An integrated approach of network pharmacology, molecular docking, and experimental verification uncovers kaempferol as the effective modulator of HSD17B1 for treatment of endometrial cancer.

BACKGROUND: Endometrial cancer (EC) is one of the most common gynecological malignancies globally, and the development of innovative, effective drugs against EC remains a key issue. Phytoestrogen kaempferol exhibits anti-cancer effects, but the action mechanisms are still unclear. METHOD: MTT assays, colony-forming assays, flow cytometry, scratch healing, and transwell assays were used to evaluate the proliferation, apoptosis, cell cycle, migration, and invasion of both ER-subtype EC cells. Xenograft experiments were used to assess the effects of kaempferol inhibition on tumor growth. Next-generation RNA sequencing was used to compare the gene expression levels in vehicle-treated versus kaempferol-treated Ishikawa and HEC-1-A cells. A network pharmacology and molecular docking technique were applied to identify the anti-cancer mechanism of kaempferol, including the building of target-pathway network. GO analysis and KEGG pathway enrichment analysis were used to identify cancer-related targets. Finally, the study validated the mRNA and protein expression using real-time quantitative PCR, western blotting, and immunohistochemical analysis. RESULTS: Kaempferol was found to suppress the proliferation, promote apoptosis, and limit the tumor-forming, scratch healing, invasion, and migration capacities of EC cells. Kaempferol inhibited tumor growth and promotes apoptosis in a human endometrial cancer xenograft mouse model. No significant toxicity of kaempferol was found in human monocytes and normal cell lines at non-cytotoxic concentrations. No adverse effects or significant changes in body weight or organ coefficients were observed in 3-7 weeks' kaempferol-treated animals. The RNA sequencing, network pharmacology, and molecular docking approaches identified the overall survival-related differentially expressed gene HSD17B1. Interestingly, kaempferol upregulated HSD17B1 expression and sensitivity in ER-negative EC cells. Kaempferol differentially regulated PPARG expression in EC cells of different ER subtypes, independent of its effect on ESR1. HSD17B1 and HSD17B1-associated genes, such as ESR1, ESRRA, PPARG, AKT1, and AKR1C1\2\3, were involved in several estrogen metabolism pathways, such as steroid binding, 17-beta-hydroxysteroid dehydrogenase (NADP+) activity, steroid hormone biosynthesis, and regulation of hormone levels. The molecular basis of the effects of kaempferol treatment was evaluated. CONCLUSIONS: Kaempferol is a novel therapeutic candidate for EC via HSD17B1-related estrogen metabolism pathways. These results provide new insights into the efficiency of the medical translation of phytoestrogens.

Estrogen receptor-associated receptor α and peroxisome proliferator-activated receptor γ in metabolism and disease (Review).

Estrogen receptor­associated receptor α (ERRα) is an orphan nuclear receptor that lacks corresponding ligands. ERRα recruits co­regulators to regulate gene transcription and plays an important role in human physiological functions. Peroxisome proliferator­activated receptor Î³ (PPARγ) is also a nuclear receptor that regulates the expression of target genes via a ligand­dependent mechanism, thereby participating in a series of physiological processes. Both ERRα and PPARγ are involved in the process of energy metabolism and tumorigenesis. In the present review, a concise overview of the important roles governed by ERRα and PPARγ in metabolism and their association with various disease are provided.

Effects of RNAi-induced Skp2 inhibition on cell cycle, apoptosis and proliferation of endometrial carcinoma cells.

The aim of the current study was to investigate the underlying mechanism of S-phase kinase associated protein 2 (Skp2) gene inhibition by lentivirus-mediated RNA interference (RNAi) on the cell cycle, apoptosis and proliferation of endometrial carcinoma HEC-1-A cells. A lentivirus shRNA vector targeting Skp2 was constructed and transfected into HEC-1-A cells. HEC-1-A cells transfected with a scramble sequence were used as negative controls. The mRNA and protein expression of Skp2, p27, cyclin D1 and caspase-3 were detected via reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The effects of Skp2 inhibition on the cell cycle, apoptosis and proliferation of HEC-1-A cells were detected using flow cytometry and a cell counting kit-8. Skp2 co-expression data was analyzed using Oncomine and TCGA databases. The positive recombinant viral clones were identified via PCR and confirmed via sequencing. The mRNA and protein expression of Skp2 were significantly decreased in HEC-1-A cells transfected with the lentiviral vectors compared with the negative control. In addition, there were no significant changes in the mRNA expression of p27 and cyclin D1; however, the protein levels of p27 and cyclin D1 were upregulated and downregulated, respectively, in HEC-1-A cells transfected with lentiviral vectors compared with negative controls. RNAi-induced Skp2 inhibition exerted an anti-proliferative effect by inducing cell cycle arrest, however cell apoptosis was not significantly affected. In the TCGA database, Skp2 expression positively associated with IGF2R, IGF2BP3, IGFBP1 and CCNF, while Skp2 expression negatively associated with IGF2, IGFBP6, IGFBP7 and IGFBP3. RNAi-induced Skp2 inhibition upregulated the protein expression of p27 and downregulated the protein expression of cyclin D1. The expression of Skp2 in endometrial cancer may therefore be regulated by the insulin-like growth factor 1 receptor signaling pathway.

[Expression and significance of matriptase in ovarian cancer cells with diverse metastatic potential].

OBJECTIVE: To study the expression and significance of matriptase in different metastatic potential of human ovarian cancer cells. METHODS: High-metastatic human ovarian cancer cell HO8910PM and ovarian cancer cell HO8910 were collected.The ability of metastatic of the former was stronger than that of the latter. Compared the ability of invasion and migration in HO8910PM and HO8910 by scratch assay and by millicell chamber artificial reconstituted basement membrane invasion assay. Detected the matriptase mRNA and protein expression levels in HO8910PM and HO8910 through reverse transcription(RT)-PCR and immunocytochemistry methods. RESULTS: The 24 hours' migration distance (347 ± 8) µm of HO8910PM cells were significantly higher than that in HO8910 group (154 ± 10) µm (P < 0.01);The number of HO8910PM cells that penetrated the matrigel after 24 hours' incubation were significantly higher than that in HO8910 group (90.7 ± 2.1 vs 63.3 ± 1.5,P < 0.01). The expression of matriptase mRNA in HO8910PM cells was higher than that in HO8910 group (0.72 ± 0.03 vs 0.38 ± 0.04,P < 0.01). The migration was positively correlated with the matriptase mRNA expression levels (r = 0.992, P < 0.01); and the invasiveness was also positively correlated with the matriptase mRNA expression levels (r = 0.973, P < 0.01). As far protein level,the expression of matriptase protein in HO8910PM cells was higher than that in HO8910 group (15.6 ± 0.8 vs 7.6 ± 1.3,P < 0.01). The migration was positively correlated with matriptase protein expression levels (r = 0.971, P < 0.01); And the invasiveness was also positively correlated with the matriptase protein expression levels (r = 0.958, P < 0.01). CONCLUSIONS: The relationship between the expression levels of matriptase and the metastatic of ovarian cancer cells may be correlative. The function of matriptase in ovarian cancer cells metastatic mechanism still need to be confirmed.

[Analysis different transcriptional factors in different phenotype endometrial cancer cells].

OBJECTIVE: To analysis the activity of transcriptional factors in endometrial cancer cell lines with different estrogen receptor subtypes. METHODS: The mRNA levels of estrogen receptor (ER) was detected by quantitative RT-PCR, and the activity of transcriptional factors was also analysed by 345-channel protein/DNA array in RL-952 (the expression status of ERalpha and ERbeta both positive), HEC-1A [ERalpha (+/-), while ERbeta negative] and HEC-1B (ERalpha and ERbeta both negative). The transcription factors of NFkappaBp65 and p38MAPK with different activity were tested by enzyme-linked immunosorbent assay (ELISA) to confirm the results of protein/DNA array. RESULTS: The mRNA levels of ERalpha in RL-952, HEC-1A and HEC-1B were (6780+/-282), (684+/-84) and (168+/-38) copy/ng, respectively. Among 345 candidate transcriptional factors, there were 28 factors associated with ER status. Compared with RL-952 cells, 13 transcriptional activity factors were concomitantly up-regulation, while 15 concomitantly down-regulation in HEC-1A and HEC-1B cells. Transcriptional activities of TTF (1)-1, NRF-1, TCE were significantly correlated with the high-expression status of ERalpha mRNA (r=0.523, P=0.037), while RFX123 and Ikaros were significantly correlated with the low-expression status of ERalpha mRNA (r=-0.312, P=0.041). CONCLUSION: Transcriptional factors of TTF (1)-1, NRF-1, TCE may be associated with ER-mediated signal pathway, while RFX123 and Ikaros may be associated with non ER-mediated signal pathway in endometrial cancer.

Different biological effect of estrogen receptor-related receptor α in estrogen receptor-positive and -negative endometrial carcinoma.

Estrogen receptor-related receptor α (ERRα) has been identified as a nuclear transfactor closely related to estrogen receptor α (ERα). ERRα interferes with ER-mediated signaling pathways through competition with ERα for the common DNA sites and coregulators. Thus, it may participate in the tumorigenesis of estrogen-related cancers. To elucidate the roles of ERRα in endometrial carcinogenesis and the crosstalk between ERα and ERRα, endometrial carcinoma Ishikawa and HEC-IA cells were treated with different concentrations of 17ß-E2 and/or the pure anti-estrogen drug ICI182,780. Using semi-quantitative RT-PCR and Western blot analysis, we found that 17ß-E2 down-regulated the expression of ERRα in ER-positive Ishikawa cells, while up-regulating the expression of ERRα in ER-negative HEC-IA cells. Down-regulation in Ishikawa cells was furthermore found to be largely abrogated by ICI182,780. Additionally, we constructed endometrial carcinoma cell lines with overexpression of ERRα by stable transfection, renaming these Ishikawa/ERRα and HEC-IA/ERRα, respectively. To investigate the effect of ERRα overexpression on the biological behavior of the cells, MTT assay and flow cytometry analysis were performed in the constructed cell lines. In ER-positive Ishikawa cells, the overexpression of ERRα inhibited cell growth in the presence of 17ß-E2, an inhibitory effect that might be due to a G0-G1 cell cycle arrest. In contrast, overexpression of ERRα stimulated cell proliferation in ER-negative HEC-IA cells independently of 17ß-E2. This accelerated action was associated with changes in cell cycle distribution. Our study demonstrates that, in addition to ER, ERRα seems to be an important regulator in endometrial carcinogenesis, playing different roles in estrogen-dependent and -independent endometrial carcinomas. ERRα might modulate the ER-mediated pathways via interference with ERα transcription in endometrial carcinoma cells.

[Overexpression of estrogen receptor-related receptor alpha can stimulate estrogen receptor negative endometrial cancer cell proliferation].

OBJECTIVE: To investigate the role of human estrogen receptor-related receptor (ERR) alpha, a submember of orphan receptors, in the tumorigenesis of endometrial cancer. METHODS: Plasmid of pSG-ERRalpha was transfected into endometrial cancer cell lines HEC-1A, HEC-1B, and Ishikawa. Real-time quantitative RT-PCR and western blot were used to analyze the mRNA and protein expression of ERRalpha in endometrial cancer cell. Flow cytometry was used to analyze the cellular growth. RESULTS: Expressions of the ERRalpha were significantly increased in the endometrial cancer cells transfected with pSG-ERRalpha plasmid; expression of the ERRalpha mRNA in HEC-1A cell was 9644.4 copies/ng, HEC-1B: 9835.3 copies/ng, and Ishikawa: 8008.6 copies/ng (P < 0.01); expression of the ERRalpha protein in HEC-1A cell was 1.128, HEC-1B: 1.104, and Ishikawa: 1.008 (P < 0.05). Flow cytometry showed over-expression of ERRalpha was accompanied by increased HEC-1A and HEC-1B cells in S and G(2)/M phase (P < 0.01), while this could not be observed in the estrogen receptor (ER) positive endometrial cancer cell line Ishikawa. Furthermore, cellular growth analysis showed that over-expression of ERRalpha induced cell growth increase of the ER negative endometrial cancer cells HEC-1A and HEC-1B (P < 0.05). CONCLUSION: Over-expression of ERRalpha could stimulate ER negative endometrial cancer cell proliferation independent of estrogen-ER pathway.

The telomerase activity and expression of hTERT gene can serve as indicators in the anti-cancer treatment of human ovarian cancer.

OBJECTIVE: The aim of this study was to evaluate the clinicopathological significance of telomerase activity and expression of hTERT gene in human ovarian cancer. The potential value of them as indicators for chemotherapy in ovarian cancer cells was also studied. MATERIALS AND METHODS: A total of 73 samples and ovarian cancer cell lines HO-8910 and COC1 were studied. Telomerase activity was detected by PCR-TRAP-ELISA assay and the expression of the hTERT mRNA was analyzed by semi-quantitative RT-PCR. Alteration of the telomerase activity and hTERT mRNA were also analyzed in the ovarian cancer cells treated with different concentration and different time of cisplatin. Cytogenetic analysis was performed to compare the telomere status in the OH-8910 cells pre- and post-cisplatin treatment. The associations between these two markers and cisplatin induced-apoptosis were respectively analyzed in COC1 cells by the flow cytometry. RESULTS: Telomerase activity are highly increased in malignancy (0.795+/-0.168(A450-655 nm)) than borderline (0.389+/-0.174(A450-655 nm)), benign tumors (0.236+/-0.102(A450-655 nm)) and normal ovary (0.213+/-0.070(A450-655 nm)) (p < 0.05). Twenty samples showed detectable levels of hTERT. The hTERT gene positive lesion showed significantly higher telomerase activity than negative (p = 0.004). There is a significant correlation between the telomerase activity and expression of hTERT (r = 0.921). Both telomerase activity and expression of hTERT can reflect the chemotherapeutic effect of cisplatin in a time-dependent and dose-dependent manner. Treatment with 10 microM cisplatin, the hTERT mRNA decreased after 12h and completely disappeared after 48 h, whereas the telomerase activity did not decrease until 24h. Results from cytogenetic analysis and flow cytometry assay confirmed that the alterations of these two markers are associated with the anti-cancer treatment of cisplatin. CONCLUSION: Expression of hTERT gene is rate-limiting with the activation of telomerase. Both of they may be useful in the predicting of chemotherapeutic effect in ovarian cancer.

[Expression of estrogen receptor-related receptor alpha and estrogen receptor alpha in endometrial carcinoma and the primary investigation of their clinical significance].

OBJECTIVE: To explore the roles of estrogen receptor-related receptor (ERR) alpha and estrogen receptor alpha in endometrial carcinoma and their clinical values. METHODS: Thirty-five cases of endometrial carcinoma were examined by immunohistochemistry. Clinicopathologic features including FIGO stage, histological grade, myometrial invasion and nodal metastasis were reviewed. RESULTS: The expression rate of ERalpha in patients with FIGO stage I endometrial carcinoma was more than that in stage II-IV(P = 0.005). The expression rate of ERRalpha in endometrial carcinoma patients at FIGO stage I was lower than that at stages II-IV(P = 0.007). The percentage of patients at FIGO stageI or with G1-2 or myometrial invasion < 1/2 in ERalpha (+) and ERRalpha (-) groups was higher than that in ERalpha (-) and ERRalpha (+) groups(P = 0.000, P = 0.031 and P = 0.022). CONCLUSION: ERalpha may be a biomarker of good prognosis while ERRalpha may serve as a biomarker of poor prognosis in endometrial carcinoma. The combinative measure of ERalpha and ERRalpha may improve the prognostic value.

[Regulatory effect of 17beta-estradiol on expression of orphan nuclear receptor ERRalpha in endometrial carcinoma cell lines].

BACKGROUND & OBJECTIVE: Estrogen receptor-related receptor alpha (ERRalpha), a member of the subfamily of orphan nuclear receptors, could compete with estrogen receptor alpha (ERalpha) to bind the same target genes and interfere in ER signal pathway. Therefore, it might be associated to the tumorigenesis of endometrial carcinoma. This study was to explore the regulatory effect of 17beta-estradiol (17beta-E(2)) on ERRalpha expression, and to elucidate the relationship between ERRalpha and ER signal pathway in endometrial carcinoma cell lines. METHODS: ERalpha-positive cell line Ishikawa and ERalpha-negative cell line HEC-IA were treated with different concentrations of 17beta-E(2) (1x10(-10) mol/L, 1x10(-8) mol/L, and 1x10(-6) mol/L) for 24 h and 48 h, respectively. The levels of ERRalpha mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. 17beta-E(2) (1x10(-8) mol/L) and complete ER inhibitor ICI182,780 (1x10(-6) mol/L) were given concomitantly to observe the change of ERRalpha expression. RESULTS: The levels of ERRalpha mRNA and protein in Ishikawa cells were down-regulated after stimulated for 24 h and 48 h by different concentrations of 17beta-E(2). The maximal effect was observed at the concentration of 1x10(-8) mol/L. When 17beta-E(2) and ICI182,780 were given simultaneously to Ishikawa cells, this down-regulation was blocked. However, the level of ERRalpha mRNA, not protein, in HEC-IA cells was up-regulated after stimulated by different concentrations of 17beta-E(2) for 24 h. After stimulated by 17beta-E(2) for 48 h, the level of ERRalpha protein was up-regulated, which could not be blocked by ICI182,780. CONCLUSIONS: 17beta-E(2) can down-regulate the expression of ERRalpha in Ishikawa cells, which is mediated by ERalpha. 17beta-E(2) can up-regulate the expression of ERRalpha in HEC-IA cells, but this regulation cannot be blocked by ICI182,780.

17 beta-estradiol activates PI3K/Akt signaling pathway by estrogen receptor (ER)-dependent and ER-independent mechanisms in endometrial cancer cells.

Cellular response to estrogen is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transducing pathways. The main aims are to study if PI3K/Akt signaling pathway can be activated by 17beta-estradiol (E2) via non-nuclear action and to investigate the relationship of the action of E2 and ER in endometrial cancer cells expressing with different status of ER. The levels of phosphorylated Akt (Ser473) (P-Akt) and total Akt were examined by western blot and Akt kinase activity was measured in cells after stimulation with 1 microM E2 at different time points. Inhibitory role of LY294002 on activation of Akt induced by E2 and its estrogen antagonist, ICI182780 were also tested. P-Akt/Akt was used as a measure of activation of Akt. We found that maximum P-Akt/Akt and Akt kinase activity took place at 30 min in Ishikawa cells and 15 min in HEC-1A cells and the activation persisted for at least 2 h after stimulation with 1 microM E2. The activation of Akt elicited gradually with increasing doses of E2. PI3K inhibitor, LY294002, stopped the activating Akt in a dose-dependent manner and 50 microM LY294002 completely blocked the activation of Akt induced by E2. ICI182780 could block the activation of PI3K/Akt in ER-positive Ishikawa cells but not in HEC-1A cells with poor-expressed ER. This study demonstrated that E2 is able to promptly activate PI3K/Akt signal pathway in Ishikawa cells in an ER-dependent manner and ER-independent in HEC-1A cells. Blockage of PI3K/Akt cascade may become a potential and effective way to control endometrial carcinoma, especially in ER-negative cancers, which show no response to endocrinal therapy.

[Expression of estrogen receptor-related receptor isoforms in endometrial carcinoma tissues and its clinical significance].

OBJECTIVE: To investigate the role of estrogen receptor-related receptor (ERR), a subfamily of orphan receptors, in the tumorigenesis of endometrial carcinoma and to evaluate the clinical significance. METHODS: A total of 46 cases of endometrial carcinoma tissues and 24 cases of normal endometrium tissues were collected. Semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry were used to measure the expressions of ERR. All samples were classified into two groups according to ERalpha status. RESULTS: It was positively correlated between the ERR (alpha, beta, gamma) mRNA and protein expression (r = 0.462, P = 0.009; r = 0.689, P = 0.016 and r = 0.673, P = 0.001). The expression rate and level of ERRalpha mRNA in ERalpha-positive endometrial carcinoma (42%, 0.24 +/- 0.18) were lower than in normal endometrium (78%, 0.42 +/- 0.23; P = 0.019, P = 0.021). No significant difference was observed in the expression of ERRbeta mRNA between endometrial carcinoma (23%, 0.21 +/- 0.16) and normal endometrium (22%, 0.27 +/- 0.15; P > 0.05). In ERalpha-positive endometrial carcinoma, the expression level of ERRgamma mRNA (0.93 +/- 0.24) was higher than normal endometrium (0.72 +/- 0.21; P = 0.023). The numbers of patients of Federal International Gynecological Oncology (FIGO) stage II-IV and of deep myometrial invasion in ERRalpha mRNA positive endometrial carcinoma group were more than those in ERRalpha mRNA negative group (P = 0.017 and P = 0.033). ERRgamma mRNA positive patients had a lower occurrence of lymph metastases than negative patients (P = 0.021). CONCLUSIONS: ERR may be closely related with the tumorigenesis of endometrial carcinoma, similar to ER. ERRalpha may serve as a biomarker of poor prognosis and ERRgamma as a favorable biomarker in endometrial carcinoma.

[Role of estrogen receptor-related receptors alpha, beta and gamma in ovarian cancer cells].

OBJECTIVE: To investigate the role of human estrogen receptor-related receptors (ERRs) in ovarian cancer. METHODS: A green fluorescent protein (GFP) reporter plasmid of ERRalpha was constructed and transfected into ovarian cancer cell lines SKOV3 and OVCAR3. RT-PCR was used to analyze the expression of ERRalpha, ERRbeta, and ERRgamma mRNA in 33 primary ovarian cancer samples and 12 normal ovarian epithelial cells. Progression-free survival time and overall survival time of patients with different expression of ERRs were analyzed by Kaplan-Meier method. RESULTS: GFP-ERRalpha fusion protein showed ERRalpha expressed in the ovarian cancer cell nucleus. Expressions of the ERRalpha and ERRgamma were increased in the ovarian cancers (58% and 48%) compared with the normal ovarian tissues (P < 0.05); ERRbeta was weakly expressed in the ovarian cancer (9%) as well as in the normal tissues (0). Expression of ERRalpha was positively correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage (r = 0.639, P = 0.017) and cell grading (r = 0.520, P = 0.022). Survival analysis showed that ERRalpha positive group had a poorer overall survival (19.0 months vs. 31.5 months, P < 0.05) and ERRgamma positive group had a longer progression-free survival (18.0 months vs. 13.5 months, P < 0.05). CONCLUSIONS: ERRalpha chiefly expresses in the cell nucleus. ERRalpha and ERRgamma may play important roles in ovarian cancer.

[Regulation of orphan receptor ERR alpha by estrogen and progesterone in endometrial carcinoma cell line].

OBJECTIVE: To explore the relationship between expression of ERR alpha mRNA and estrogen and progesterone and to elucidate the function of ERR alpha in endometrial carcinoma. METHODS: The expression of ERR alpha mRNA was examined by reverse transcription polymerase chain reaction. Endometrial carcinoma cell line Ishikawa was dealt with different concentrations of 17beta-estradiol (10(-10) mol/L, 10(-8) mol/L and 10(-6) mol/L) for 15 min, 30 min and 24 h and 17beta-estradiol and ER inhibitor-ICI182780 were given concomitantly to observe the change of ERR alpha mRNA. Different concentrations of progesterone (10(-8) mol/L, 10(-7) mol/L, 10(-6) mol/L and 10(-5) mol/L) were also given to Ishikawa cell line for 24 h to observe the change of ERR alpha mRNA. RESULTS: The expression level of ERR alpha mRNA was slightly higher than that of the control group after being stimulated for 15 min, 30 min and 24 h by 10(-10) mol/L 17beta-estradiol. However the expression level of ERR alpha mRNA was significantly lower than that of the control group after being stimulated for 15 min, 30 min and 24 h by 10(-8) mol/L and 10(-6) mol/L of 17beta-estradiol. When 10(-6) mol/L of ICI182780 and 10(-8) mol/L of 17beta-estradiol were given simultaneously to Ishikawa cell line, this down-regulation was blocked. After being stimulated for 24 h by 10(-7) mol/L, 10(-6) mol/L and 10(-5) mol/L of progesterone, the expression levels of ERR alpha mRNA were significantly higher than that of the control group, but no change was found in 10(-8) mol/L of progesterone. CONCLUSION: 17Beta-estradiol can down-regulate the expression of ERR alpha mRNA and this regulation is mediated by estrogen receptor. Progesterone can up-regulate the expression of ERR alpha mRNA.

[Activation of phosphatidylinositol 3-kinase-protein kinase B (PI3K-PKB) induced by 17beta-estradiol in endometrial carcinoma cell (Ishikawa)].

OBJECTIVE: Cellular response to estradiol is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transduction pathways such as mitogen activated protein kinase (MAPK) pathway. However, the signal transduction of estrogen involving phosphatidylinositol 3-kinase-protein kinase B (PI3K-PKB) is not clear in endometrial carcinoma. Our purpose was to study if PI3K-PKB signaling pathway could be activated rapidly by 17beta-E(2) through non-nuclear action and also, whether PI3K inhibitor, LY294002, could inhibit such non-nuclear action of 17beta-E(2) in endometrial carcinoma cell line Ishikawa. METHODS: Levels of phosphorylated PKB (Ser473 site, p-PKB) and total PKB were examined by western blotting in Ishikawa cells after stimulation with 17beta-E(2) at 1 x 10(-6) mol/L for different time periods and at varied doses for 30 min. Optimal time and appropriate dose for 17beta-E(2) to activate PKB in Ishikawa cells were observed. Inhibitory effect of LY294002 on activation of PKB induced by 17beta-E(2) was also studied. p-PKB/PKB ratio was used to indicate levels of activation of PKB. RESULTS: p-PKB/PKB at 15 min (0.533 +/- 0.029) was significantly higher than the control (0.361 +/- 0.029, P < 0.05). Maximal activation of PKB (maximal levels of p-PKB/PKB) took place at 30 min (0.666 +/- 0.021, P < 0.001) and the activation persisted at least 2 h after stimulation with 1 x 10(-6) mol/L 17beta-E(2) in Ishikawa cells. With increased doses of 17beta-E(2) (vehicle, 1 x 10(-10), 1 x 10(-8), 1 x 10(-6), 1 x 10(-4) mol/L), the activation of PKB (p-PKB/PKB was 0.300 +/- 0.098, 0.312 +/- 0.081, 0.625 +/- 0.100, 0.771 +/- 0.041, 0.902 +/- 0.043 accordingly) increased gradually (in comparison with vehicle, P > 0.05, < 0.05, < 0.05, < 0.001 accordingly). PI3K inhibitor, LY294002 could inhibit the activation of PKB induced by 17beta-E(2) in Ishikawa cells. Levels of PKB (p-PKB/PKB) decreased (0.443 +/- 0.032, 0.415 +/- 0.032, 0.111 +/- 0.035, 0, 0) gradually with increased concentrations (vehicle, 0.1, 10, 50, 100 micro mol/L) of LY294002 (compared with vehicle, P > 0.05, < 0.05, < 0.001, < 0.001) and 50 micro mol/L LY294002 completely blocked the activation of PKB induced by 17beta-E(2). CONCLUSIONS: 17beta-estradiol, by non-nuclear action, can activate promptly PI3K-PKB signaling pathway in endometrial carcinoma cell line, Ishikawa. This action of 17beta-E(2) is PI3K dependent and can be inhibited or blocked by LY294002.

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

OBJECTIVE: To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. METHODS: (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. RESULTS: (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P < 0.01) and 1.9-fold (128 +/- 37, 349 +/- 30, P < 0.05) in ERalpha and ERbeta, respectively, in pcDI-[(12)Asp]K-ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P < 0.05) and 1.8:1 (493 +/- 20, 284 +/- 20, P < 0.01), respectively; In HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P < 0.01) and 2.4:1 (405 +/- 33, 165 +/- 15, P < 0.01), respectively. (3) In low serum (2%) culture condition, estradiol (E(2)) stimulated luciferase activity with an increase of 13-fold (130 +/- 42, 1681 +/- 242, P < 0.01) in pcDI-[(12)Asp] K-ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P < 0.001) in HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P < 0.01) and 6-fold (2148 +/- 259, 12,705 +/- 2670, P < 0.001), respectively. rafS621A mutant had suppressive effects on luciferase activities in HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P < 0.05) and 6.4:1 (1949 +/- 212, 304 +/- 60, P < 0.01), respectively. CONCLUSIONS: (1) [(12)Asp]K-ras4B can enhance the expression of ERalpha and beta proteins. This may be correlated with [(12)Asp]K-ras4B/raf signaling pathway. (2) The effect of mutant-type [(12)Asp]K-ras4B gene on ERs transcriptional activity in HEC-1A cells appears to need E(2).