Koji Hisamoto

Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel.

We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-Akt-BAD cascade, ERK-BAD cascade, and Akt-Raf-1 cascade in the paclitaxel-resistant SW626 human ovarian cancer cell line, which lacks functional p53. Treatment of SW626 cells with paclitaxel activates Akt and ERK with different time frames. Interference with the Akt cascade either by treatment with PI-3K inhibitor (wortmannin or LY294002) or by exogenous expression of a dominant negative Akt in SW626 cells caused decreased cell viability following treatment with paclitaxel. Interference with the ERK cascade by treatment with an MEK inhibitor, PD98059, in SW626 cells also caused decreased cell viability following treatment with paclitaxel. Treatment of cells with paclitaxel also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. The phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin or cotransfection with the dominant negative Akt. On the other hand, the phosphorylation of BAD at Ser-112 was blocked by PD98059. We further examined the role of BAD in the viability following paclitaxel treatment using BAD mutants. Exogenous expression of doubly substituted BAD2SA in SW626 cells caused decreased viability following treatment with paclitaxel. Moreover, because paclitaxel-induced apoptosis is mediated by activated Raf-1 and the region surrounding Ser-259 in Raf-1 conforms to a consensus sequence for phosphorylation by Akt, the regulation of Raf-1 by Akt was examined. We demonstrated an association between Akt and Raf-1 and showed that the phosphorylation of Raf-1 on Ser-259 induced by paclitaxel was blocked by treatment with wortmannin or LY294002. Furthermore, interference with the Akt cascade induced by paclitaxel up-regulated Raf-1 activity, and expression of constitutively active Akt inhibited Raf-1 activity, suggesting that Akt negatively regulates Raf-1. Our findings suggest that paclitaxel induces the phosphorylation of BAD Ser-112 via the ERK cascade, and the phosphorylation of both BAD Ser-136 and Raf-1 Ser-259 via the PI-3K-Akt cascade, and that inhibition of either of these cascades sensitizes ovarian cancer cells to paclitaxel.

Tamoxifen regulates human telomerase reverse transcriptase (hTERT) gene expression differently in breast and endometrial cancer cells

Tamoxifen is widely applied as an antiestrogenic agent for adjuvant therapy in the treatment of breast cancer, while its estrogen-agonistic activity occasionally causes proliferative disorders or carcinogenesis at other sites, such as the uterus. We reported that estrogen activates telomerase in breast and endometrial cancer cells. The present study examines the effects of tamoxifen on the gene expression of human telomerase reverse transcriptase (hTERT) in breast and endometrial cancer cells. Tamoxifen inhibited the cell growth of MCF-7 cells, as well as hTERT mRNA expression in the presence of estrogen (E2), antagonizing the E2 effects. In contrast, tamoxifen stimulated the growth of Ishikawa cells and activated hTERT mRNA expression in the absence or presence of E2, exhibiting estrogen-agonistic action. Transient expression assays revealed that these actions of tamoxifen are achieved by transcriptional regulation of the hTERT promoter. An estrogen responsive element (ERE) in the hTERT 5′ regulatory region was partly responsible for both the E2-antagonistic and -agonistic actions of tamoxifen. Tamoxifen activated the MAP kinase cascade in Ishikawa cells, but not in MCF-7 cells, and the activation of hTERT mRNA expression was effectively blocked by MEK inhibitor, suggesting that the MAP kinase pathway is involved in the tamoxifen-induced activation of hTERT. These findings indicate that tamoxifen regulates hTERT expression in a cell-type specific manner. Tamoxifen-induced activation of hTERT may be one component of estrogen agonistic function of tamoxifen that is involved in endometrial carcinogenesis induced by this agent.

Rapid changes of flow-mediated dilatation after surgical menopause

OBJECTIVES Estrogen acts directly on endothelial nitric oxide synthase through a non-genomic mechanism, resulting in rapid dilatation of blood vessels. In this study, we examined the change of endothelial function after surgical menopause. METHODS In 20 subjects who underwent gynecological operations (ovariectomy (OVX) 12, sham (SHAM) operation 8), postoperative changes of flow-mediated dilatation (FMD) of the brachial artery were examined using ultrasonography. Postoperative changes of the response to nitroglycerin (NTG) were also studied in these patients. RESULTS In the OVX group, significant decreases of FMD were observed 1 week after the operation, although no changes were observed in the response to NTG. In the SHAM group, no remarkable changes of FMD or the response to NTG were observed after the operation. CONCLUSIONS OVX influences endothelium-dependent vasodilatation within as little as 1 week. Therefore, it may be important to address the rapid changes of circulation after surgical menopause in order to prevent cardiovascular disease.

Cesarean scar pregnancies successfully treated with methotrexate.

Three cases of cesarean scar pregnancy successfully treated with methotrexate are described. The diagnosis was confirmed by transvaginal sonographic examinations showing a well‐formed gestational sac in the myometrium of the lower uterine segment. Initial treatment with a systemic injection of 50 mg of methotrexate was not sufficient, and multiple doses were required to obtain a complete remission in two cases. In a case with a β‐hCG level of more than 20,000 mIU/mL with a viable embryo in a gestational sac, a combination of systemic and local treatment with methotrexate was required. It took 7–11 weeks for the β‐hCG level to become undetectable and 12–17 weeks for the cesarean scar pregnancy mass to disappear completely.

Estrogen and raloxifene inhibit the monocytic chemoattractant protein-1-induced migration of human monocytic cells via nongenomic estrogen receptor alpha.

Objective: To investigate the effects of estradiol (E2) and raloxifene on the migration of human monocytic THP-1 cells to endothelium. Design: A prospective comparative study. THP-1 cells, a human acute monocytic leukemia cell line, were used for the study. Migration assays were performed using transwell inserts. THP-1 cells were exposed to E2 or raloxifene in the presence of monocytic chemoattractant protein-1 (MCP-1), a major chemoattractant for monocytes. The cells were transfected with small interfering RNA (siRNA) against estrogen receptor (ER) &agr; and ER&bgr; for gene silencing. ER expression was evaluated by Western blot analysis. Results: MCP-1 induced the migration of the cells for 90 minutes. The addition of E2 or raloxifene significantly inhibited the MCP-1-induced migration for 90 minutes. Preincubation of THP-1 cells with an ER antagonist, ICI 182780, significantly attenuated the inhibitory effects of E2 and raloxifene. Whereas transfection with siRNA of ER&agr; significantly attenuated the inhibition by E2 of MCP-1-induced monocyte migration, transfection with control siRNA or siRNA of ER&bgr; had no effect on the rapid inhibitory action of E2. Moreover, preincubation of THP-1 cells with a transcriptional inhibitor, actinomycin D, had no effect on the rapid inhibitory action of E2. Conclusions: Our findings suggest that both E2 and raloxifene inhibited the MCP-1-induced monocyte migration through nongenomic ER&agr;. This result may explain one of the antiatherosclerotic effects of E2 and raloxifene on vasculature.

Primary Neuroendocrine Carcinoma of the Uterine Cervix Treated With Complete Surgical Resection and Adjuvant Combination Chemotherapy

We describe our experience with one case of cervical large cell neuroendocrine carcinoma (LCNC), with an attempt of an adjuvant chemotherapy after complete surgery. The patient was a 66-year-old female (gravida 3, para 2) presenting with genital bleeding. A cervical mass was diagnosed as high-grade neuroendocrine carcinoma. Radical hysterectomy, pelvic lymph node dissection and bilateral salpingo-oophorectomy were performed as primary treatment for the cervical cancer. The surgical specimen of the uterus had an enlarged cervix of 4 cm in diameter with parametrial invasion. Microscopically, the surgical specimen exhibited invasive proliferation of relatively large tumor cells. Peripheral nuclear palisading and central necrosis were also histologically observed. Tumor cells had abundant cytoplasm with vesicular nuclei. The final pathological conclusion was high-grade neuroendocrine carcinoma (LCNC). The postoperative diagnosis was cervical cancer, high-grade neuroendocrine carcinoma (LCNC), pT2bN0M0, FIGO stage IIB, ly (+), v (+). Adjuvant treatment with cisplatin and irinotecan was planned at 4-week intervals. With the completion of three cycles of adjuvant chemotherapy, there was no evidence of recurrent disease. We determined the adjuvant therapy to be effective and well tolerated, and the therapy is now planned to be continued. In conclusion, we experienced a rare case of uterine cervical LCNC. It was surgically resected completely, and adjuvant chemotherapy has been continued. Adjuvant combination chemotherapy with cisplatin and irinotecan is expected to improve the prognosis of cervical LCNC. J Clin Gynecol Obstet. 2017;6(1):23-27 doi: https://doi.org/10.14740/jcgo432w

Case of uterine papillary serous carcinoma following tamoxifen treatment that could not be diagnosed during screening

This case involved a 69‐year‐old woman who had been taking tamoxifen for 5 years after breast cancer surgery. She was referred to our clinic for endometrial cancer screening when tamoxifen was first prescribed. Subsequently, transvaginal ultrasonography and endometrial cytology were performed every 6 months. Despite these regular examinations, stage IVb papillary serous carcinoma was detected 8 months after the end of tamoxifen administration. Total abdominal hysterectomy was performed, but only a small polyp was seen upon macroscopic examination of the uterus. However, papillary serous carcinoma was found microscopically in almost all lymphovascular spaces in the uterus from the endometrium to the serosa. On the surface of the polyp, only endometrial intraepithelial carcinoma with positive immunostaining for p53 was detected. Chemotherapy, including a platinum compound, was administrated, but unfortunately it was ineffective and the patient died of her disease 14 months after the operation.