Kenichiro Morishige

Maintenance Treatment with Bevacizumab Prolongs Survival in an In vivo Ovarian Cancer Model

Purpose: Vascular endothelial growth factor (VEGF) plays a central role in tumor angiogenesis and is regarded as a promising therapeutic target. We hypothesized that treatment with bevacizumab, a humanized recombinant anti-VEGF monoclonal antibody, could enhance antitumor response to cisplatin and prolong survival in a murine ovarian cancer model. Experimental Design: We conducted an MTS assay to examine the effect of bevacizumab on proliferation of the VEGF producing human ovarian cancer cell lines in vitro. Next, the antiangiogenic activity of bevacizumab was investigated by in vivo angiogenesis and wound healing assays. We then determined the toxicity and antitumor response of bevacizumab and cisplatin as single agents or in combination in xenograft models of ovarian cancer. Finally, using the same xenograft model, we examined the effect of these regimens, as well as bevacizumab maintenance therapy, on survival. Results: Bevacizumab had no effect on the proliferation of ovarian cancer cells in vitro but significantly inhibited angiogenesis and delayed wound healing in vivo. Bevacizumab inhibited i.p. tumor growth and ascites production in the nu/nu mouse xenograft model and enhanced the therapeutic efficacy of cisplatin. Combination therapy with bevacizumab and cisplatin for 3 weeks was associated with complete disappearance of all macroscopic evidence of disease. Moreover, maintenance treatment with bevacizumab after 3 weeks of induction combination therapy inhibited recurrence and significantly prolonged survival. Conclusions: Bevacizumab has significant antitumor activity not only as a single agent or in combination with cisplatin but may also prolong survival when used as maintenance therapy after a complete response to cisplatin-based chemotherapy.

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.

The use of serum TA-4 in monitoring patients with malignant transformation of ovarian mature cystic teratoma

The development of cancer in mature cystic teratomas of the ovary is rare and sometimes difficult to detect because of sampling errors. Six cases of squamous cell carcinoma arising in ovarian mature cystic teratomas were studied, five of which showed an elevated level of a squamous cell carcinoma‐associated antigen, TA‐4, in the sera obtained preoperatively; the preoperative determination was not performed in the sixth case. However, no elevated TA‐4 level was detected in the sera of 28 patients with mature cystic teratomas of the ovary. Moreover, serial determination of the serum TA‐4 level showed a good correlation between the clinical course and the serum TA‐4 level. Interestingly, an abnormal TA‐4 level preceded the clinical detection of recurrence by 2 months in two patients. Thus, determination of the serum TA‐4 concentration may be useful for diagnosing and monitoring patients with squamous cell carcinoma arising in mature cystic teratomas of the ovary.

Metal Transcription Factor-1 Is Involved in Hypoxia-Dependent Regulation of Placenta Growth Factor in Trophoblast-Derived Cells

Placenta growth factor (PlGF) is a placental angiogenic factor. Metal-responsive transcription factor (MTF)-1 was reported to take part in the hypoxic induction of PlGF in RAS-transformed mouse fibroblasts. We contrarily showed that PlGF mRNA and protein levels decreased under hypoxia in a choriocarcinoma BeWo cell line derived from trophoblast. In this report, we examined whether hypoxia-dependent regulation of the PlGF gene in these cells also depends on MTF-1. We analyzed the effect of hypoxia on MTF-1 expression, and it was revealed to be decreased. Moreover, MTF-1 small interfering RNA treatment decreased PlGF mRNA level. To investigate the transcription of PlGF under hypoxia, we cloned promoter region of the human PlGF. Promoter deletion analysis suggested that triple repeats of metal-responsive element located between -511 and -468 bp in the promoter are important for the hypoxic regulation of PlGF. Treatment with MTF-1 small interfering RNA resulted in the significant decreased luciferase activity in PlGF reporter constructs. Chromatin immunoprecipitation showed the binding of the MTF-1 protein to the promoter region. We examined MTF-1 immunoreactivity in trophoblasts of term placental tissue from patients with normal pregnancies and preeclampsia, which represents a condition of placental hypoxia. Immunoreactivity of the MTF-1 protein was decreased in placentas from pregnant women with preeclampsia when compared with those from normal pregnant women. Taken together, these findings suggest that MTF-1 is involved in hypoxia-dependent regulation of PlGF in trophoblast-derived cells.

Ovariectomy increases the level of estrogen receptor mRNA and estrogen receptor binding sites in female rat adipose tissue

The roles of estrogen in the changes in estrogen receptor (ER) mRNA and ER binding sites in rat adipose tissue were studied in female rats. To elucidate the mechanism(s) behind ovariectomy (OVX)-induced obesity, the levels of ER mRNA and ER binding sites in adipose tissue were analyzed three weeks after OVX using Northern blot analysis of ER mRNA and the [3H]E2 binding assay, respectively. OVX induced an increase in body weight, and replacement of estradiol (E2) prevented that increase. Significant increases in the amounts of ER mRNA and in [3H]E2-specific binding were observed after OVX, and E2 replacement reduced both of those increases. These results suggest that E2 may regulate rat obesity directly through ER in adipose tissues.

Epidermal growth factor attenuates cell proliferation by down-regulating the transforming growth factor-β receptor in the osteoblastic cell line MC3T3-E1

We investigated the role of transforming growth factor beta 1 (TGF-beta 1) in growth regulation of the murine osteoblastic cell line, MC3T3-E1. We detected TGF-beta activity in the conditioned medium of MC3T3-E1 cells and its activity was increased by acid treatment of the bioassay system using CC1 64 cells. Northern blot analysis revealed the expression of TGF-beta 1 precursor messenger ribonucleic acid (mRNA). MC3T3-E1 cells possessed a single class of high affinity TGF-beta 1 receptor (2.8 x 10(4) sites per cell, Kd = 196 pM). Cross-linking studies revealed three specific TGF-beta receptors with molecular masses of 65, 95 and 280 kDa. Both TGF-beta and epidermal growth factor (EGF) stimulated [3H]-thymidine incorporation into cells. EGF decreased the number of TGF-beta receptor dose-dependently, and pretreatment of cells with 10 nM EGF attenuated cell growth by TGF-beta. All these data suggested that TGF-beta might be an autocrine growth factor in murine osteoblastic MC3T3-E1 cells and that EGF might modulate the growth of cells by down-regulating the TGF-beta receptor level.

Effects of bezafibrate and simvastatin on plasma lipoproteins in hypercholesterolemia resistant to hormone replacement therapy

OBJECTIVES Estrogen replacement therapy has favorable effects on serum lipoprotein levels in postmenopausal women with hypercholesterolemia. However, there are some patients who fail to respond to hormone replacement therapy (HRT) to lower the serum cholesterol level. In these cases, a conventional lipid-lowering therapy will be applied in addition to HRT, while the effects of these drugs are not well understood. In this study, we studied the effects of simvastatin and bezafibrate administered in addition to HRT. METHODS Patients who were hypercholesterolemic even after HRT were randomly assigned to three treatment groups: HRT only (control group, n=10), HRT+simvastatin (10 mg/day, n=10), or HRT+bezafibrate (400 mg/day, n=10). Serum lipids and lipoprotein levels were measured throughout 12 weeks. RESULTS The serum triglyceride levels were decreased by 24+/-28 and 38+/-13% in the HRT+simvastatin and HRT+bezafibrate groups, respectively. HRT+simvastatin decreased the total cholesterol (21+/-10%) and low-density lipoprotein cholesterol (28+/-12%) levels without affecting the high-density lipoprotein cholesterol (HDL-C) level, while HRT+bezafibrate increased the HDL-C level (12+/-11%). CONCLUSIONS Treatment with simvastatin or bezafibrate in addition to HRT should be considered in cases of postmenopausal hypercholesterolemia in which HRT alone fails to lower the serum lipoprotein levels.