Expression of mitotic-arrest deficiency 2 predicts the efficacy of neoadjuvant chemotherapy for locally advanced uterine cervical cancer.

We previously reported satisfactory therapeutic results when using cisplatin-based cyclic balloon-occluded arterial infusion chemotherapy as neoadjuvant chemotherapy (NAC), which enabled hysterectomy to be performed for patients with locally advanced cervical cancer. Mitotic arrest deficiency 2 (MAD2) is a key component of the mitotic spindle checkpoint pathway. The expression of MAD2 is associated with tumor progression and resistance to chemotherapy. Therefore, the aim of the present study was to examine whether the expression of MAD2 is related to the efficacy of NAC for locally advanced uterine cervical cancer. We reviewed 53 cases of locally advanced uterine cervical cancer (stage IIIa-IIIb; based on the International Federation of Gynecology and Obstetrics criteria). These patients were initially treated at Osaka City University Medical School Hospital, Japan, from 1995 to 2008 and were under 70 years old. Tumor samples were obtained by biopsy prior to NAC. Cases were divided into two groups: one group in which NAC was effective, surgery was possible and radiotherapy was performed (NAC+OP+R group; n=33), and another group in which NAC was ineffective and radiation therapy was performed (NAC+R group; n=20). MAD2 expression was examined in paraffin-embedded sections using the avidin-biotin peroxidase complex method. The results showed that MAD2 expression was significantly higher in the NAC+R group compared to the NAC+OP+R group (P<0.001). There was no significant difference in overall survival between the two groups, although the prognosis for the NAC+OP+R group tended to be slightly better (P=0.064). Taken together, these results suggest that the expression of MAD2 may predict the efficacy of NAC as a treatment for locally advanced uterine cervical cancer.

Mitotic arrest deficiency 2 induces carcinogenesis in mucinous ovarian tumors.

Mitotic arrest deficiency 2 (MAD2) is a key component of the mitotic spindle checkpoint pathway. A compromised mitotic spindle checkpoint results in an abnormal number of chromosomes. This is referred to as chromosomal instability, and has been reported in most types of human cancer. The aim of this study was to examine the expression of MAD2 in mucinous ovarian tumors exhibiting varying degrees of malignancy. We reviewed 128 cases of mucinous ovarian tumors initially treated at Osaka City University Medical School Hospital, Japan. Tumor samples were obtained following surgery. The cases were divided into three groups: benign (group B; n=30), borderline malignant (group BM; n=55) and malignant (group M; n=43). MAD2 expression was examined in paraffin-embedded sections using the avidin-biotin peroxidase complex method. Results showed MAD2 expression to be significantly greater in group M compared to groups B and BM (P<0.05). In addition, there was a moderate correlation between MAD2 expression and the degree of malignancy (r=0.51, P<0.05). However, when the samples in group M were classified according to a low or high expression of MAD2, no difference was observed in terms of overall survival. These findings suggest that the overexpression of MAD2 may be correlated to carcinogenesis in mucinous ovarian tumors.

Reference ranges for time-related analysis of ductus venosus flow velocity waveforms in singleton pregnancies.

OBJECTIVES: To investigate time intervals of ductus venosus (DV) flow velocity waveforms (FVW) in correlation to fetal heart rate and gestational age and to construct reference ranges for the second and third trimester. Furthermore, we investigate time intervals of FVW through the tricuspid valve. METHODS: Flow velocity waveforms of the DV and through the tricuspid valve were recorded in 135 normal singleton fetuses between 17 and 38 weeks' gestation. Time intervals for systolic (S) and early diastolic (D) peaks were analyzed regarding acceleration time (acc-S for S, acc-D for D) and deceleration time (dec-S for S, dec-D for D), respectively. Similarly, time intervals for both peaks of right ventricular inflow were analyzed regarding acceleration time (acc-E for E-wave, acc-A for A-wave) and deceleration time (dec-E for E-wave, dec-A for A-wave), respectively. RESULTS: In the DV, acc-D and dec-D increased significantly with gestational age. In tricuspid valve, acc-E and acc-A showed a significant increase with gestational age. All parameters except acc-S showed significant negative correlations with fetal heart rate. CONCLUSION: With advancing gestational age, prolongation of the diastolic phase of DV-FVW and of the E-wave of tricuspid flow was observed, suggesting maturation of ventricular diastolic function. Time-related analysis of Doppler signals of DV-FVW may provide detailed insights into fetal cardiac function.

An upstream estrogen response element linked to exogenous p53 tumor suppressor gene expression differentiates effects of the codon 72 polymorphism.

The objective of this study was to assess the effects of an upstream estrogen response element (ERE) on exogenous p53 tumor suppressor gene with a codon 72 polymorphism about which there have been controversial reports in relation to cancer risk. The p53 gene (bases 166-1143 from start codon) with the codon 72 polymorphism, inserted into the pIRES-hrGFP II plasmid with or without upstream ERE, were transfected into HHUA endometrial cancer cells expressing the estrogen receptor. The ERE-linked p53 gene with the proline variant at codon 72 showed lower transfection rates than the gene without ERE or with the arginine variant at codon 72. p21 expression was significantly higher in HHUA cells transfected with the proline variant gene than in those transfected with the arginine variant gene. We consider that the presence of an upstream ERE promotes the transcriptional effects of the exogenous p53 gene with the proline variant, which strengthens the expression of p21, and results in lower transfection rates through cell cycle inhibition.

Extragastrointestinal stromal tumor originating from the vulva.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. They have gain-of-function mutations of the c-kit receptor tyrosine kinase gene and have been suggested to originate from the interstitial cells of Cajal. A small percentage of GISTs form extragastrointestinal masses. We report a rare case of a GIST originating from the vulva. A 55-year-old woman presented with a vulvar tumor. The tumor was initially diagnosed as a leiomyosarcoma following the first resection. Following a second recurrence the patient was administered chemotherapy. A third recurrence occurred and the patient underwent a third resection. Histology revealed that a bundle of fibrous tumor cells had invaded the connected tissue and muscular coat, and some spindle-shaped and blunt-ended nuclei were detected. Furthermore, immunohistochemical evaluation revealed that the tumor cells exhibited strong and diffuse staining for c-kit and CD34. The recurrent tumor was diagnosed as a GIST and a reevaluation of the original specimens also revealed a GIST. The patient was treated with imatinib, and is currently healthy with no evidence of recurrence at 20 months after the last surgery.

Acceleration time of fetal arterial blood flow velocity waveforms: a preliminary study.

BACKGROUND: Doppler ultrasonography has been widely used for fetal estimation, but most of them were estimated by the resistance and pulsatility indices. Acceleration time is one of parameters of flow velocity waveforms, but only few reports had discussed acceleration time of fetal circulation. METHODS: We analyzed Doppler flow velocity waveforms of fetal middle cerebral artery, descending aorta and abdominal umbilical artery obtained from 70 normal pregnant women between 18 and 40 weeks. Acceleration time was cross-sectionaly examined throughout pregnancy course. RESULTS: The acceleration times revealed no remarkable changes throughout pregnancy course in the middle cerebral artery, but significant increases were observed in the acceleration time of the descending aorta between 18-23 and 24-32 weeks. It also significantly decreased after 33 weeks compared to that of 18-32 weeks in the umbilical artery. CONCLUSIONS: The acceleration time in fetal descending aorta is significantly shorter than that in the abdominal umbilical artery between 18 and 32 weeks of gestation. The umbilical arterial acceleration time apparently decreases throughout pregnancy course. Its values are almost double for that in the descending aorta between 18 and 23 weeks, but it becomes to be equal to the aortic acceleration time. The continuous decrease of the umbilical arterial acceleration time might be a result of a relative increase of placental flow.

Multiple organ failure of tumor-bearing rabbits in cancer cachexia is caused by apoptosis of normal organ cells.

In cancer-bearing animals, we previously demonstrated that skeletal muscle apoptosis may be involved in muscle wasting and that Bax may play a role in skeletal muscle cell apoptosis at an early stage. In this study, we investigated the occurrence of apoptosis in the liver, kidney, spleen, lung and heart during cancer cachexia as well as the associations between apoptosis and the expression levels of Bcl-2, Bcl-xL and Bax proteins. We also examined the relationship between normal organ apoptosis in cancer cachexia and multiple organ failure. We further studied the changes in body weight, lean body mass (LBM), apoptotic index (AI), DNA laddering pattern and expression levels of Bax, Bcl-2 and Bcl-xL in the liver, kidney, spleen, lung and heart in VX2 carcinoma-bearing rabbits. In the early stage of tumor bearing (20 days after implantation), the LBM was significantly reduced by 19.06+/-1.02% in the tumor-bearing group compared to an increase of 1.66+/-0.83% in the control group. The apoptotic indices of the liver, kidney, lung and spleen in the tumor-bearing group increased by 14.2+/-1.8%, 34.4+/-2.0%, 66.7+/-6.0% and 24.8+/-3.8%, respectively and were significantly higher than the corresponding indices in the control group. DNA laddering patterns characteristic of DNA fragmentation were visible on day 50 in the liver, kidney, spleen and lung in the tumor-bearing group. The expression of Bax increased in the tumor-bearing group and coincided with the occurrence of apoptosis. However, no significant changes were noted in the expression levels of Bcl-2 and Bcl-xL. These findings suggest the possibility that normal organ cell apoptosis related to Bax not only causes body weight loss but also multiple organ failure in cancer cachexia.

Uterine artery flow velocity waveforms during uterine contractions: differences between oxytocin-induced contractions and spontaneous labor contractions.

AIM: To clarify the effects on uterine arterial flow velocity waveforms of uterine contractions following oxytocin infusion and during spontaneous labor. METHODS: Uterine arterial flow velocity waveforms were obtained by pulsed Doppler methods from 22 women during an oxytocin challenge test (OCT), 26 women during oxytocin-induced labor, and 40 women during spontaneous labor. Mean resistance index (RI) for bilateral arteries was used for analyses. After the onset of labor, flow velocity waveforms were assessed according to cervical dilatation. During OCT, Doppler flow velocimetry was performed when three uterine contractions occurred per 10-min period. RESULTS: RI values did not differ significantly between induced and spontaneous labor during relaxations at any level of cervical dilatation. However, during contractions, RI was significantly higher for induced labor than for spontaneous labor. Absence or reversal of flow was more frequent in the OCT group than in the induced labor group (P < 0.0001). However, no significant differences were found between spontaneous and induced labor groups. CONCLUSION: Interactions between the contracting uterine body and the relaxing lower segment in oxytocin-induced labor might be associated with differences in uterine arterial flow during contraction between oxytocin-induced and spontaneous labor. However, changes in the intensity of uterine contractions during labor progression might differ between oxytocin-induced and spontaneous labor.

Protein S deposition at placenta: a possible role of protein S other than anticoagulation.

Protein S is an antithrombotic cofactor for protein C that also has multifunctional anti-inflammatory, cellular protective, apoptotic and mitogenic properties. Protein S levels are thought to decrease during pregnancy, but the underlying mechanism remains unknown. We compared protein S concentrations throughout normal pregnancy with those of nonpregnant women and measured plasma C4b-binding protein levels in nonpregnant women and in pregnant women at the 40th gestational week. We also examined protein S and C4b-binding protein in the placenta by immunohistochemical staining at early (20th gestational week) and late (40th gestational week) stages of pregnancy. Plasma protein S activity and free protein S-antigen levels significantly decreased from the 10th gestational week and total protein S antigen decreased from the 20th. C4b-binding protein levels between pregnant and nonpregnant women did not significantly differ. The stainable portion of protein S was located at the fetomaternal interface, particularly at degenerative villi. C4b-binding protein was weakly stained at the same areas as protein S. Neither protein S nor C4b-binding protein were stained at normal villi. These results indicated that protein S can protect or restore damaged villi via a physiological effect in addition to its anticoagulation properties.

Estradiol-17beta regulates vascular endothelial growth factor and Bcl-2 expression in HHUA cells.

HHUA, a rare endometrial cancer cell line expressing the estrogen receptor (ER), was adopted to investigate the expression of vascular endothelial growth factor (VEGF), erythropoietin (Epo), Bcl-2 and p53 under the administration of estradiol-17beta (E2). Based on quantitative real-time reverse transcription polymerase chain reaction assays, both VEGF and Bcl-2 mRNA levels decreased in a dose-dependent manner, although VEGF levels were increased in a time-dependent manner; no significant change was found for Epo and p53. An immunocytochemical study also showed the suppressed expression of VEGF and Bcl-2 under E2 induction. Both ERalpha and ERbeta mRNAs were detected in HHUA cells with ERbeta expression being predominantly higher than that of ERalpha, which is the converse of the pattern seen in normal endometria. The present study shows the E2-downregulated expression of VEGF and Bcl-2, and reveals a disrupted balance of ERalpha and ERbeta expression, which should be taken into consideration to understand the particularity of E2-regulated gene expression in HHUA cells.

Vascular endothelial growth factor, matrix metalloproteinases, and cyclooxygenase-2 influence prognosis of uterine cervical cancer in young women.

Recent changes in the lifestyle of young women have led to an increase in the rate of uterine cervical cancer. We investigated the clinicopathological characteristics of uterine cervical cancer in young women, and examined the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). Tumor samples from 439 patients with uterine cervical cancer, who were initially treated at Osaka City University Medical School Hospital, Japan between 1995 and 2004, were stained immunohistochemically. The patients were classified into two groups according to age at onset: group Y included women aged < or =35 years, and group O included women aged > or =36 years. Group Y had more cases of squamous cell carcinoma, while group O had more advanced cases (P<0.05). Advanced cases (beyond stage Ib2) had a significantly worse prognosis in group Y than in group O (P<0.05). There were no differences between the two groups in the expressions of VEGF, MMP-2 and COX-2. However, in advanced cases (beyond stage Ib2), the expression of VEGF, MMP-2 and COX-2 was significantly greater in group Y than in group O (P<0.05). The above findings suggest that the expression of VEGF, MMPs and COX-2 is related to a worse prognosis for advanced uterine cervical cancer in young women.

Expression of glucose transporters in epithelial ovarian carcinoma: correlation with clinical characteristics and tumor angiogenesis.

We immunohistochemically examined the expression of the glucose transporters (GLUT)1, GLUT3 and GLUT4, in 154 tumor samples of epithelial ovarian carcinoma. In addition, we investigated the correlations between the expression of GLUTs and the vascular endothelial growth factor (VEGF), and microvessel count and clinical parameters. The rates of expression of GLUT1, GLUT3 and GLUT4 were 98.7%, 92.8% and 84.4%, respectively. GLUT1 and GLUT4 were both strongly expressed in serous adenocarcinoma, but weakly expressed in clear cell adenocarcinoma. The expressions of GLUT1 and GLUT4 correlated with the clinical disease stage. The expressions of GLUT1, GLUT3 and GLUT4 correlated positively with VEGF expression. The expression status for GLUT1, GLUT3, GLUT4 and VEGF did not represent a prognostic factor. These findings suggest that characteristic differences in the patterns of glucose uptake can exist according to the histological type and that GLUT1, GLUT3 and GLUT4 could be related to tumor angiogenesis in epithelial ovarian carcinoma.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in uterine endometrial carcinoma and a correlation between expression of matrix metalloproteinase-7 and prognosis.

Matrix metalloproteinases (MMPs) are associated with invasion and metastasis of several human malignant tumors, in particular MMP-7, which is mainly produced by the cancer cell itself. We examined the expression of MMP-2, 7 and 9, and tissue inhibitors of metalloproteinase (TIMP)-1 and 2 in uterine endometrial carcinoma, and compared the expression with clinicopathological characteristics in uterine endometrial carcinoma (UEC). A group of 256 patients with UEC received surgery at the Osaka City University Medical School Hospital, and 196 tumor samples were immunohistochemically stained to examine the expression of MMP-2, 7 and 9, and TIMP-1 and 2. Additionally, the invasion ability of cell stain established from UEC was examined using an in vitro invasion assay. The expression of MMP-2, 7 and 9, and TIMP-1 and 2 was observed in the cytoplasm, and the expression of MMP-2 and 7, and TIMP-1 and 2 was observed in stromal cells around the tumor cells. The expression of MMP-7 was significantly stronger in higher-grade than lower-grade tumors (P<0.05). The invasion assay showed that the invasion of cells derived from UECs was significantly inhibited by TIMP-1 and 2. The disease-free interval was significantly shorter when MMP-7 expression was intense. This increased expression of MMP-7 in high grade UECs may be associated with tumor invasion and metastasis, and MMP-7 could serve as a prognostic maker in UEC.

Multiple roles of Rbx1 in the VBC-Cul2 ubiquitin ligase complex.

The importance of the ubiquitin system largely depends on ubiquitin ligases, E3s, as they determine the specificity of the system. Rbx1/ROC1/Hrt1, a RING finger protein, functions as an important component of the cullin-containing SCF and VBC-Cul2 ligases. Modification of cullins by NEDD8 (NEDDylation), has been shown to be essential for the E3 activity of both SCF and VBC-Cul2, and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation. RING finger is composed of eight cysteine and histidine residues that bind to zinc ions. Rbx1 is a highly evolutionarily conserved protein; however, the eighth coordination residue in its RING finger is aspartate (D97) rather than cysteine. Substitution of D97 with each of the other 19 amino acids demonstrates that aspartate is superior to cysteine in cullin NEDDylation. Interestingly, however, different D97 mutants demonstrate different activities towards 6 cullins tested. Importantly, we were able to discriminate between the NEDDylating activity of Rbx1 and its involvement in the ubiquitylation reaction within the context of VBC-Cul2. Moreover, while Rbx1 is not involved in governing the stability of SCF, Rbx1 mutants destabilize VBC-Cul2. Taken together, these results indicate that various mechanisms regulate both the activities and the stability of cullin-based ligases.

Expression of apoptosis-related proteins in advanced uterine cervical cancer after balloon-occluded arterial infusion chemotherapy as an indicator of the efficiency of this therapy.

We previously reported satisfactory therapeutic results of cisplatin-based cyclic balloon-occluded arterial infusion chemotherapy (BOAI) as neoadjuvant chemotherapy, which enabled treatment by hysterectomy in patients with advanced cervical cancer. We also reported expression of apoptosis among these patients and determined that the bax gene is related to this apoptosis. In the present study, we investigated the relationship between the effectiveness of BOAI therapy and expression of apoptosis regulatory genes and proteins in these cases. The subjects were 27 women with advanced cervical cancer classified as FIGO (International Federation of Gynecology and Obstetrics) stage III or higher who were admitted to the Department of Gynecology, Osaka City University Medical School Hospital between 2000 and 2003. All patients were treated by BOAI, and expression of cancer cell apoptosis was examined by the TUNEL method, expression of bax, bcl-2 and bcl-xL proteins were examined by immunohistochemistry, and expression of bax, bcl-2 and bcl-xL mRNA was examined by quantitative RT-PCR before and 3 days after BOAI. The effectiveness of BOAI therapy was thus determined. The 20 patients in whom BOAI was effective showed significantly higher expression of the bax protein and gene after BOAI, and cancer cell apoptosis was accelerated. On the other hand, the 7 patients in whom BOAI was ineffective showed significantly higher expression of the bcl-xL protein and gene after BOAI. These results suggest that bax/bcl-xL expression can be used as an indicator of the effectiveness of BOAI therapy.

Association of HPV infection with prognosis after neoadjuvant chemotherapy in advanced uterine cervical cancer.

Whether the human papillomavirus (HPV) status of the tumor affects the sensitivity to neoadjuvant chemotherapy, and the prognosis in advanced uterine cervical cancer (FIGO stage III or higher) remains unknown. We examined the HPV status of 43 patients who had received CDDP therapy by balloon-occluded arterial infusion (BOAI), as neoadjuvant chemotherapy for advanced uterine cervical cancer (squamous cell carcinoma) stage III or higher. DNA was extracted from formalin-fixed, paraffin-embedded tumor samples obtained by punch biopsy before the neoadjuvant chemotherapy. The detection of HPV and its typing were analyzed by a polymerase chain reaction (PCR)-based assay using consensus primers for the L1 consensus regions. HPV DNA was detected in all 43 patients (100%): 29 cases with HPV 16 (67.4%), 5 cases with HPV 33 (11.6%), 4 cases with HPV 31 (9.3%), 3 cases with HPV 35 (7.0%), 1 case with HPV 18 (2.3%) and 1 case with HPV 58 (2.3%). The HPV types were divided into 3 groups, HPV 16, HPV 33 and other HPV types (HPV 18, 31, 35, 58), and comparisons and examinations were performed among the 3 groups. Although the rates of tumor reduction and operation accomplishment after 3 courses of BOAI showed no significant differences among the 3 groups, there were significant differences in the survival rates. The survival rate of advanced uterine cervical cancer patients with HPV 33 infection was the highest, followed by that of patients with HPV 16 infection. The survival rates of patients with the other types of HPV infection were the worst among the 3 groups and significantly lower than those of patients with HPV 16 or HPV 33 infection. The differences in the curative effect after BOAI may depend on the different characters of the HPV types.

VEGF mRNA in adipocytes increase with rebound weight-gain after diet-restriction.

The mechanism of rebound body weight-gain after a restricted-diet state is unclear. We investigated the expression of angiogenic factors in human adipocytes with a changing nutritional state in culture medium, and attempted to ascertain the mechanisms involved in rebound weight-gain. Adipocytes were divided into three groups; the first group (control group) was cultured in medium with 10% fetal calf serum (FCS), the second (DR3% group) was cultured in medium with 3% FCS, and the third (DR6% group) was cultured in medium with 6% FCS. After being cultured for 48 h, each was next cultured with 12% FCS for a further 48 h. When made to change from a low nutrition state to a higher one, adipocytes changed from hypotrophic to hypertrophic. Simultaneously, vascular endothelial growth factor (VEGF) in the culture medium increased significantly. When investigated immunohistochemically, the expression of VEGF was similarly shown in the cytoplasm of adipocytes. The same tendency with the same quantity of mRNA was shown by RT-PCR. These results show that VEGF produced and secreted from adipocytes increases, when the cultivation state of adipocytes is changed from a low nutritional state to a higher one. VEGF produced and secreted from adipocytes may be related to rebound weight-gain.