Yuji Takei

Effect of Recombinant Placental Growth Factor 2 on Hypertension Induced by Full-Length Mouse Soluble fms-Like Tyrosine Kinase 1 Adenoviral Vector in Pregnant Mice

The first aim of our study was to develop a pregnant mouse model for preeclampsia using adenoviral vector containing mouse full-length soluble fms-like tyrosine kinase 1 (sFlt-1) but not truncated sFlt-1. The second aim was to evaluate effects of recombinant mouse (rm) vascular endothelial growth factor (VEGF) and rm placental growth factor (PlGF) on a preeclampsia model induced by adenoviral vector containing mouse full-length sFlt-1. We injected adenoviral vector containing mouse full-length sFlt-1 on day 8.5 or 9.5 of gestation into pregnant Institute of Cancer Research mice, resulting in hypertension, proteinuria, and similar glomerular histological changes as those seen in human preeclamptic women with glomerular endotheliosis on day 16.5 or 17.5 of gestation. The preeclampsia models were treated with 100 &mgr;g/kg of rmVEGF164 (n=5), 100 &mgr;g/kg of rmPlGF-2 (n=5), or vehicle (n=7) twice a day for 2 days IP. The rmVEGF164 treatment significantly decreased the mean blood pressure on day 16.5 or 17.5 of gestation compared with the vehicle treatment (85±4 versus 97±2 mm Hg; P=0.018). The rmPlGF-2 treatment also significantly decreased the mean blood pressure on day 16.5 or 17.5 of gestation compared with the vehicle treatment (86±3 versus 97±2 mm Hg; P=0.018). However, proteinuria was not affected by either rmVEGF164 or rmPlGF-2. In conclusion, we, for the first time, created a mouse preeclampsia model using mouse full-length sFlt-1. VEGF and PlGF may be promising for ameliorating hypertension in women with preeclampsia. Additional study of PlGF as a potential drug for preeclampsia is warranted.

Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cell line by overexpression of bikunin.

Bikunin (bik), a Kunitz‐type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell‐associated plasmin activity and suppression of urokinase‐type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik+ clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion‐blocking treatments of bik+ clones abrogated bik‐mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA‐plasmin‐MMP‐2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik+ clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik+ clones.

Inhibition of peritoneal dissemination of ovarian cancer by tyrosine kinase receptor inhibitor SU6668 (TSU‐68)

SU6668 (TSU‐68) is a small‐molecule synthetic inhibitor of the angiogenic related receptor tyrosine kinases Flk‐1/KDR, PDGFRβ, and FGFR1. Using a mouse model of peritoneally disseminated ovarian cancer, we investigated whether SU6668 inhibits peritoneal dissemination and prolongs survival time. BALB/c nude mice were intraperitoneally (i.p.) inoculated with SHIN‐3 (VEGF‐hypersecretory) or KOC‐2S (PDGF‐hypersecretory) ovarian serous adenocarcinoma cells with marked peritoneal dissemination ability. From the day after i.p. inoculation of tumor cells, SU6668 was orally administered 6 times weekly at a daily dose of 100 mg/kg or 400 mg/kg. The SU6668‐administered group and the vehicle‐administered control group were compared for the number of tumor vascular endothelial cells, weight of peritoneally disseminated tumors, amount of ascitic fluid and survival time. As a result, these 3 parameters were significantly smaller in the SHIN‐3‐inoculated, SU6668‐administered mice than in the control group (p = 0.03, p = 0.002, and p = 0.02, respectively). The mean survival time was significantly longer, at 58.1 ± 11.2 days, in the SU6668‐administered mice than that (34.5 ± 8.8 days) in the control group (p = 0.002). Similarly, in the KOC‐2S‐inoculated mice, the oral administration of SU6668 significantly reduced these 3 parameters (p = 0.04, p = 0.04, and p = 0.03, respectively), and significantly prolonged survival (16.6 ± 1.7 days vs. 11.0 ± 0.7 days, p = 0.008). Thus, the oral administration of SU6668 inhibited angiogenesis and peritoneal dissemination and prolonged survival in mice with peritoneally disseminated ovarian cancer. These effects were observed with both the VEGF‐ and PDGF‐hypersecretory cell lines. Our results suggest that molecular targeting with oral SU6668 will become a new therapeutic strategy targeting peritoneally disseminated ovarian cancer.

Relationships between Chlamydia trachomatis antibody titers and tubal pathology assessed using transvaginal hydrolaparoscopy in infertile women.

Problem: Since transvaginal hydrolaparoscopy (THL) was introduced as the first‐line procedure in the early stages of the exploration of the adnexal structures in infertile women, it has been shown that THL is a less traumatic and a more suitable outpatient procedure than diagnostic laparoscopy. This study was performed to investigate the relationships between Chlamydia trachomatis antibody titers and tubal pathology assessed using THL in infertile women.

In vivo delivery of siRNA targeting vasohibin-2 decreases tumor angiogenesis and suppresses tumor growth in ovarian cancer

Vasohibin‐2 (VASH2) is a homolog of vasohibin‐1 and exhibits pro‐angiogenic activity. We recently reported that VASH2 is expressed in certain ovarian cancers and promotes tumor growth through angiogenesis. To further demonstrate the effectiveness of molecular targeting of VASH2 for anticancer treatment, we applied in vivo delivery of siRNA targeting VASH2 (siVASH2) using atelocollagen to a xenograft model of ovarian cancer. We inoculated mice s.c. with DISS and SKOV‐3, two representative human ovarian serous adenocarcinoma cell lines. When tumors were measurable, we initiated treatment with control or siVASH2 mixed with atelocollagen, which enveloped the whole tumor. Treatment with siVASH2 significantly inhibited s.c. tumor growth by abrogating tumor angiogenesis. We confirmed that expression of VASH2 mRNA in the tumor was downregulated by siVASH2 treatment. In addition, the siVASH2‐treated tumor contained more blood vessels covered with pericytes, indicating that knockdown of VASH2 contributes to the normalization of tumor blood vessels. Based on these results, VASH2 may be a promising molecular target for ovarian cancer treatment.

Gynecologic Cancer InterGroup (GCIG) consensus review for small cell carcinoma of the cervix.

Abstract Small cell carcinoma of the cervix (SCCC) is a rare histological entity of uterine cervical cancer. Compared with other common histological types, squamous cell carcinoma or adenocarcinoma, the outcome of SCCC is poor because of the high incidence of nodal or distant metastasis even with early stage. In this review, current consensus of epidemiology, pathology, and initial treatment for SCCC will be discussed.

Indoleamine-2,3-dioxygenase, an immunosuppressive enzyme that inhibits natural killer cell function, as a useful target for ovarian cancer therapy

This study examined the role of the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO) in ovarian cancer progression, and the possible application of this enzyme as a target for ovarian cancer therapy. We transfected a short hairpin RNA vector targeting IDO into the human ovarian cancer cell line SKOV-3, that constitutively expresses IDO and established an IDO downregulated cell line (SKOV-3/shIDO) to determine whether inhibition of IDO mediates the progression of ovarian cancer. IDO downregulation suppressed tumor growth and peritoneal dissemination in vivo, without influencing cancer cell growth. Moreover, IDO downregulation enhanced the sensitivity of cancer cells to natural killer (NK) cells in vitro, and promoted NK cell accumulation in the tumor stroma in vivo. These findings indicate that downregulation of IDO controls ovarian cancer progression by activating NK cells, suggesting IDO targeting as a potential therapy for ovarian cancer.

Suppression of ovarian cancer by muscle-mediated expression of soluble VEGFR-1/Flt-1 using adeno-associated virus serotype 1-derived vector

Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt‐1 (sFlt‐1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt‐1 in tumor suppression by means of anti‐angiogenesis. We previously demonstrated the efficacy of sflt‐1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle‐mediated expression of sFlt‐1 in tumor‐bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt‐1‐expressing ovarian cancer (SHIN‐3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno‐associated virus vectors encoding sflt‐1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt‐1 levels were constantly observed, and the growth of both subcutaneously‐ and intraperitoneally‐inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt‐1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer.

Overexpression of PTEN in ovarian cancer cells suppresses i.p. dissemination and extends survival in mice

The main mode of progression of ovarian cancer is peritoneal dissemination, and its inhibition may lead to improved outcome. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) reportedly inhibits the proliferation, migration, and invasion of cancer cells. The purpose of this study is to explore the possibility of PTEN gene therapy for ovarian cancer. We transfected the ovarian cancer cell line SHIN-3 [vascular endothelial growth factor (VEGF)–hypersecretory cell line] with PTEN or luciferase (LUC)–expressing plasmid. After selection, PTEN-overexpressing cells (SHIN-3/PTEN) and control cells (SHIN-3/LUC) were obtained. SHIN-3/PTEN implanted s.c. into nude mice was examined for the change in tumor diameter and the number of new blood vessels. Mice with peritoneally disseminated tumors created by i.p. inoculation of the same cells were examined for changes in body weight and abdominal circumference and for survival time. The growth of s.c. SHIN-3/PTEN was significantly lower than that of control (P < 0.001). Compared with controls, mice with i.p. inoculated SHIN-3/PTEN showed significantly smaller increases in the body weight and abdominal circumference (P < 0.01) and a significantly longer survival time (P < 0.05). VEGF concentration in the supernatant of SHIN-3/PTEN was about half that of controls (P < 0.05). The number of new blood vessels in SHIN-3/PTEN was significantly smaller than that in controls (P < 0.001). Overexpression of PTEN suppressed tumor growth and peritoneal dissemination of VEGF-hypersecretory ovarian cancer cells and prolonged the survival time of the mice with peritoneal disseminated tumor. PTEN gene therapy could have therapeutic potential for ovarian cancer and exerts some of this effect by inhibiting angiogenesis. [Mol Cancer Ther 2008;7(3):704–11]

Downregulation of indoleamine-2,3-dioxygenase in cervical cancer cells suppresses tumor growth by promoting natural killer cell accumulation

This study examined the role of the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO) in cervical cancer progression and the possible use of this enzyme for cervical cancer therapy. We analyzed IDO protein expression in 9 cervical cancer cell lines (SKG-I, -II, -IIIa, -IIIb, SiHa, CaSki, BOKU, HCS-2 and ME-180) stimulated with interferon-γ. IDO expression was observed in all cell lines except for SKG-IIIb. We transfected the human cervical cancer cell line CaSki that constitutively expresses IDO with a short hairpin RNA vector targeting IDO, and established an IDO-downregulated cell line to determine whether inhibition of IDO mediates cervical cancer progression. IDO downregulation suppressed tumor growth in vivo, without influencing cancer cell growth in vitro. Moreover, IDO downregulation enhanced the sensitivity of cervical cancer cells to natural killer (NK) cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. These findings indicate that downregulation of IDO controls cervical cancer progression by activating NK cells, suggesting IDO as a potential therapy for cervical cancer.