Apoptosis In Ishikawa Cells Research Articles

Significance of Nuclear p-Akt in Endometrial Carcinogenesis: Rapid Translocation of p-Akt Into the Nucleus by Estrogen, Possibly Resulting in Inhibition of Apoptosis

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. 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. 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. 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.

5-Aza-2′-deoxycytidine is a potent inhibitor of DNA methyltransferase 3B and induces apoptosis in human endometrial cancer cell lines with the up-regulation of hMLH1

The aim of our study was to evaluate the effects of 5-aza-2'-deoxycytidine (5-azadC) on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of hMLH1 and DNMT3B in human endometrial cancer cell lines. Ishikawa, HHUA, and KLE cell lines were used. After treatment with 5-azadC, cells were measured by MTT to detect the growth inhibition. Flow cytometry analysis was used to evaluate the cell cycle distribution and apoptosis effect. The expression of hMLH1 and DNMT3B was performed by real-time PCR and Western blotting analysis. The methylation status of the hMLH1 gene was monitored by methylation-specific PCR. We confirmed that 5-azadC treatment resulted in growth inhibition, G(2) arrest, and cell apoptosis in human endometrial cancer cell lines. Furthermore, the data obtained by real-time PCR and Western blotting analysis demonstrated that the expression of hMLH1 was up-regulated by 5-azadC treatment in Ishikawa cells, accompanied by down-regulation of DNMT3B expression, when 5-azadC led to cell inhibition, G(2)/M arrest, and apoptosis. Our results suggested that 5-azadC is a potent inhibitor of DNA methyltransferase 3B and induces apoptosis in Ishikawa cells with the up-regulation of hMLH1.

X-Linked Inhibitor of Apoptosis Protein Levels and Protein Kinase C Activity Regulate the Sensitivity of Human Endometrial Carcinoma Cells to Tumor Necrosis Factorα-Induced Apoptosis

Endometrial carcinomas are often chemoresistant. TNFalpha shows potent antitumor activity against various cancers, and if it demonstrates good antitumor activity against endometrial cancer, the cytokine could represent a valuable alternative therapeutic approach. We have tested the ability of TNFalpha to induce apoptosis in endometrial carcinoma cells, and examined a putative role for X-linked inhibitor of apoptosis protein (XIAP) in regulating cellular sensitivity to the cytokine. Exposure to TNFalpha triggered TNF-R1-dependent activation of caspases-8, -9, and -3, down-regulated Akt and XIAP proteins and induced dose-dependent and time-dependent apoptosis in Ishikawa cells. On the opposite, TNFalpha up-regulated XIAP in Hec-1A cells; in these cells, the cytokine induced delayed TNF-R1-dependent activation of caspase-8, and failed to activate caspases -9 and -3 and to induce apoptosis. However, XIAP small interfering RNA restored TNFalpha-induced caspase signaling and apoptosis in Hec-1A cells; XIAP small interfering RNA also increased TNFalpha-induced apoptosis in Ishikawa cells. In addition, inhibition of protein kinase C activity enhanced TNFalpha-induced down-regulation of XIAP and potentiated apoptosis induction, in both Ishikawa and Hec-1A cells. Finally, we found XIAP immunoreactivity in epithelial cells from a large number of human endometrial tumor tissue samples, indicating that XIAP is produced by endometrial tumor cells in vivo. This could allow XIAP to play a putative in vivo role in counteracting TNFalpha-induced apoptosis in endometrial tumor cells; in this case, direct or indirect targeting of XIAP should potentiate the antitumor effect of TNFalpha.

Inhibition of survivin reduces cell proliferation and induces apoptosis in human endometrial cancer

Endometrial cancer is a common gynecologic malignancy among women. The molecular mechanisms involved in the progression of endometrial cancer are unclear, which has hampered the development of an effective treatment. Survivin, a newly identified member of the inhibitor of apoptosis (IAP) family, regulates 2 critical processes in neoplastic transformation: cell proliferation and apoptosis. Survivin mRNA and protein expression levels were analyzed in human normal cycling endometrium, atypical endometrium, and endometrial adenocarcinoma by immunohistochemical, reverse-transcriptase polymerase chain reaction (RT-PCR), and Western blot analyses. To study the biological function of survivin in endometrial cancer, RNA interference was applied to knock down survivin expression in the Ishikawa endometrial cancer cell line by recombinant plasmids producing survivin small hairpin RNA. Furthermore, the signal pathway that regulates survivin expression was investigated. Higher levels of survivin mRNA and protein expression were observed in endometrial adenocarcinomas than in atypical or normal endometrium. Immunohistochemical staining revealed that 83.3% (50 of 60) of endometrial adenocarcinoma samples, 55.0% (11 of 20) of atypical endometrium samples, and 25.0% (5 of 20) of normal endometrium samples were positive for survivin protein. Inhibition of survivin by RNA interference reduced cell proliferation and induced apoptosis in Ishikawa cells by down-regulating cyclin D1 and phosphorylated RB and activating caspase-3 and caspase-8. The authors also found that the MAPK pathway was a signal transduction pathway upstream of survivin. Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) upregulated survivin protein expression by activating the MAPK pathway in endometrial cancer cells. Survivin is an attractive target for endometrial cancer treatment. Growth factors could regulate survivin expression by activating the MAPK pathway.

Mechanisms involved in the evolution of progestin resistance in human endometrial hyperplasia—precursor of endometrial cancer

Background Successful treatment of endometrial hyperplasia with progestins is commonly accompanied by the finding of an inactive or suppressed endometrium after therapy. However, approximately 30% of the endometrial hyperplasia cases do not respond to progestins and hyperplastic glands persist. The Fas/FasL system is known to play a role in tissue remodeling as a result of changes in menstrual hormone levels. The aims of this study are to examine Fas/FasL expression in endometrial hyperplasia of pre- and postprogestin treatment samples and to study the Fas/FasL regulation in vitro with Ishikawa cells after progestin stimulation. Design Pre- and posttreatment paraffin-embedded endometrial hyperplasia tissue samples from 26 women were examined by immunohistochemistry for changes in Fas/FasL expression related to the administration of progestins. Among 26 patients, 18 were successfully treated with progestins and 8 failed treatment. Fas/ FasL positivity was defined by the presence of 10% or more immunoreactive epithelial cells in each specimen. In positive cases, a percentage or an immunoscore of immunoreactive cells was given by counting 500 cells. Cell viability was evaluated by the MTT assay. The in vitro effects of progesterone on Fas/FasL expression and apoptosis in Ishikawa cells were examined by using Western blot and TUNEL assays, respectively. Results Fas immunoreactivity was present in 4/26 (15%) preprogestin cases with an average of 16% of the epithelial cells expressing Fas. FasL was expressed in 21/26 (80%) pretreatment cases with an average of 42% of the hyperplastic glandular cells being positive. In postprogestin cases, an increase of Fas expression (14/18, 77%) with an average of 47% stained cells was seen in responders ( P < 0.001), while FasL was found in 16/18 (89%) responders with an average of 65% of cells positive ( P = 0.587). In nonresponders, no significant changes in Fas/FasL expression were detected compared to pretreatment samples. With in vitro Ishikawa cells, a slight increase (10–20%) of Fas and FasL protein expression was detected after 24 h of progesterone treatment, but a more significant increase (220–343%) of both Fas and FasL expression was found after 48 h of withdrawing progesterone, which parallels apoptotic activity. Conclusions The Fas/FasL system may be involved in the development of endometrial hyperplasia. Part of the molecular mechanisms of progestin therapy for endometrial hyperplasia is through upregulation of Fas/FasL expression. Dysregulation of Fas/FasL expression in hyperplastic endometrium may be part of the molecular mechanisms for nonresponders to progestin treatment. Intermittent, rather than continuous, progestin treatment may be more effective clinically for the treatment of endometrial hyperplasia.

PTEN augments staurosporine-induced apoptosis in PTEN-null Ishikawa cells by downregulating PI3K/Akt signaling pathway.

Staurosporine is a potent apoptosis inducer, but its mechanism remains to be clarified. We investigated the involvement of PTEN in staurosporine-induced apoptosis. Ishikawa cells, from an endometrial carcinoma cell line, expressed a high amount of PTEN mRNA but did not express the PTEN protein because of protein truncations. We isolated clones expressing the steady-state level of the PTEN protein from PTEN-null Ishikawa cells by transfection. The obtained clones showed reduced proliferative activity and reduced anchorage-independent cell growth with the augmented p27(Kip1). These cell lines were sensitized to apoptosis by staurosporine. A low concentration of UCN-01 did not affect apoptosis, but a high concentration augmented apoptosis in the PTEN-expressing clone. Alpha-sphingosine and H-7 did not affect apoptosis in these cell lines. PI3K inhibition augmented staurosporine-induced apoptosis in the parental cell line, but not in the PTEN-expressing clone. In the clone, phosho-Akt/PKB and phospho-Bad (Ser-136) were downregulated. Staurosporine reduced the levels of phospho-Akt/PKB and phospho-Bad (Ser-136) in all the cell lines, but the reduction was most significant in the PTEN-expressing clone. These results suggest that inhibition of the PI3K/Akt/PKB signaling pathway might be associated with staurosporine-induced apoptosis in Ishikawa cells.