Jun Kawagoe

Induction of hTERT expression and phosphorylation by estrogen via Akt cascade in human ovarian cancer cell lines

We examined the mechanism by which estrogen regulates telomerase activity in Caov-3 human ovarian cancer cell lines, which express ER, to determine whether the regulation affects the expression and/or phosphorylation of the telomerase catalytic subunit (hTERT). 17β-Estradiol (E2) induced telomerase activity and hTERT expression. Transient expression assays using luciferase reporter plasmids containing various fragments of hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the E2-induced activation of the hTERT promoter. Either pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, or transfection with a dominant-negative Akt attenuated the E2-induced activation of the hTERT promoter. In addition, estrogen induced the phosphorylation of IκB inhibitor protein via the Akt cascade, and cotransfection with a dominant-negative subunit of NFκB attenuated the response of the ERE-deleted hTERT promoter to E2. Moreover, E2 induced the phosphorylation of hTERT, the association of 14-3-3 protein and NFκB with hTERT, and nuclear accumulation of hTERT in an Akt-dependent manner. These results indicate that E2 induces telomerase activity not only by transcriptional regulation of hTERT via an ERE-dependent mechanism and a PI3K/Akt/NFκB cascade, but also by post-transcriptional regulation via Akt-dependent phosphorylation of hTERT. Thus, the phosphorylation of Akt is a key event in the induction of telomerase activity by E2 in human ovarian cancer cells.

Inhibition of inhibitor of nuclear factor-κB phosphorylation increases the efficacy of paclitaxel in in vitro and in vivo ovarian cancer models

We investigated whether inhibition of nuclear factor-κB (NFκB) increases the efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. Treatment of paclitaxel-sensitive Caov-3 cells with paclitaxel transiently activated the phosphorylation of Akt, the phosphorylation of IκB kinase (IKK), and the phosphorylation of inhibitor of NFκB (IκBα). Paclitaxel also caused a transient increase in NFκB activity, followed by a decrease in NFκB activity. We show an association between Akt and IKK and show that the phosphorylation of IKK induced by paclitaxel is blocked by treatment with a phosphatidylinositol 3-kinase inhibitor (wortmannin or LY294002). Furthermore, interference of the Akt signaling cascade inhibits the transient induction of IκBα phosphorylation and NFκB activity by paclitaxel. Inhibition of NFκB activity by treatment with an IκBα phosphorylation inhibitor (BAY 11-7085) attenuated both basal and transient induction of IκBα phosphorylation by paclitaxel. Treatment with BAY 11-7085 also enhanced the inhibition of NFκB activity by paclitaxel for up to 24 hours. In addition, treatment with BAY 11-7085 decreased the viability of cells treated with paclitaxel. Moreover, treatment with BAY 11-7085 increased the efficacy of paclitaxel-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated intraperitoneally with Caov-3 cells. These results suggest that paclitaxel transiently induces NFκB activity via the phosphatidylinositol 3-kinase/Akt cascade and that combination therapy with paclitaxel and an NFκB inhibitor would increase the therapeutic efficacy of paclitaxel.

Fasudil inhibits vascular endothelial growth factor–induced angiogenesis in vitro and in vivo

Vascular endothelial growth factor (VEGF)–induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion, stress fiber formation, and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study, we investigated the antiangiogenic effect of fasudil, one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration, viability, and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation, focal adhesion assembly, and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore, fasudil inhibited VEGF-induced phosphorylation of myosin light chain, which is one of the main substrates of ROCK. Therefore, the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis, which resulted in the decrease of cell viability. Moreover, fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore, fasudil might be useful for the treatment of angiogenesis-related diseases, especially cancer. [Mol Cancer Ther 2007;6(5):1517–25]

Poor Embryo Development in Mouse Oocytes Aged In Vitro Is Associated with Impaired Calcium Homeostasis

Abstract We examined whether impairment of intracellular Ca2+ homeostasis is related to poor embryo development in in vitro-aged oocytes. We found that in vitro aging of mouse oocytes affected the patterns of Ca2+ oscillations at fertilization: these Ca2+ oscillations were lower in amplitude and higher in frequency compared with oocytes without in vitro aging. We also observed that the intracellular Ca2+ store was decreased in in vitro-aged oocytes. A decrease in the Ca2+ store induced by thapsigargin, a specific endoplasmic reticulum (ER) membrane Ca2+-ATPase inhibitor, resulted in a lower fertilization rate and in poorer embryo development. The frequency of Ca2+ oscillations was significantly increased at fertilization, whereas their amplitude was decreased in thapsigargin-treated oocytes. These results suggest that impairment of intracellular Ca2+ homeostasis (such as a decrease in the ER Ca2+ store) caused an alteration in Ca2+ oscillations and the poor embryo development in in vitro-aged oocytes. Because embryo fragmentation is closely related to apoptosis, we examined expression of BAX (a proapototic protein) and BCL2 (an antiapoptotic protein) in in vitro-aged oocytes. Although BCL2 was strongly expressed in oocytes without in vitro aging, expression of BCL2 was significantly reduced in oocytes of other culture conditions and treatments such as those in in vitro aging and those that were pretreated with H2O2 or thapsigargin. Acting together, alteration in Ca2+ oscillations and decrease in BCL2 expression in in vitro-aged oocytes may lead to poor embryo development.

Raloxifene inhibits estrogen-induced up-regulation of telomerase activity in a human breast cancer cell line.

The mechanism by which raloxifene acts in the chemoprevention of breast cancer remains unclear. Because telomerase activity is involved in estrogen-induced carcinogenesis, we examined the effect of raloxifene on estrogen-induced up-regulation of telomerase activity in MCF-7 human breast cancer cell line. Raloxifene inhibited the induction of cell growth and telomerase activity by 17β-estradiol (E2). Raloxifene inhibited the E2-induced expression of the human telomerase catalytic subunit (hTERT), and transient expression assays using luciferase reporter plasmids containing various fragments of the hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the action of raloxifene. E2 induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, attenuated the E2-induced increases of the telomerase activity and hTERT promoter activity. Raloxifene inhibited the E2-induced Akt phosphorylation. In addition, raloxifene also inhibited the E2-induced hTERT expression via the PI3K/Akt/NFκB cascade. Moreover, raloxifene also inhibited the E2-induced phosphorylation of hTERT, association of NFκB with hTERT, and nuclear accumulation of hTERT. These results show that raloxifene inhibited the E2-induced up-regulation of telomerase activity not only by transcriptional regulation of hTERT via an estrogen-responsive element-dependent mechanism and the PI3K/Akt/NFκB cascade but also by post-translational regulation via phosphorylation of hTERT and association with NFκB.

Fasudil-induced hypoxia-inducible factor-1α degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells

Hypoxic response of endothelial cells (EC) is an important component of tumor angiogenesis. Especially, hypoxia-inducible factor-1 (HIF-1)–dependent EC-specific mechanism is an essential component of tumor angiogenesis. Recently, the Rho/Rho-associated kinase (ROCK) signaling has been shown to play a key role in HIF-1α induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1α expression through the Rho/ROCK signaling in human umbilical vascular ECs (HUVEC). Pull-down assay showed that hypoxia stimulated RhoA activity. Under hypoxic conditions, HUVECs transfected with small interfering RNA of RhoA and ROCK2 exhibited decreased levels of HIF-1α protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1α mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1α expression without altering HIF-1α mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1α expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1α expression is through preventing HIF-1α degradation by activating the Rho/ROCK signaling in ECs. Furthermore, hypoxia induced both vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression through the Rho/ROCK/HIF-1α signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1α signaling is an essential mechanism for hypoxia-driven, VEGF-mediated autocrine loop in ECs. Therefore, fasudil might have the antimigratory effect against ECs in tumor angiogenesis. [Mol Cancer Ther 2008;7(6):1551–61]

Raloxifene Increases Proliferation and Up-regulates Telomerase Activity in Human Umbilical Vein Endothelial Cells

Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) α and ERβ. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3′-azido-3′-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IκB. Moreover, cotreatment with an IκBα phosphorylation inhibitor, BAY-11–7082, or a specific NFκB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFκB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFκB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene.

Impact of Menopause on the Augmentation of Arterial Stiffness with Aging

Background/Aims: Aortic stiffness, determined by pulse wave velocity (PWV), is an independent marker of cardiovascular risk. PWV is mainly influenced by age-associated alterations in arterial wall structure and blood pressure. The present study was conducted to assess the impact of menopause on the brachial-ankle PWV (baPWV) in healthy women. Methods: Fifty premenopausal women aged 22–54 years and 40 postmenopausal women aged 40–73 years were recruited for this study. Subjects with hypertension, diabetes, and hyperlipidemia were strictly excluded. The results of baPWV were analyzed chronologically by 10- or 5-year age intervals. Results: There was no significant difference in baPWV between premenopausal and postmenopausal women in their 40s and 50s. The baPWV of postmenopausal women aged over 60 years was significantly higher than that of postmenopausal women in their 50s. To clarify the age-dependent elevation in baPWV in detail, women their 50s and 60s were divided into subgroups by 5-year age intervals. There was no significant difference in baPWV among the 50–54-, 55–59- and 60–64-year subgroups. baPWV significantly increased in the 65–69- year subgroup (p< 0.05). There was a significant relationship between baPWV and age in premenopausal (r = 0.452, p = 0.001) and postmenopausal (r = 0.581, p < 0.0001) women. The slope of the regression line for baPWV plotted against age was steeper in postmenopausal than in premenopausal women. Conclusions: This study produces suggestive evidence that menopause amplifies the age-dependent increase in arterial stiffness.

Estrogen induces neurite outgrowth via Rho family GTPases in neuroblastoma cells.

Estrogen (E2) has direct in vivo and in vitro effects, such as inducing neurite outgrowth, on neurons. We investigated the morphological changes and intracellular signaling pathway induced by E2 in neuroblastoma (SH-SY5Y) cells. The effect of medroxyprogesterone acetate (MPA) or progesterone (P4) on the E2-induced neurite outgrowth was also examined using SH-SY5Y cells. Neurite outgrowth was induced by E2 in association with the phosphorylation of Akt, and these effects of E2 were abolished by MPA but not by P4. Progesterone receptor antagonist RU486 blocked the inhibitory effects of MPA. Estrogen receptor antagonist ICI 182,780 and phosphatidylinositol 3-kinase inhibitor LY294002 inhibited the E2-induced neurite outgrowth. Because the Rho family of small GTPases has been shown to be involved in the regulation of neurite outgrowth, we examined the cross-talk among Rac1, Cdc42 and RhoA in the E2-induced neurite outgrowth. E2 immediately increased the Rac1 and Cdc42 activity and decreased the RhoA activity. E2-induced neurite outgrowth was attenuated in cells expressing dominant-negative mutants for Rac1 or Cdc42. These results suggest that regulation of Rho family GTPase activity by E2 is important for the neurite outgrowth in neuroblastoma cells, and that MPA may have an antagonistic effect against E2.

Raloxifene improves the ovariectomy-induced impairment in endothelium-dependent vasodilation.

Objective:To examine the effect of raloxifene on the endothelial dysfunction caused by surgical menopause. Design:Ten premenopausal women who underwent gynecological surgery with ovariectomy were divided into two groups. Five participants used raloxifene (60 mg/d) for 7 days staring 1 week after the surgery, and the other five participants did not use raloxifene. We examined the changes in flow-mediated dilatation (FMD) of the brachial artery using ultrasonography. Vasodilation in response to nitroglycerin was also studied. We also measured the brachial-ankle pulse wave velocity to examine the change in arterial stiffness in these participants before and after surgical menopause. Results:In both the raloxifene and control groups, a significant decrease in FMD was observed 1 week after the surgery. Although no further changes in FMD were observed in the control group at 2 weeks after surgery, FMD was significantly increased in the raloxifene group. No remarkable changes in nitroglycerin or brachial-ankle pulse wave velocity were observed after surgery in either group. Conclusions:Raloxifene rapidly restored FMD that was impaired after surgical menopause. Therefore, raloxifene may be effective for ameliorating and maintaining endothelial function in premenopausal women who undergo ovariectomy.