Normal Endometrial Stromal Cells Regulate Survival And Apoptosis Signaling In Endometrial Adenocarcinoma Cells

Objective:Stroma-tumor communication plays an important role in tumorigenesis. Communications between tumor cells and microenvironments have invited increasing interest in cancer research, and the underlying mechanisms are cell-cell and cell-matrix interactions. Endometrial adenocarcinoma originates in endometrial epithelium and in vitro studies have shown that endometrial stroma cell and ECM can induce diferentiation and suppress proliferation in endometrial cancer cells. However, the molecular mechanisms and intracellular signaling in this tumor-stroma communication remains unlear. Phosphoinositide-3-kinase (PI3K)/ AKt (protein kinase B) signaling pathway plays an important role in proliferation regulation in endometrial cancers. PI3K dependent AKt activation can be negatively regulated by tumor suppressor PTEN, the mutations and loss of which are frequently observed in endometrial carcinomas. Survivin is a human gene of the inhibitor of apoptosis (IAP) family and its overexpression can be detected in many human cancers, including endometrial andenocarcinoma. Some growth factors can activate Survivin via the PI3K/AKt pathway. and PI3K inhibition can decrease the Survivin levels. Survivin inhibition can reduce cell proliferation and induce apoptosis in endometrial cancer cell lines.MAP (mitogen-activated protein kinase)/ERKs (extracellular signal-regulated kinases)/Survivin pathway is also reported to be involved in the regulation of cell proliferatin/differentiation and ERKs is overexpressed in endometrial adenocarcinoma. In the current study, we investigated the effect of normal stromal cells on the survival and apoptosis of endometrial cancer cells and further explored the possible signaling pathways implied in this communication. Using primarily cultured endometrial stromal cells and an endometrial adenocarcinoma cell line, Ishikawa cells, we established a 2D-coculture system to observe the stromal cell - tumor cell crosstalk in endometrial carcinomas, to determine the effect of normal stromal factors on Survivin expression in endometrial cancer cells, and to explore the pathways that might be involved in Ishikawa cells Survivin regulation in this stroma-tumor cells interaction.Materials and Methods:1. Tissue collection and cell cultureNormal endometrial biopsies were obtained from 12 women of reproductive age (mean age:45.6±3.378) undergoing bilateral tubal ligation. The patients had regular menstrual cycles and biopsies were all in their proliferative phase. All the participants did not receive any hormonal therapy for at least 6 months before their operations. Informed consents were obtained from all participants prior to biopsy and the use of human tissues was approved by the institutional review board of Provincial Hospital Affiliated to Shandong University.Normal stromal cells (NSCs) were isolated from the tissue using 0.1% collagenase IA and sieves, and were characterized by immunocytochemistry analysis with mouse anti-human vimentin antibody.2. Preparation of conditioned culture mediumConditioned culture medium from NSCs.2×105 NSCs of passage 1-2 were plated on top of a thin Matrigel layer (0.5 mm) in 24 well plates.2 ml phenol red-free DMEM/F12 (1:1, v/v) supplemented with 2% charcoal-stripped serum, which was called basic medium (BM), was added to each well. At day 5 the supernatants were collected and the cell debris were removed by centrifugation.Conditioned culture medium from HELF cells.2×105 HELF cells were plated on top of a thin Matrigel layer in 24 well plates.2 ml BM was added to each well. At day 5 the supernatants were collected and the cell debris were removed by centrifugation.3. Methyl thiazolyl tetrazolium (MTT) assay The proliferation of Ishikawa cells was assessed by MTT test. Cell suspension was added to each well of the 96-well flat-bottom plates. After cell adherence, the medium was removed and replaced with CM or experimental medium for 5 days. Following incubation, MTT working solution was added to each well and the absorbance of each well was measured with a microculture plate reader at 570 nm.4. Colony formation assay of Ishikawa cellsIshikawa cells were plated on 12-well plates with culture medium. After cell adherence, CM or experimental medium was added to each dish. Cultures were maintained for 5 days, then the dishes were fixed and stained with methylene blue. Colonies with more than 75 cells were counted.5. Immunocytochemistry and immunohistochemistryAfter treatment, Ishikawa cells on slips were fixed with 4% paraformaldehyde for half an hour and tritonX-100 for 20min. Endogenous peroxidase was blocked by incubation in 0.3% H2O2 for 10 min. Next the slips were incubated with rabbit anti-human Survivin as primary antibodies for 2h, and HRP-conjugated goat anti-rabbit IgG as the second antibody. HRP activity was detected using DAB as substrate for 2 min following the manufacturer's instructions.Tissue samples used for immunohistochemistry were normal endometria (n=21) and endometrial adenocarcinoma tissue (n=20). Fresh tissues were fixed with 4% paraformaldehyde for 24 hr and embedded in paraffin. Samples were cut into 4μm sections and mounted onto glass slides. After blocking with 0.3% H2O2 and antigen retrieval with microwave oven, the sections were incubated with the primary rabbit anti-human Survivin antibody overnight. A HRP-conjugated goat anti-rabbit IgG was used as second antibody. HRP activity was detected using diaminobenzidine tetrahydrochloride (DAB) as substrate for 2 min. The immunohistochemical score was estimated as series of the percentage of positively staining glandular epithelial cells. The immunoactivity was shown as quantity score×staining intensity.6. Total protein extraction and Western blottingCells were collected by trypsinization. Total protein was extracted using the Mammalian Cell Extraction Kit.50μg of total protein extracted from cells were transferred to Hybond-P polyvinylidene difluoride membranes. After transfer, the membranes were incubated with rabbit anti-human AKt, p-AKt, Survivin, andβ-actin monoclonal antibodies, then were incubated with IIRP-conjugated goat anti-rabbit secondary antibodies. The immunoblots were developed with the ECL Plus western blotting agent and exposed to X-ray film. Western blot densitometric analysis was performed using AlphaImager 2200 gel documentation system with image analysis software.7.Enzyme linked immunosorbent assay (ELISA)Ishikawa cells were plated and treated in 24-well flat-bottom plates.100μl of each diluted cell lysate was incubated with Detection Antibody for 1 hour at 37℃and then, HRP-linked secondary antibody was added. The absorbance was read at 450 nm within 30 minutes after adding STOP Solution.8.Statistical AnalysisStatistical analyses were performed with ANOVA using SPSS 11.5 (SPSS Inc., Chicago, IL). Values are expressed as mean±SD. Differences between two and multiple groups were determined by analysis of variance (ANOVA). A p-value <0.05 was considered statistically significant.Results:1.17β-estradiol-progesterone promotes Ishikawa cells proliferation by evoking cell survival signaling. Combined 17β-estradiol-progesterone (E+P) significantly enhanced Ishikawa cell proliferation (p＜0.01). Through immunohistochemistry and immunocytochemical analysis, endometrial adenocarcinoma showed significantly higher Survivin expression than normal enodmetrium (p<0.01). Also the Survivin immunostaining in Ishikawa cells was strong and mostly localized to the nuclei. Western blot analysis showed that combined E+P induced a significant Survivin expression increase in Ishikawa cells (p<0.01). MTT test and colony counting showed that both LY294002, a specific PI3K inhibitor, and U0126, a specific MAPK inhibitor, significantly inhibited basic and hormone-induced cell proliferation in Ishikawa cells (p<0.01). Western blot analysis showed that LY294002 treatment significantly decreased basic and hormone-induced Survivin expression (p<0.01). Via ELISA analysis, we also found that U0126 significantly decreased basic and hormone-induced Survivin expression in Ishikawa cells (p<0.01). Relationship analysis showed that AKt phosphorylation (r2=0.916, p＜0.001), ERK phosphorylation (r2=0.371, p＜0.001), and Survivin expression (r2=0.758, p＜0.001) were all positively correlated with cell proliferation in Ishikawa cells.2. Paracrine factors from NSCs abolish hormone-stimulated cell growth in Ishikawa cells. MTT test showed that CM by NSCs efficiently decreased both the baseline cell proliferation (p<0.01) and hormone-induced cell proliferation (p<0.01) in Ishikawa cells, while CM by HELF, a human embryonic lung fibroblast cell line, did not induce proliferation inhibition in Ishikawa cells.3. Paracrine factors from NSCs decrease Survivin expression in Ishikawa cells. Data by western blot analysis showed that the Survivin expression in Ishikawa cells was decreased by 23% in the presence of CM by NSCs. CM by NSCs also blocked hormone-induced Survivin expression by 82%.4. PI3K/AKt signaling, but not MAPK/ERK signaling, is responsible for the Survivin reduction by normal stromal factors. Using western blot, both basic and E+P stimulated AKt phosphorylation in Ishikawa cells were reduced by the addition of stromal CM to the culture (p<0.01), but unexpectedly, no effect on ERK phosphorylation was noted in the presence of CM by NSCs (p>0.05).Conclusions:1. There are two cell survival pathways in Ishikawa cells:the PI3K/AKt/Survivin pathway, and the MAPK/ERK/Survivin pathway,and combined estrogen-progesterone (E+P) treatment can promote cell proliferation in Ishikawa cells via the two different pathways.2. Both PI3K inhibition and MAPK inhibition repeatedly and significantly abolished E+P-stimulated and decreased background cell growth in Ishikawa cells, indicating that PI3K/AKt signaling and MAPK/ERK signaling are essential in E+P-stimulated cell growth. 3. Survivin expression was increased along with AKt phosphorylation and ERK phosphorylation under E+P treatment. Survivin inhibition reduces cell proliferation and induces apoptosis in endometrial cancer, indicating the essential role of Survivin in endometrial cancer.4. Paracrine factors from endometrial stromal cells grown on Matrigel decrease E+P-stimulated cell growth and E+P-stimulated activity of PI3K/AKt/Survivin signaling in Ishikawa cells. This suggests that Paracrine factors from endometrial stromal cells grown on Matrigel can inhibit E+P-stimulated cell growth via the survival signaling pathway.5. It is PI3K/AKt signaling, not MAPK/ERK signaling, that is responsible for the decrease in Survivin expresion by paracrine factors from normal endometrial stromal cells.6. The growth regulation by paracrine factors from NSCs cultured on Matrigel was specific to endometrial stromal cells.