Wen-Shu Sun

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.

Revised Guidelines for Neonatal Screening Programmes for Primary Congenital Hypothyroidism

Since the first guidelines for neonatal screening for congenital hypothyroidism (CH) were issued by ESPE in 1993 [1], there have been considerable advances in our understanding of CH and our appreciation of the various geographical and logistic difficulties involved. Therefore, an updating of the guidelines is overdue. Experience from countries where screening began in the late 1970s and early 1980s has indicated that treatment should be started no later than the first 2 weeks of life using a ‘high’ dosage regime of L-thyroxine (10–15 Ìg/kg/day). It has also been shown that the quality of long-term outcome is closely related to the quality of follow-up. In Eastern Europe, screening programmes for CH have either been started or will start soon in almost all countries, and although many programmes are operating satisfactorily, it is important to standardise screening procedures and management of suspected cases as much as possible in order to optimise outcome. A degree of uniformity throughout Europe would not only facilitate early detection and treatment of individual patients but give insight into the economic and epidemiological aspects of the screening programmes as well as the epidemiological aspect of CH.

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 PGE2 production were upregulated by ASA and indomethacin in all three cell lines. However, NS398 did not alter COX‐2 protein expression or PGE2 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.

Dominant expression of progesterone receptor form B mRNA in ovarian endometriosis.

This study was designed to examine the biological implications of progesterone receptor form A (PR-A) and B (PR-B) mRNA expressions in human ovarian endometriosis (ectopic endometrium). A high ratio of PR-B to PR-AB (PR-A+PR-B) mRNA expression was found in 8 of 14 cases of endometriosis, compared with the ratio in eutopic endometrium. The mean ratio in ectopic endometria was significantly (p < 0.01) higher than in eutopic endometria. The ratio in eutopic and ectopic endometria showed no significant change during the menstrual cycle. The mean ratio in ectopic endometria in the proliferative and secretory phases of the endometrium was significantly (p < 0.01) higher than in eutopic endometria. In conclusion, PR-B mRNA was relatively highly expressed in some endometriomas, which might lead to aberrations in the control of progestational effects involving responsiveness to sex steroidal growth regulation.

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.

Evidence for the Synthesis of Corticosteroid-Binding Globulin in Human Placenta

We demonstrated the expression of corticosteroid-binding globulin (CBG) in human placenta using reverse transcription-polymerase chain reaction-Southern blot analysis and immunohistochemical and immunoblotting studies. In the RT-PCR-Southern blot analysis, only one predicted PCR product was detected without nonspecific products in all samples of human placenta and 3A (tPA-30-1) human placental cells. In Western blot analysis, polyclonal anti-CBG antibodies recognized a protein of approximately 55 kD in the protein extracts prepared from 3A (tPA-30-1) cells. Additionally, CBG mRNA expression was demonstrated by in situ hybridization in the syncytiotrophoblasts. Immunohistochemical studies performed on the placenta demonstrated the presence of specific immunoreactivity in the syncytiotrophoblast layer surrounding the chorionic villi. These findings suggest that CBG is synthesized in human placenta during pregnancy in addition to its synthesis in the liver.

Identification of exon-deleted progesterone receptor mRNAs in human uterine endometrial cancers

We demonstrated the expression of various exon-deleted progesterone receptor (PR) variant mRNAs in human uterine endometrial cancers using the reverse transcription-polymerase chain reaction-DNA sequencing analyses. In addition to PR wild-type mRNA, exon 4-deleted, exon 6-deleted, exon 3,4-deleted, exon 5,6-deleted, exon 4,5,6-deleted and exon 3,4,5,6-deleted PR variant mRNAs were identified. The exon 6-deleted and exon 5,6-deleted PR variant mRNAs lacked encoding for the steroid-binding domain. The exon 4-deleted, exon 3,4-deleted, exon 4,5,6-deleted and exon 3,4,5,6-deleted PR variant mRNAs lacked encoding for the DNA-binding domain in addition to encoding for the steroid-binding domain. While the exon 4-deleted, exon 6-deleted and exon 3,4-deleted PR variant mRNAs were observed in all samples analyzed, the exon 5,6-deleted, exon 4,5,6-deleted and/or exon 3,4,5,6-deleted PR variant mRNAs could not be detected in some cases, especially in poorly differentiated adenocarcinoma as compared with well-differentiated and moderately differentiated adenocarcinomas. The present study demonstrates the coexpression of PR exon-deleted variant mRNAs with the wild-type in uterine endometrial cancers. All translated variant proteins might possess functional diversity and might modify the progestational action of wild-type PR, and the expression of some PR variant mRNAs may be lost as endometrial cancer cells undergo dedifferentiation.

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.

Levels of Corticosteroid-Binding Globulin mRNA in Human Ovarian Cancers

To understand the role of corticosteroid-binding globulin (CBG) in the intracellular steroidal actions in human ovarian cancers, the level of CBG mRNA expression was evaluated in normal ovarian tissues and in ovarian cancers using competitive reverse transcription-polymerase chain reaction-Southern blot analysis. The expression of CBG mRNA was detected in all normal ovaries and ovarian cancers analyzed. There were no significant differences in the mean CBG mRNA levels between normal ovaries and ovarian cancers. The expression in normal ovaries was significantly higher (p < 0.01) in the premenopause than in the postmenopause. A high expression of CBG mRNA was observed in 11 out of 29 cases (38%) of ovarian cancer in comparison with normal ovaries. There was no difference in the expression among the histological classifications or clinical stages of ovarian cancers. These data suggest that human normal ovaries and ovarian cancers might synthesize CBG intracellularly, ovarian cancers might conserve a progesterone-associated property via CBG, and the regulation of intracelluar CBG expression might be changed in some cancers.

Expression of sex hormone-binding globulin exon 7 splicing variant mRNA in secondary spreading lesions of gynecological cancers

This study was designed to determine the clinical implications of intracellular expression of sex hormone-binding globulin (SHBG) wild-type and exon 7 splicing variant mRNAs in secondary spreading lesions of gynecologic cancers using the reverse transcription-polymerase chain reaction-Southern blot and DNA sequencing analyses. Compared with primary cancers, a relative increase in SHBG variant mRNA to wild-type mRNA expression was observed (4/10 cases of uterine endometrial cancers, 5/10 cases of uterine cervical cancers, 6/10 cases of ovarian cancers) or the expression of SHBG wild-type and variant mRNAs could not be detected (5/10 cases of uterine endometrial cancers, 3/10 cases of uterine cervical cancers, 4/10 cases of ovarian cancers). On the other hand, alteration to a relative increase in SHBG wild-type mRNA expression in the metastatic lesions occurred in only 3 cases (1/10 cases of uterine endometrial cancers and 2/10 cases of uterine cervical cancers) analyzed. These results suggest that the transcription of SHBG mRNA and the regulation of its splicing might be altered with metastatic potential, and this status might be involved in a change in steroidal dependency of metastatic lesions.