Inhibitory Effects of Anti-VEGF Antibody on the Growth and Angiogenesis of Estrogen-induced Pituitary Prolactinoma in Fischer 344 Rats: Animal Model of VEGF-targeted Therapy for Human Endocrine Tumors

Estrogen-induced pituitary prolactin-producing tumors (PRLoma) in F344 rats express a high level of vascular endothelial growth factor (VEGF) associated with marked angiogenesis and angiectasis. To investigate whether tumor development in E2-induced PRLoma is inhibited by anti-VEGF monoclonal antibody (G6-31), we evaluated tumor growth and observed the vascular structures. With simultaneous treatment with G6-31 for the latter three weeks of the 13-week period of E2 stimulation (E2+G6-31 group), the following inhibitory effects on the PRLoma were observed in the E2+G6-31 group as compared with the E2-only group. In the E2+G6-31 group, a tendency to reduction in pituitary weight was observed and significant differences were observed as (1) reductions in the Ki-67-positive anterior cells, (2) increases in TUNEL-positive anterior cells, and (3) repair of the microvessel count by CD34-immunohistochemistry. The characteristic “blood lakes” in PRLomas were improved and replaced by repaired microvascular structures on 3D observation using confocal laser scanning microscope. These inhibitory effects due to anti-VEGF antibody might be related to the autocrine/paracrine action of VEGF on the tumor cells, because VEGF and its receptor are co-expressed on the tumor cells. Thus, our results demonstrate that anti-VEGF antibody exerted inhibitory effects on pituitary tumorigenesis in well-established E2 induced PRLomas

Differential Expression of Survivin in Mammary Gland Diseases

Objective: Unlike other inhibitors of apoptosis proteins (IAP), survivin is expressed during fetal development, cannot be detected in normal adult tissues, and is re-expressed in most of the common human cancers. However, the clinicopathological significance of the expression of survivin in human breast carcinoma has not been fully elucidated. Therefore, we examined the accumulation of survivin in pathological specimens of mammary gland diseases to identify new protein markers that may lead to improvements in patient management.Methods: We investigated the expression of survivin using immunohistochemistry in 180 cases of breast disease accessioned in the Department of Pathology, International University of Health and Welfare Hospital and Nissan Tamagawa Hospital between 2003 and 2013.Results: Ninety-eight out of 141 malignant tumor cases (69.5%) stained positive for survivin, with no significant staining being detected in the remaining cases. Staining for survivin was completely absent in the epithelial cells of 34 of 39 cases of benign disease. No correlation was observed between the expression of survivin and major prognostic factors in breast carcinomas including patient age, tumor size, histological grade, axillary lymph node metastasis, local recurrence, and visceral metastasis status. However, a significant difference was observed in the expression of survivin between malignant and benign tumors and other benign diseases.Conclusion: The results of the present study suggest that the expression of survivin is strongly selective for cancer cells and may be useful for identifying and quantifying human breast cancer

Immunohistochemical Analysis of Neuroendocrine (NE) Differentiation in Testicular Germ Cell Tumors (GCTs): Use of Confocal Laser Scanning Microscopy (CLSM) to Demonstrate Direct NE Differentiation from GCTs

Neuroendocrine (NE) differentiation is infrequent in testicular tumors and its histogenesis is not well understood. The present study is aimed at elucidating the pathway of neuroendocrine differentiation in germ cell tumors (GCTs) of the testis. In the analysis of 46 germ cell tumor components from 23 testicular tumors, we focused on GCTs with neuroendocrine differentiation, 7 teratoma, 1 embryonal carcinoma and 1 neuroendocrine carcinoma by immunohistochemical study and confocal laser scanning microscopy (CLSM) analysis. NE marker positive cells were noted in the tumor with collision of teratoma and embryonal carcinoma (E&T tumor), in the immature columnar cells of transitional form of embryonal carcinoma to teratoma (E-T cells) and neuroendocrine carcinoma cells, in addition to the well known mature intestinal mucosa in teratoma. Double staining for a NE marker (CGA) and a germ cell marker (PLAP) demonstrated the localization of both proteins in the same E-T cells confirmed by CLSM. Another finding, indicating the intimate relation between embryonal carcinoma and neuroendcrine differentiation, is that neuroendocrine carcinoma expressed a marker of embryonal carcinoma, CD30. The present results indicated that the NE cells might be differentiated from embryonal carcinoma, a view that has not been proposed before, but that is made in the present study using CLSM

The Expression of Wnt4 Is Regulated by Estrogen via an Estrogen Receptor Alpha-dependent Pathway in Rat Pituitary Growth Hormone-producing Cells

Wnt signaling is important in many aspects of cell biology and development. In the mouse female reproductive tract, Wnt4, Wnt5a, and Wnt7a show differential expression during the estrus cycle, suggesting that they participate in female reproductive physiology. Although the pituitary is a major gland regulating reproduction, the molecular mechanism of Wnt signaling here is unclear. We elucidated the subcellular distribution of Wnt4 in the pituitary of estrogen-treated ovariectomized female rats. Expression of Wnt4 mRNA increased dramatically, particularly in proestrus compared with estrus and metestrus. Wnt4 protein was observed in the cytoplasm of almost all growth hormone (GH)-producing cells and in only a few thyroid-stimulating hormone β (TSHβ)-producing cells. In rat GH-producing pituitary tumor (MtT/S) cells, estrogen-induced expression of Wnt4 mRNA was completely inhibited by estrogen receptor antagonist ICI 182,780 in vitro. Thus, rat pituitary GH cells synthesize Wnt4 and this is induced by estrogen mediated via an estrogen receptor alpha-dependent pathway

Sevoflurane Stimulates MAP Kinase Signal transduction through the Activation of PKC α and βII in Fetal Rat Cerebral Cortex Cultured Neuron

Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral ­cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (α, βI, βII, γ, δ, ɛ and ζ) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC α and PKC βII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC α and βII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5–10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10–15 min after sevoflurane treatments. That of ERK proteins was induced at 15–60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the ­localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0.25 mM sevoflurane-treatment. These results demonstrated that sevoflurane induced the phosphorylation of the MAP kinase cascade through the activation of the PKC α and PKC βII species

Neuroendocrine Carcinoma of the Stomach: A Case Study

Gastric neuroendocrine carcinomas are rare and have a poor prognosis, and the diagnostic criteria for this disease have recently changed. We herein report a case of sporadic gastric neuroendocrine carcinoma. A 75-year-old man was referred to our hospital with epigastric pain. Endoscopic examination revealed a localized ulcerative lesion (diameter, 4 cm) at the upper stomach. The diagnosis on biopsy was neuroendocrine carcinoma. Total gastrectomy with D2 lymphadenectomy, splenectomy, and cholecystectomy was performed. Pathologically, the tumor infiltrated the subserosal layer, and 6/49 lymph nodes were involved. The tumor was uniform in shape and arranged in a rosette-like structure to form solid nests, with medium-sized, round-to-cuboid-shaped tumor cells and intense mitosis 46/10 HPF. It was positive for synaptophysin and chromogranin A, and the Ki-67 labeling index was 70–80%. The diagnosis of neuroendocrine carcinoma was made according to the WHO 2010 criteria. The patient was followed up for three years without recurrence

Treatment Strategy for Recurrent and Refractory Epithelial Ovarian Cancer: Efficacy of High-Dose Chemotherapy with Hematopoietic Stem Cell Transplantation

According to population statistics in Japan, approximately 3,800 women die of ovarian ­cancer annually, and approximately 6,000 are affected by this disease. Ovarian cancer is ­referred to as a “silent tumor”, since patients have few subjective symptoms and by the time symptoms are observed, the cancer has progressed to Stage III or IV in about half of the patients. The basic treatment for advanced epithelial ovarian cancer is to remove as much of the tumor as possible, and subsequently to perform anticancer therapy using drugs such as cisplatin, carboplatin and paclitaxel, all of which have been shown to be effective for epi­thelial ovarian cancer. However, the 5-year survival rate in advanced ovarian cancer patients is still only about 20%, and a treatment that leads to long-term survival has yet to be developed. Here, we review the available treatments for ovarian cancer, and present the results of high-dose chemotherapy (HDC) performed in our hospital for recurrent and refractory ­ovarian cancer

Analysis of mTOR Inhibition-Involved Pathway in Ovarian Clear Cell Adenocarcinoma

This study was designed to clarify the mechanism of the mammalian target of rapamycin (mTOR)-hypoxia inducible factor-1 (HIF-1) pathway using the cultured cell strain derived from human ovarian clear cell adenocarcinoma (CCA). Everolimus (a derivative of rapamycin)-treated cells and non-treated cells did not show any difference in mTOR expression. But, phosphorylated-mTOR (p-mTOR) expression significantly decreased in the treated cells, and mTOR-related factors such as phosphorylated-4E-BP1 (p-4E-BP1), HIF-1α, and vascular endothelial growth factor (VEGF) in the downstream region of mTOR revealed a marked decrease in expression. The analysis of influences of the drug on the HIF-1α degradation system showed an increase in von-Hippel Lindau (VHL) expression in the treated cells. Increase of cleaved caspase-3, one of key factors involved in apoptosis, was also shown in the treated cells. In the next step, using nude mice implanted with RMG-1 cells, a decrease in tumor size was demonstrated in 4 of the 7 mice which were orally administered with everolimus. As a result, it was suggested that everolimus administration would be helpful as an anti-tumor therapy for CCA not only via down-regulation of p-mTOR but also degradation of HIF-1α by VHL and induction of apoptosis by cleaved caspase-3

An Up-to-Date Anti-Cancer Treatment Strategy Focusing on HIF-1α Suppression: Its Application for Refractory Ovarian Cancer

Hypoxia inducible factor-1α (HIF-1α) predominantly determines the transcriptional activity of HIF-1, which induces the certain genetic expressions to participate in the proliferation and progression of the tumor. It is supposed that HIF-1α is also an extremely important factor in cancer treatment. Based on the results of our recent analyses using ovarian tumors, which indicated the close association of HIF-1α expression with the acquisition of malignancy and the characterization of histology, we further investigated the possibility of a new strategy of cancer therapy that targeted HIF-1α inhibition in the ovarian carcinoma. The cell line HUOCA-II, which originates from the refractory ovarian clear cell adenocarcinoma, was treated with rapamycin. The inhibitory effect of HIF-1α was analyzed by immunohistochemistry and western blotting. It was demonstrated that inhibition of HIF-1α and vascular endothelial growth factor (VEGF) expressions would lead to the down-regulation of tumor cell proliferation. Interestingly, there was little or no change in GLUT-1 expression by rapamycin administration. Thus, the inhibition of GLUT-1 may also be a key for the new strategy of cancer therapy as well as HIF-1α and VEGF