Clinical implications of steroid receptor coactivator (SRC)-3 in uterine endometrial cancers.

Estrogen is recognized as a significant modifier in the development, growth and invasion of uterine endometrial cancer. Steroid receptor coactivator-3 (SRC-3; AIB1, ACTR, RAC3, TRAM-1, and pCIP) is a member of the p160 family of coactivator for nuclear hormone receptors including estrogen receptor (ER). It is reported that SRC-3 is overexpressed in various cancers. However, SRC-3 expression manner in uterine endometrial cancer is not fully understood. In this study, we showed SRC-3 mRNA expression correlates with clinical stage, depth of myometrial invasion and dedifferentiation. The prognosis of the 25 patients with higher expression of SRC-3 mRNA in uterine endometrial cancers was extremely poor (36%), whereas the 24-month survival rate of the 15 patients with lower expression of SRC-3 mRNA was 96%. These data indicate that SRC-3 might be an important indicator of uterine endometrial cancer advancement and survival.

Significant anti-proliferation of human endometrial cancer cells by combined treatment with a selective COX-2 inhibitor NS398 and specific MEK inhibitor U0126.

The extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway plays a critical role in the anticancer action in vitro. ERK1/2 activation or phosphorylation is responsible for increased cyclooxygenase-2 (COX-2) protein expression in some cancer cells treated with selective COX-2 inhibitor NS398. We determined the effect of NS398 on ERK signaling and the synergistic effect of combined treatment with NS398 and a specific MEK inhibitor U0126 on three human endometrial cancer cell lines: Ishikawa, HEC-1A and AN3CA cells. Results showed that NS398 and U0126 individually, and especially the combination of both exhibited profound anti-proliferation of all three cell lines in a time- and concentration-dependent manner by [3-(4, 5)-dimethylthiazol-z-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. The phosphorylated ERK1/2 was up-regulated in HEC-1A and AN3CA cells, but the COX-2 protein expression was unchanged in the three cancer cell lines treated with NS398 alone. However, both phosphorylated ERK1/2 and COX-2 protein expression were concentration-dependently decreased in all three cell types by combined treatment with NS398 and U0126 assessed by western blot analysis. Simultaneously, the combination of NS398 and U0126 resulted in 2-fold increase in apoptosis of all three lines over that by the individual alone, and enhanced G0/G1 phase arrest of Ishikawa and HEC-1A cells induced by U0126 treatment determined by flow cytometry. The synergistic and complementary effects of combining NS398 and U0126 were found to be associated with activation of caspase-3, alterations of Bcl-2 family proteins and cell cycle regulatory proteins detected by western blot analysis. Taken together, these findings correlate with blocking MEK-ERK signaling cascade and down-regulating COX-2 protein expression in endometrial cancer cells with combination treatment of NS398 and U0126, suggesting that the combinatory use of NS398 and specific MEK inhibitors may be valuable for chemotherapy or chemoprevention of human endometrial cancer.

Non-steroidal anti-inflammatory drugs inhibit cellular proliferation and upregulate cyclooxygenase-2 protein expression in endometrial cancer cells.

We determined the effects of several non-steroidal anti-inflammatory drugs (NSAIDs), aspirin (acetylsalicylic acid, ASA), indomethacin and a cyclooxygenase-2 (COX-2)-selective inhibitor (NS398), on cellular proliferation and regulation of COX-2 protein expression in endometrial cancer cells in vitro, and investigated their modes of action. All three NSAIDs markedly inhibited the proliferation of Ishikawa, HEC-1A and AN3CA endometrial cancer cell lines in a time- and concentration-dependent manner. ASA and indomethacin triggered apoptosis in cells of all three lines through release of cytosolic cytochrome c, activation of caspase-9 and-3, and cleavage of poly(ADP-ribose) polymerase (PARP), but NS398 induced minimal apoptosis only in Ishikawa cells. ASA altered the cell cycle distribution, with G2/M phase accumulation of cells, and induced overexpression of Ki-67 protein. Both ASA and indomethacin reduced the protein levels of Bcl-2 and Bcl-xl, but upregulated those of Bax and Bcl-xs. COX-2 protein expression and PGE(2) production were upregulated by ASA and indomethacin in all three cell lines. However, NS398 did not alter COX-2 protein expression or PGE(2) production in these cells. These results indicate that NSAIDs inhibit proliferation of endometrial cancer cells independently of the reduction of COX-2 protein expression. A cytochrome c-dependent apoptotic pathway and/or cell cycle arrest may contribute to the inhibitory effects of these NSAIDs.

In vitro stimulation of granulosa cells by a combination of different active ingredients of unkei-to.

Unkei-to is widely used in traditional Japanese herbal medicine for its ovulation-inducing effect. In the present study, we investigated the in vivo effects of unkei-to and its compounds on the steroidogenesis and cytokine secretion in human granulosa cells. Unkei-to stimulated the secretions of 17beta-estradiol and progesterone from highly luteinized granulosa cells obtained from in vitro fertilization patients; the stimulated effect on estradiol secretion occurred with 0.3 microg/ml, while a significant effect on progesterone secretion was obtained at 10 microg/ml. The unkei-to stimulation of estradiol secretion could be accounted for by the effects of its ingredients, Shakuyaku (paeoniae radix, Paeonia lactiflora Pallas) and Keihi (cinnamomi cortex, Cinnamomum cassia Blume); while dose response curves for unkei-to and Keihi to induce progesterone production were superimposable. Exposure of the cells to unkei-to caused dose-dependent increases in the concentrations of interleukin (IL)-1beta, IL-6 and IL-8 in the culture medium. Similar results were obtained when cells were incubated with the ingredient Ninjin (ginseng radix, Panax ginseng C.A. Meyer), but not Shakuyaku and Keihi. These results indicate that unkei-to has direct stimulatory effects on human granulosa cells to stimulate the steroidogenesis and secretion of cytokines (IL-1, IL-6 and IL-8). The various beneficial actions of unkei-to on the ovary may result from a combination of different ingredient herbs with different stimulatory effects on both steroidogenesis and the ovulatory process within the ovary, as well as stimulatory effect on the hypothalamus-pituitary axis.

Coexpression of Gas6/Axl in human ovarian cancers.

OBJECTIVES: Gas6, the protein product of the growth arrest-specific gene 6 (gas6), a member of the vitamin K-dependent protein family, was identified as a ligand for the Axl/Sky family of receptor tyrosine kinases. The aim is to study for the presence of Gas6 and its receptor Axl and Sky related to specific growth in the ovarian cancers, and to evaluate their plausible growth potential and mechanism. METHODS: In ovarian cancers of 90 cases, the histoscores and mRNA levels of Gas6, Axl and Sky were determined by immunohistochemistry and competitive reverse transcription-polymerase chain reaction-Southern blot analysis using the recombinant RNA, respectively. RESULTS: The histoscores and mRNA levels of Gas6 and Axl in ovarian cancers were significantly higher than in normal ovaries, regardless of histopathological type or clinical stage of ovarian cancers. CONCLUSIONS: Gas6/Axl pathway could play a role in the complex events taking place during the early changes of ovarian cancer progression.

Translocation of lysophosphatidic acid phosphatase in response to gonadotropin-releasing hormone to the plasma membrane in ovarian cancer cell.

OBJECTIVE: Lysophosphatidic acid mediates proliferative and/or morphologic effects on multiple cell lineages, which include ovarian cancer cells. Lysophosphatidic acid hydrolysis limits the duration of lysophosphatidic acid's action. We examined hormonal translocation of lipid phosphate phosphatase type 3 to the plasma membrane in gonadotropin-releasing hormone-responsive ovarian cancers. STUDY DESIGN: Ovarian cancers that were removed surgically and the ovarian cancer cell lines Caov-3 and SK-OV-3 were examined. Lipid phosphate phosphatase type 3 protein and activity in plasma membranes were assessed by immunohistochemical staining with lipid phosphate phosphatase type 3-specific antibodies and by the measurement of the conversion of exogenous [(3)H-oleoyl]lysophosphatidic acid to mono[(3)H-oleoyl]glycerol, respectively. RESULTS: In ovarian cancers that were removed surgically, the cell surface staining and activity measurements indicated that a portion of the enzyme was localized to the plasma membrane. In Caov-3 cells and SK-OV-3 cells, lipid phosphate phosphatase type 3 protein was present both in the cytoplasm and at the plasma membrane. Treatment of the cells with a gonadotropin-releasing hormone agonist buserelin produced a rapid and progressive translocation of lipid phosphate phosphatase type 3 protein to the plasma membrane, with a concomitant loss of cytoplasmic staining. The enzyme activity in plasma membrane was also increased when the cell lines were exposed to the gonadotropin-releasing hormone agonist in intact cells before the assay of the cell membranes. CONCLUSION: These findings support the presence of lipid phosphate phosphatase type 3 in plasma membrane of ovarian cancers and provide for the ability of agonists (such as gonadotropin-releasing hormone) to induce the translocation of lipid phosphate phosphatase type 3 to plasma membrane in ovarian cancer cells.

Expression of estrogen receptor beta exon-deleted variant mRNAs in ovary and uterine endometrium.

Various estrogen receptor beta exon-deleted variant (ER-beta EDV) mRNAs were expressed in human ovary and uterine endometrium. Estrogen receptor beta (ER-beta) completely or partially deleted exon n is expressed as ER-beta EnDV or En'DV, respectively. The mRNAs for ER-beta single exon-deleted variant (EDV), ER-beta E2DV, E4DV, E5DV and E6DV; for ER-beta double exon-deleted variants, ER-beta E1'+2DV, E4+5DV and E5+6DV; and for ER-beta triple exon-deleted variants, ER-beta E2'+3+4DV and E4+5+6DV were detected. In ER-beta E2DV, E4+5DV, E5DV and E6DV mRNAs, the new stop codon is made in the exon following the deleted exon(s), and the new proteins may lack the corresponding domains. In ER-beta E1'+2DV, E2'+3+4DV, E4DV, E4+5+6DV and E5+6DV mRNAs, the original stop codon is still present, and the new proteins may conserve the new short amino acid sequences surrounding the deleted exons. ER-beta E1'+2DV, E2DV, E2'+3+4DV, E4DV, E4+5DV and E4+5+6 are unlikely to work as a transcription factors. On the other hand, ER-beta E5DV, E6DV and E5+6DV lack only the ligand-binding domain, and might work as dominant positive or negative factors. Therefore, ER-beta E5DV, E6DV and E5+6DV, constitutively expressed in human ovary and uterine endometrium might, in part regulate estrogen responsiveness.