Antiangiogenic Therapy with Human Apolipoprotein(a) Kringle V and Paclitaxel in a Human Ovarian Cancer Mouse Model.

INTRODUCTION: The present study compared the effect of combination therapy using human apolipoprotein(a) kringle V (rhLK8) to conventional chemotherapy with paclitaxel for human ovarian carcinoma producing high or low levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Human ovarian carcinoma cells producing high (SKOV3ip1) or low (HeyA8) levels of VEGF were implanted into the peritoneal cavity of female nude mice. Seven days later, mice were randomized into four groups: control (vehicle), paclitaxel [5 mg/kg, weekly intraperitoneal (i.p.) injection], rhLK8 (50 mg/kg, daily i.p. injection), or the combination of paclitaxel and rhLK8. Mice were treated for 4 weeks and examined by necropsy. RESULTS: In mice implanted with SKOV3ip1 cells, rhLK8 treatment had no significant effect on tumor incidence or the volume of ascites but induced a significant decrease in tumor weight compared with control mice. Paclitaxel significantly reduced tumor weight and ascites volume, and combination treatment with paclitaxel and rhLK8 had an additive therapeutic effect. Similarly, in HeyA8 mice, the effect of combination treatment on tumor weight and tumor incidence was statistically significantly greater than that of paclitaxel or rhLK8 alone. Immunohistochemical analysis showed a significant decrease in microvessel density and a marked increase of apoptosis in tumor and tumor-associated endothelial cells in response to combination treatment with paclitaxel and rhLK8. CONCLUSION: Collectively, these results suggest that antiangiogenic therapy with rhLK8 in combination with taxane-based conventional chemotherapy could be effective for the treatment of ovarian carcinomas, regardless of VEGF status.

Characterization of CD45-/CD31+/CD105+ circulating cells in the peripheral blood of patients with gynecologic malignancies.

PURPOSE: Circulating endothelial cells (CEC) have been widely used as a prognostic biomarker and regarded as a promising strategy for monitoring the response to treatment in several cancers. However, the presence and biologic roles of CECs have remained controversial for decades because technical standards for the identification and quantification of CECs have not been established. Here, we hypothesized that CECs detected by flow cytometry might be monocytes rather than endothelial cells. EXPERIMENTAL DESIGN: The frequency of representative CEC subsets (i.e., CD45(-)/CD31(+), CD45(-)/CD31(+)/CD146(+), CD45(-)/CD31(+)/CD105(+)) was analyzed in the peripheral blood of patients with gynecologic cancer (n = 56) and healthy volunteers (n = 44). CD45(-)/CD31(+) cells, which are components of CECs, were isolated and the expression of various markers (CD146, CD105, vWF, and CD144 for endothelial cells; CD68 and CD14 for monocytes) was examined by immunocytochemistry. RESULTS: CD45(-)/CD31(+)/CD105(+) cells were significantly increased in the peripheral blood of patients with cancer, whereas evaluation of CD45(-)/CD31(+)/CD146(+) cells was not possible both in patients with cancer and healthy controls due to the limited resolution of the flow cytometry. Immunocytochemistry analyses showed that these CD45(-)/CD31(+)/CD105(+) cells did not express vWF and CD146 but rather CD144. Furthermore, CD45(-)/CD31(+)/CD105(+) cells uniformly expressed the monocyte-specific markers CD14 and CD68. These results suggest that CD45(-)/CD31(+)/CD105(+) cells carry the characteristics of monocytes rather than endothelial cells. CONCLUSIONS: Our data indicate that CD45(-)/CD31(+)/CD105(+) circulating cells, which are significantly increased in the peripheral blood of patients with gynecologic cancer, are monocytes rather than endothelial cells. Further investigation is required to determine the biologic significance of their presence and function in relation with angiogenesis.

Immunoglobulin Fc domain fusion to apolipoprotein(a) kringle V significantly prolongs plasma half-life without affecting its anti-angiogenic activity.

Angiogenesis is crucial for tumor growth and metastasis. Blocking this process is, therefore, a potentially powerful approach for the treatment of cancer. Human apolipoprotein(a) kringle V (rhLK8) is an angiogenesis inhibitor and is currently under development as an anti-cancer therapeutic. However, a relatively short in vivo half-life limits its widespread clinical use. This study was performed to evaluate whether fusion of an Fc domain to rhLK8 can extend plasma half-life. RhLK8-Fc fusion protein was expressed in CHO DG44 cells as a dimer and was readily purified by protein G affinity chromatography. The anti-angiogenic activity of rhLK8-Fc was similar to that of rhLK8, as determined by migration and tube formation assays with endothelial cells in vitro and a chorioallantoic membrane assay in vivo. Pharmacokinetic profiles in mice after single intravenous administration of rhLK8 or rhLK8-Fc showed that Fc fusion significantly increased the elimination half-life (t(½)) and the systemic exposure (AUC(inf)) of the protein, in parallel with a significant decrease in total clearance (CL). These data suggest that Fc fusion to rhLK8 is a powerful strategy for extending the plasma half-life of rhLK8 without affecting its anti-angiogenic activity, and could thus improve the clinical applicability of rhLK8.

Proteomic analysis of boar spermatozoa and quantity changes of superoxide dismutase 1, glutathione peroxidase, and peroxiredoxin 5 during epididymal maturation.

Mammalian spermatozoa and their various proteins undergo various modifications during maturation in the epididymis. To characterize proteins that change in quantity during this maturational process, boar spermatozoa were collected from various regions of the epididymis, and extracts were analyzed by two-dimensional gel electrophoresis (2-DE). A number of proteins were identified as changing in quantity, and MALDI-MS analysis revealed that superoxide dismutase 1 (SOD1) from the acrosomal proteins of spermatozoa, and glutathione peroxidase (GPX) and peroxiredoxin 5 from the membranous fraction increased during the epididymal transit of spermatozoa. These proteins are antioxidants that remove reactive oxygen species (ROS); they are presumed to protect spermatozoa during epididymal transit and storage. Western blot analysis of SOD1, GPX and peroxiredoxin 5 showed that these protein levels increased as the spermatozoa traveled from the caput to the cauda epididymis. Activity analysis showed that total SOD activity also increased. Therefore, we conclude that several antioxidant proteins increase during the transit of boar spermatozoa through the epididymis, ultimately contributing to the maturation and/or survival of sperm.

Expression of p57kip2 in germ cells and Leydig cells in human testis.

p57kip2, a KIP family cyclin-dependent kinase (Cdk) inhibitor, blocks the cell cycle by acting on multiple cyclin-Cdk complexes. To investigate the role of p57kip2 in human fertility, the expression of p57kip2 was investigated in testes from normal and obstructive azoospermic male patients who were positive for p57kip2 mRNA. In the seminiferous tubule, strong immunoreactivity of p57kip2 was found in nuclei of early spermatocytes, but not in the spermatogonia. The p57kip2 immunoreactivity in spermatocytes was markedly heterogeneous. Preleptotene spermatocytes showed strong p57kip2 immunoreactivity, but no visible signal was found in late pachytene spermatocytes. Nuclei of the elongating spermatids was also positive for p57kip2 immunoreactivity. Taken together, this suggests that p57kip2 may play a role in the regulation of meiotic progression of early spermatocytes and cell cycle arrest and differentiation of spermatids. p57kip2 immunoreactivity was found in the perinuclear region of the peritubular cells, but not in the Sertoli cells. In Leydig cells, moderate immunoreactivity of p57kip2 was largely found in the cytoplasm, suggesting the noble function of p57kip2 in the differentiation of adult Leydig cells.

Molecular characterization of the 32 kDa boar sperm protease.

The purified 32 kDa boar sperm protease, known as the 32 kDa sperminogen, was identified by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis following its partial peptide sequencing. The 32 kDa boar sperm protease was purified from the acid extracts of boar spermatozoa and subjected to CNBr-digestion. The most prominently digested 30 kDa product was purified by HPLC and its peptide sequence was analyzed. NCBI Blast search of the analyzed 21 amino acid sequence revealed that the sequence matched 91% with that of proacrosin. DNA primer was deduced from the analyzed peptide sequence and the 32 kDa protease was further identified by RT-PCR. Upon RT-PCR, 1 Kbp DNA fragment was amplified, which is the expected length if the product was amplified from the proacrosin mRNA, implying that there is no separate mRNA for the 32 kDa sperminogen. To confirm these results, Northern blot analysis was performed. Four DNA probes generated from the exons of proacrosin genomic DNA sequence all detected a single species of mRNA, suggesting that there is no separate mRNA for the 32 kDa sperminogen which might be produced either from the potential separate 32 kDa sperminogen gene or by differential splicing from proacrosin mRNA. These results strongly suggest that the 32 kDa protease is part of the proacrosin/acrosin system, and that the 32 kDa sperminogen might be formed from post-translational processing of proacrosin.

RU486 suppresses progesterone-induced acrosome reaction in boar spermatozoa.

The effects of progesterone on the acrosome reaction, as well as the effects of RU486 on the progesterone-induced acrosome reaction in capacitated boar spermatozoa, were investigated. Progesterone, a major steroid that is secreted by the cumulus cells of oocyte, clearly induced the acrosome reaction in a dose-dependent manner in capacitated boar spermatozoa, even though it failed to show similar effects in non-capacitated spermatozoa. RU486, a potent antiprogestin, significantly reduced the effects of progesterone on the progesterone-induced acrosome reaction; however, when treated alone, it showed no inhibitory effects on the acrosome reaction. The inhibitory effects of RU486 were also shown to be dose dependent. These results imply that in addition to the wellknown inducer of the acrosome reaction, zona pellucida, progesterone can also induce the acrosome reaction through its specific receptors on spermatozoa after the spermatozoa undergo capacitation.