggest that atomic force microscopy can be used to analyze anticancer drug activity in cancer cells.

Altered claudin-4 expression in progesterone-treated endometrial adenocarcinoma cell line Ishikawa.

OBJECTIVE: To detect the expression change of claudin-4 in Ishikawa endometrial adenocarcinoma cell line in response to progesterone. To determine whether claudin-4 is involved in the anticancer effect of progesterone. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the 50% inhibitory concentration (IC50) of megestrol acetate (MA) in treating Ishikawa cells. After the Ishikawa cells were treated with MA at IC50, cell apoptosis was examined by flow cytometry and transmission electron microscopy. The messenger RNA and protein expression levels of claudin-4 were further quantified by real-time polymerase chain reaction and Western blot. The localization of claudin-4 was examined by immunofluorescent staining. RESULTS: The IC50 of MA on Ishikawa cells was 15 mg/L incubated for 72 hours. Apoptosis percentage was elevated from 0.07% ± 0.02% to 3.93% ± 0.81% after MA treatment. The expression of claudin-4 at both protein and messenger RNA levels was significantly decreased after the treatment of MA (P < 0.05). The localization of claudin-4 transferred from cytomembrane to cytoplasm and nucleus. CONCLUSION: Megestrol acetate can inhibit the growth of Ishikawa cells. It may work through decreasing claudin-4 expression and cell apoptosis. The localization change of claudin-4 may also be involved in the anticancer effect of progesterone.

Karyometry in atypical endometrial hyperplasia: a Gynecologic Oncology Group study.

OBJECTIVES: Treatment for atypical endometrial hyperplasia (AEH) is based on pathologic diagnosis. About 40% of AEH is found to be carcinoma at surgery. This study's objective is to derive an objective characterization of nuclei from cases diagnosed as AEH or superficially invasive endometrial cancer (SIEC). METHODS: Cases from GOG study 167A were classified by a central pathology committee as AEH (n=39) or SIEC (n=39). High resolution digitized images of cell nuclei were recorded. Features of the nuclear chromatin pattern were computed. Classification rules were derived by discriminant analysis. RESULTS: Nuclei from cases of AEH and SIEC occupy the same range on a progression curve for endometrial lesions. Cases of AEH and SIEC both comprise nuclei of two phenotypes: hyperplastic characteristics and premalignant/neoplastic characteristics. The principal difference between AEH and SIEC is the percentage of premalignant/neoplastic nuclei. When this percentage approaches 50-60% superficial invasion is likely. SIEC may develop already from lesions at the low end of the progression curve. CONCLUSIONS: AEH comprises cases which may constitute a low risk group involving <40% of AEH cases. These cases hold a percentage of <20% of nuclei of a preneoplastic phenotype. AEH cases from the central and high end of progression have >40% of nuclei of preneoplastic phenotype. Nuclei of the preneoplastic phenotype in AEH lesions are almost indistinguishable from nuclei in SIEC, where this percentage exceeds 60%. The percentage of nuclei of the preneoplastic phenotype in AEH esions might serve as criterion for assessment of risk for the development of invasive disease.

Morphological characteristics and co-stimulatory molecule (CD80, CD86, CD40) expression in tumor infiltrating dendritic cells in human endometrioid adenocarcinoma.

OBJECTIVE: The purpose of this study was to investigate changes of the antigen-presenting function of tumor infiltrating dendritic cells (TIDCs) in human endometrioid adenocarcinoma. STUDY DESIGN: The TIDCs from 45 cases of endometrioid adenocarcinoma were compared with 20 cases of normal human endometrial tissue, using transmission electron microscopic examination, and the expression of CD80, CD86, and CD40 was analyzed by flow cytometry. RESULTS: In comparison with the control group, the ultrastructure of TIDCs in human endometrioid adenocarcinoma showed the following differences: numerous TIDCs were small in volume and round in shape but some were oval and multi-angular. The cytoplasmic processes were obviously decreased in number and stubbed. Round primary lysosomes with high electron-dense granules, and secondary lysosomes with high or low electron-dense granules were seen frequently in the cytoplasm. TIDCs contained much rough endoplasmic reticulum (RER). Vacuoles with flocculent electron-dense granules were rare. High electron-dense contents in the granules were near one side and the other side was bright. The nucleus became markedly small in volume, nephroid or hoofed in shape. The nucleus had little euchromatin and lots of heterochromatin under the nuclear membrane. The levels of expression of CD80, CD86 and CD40 on TIDCs were low or even nonexistent. The expression levels of CD80, CD86 and CD40 on DCs in human normal endometrium were significantly higher than those on TIDCs in endometrioid adenocarcinoma. CONCLUSION: It is suggested that morphological differences and low expression of co-stimulatory molecules on TIDCs in endometrioid adenocarcinoma reflected the functional changes of the TIDCs in uptake, processing and presenting antigen, which may lead to the occurrence of tumor immune escape.

Importance of the cytoplasmic super-oxide dismutase in the normal tissue of the endometrium and the endometrium carcinoma.

OBJECTIVES: The objective of the study is comparing pathohistological picture and test results of the activity of the enzymes of the anti-oxidative protection -cytoplasmic super-oxide dismutase (CuZnSOD) from the blood and endometrium in the promotion of the progression or regression of the hyperplasia and endometrium carcinoma. MATERIALS AND METHODS: The study has been carried out on 70 patients. We have analysed:The age patients, the supersonic test - transvaginal probe, pathohistological diagnosis (PHD) analysis of the curet of the patient-we have gathered the tissue of the normal and the pathologicaly changed endometrium from the exploratory curretage, determining the CuZnSOD in the blood and in the tissue of the normal and pathological endometrium of the uterus. The Group A has been made out of 30 of them who did not have the irregular bleeding from the uterus, and 40 of them represented the Group B with the irregular bleeding, who also had PHD confirmed hyperplasia or malign changes of the endometrium. We have tested if there has been the pathalogical changes in the small pelvis (the ovary tumor, myoma etc.) in both groups. RESULTS: Dominant age in the Group B is 41 - 50 (55%), in Group A, age difference is not that apparent (p > 0.05). The results of the arithmetic mean of the CuZnSOD in the blood (19.90%) and (29.05%) in the endometrium which is lower than the Group A (blood-29.95%, endometrium-32.56%). Lower values CuZnSOD in the blood (18.9%) and endometrium (30.09%) we have in the experimental group patients who have had bleeding as well as those beside bleeding had some other gynecological - patological proces (myoma, cyst on the ovary etc.) CONCLUSIONS: According to the facts we can see the significance of the activity of the enzymes of the anti-oxidative system in the diagnostic of the hyperplasia and endometrium carcinoma as well as the possibility of their application in the clinical practice.

Precursors of endometrial and ovarian carcinoma.

This review discusses precursor lesions of endometrial and ovarian carcinoma with an emphasis on the unique molecular alterations that have led to the development of binary classification schemes for tumors of both the endometrium and ovary. While such a system is well established for endometrial carcinoma, only recently has a binary classification scheme been proposed for ovarian carcinoma. For both, the morphologic and molecular genetic-defining characteristics of their respective precursor lesions are described in detail. Furthermore, similarities and differences of the precursor lesions of specific tumors of these two genital tract organs are also addressed with a brief discussion of the clinical implications of their diagnosis.

Prognostic markers for coexistent carcinoma in high-risk endometrial hyperplasia with negative D-score: significance of morphometry, hormone receptors and apoptosis for outcome prediction.

OBJECTIVES: Hysterectomy represents the current routine therapy for high-risk endometrial precancers. More sophisticated methods are needed for treatment decision among women who want to preserve fertility and seriously ill patients. Among women diagnosed with high-risk hyperplasia, approximately 40% show signs of endometrial cancer in the hysterectomy specimen. Thus, more sophisticated methods are needed to select the women at risk. SETTING: University Hospital of Tromsø, Regional Center for Gynecological Oncology in northern Norway. POPULATION: From 1999 to 2004, 258 consecutive patients had endometrial hyperplasia diagnosed by D-score; 57 among these were high-risk cases (D-score < 0) and 10 had coexisting endometrial carcinoma. No further cancers were detected after long-term follow-up (4-10 years). DESIGN: From the initial histological specimens, material from the 10 patients with cancer and from the 13 cases without cancer (high-risk D-score < 0) was analyzed with selected histomorphometric (architectural and nuclear) and immunohistochemical (hormone receptors and apoptotic) features blinded to the investigator. METHOD: Original slides were used for computerized histomorphometry (4-class rule and related procedures). Serial sections from the paraffin embedded material were used for immunohistochemical investigations. Immunohistochemical expression in glands and stroma was evaluated by the semi-quantitative H-score (ER-alpha, ER-beta, PR-A, PR-B, RCAS-1, Bcl-2, BAX, and Caspase-3). RESULTS: The histomorphometric 4-class rule differentiates between presence and absence of cancers with a sensitivity of 80% and specificity of 77%. Several morphometric and immunohistochemical features were significantly different in cases with cancer and hyperplasia. CONCLUSIONS: Histomorphometry seems superior in predicting coexistent carcinoma in high-risk endometrial hyperplasia and should be considered for clinical use.

[Usefulness of ultrasound endometrium thickness measurement in diagnosis of endometrium pathology in women with abnormal peri- and postmenopausal bleeding]. / Przydatnosc ultrasonograficznego pomiaru grubosci endometrium w wykrywaniu patologii blony sluzowej macicy u kobiet z nieprawidlowymi krwawieniami macicznymi w okresie peri- i postmenopauzy.

OBJECTIVE: Aim of the study was to assess the usefulness of ultrasound endometrial thickness measurement in the diagnosis of endometrial pathology in women with abnormal peri- and postmenopausal bleeding. MATERIAL AND METHODS: Material included 182 patients whose endometrium sample was obtained for analysis. Patients were divided into six groups according to histological findings. RESULTS: Correlation between thickness of endometrium and histological diagnostic was sought. Wide and partially covered range of endometrium thickness were found in the observed groups. In carcinoma group the highest were maximal and average values. The difference in average thickness of endometrium in carcinoma and hyperplasia groups when compared to the remaining groups proved to be statistically significant A value of cut down ultrasound measured endometrial thickness to exclude endometrial cancer was 9 mm. CONCLUSION: Ultrasound measurement of endometrial thickness is not sufficient to increase effectiveness of endometrial pathology diagnosis in women with abnormal peri- and postmenopausal bleeding.

[Prognostic significance of AgNORs expression in endometrial cancer]. / Rokownicze znaczenie ekspresji liczby AgNORs w komórkach raka endometrium.

OBJECTIVES: The purpose of the study was assessing prognostic value of AgNORs expression in endometrial cancer. MATERIALS AND METHODS: The study was performed in a group of 90 patients, diagnosed with endometrial cancer, treated in Clinic of Gynecological Oncology in Wroclaw. Silver nucleolar organizer regions (AgNORs) were stained in paraffin-embedded samples, using Ploton method modified by Oefner. RESULTS: The mean number of AgNORs was 2.76 +/- 0.46. To assess the possible influence of AgNORs expression, patients were divided into three groups, based on the mean number of AgNORs: I- <2.3; II- 2.3-3; III- >3. The differences in the outcome between these groups were not significantly different. The mean number of AgNORs in the group of patients with distant metastases and progression did not differ (2.72). The highest mean number of AgNORs (3.38) was observed in patients with local failure. CONCLUSIONS: At this point in time, there is inadequate evidence to use AgNORs as predictive factors in patients with endometrial cancer further studies are warranted.

Ultrastructural study of benign, low-malignant potential (LMP), and malignant ovarian tumors.

Ultrastructural characteristics of benign, low-malignant potential (LMP), and malignant ovarian tumors were investigated, considering the aspects of histologic subtypes and histologic grading. In addition, the histogenesis of ovarian cancer was histologically investigated in an attempt to elucidate whether malignant tumor was generated from benign or LMP tumor, or whether it was generated de novo from normal tissues. Although all the benign, LMP, and malignant tumors appeared to be derived from Mullerian duct in serous tumors, the origin of endometrioid or mucinous tumor could not be ultrastructurally clarified. However, there was ultrastructural similarity between benign and malignant tumors among serous, endometrioid, and mucinous tumors, and it was suggested that benign adenoma may be the developmental origin of malignant tumors regardless of the histologic subtype. In addition, the investigation of endometrioid tumors revealed that the differences of histologic grading in malignant tumors reflected the ultrastructural differences, and that G1 tumor had an ultrastructure that was more similar to that of benign and LMP tumors than to that of G2 tumor.

Induction of apoptosis or necrosis in human endometrial carcinoma cells by 2-methoxyestradiol.

BACKGROUND: We investigated the effects of 2methoxyestradiol (2-ME), an endogenous estrogenic metabolite, on human endometrial cancer HEC-1-A and RL-95-2 cell lines. MATERIALS AND METHODS: After exposure of HEC-1-A and RL-95-2 cells to 2-ME, the morphological changes were evaluated by acridine orange staining and transmission electron microscopy. Cell cycle progress, apoptosis and necrosis were assessed by flow cytometry, DNA fragmentation and Western blot. RESULTS: 2-ME inhibited cell growth by blocking the S- and G2/M-phase in both cell lines, by inducing apoptosis in HEC-1-A cells and by causing necrosis in RL-95-2 cells. Apoptosis, on HEC-1-A cells, was accompanied by an increased expression of iNOS and STAT1. This apoptotic effect was prevented by the iNOS inhibitor 1400W and eliminated by the caspase inhibitor Z-VAD-FMK. Necrosis, on RL-95-2 cells, was due to a severe disruption of the mitochondrial membrane potential. 2-ME had no significant effect on normal human endometrial cells. CONCLUSION: The data suggest that 2-ME has an antitumor effect on human endometrial carcinoma cells (HEC-1-A and RL-95-2) and may contribute as a new therapeutic agent for endometrial cancer patients.

Retinoic acid signaling through PI 3-kinase induces differentiation of human endometrial adenocarcinoma cells.

The specific signals required for actin polymerization in response to extracellular factors remain unknown. However, in many cell types, there is a correlation between actin polymerization, activation of phosphatidylinositol 3-kinase (PI 3-kinase), and the production of the second messenger phosphatidylinositol-3,4,5-triphosphate. Increased levels of PI 3-kinase have been detected during cell growth and transformation. However, PI 3-kinase is also activated during differentiation, suggesting that PI 3-kinase and its lipid products also play a role in the regulation of cellular differentiation. The newly characterized CAC-1 cell line established from a poorly differentiated human endometrial adenocarcinoma (Exp. Mol. Pathol. 69 (2000), 175) was used as a model to investigate the role of PI 3-kinase in differentiation induction. CAC-1 cells differentiated upon treatment with pharmacological doses of retinoids (1 micro M of 13-cis or all-trans), evidenced by actin filament reorganization, and cell enlargement. PI 3-kinase staining is primarily localized to perinuclear regions in untreated cells. However, retinoic acid treatment induced PI 3-kinase to relocalize throughout the cytoplasm. Subcellular fractionation and Western blotting confirmed that PI 3-kinase decreased in the particulate fraction, concurrent with retinoid-induced differentiation. Interestingly, pretreatment with the PI 3-kinase inhibitor wortmannin (100 nM) prior to retinoic acid treatment prevented retinoic acid-induced actin reorganization and cell enlargement. To distinuish whether retinoid regulation of PI 3-kinase is mediated through traditional nuclear retinoic acid receptors, the levels of retinoic acid receptor-beta (RAR-beta) protein were evaluated. Retinoid treatment did not alter RAR-beta protein levels compared to controls. These data suggest that PI 3-kinase activity and cytoplasmic relocalization are required for retinoid-induced differentiation of poorly differentiated human endometrial adenocarcinoma cells.